Redox Proteomics - American Chemical Society

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Redox Proteomics: Chemical Principles, Methodological Approaches and Biological/Biomedical Promises Angela Bachi,§ Isabella Dalle-Donne,† and Andrea Scaloni*,‡ §

Biological Mass Spectrometry Unit, San Raffaele Scientific Institute, 20132 Milan, Italy Department of Biosciences, University of Milan, 20133 Milan, Italy ‡ Proteomics & Mass Spectrometry Laboratory, ISPAAM, National Research Council, 80147 Naples, Italy †

4.2.4. Analysis of Protein Adducts Resulting from S-Nitrosylation 4.2.5. The OxMRM Method 4.3. Analysis of Protein Adducts Resulting from ROS/RNS-Induced Modifications at Methionine 4.4. Analysis of Protein Carbonylation Products 4.4.1. Analysis of Protein Adducts Resulting from Oxidation at Lysine, Arginine, Threonine, and Proline 4.4.2. Analysis of Adducts Resulting from Protein Reaction with Lipooxidation End-Products 4.4.3. Analysis of Protein Glycooxidation EndProducts 4.5. Analysis of Protein Adducts Resulting from ROS/RNS-Induced Modifications at Tyrosine 4.6. Analysis of Protein Adducts Resulting from ROS/RNS-Induced Modifications at Tryptophan and Histidine 4.6.1. Analysis of Protein Adducts Resulting from Tryptophan Oxidation 4.6.2. Analysis of Protein Adducts Resulting from Tryptophan Nitration 4.6.3. Analysis of Protein Adducts Resulting from Tryptophan Halogenation 4.6.4. Analysis of Protein Adducts Resulting from Histidine Oxidation 5. Concluding Remarks and Future Perspectives Author Information Corresponding Author Notes Biographies Acknowledgments References

CONTENTS 1. Introduction 2. Reactive Oxygen and Nitrogen Species 2.1. Exogenous and Endogenous Cellular Sources of ROS and RNS 2.1.1. Superoxide Anion 2.1.2. Hydrogen Peroxide and Hydroxyl Radical 2.1.3. Nitric Oxide 2.1.4. Peroxynitrite 2.1.5. Derivatives from Lipid Peroxidation 2.2. Molecular Systems Involved in the Maintenance of Cell Redox Homeostasis 2.3. Biological Effects Associated with ROS/RNS Activity 2.4. Oxidative/Nitrosative Damage and Pathological Aspects 3. Proteins as Targets of ROS/RNS Activity 3.1. Cysteine Susceptibility to Redox Reactions 3.1.1. Protein S-Glutathionylation 3.1.2. Protein S-Nitrosylation 3.2. Methionine Sulfoxidation 3.3. Protein Carbonylation 3.4. Tryptophan and Histidine Oxidation 3.5. Tyrosine and Tryptophan Nitration 3.6. Tyrosine Halogenation 4. Proteomic Analysis of Oxidized, Nitrosylated, and Nitrated Proteins 4.1. Proteomic Methods 4.2. Analysis of Protein Adducts Resulting from ROS/RNS-Induced Modifications at Cysteine 4.2.1. Analysis of Protein Adducts Resulting from Disulfide Bond Formation 4.2.2. Analysis of Protein Adducts Resulting from S-Glutathionylation 4.2.3. Analysis of Protein Adducts Resulting from Cysteine Oxidation

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1. INTRODUCTION Inevitable byproducts of aerobic metabolism are free radicals and other highly reactive oxidants and nitrosants that are continuously formed within cells. Most cells generally function in a reduced state, though they can produce physiological low (i.e., subtoxic) amounts of selected reactive oxygen species (ROS) and reactive nitrogen species (RNS) in a regulated way and through different mechanisms, also using some of them as

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dx.doi.org/10.1021/cr300073p | Chem. Rev. XXXX, XXX, XXX−XXX

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Table 1. Main Types of Reactive Oxygen (ROS) and Nitrogen (RNS) Species ROS nonradicals hydrogen peroxide (H2O2) hypochlorous acid (HOCl) hypobromous acid (HOBr) ozone (O3) singlet oxygen (O21Δg) organic peroxides (ROOH) lipid peroxides (LOOH) radicals alkoxyl (RO•) hydroperoxyl (HO2•) hydroxyl (HO•) peroxyl (RO2•) lipid peroxyl (LO2•) superoxide (O2•−) carbonate (CO3•−) carbon dioxide (CO2•−) singlet oxygen (O2 1Σg+)

RNS nonradicals alkyl peroxynitrites (ROONO) alkyl peroxynitrates (RO2ONO) dinitrogen tetroxide (N2O4) dinitrogen trioxide (N2O3) nitrosyl cation (NO+) nitrous acid (HNO2) nitroxyl anion (NO−) peroxynitrite (ONOO−) nitrite (NO2−) nitrate (NO3−) peroxynitrous acid (ONOOH) nitrosoperoxycarbonate (ONOOCO2−) radicals nitrogen monoxide or nitric oxide (NO•) nitrogen dioxide (NO2•)

signaling molecules in many intra- or intercellular communication processes (redox signaling).1 On the other hand, excessive production of ROS and RNS, impairment of cellular antioxidant defense(s), or an imbalance between ROS/RNS and antioxidants, that is ROS or RNS concentrations exceeding the antioxidant capacity of the cell, can lead to a common pathophysiological situation termed oxidative stress. This cellular condition with altered oxidation-reduction (redox) homeostasis may determine oxidative modification of cellular macromolecules, modify their function, and eventually promote cell death. Such a limiting definition of oxidative stress, formulated in 1985 as a global imbalance of prooxidants and antioxidants,2 has been recently updated as “an imbalance between oxidants and antioxidants in favor of the oxidants, leading to a disruption of redox signaling and control and/or molecular damage”.3 Molecular identity of each ROS or RNS is often critical in determining both its chemical reactivity and its biological action in living cells. Similarly, subcellular location or microenvironment where a particular ROS or RNS is generated can determine what targets it will potentially encounter spatially and temporally. Because the sources of most ROS and RNS are localized into specific intracellular regions, ROS/RNS fluxes may not be homogeneous in concentration throughout the cell, and therefore, the amounts of ROS and RNS near a source of generation can reach a local plateau. ROS and RNS can be produced by several intracellular pathways; major processes occur in mitochondria in all eukaryotic cells as well as in chloroplasts in plant photosynthetic cells. In addition, ROS/ RNS production can be induced by a number of extracellular stimuli, including osmotic stress, UV irradiation, chemotherapeutic drugs, heavy metals, and ischemia/reperfusion.4−8 Thus, ROS and RNS have a wide variety of both physiological and toxic effects on cells. The balance between physiological functions and damage is determined by the relative rates of ROS/RNS formation and removal. Normally, ROS and RNS are removed rapidly before they cause cell dysfunction and eventually death. By contrast, oxidative stress has been shown to be involved in numerous pathological conditions, such as cancer, neurodegenerative diseases, diabetes mellitus, and atherosclerosis,6,8−11 as well as in aging.7,12

ROS, RNS, and other reactive species may determine various forms of reversible or irreversible oxidative modification to nucleic acids, carbohydrates, lipids, and proteins, which eventually can lead to loss of molecular or cellular functions. To counteract oxidative stress, organisms evolved enzymatic and nonenzymatic response machineries that are designed to remove ROS and RNS directly or to repair oxidative damage.13 In this way, ROS and RNS levels are buffered to ensure intracellular redox homeostasis, while avoiding the excessive oxidation of cellular components. Key players in these systems usually are proteins with redox-active amino acids, the side chains of which can directly react with oxidants or oxidized cellular products. Among such residues, the most commonly used is cysteine, which undergoes a variety of regulatory posttranslational modifications (PTMs).14−16 Cell adaptive responses to oxidative stress are often mediated by redox-sensitive transcription factors, whose redox-active cysteine residues are targets for redox PTMs15 that lead to protein structural and functional changes; thus, these modifications may have eventual consequences on the expression of several genes, some of which encode for enzymes directly involved in ROS/RNS scavenging.17 For instance, transcription factors such as Yap1 in Saccharomyces cerevisiae18 and FoxO4 in mammals19 can detect H2O2 through reversible cysteine oxidation (yielding disulfide bond formation) and activate genes associated with redox regulation. Therefore, ROS/RNS themselves can induce mechanisms controlling their concentration at the cellular level. Sensing is not always mediated by cysteine residues; ROS and RNS can also be sensed by metal centers. For example, Bacillus subtilis PerR senses H2O2 by iron-catalyzed histidine oxidation,20 Escherichia coli SoxR senses superoxide anion (O2•−) by oxidation at a [2Fe−2S] center,21 and E. coli NorR senses nitric oxide (NO•) by S-nitrosylation of a non-heme Fe center.22 Identification of essential ROS- and RNS-modified proteins determining loss of cell functions and, ultimately, viability remains a crucial goal. Oxidized proteins may also gain a toxic function through the formation of cytotoxic aggregates, as observed in various age-related neurodegenerative disorders.23−28 In this study, we overview proteomic techniques geared toward characterizing ROS/RNS-induced protein B


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2.1. Exogenous and Endogenous Cellular Sources of ROS and RNS

modifications involved in the regulation of a variety of cellular physiological processes, through redox-regulated signaling pathways, as well as in the pathogenesis and/or progression of diseases associated with oxidative stress. This wellconsolidated branch of proteomics, also known as redox proteomics, is aimed at identifying on holistic basis the polypeptide targets of ROS/RNS activity, their modified amino acids, how these modifications may control protein function and, by extension, the cellular pathways of which they are a part. The burgeoning field of redox proteomics is now providing a global perspective on the quantitative evaluation of protein oxidation in normal, disease-affected, genetically engineered, or chemically stressed cells.9,29−34 Nowadays, correlation between protein oxidation and human diseases is widely appreciated, although unraveling cause and effect remains a challenge. In the past few years, combined nanoliquid chromatography (nLC) and electrophoretic protein/peptide separation techniques, mass spectrometry (MS), and affinitybased methodologies have contributed in a significant way to provide a better understanding of the protein oxidative modifications present in prokaryotic and eukaryotic cells/ organisms, as well as in various biological specimens under different pathophysiological conditions.9,29−41 The main aim of this review is to provide a comprehensive picture of the most promising chemical metodologies used for identifying and characterizing all types of oxidative or nitrosative protein modifications, also highlighting the methodological limits and pitfalls emerging from current redox proteomic studies.

ROS are produced by a great variety of mechanisms and originate from many sources. Exogenous sources include O2, O3 (a powerful oxidizing agent that modifies biomolecules directly), ionizing and nonionizing radiations, air pollutants, cigarette smoke, industrial contaminants, pathogens, and food, which may contain various oxidants including peroxides, aldehydes, oxidized fatty acids, and transition metals. ROS generation occurs because the complete reduction of O2 into two H2O molecules requires four electrons but O2 accepts only one electron at a time (Figure 1A). Addition of the

Figure 1. Production of ROS. (A) The electronic structure of O2 favors its reduction by addition of one electron at a time, leading to the generation of oxygen radicals causing cellular damage. The stepwise transfer of electrons to O2 determines the formation of O2•−, then H2O2, and finally OH•. (B) The Haber−Weiss reaction and (C) the Fenton reaction for the formation of toxic OH• are also shown.

2. REACTIVE OXYGEN AND NITROGEN SPECIES ROS and RNS may be basically divided into two groups depending on their chemical nature (Table 1): (i) radical compounds, among which representative examples are superoxide ion (O2•−), hydroxyl (OH•), nitric oxide (NO•), peroxyl (RO2•), and alkoxyl (RO•) radicals, and one form of singlet oxygen (O2 1Σg+); (ii) nonradical compounds comprising a large variety of substances, such as hydrogen peroxide (H2O2), organic peroxides, aldehydes, ozone (O3), molecular oxygen (O2), and hypochlorous acid (HOCl). These compounds have distinct chemical properties, which include reactivity, half-life, and solvent solubility,6 as exemplified in Table 2. For instance,

first electron to O2 yields O2•−, which is converted into H2O2 when one additional electron is added. Then, electron exchange between O2•− and H2O2, via the Haber−Weiss reaction (Figure 1B), generates OH•, which is also produced by the reduction of H2O2 in the presence of endogenous Fe2+ or Cu+ by means of the Fenton reaction (Figure 1C). Three main endogenous cellular systems can generate ROS and RNS (Figure 2A): (i) the leakage of activated oxygen from the respiratory chain within the inner mitochondrial membrane during oxidative phosphorylation;42 (ii) monoamine oxidase (MAO)-catalyzed production of H2O2 during oxidative deamination of biogenic amines within the outer mitochondrial membrane;42 (iii) oxygen-metabolizing enzymatic reactions catalyzed by xanthine oxidase (XO), cytochromes P450, NADPH oxidases (NOXs), dual oxidases (DUOXs), myeloperoxidase (MPO), eosinophil peroxidase (EPO), lipoxygenases, and nitric oxide synthases (NOSs). Expression of multiple isoforms of these enzymes in a variety of tissues and cells provides evidence that the deliberate production of low levels of oxidants is a key feature of many cells.43,44 On the other hand, metals, due to their capacity to lose electrons, are thought to be primarily toxic by virtue of their potential to generate ROS. Thus, exposure to high concentrations of heavy metals can result in ROS accumulation and, potentially, oxidative damage.4 Among the main ROS/RNS-generating enzymatic systems, the NADPH oxidase (NOX) family of enzymes produces O2•− in a highly regulated manner by catalyzing electron transfer from NADPH onto molecular O2. The NOX family includes seven members (NOX1−5 and DUOX1 and 2) having different tissue distribution and cell-type-specific subcellular localization.43 NOXs appear to be the only enzymes whose primary function is ROS generation, mainly for phagocytic

Table 2. Estimated Half-Life of Some ROS and RNS in Chemical and Biological Environments species •

HO RO• CO3•− NO2• ONOO− NO• 1 O2 RO2• H2O2, HOCl

T1/2 (s) 1 mM); thus, nitrosoperoxocarbonate (ONO2CO2−) may be the most physiologically relevant nitrating agent. However, it is very short-lived (109 M−1 s−1) of SODs in the cytosol, mitochondria, and extracellular compartments further limit O2•− activity within a very short distance from its generation site. Its instability and inability to diffuse through membranes make this ROS a poor signaling molecule. O2•− oxidizes [Fe− S] clusters at a rate that is almost diffusion-limited and releases iron. O2•− can also react with thiols in vitro, oxidizing them to thiyl radical (RS•), but the slow reaction rates observed suggest that these processes cannot occur in vivo.68 2.1.2. Hydrogen Peroxide and Hydroxyl Radical. Dismutation of O2•−, both inside and outside cells, produces H2O2, and this process can occur spontaneously or enzymatically through the action of SODs;6 as a matter of fact, O2•− is the immediate precursor of most of H2O2 produced by cells. Additional sources of H2O2 are two NADPH oxidases, DUOX 1 and 2, which catalyze two-electron reduction of oxygen,43 and a number of flavoprotein enzymes involved in metabolism directly generating H2O2, such as p66Shc and amine oxidase (in mitochondria), peroxisomal oxidases (in peroxisomes), sulfhydryl oxidase Ero1 and quiescin-sulfhydryl oxidase isozymes (in ER), and amino acid oxidases, cyclooxygenase, lipid oxygenase, and xanthine oxidase (in the cytosol). Notably, H2O2 is the only species among ROS that is generated by several specific enzymes, which suggests that its intracellular concentration is tightly regulated and may serve specific cellular functions.69 The finding that p66Shc functions as a redox enzyme, which generates mitochondrial H2O2 (accounting for ∼30% of the total pool of intracellular H2O2) to trigger mitochondrial swelling and apoptosis,67 provides evidence that H2O2 generation by mitochondria is not just the byproduct of respiration. For this function, p66Shc uses reducing equivalents of the mitochondrial ETC through the direct oxidation of cytochrome c. H2O2 is an uncharged, nonradical, and weak oxidant molecule with a relatively long half-life (cellular half-life ∼1 ms, 10−5 s, steady-state levels ∼10−7 M);69 these properties permit its diffusion within cells and across cellular membranes.6 However, recent evidence suggests that some mammalian and plant membranes are poorly permeable to H2O2 and that its transport may be regulated by aquaporins, a class of membranespanning proteins facilitating the diffusion of water and other substrates with varying specificity.70 A further study has demonstrated that some, but not all, aquaporins can facilitate the uptake of H2O2 in mammalian cells. In particular, aquaporin-3, which is widely expressed in many mammalian tissues, can facilitate the cellular uptake of H2O2 and modulate downstream intracellular signaling pathways that rely on it as a physiological messenger.71 Regardless of specific cellular sources, H2O2 diffuses into the cytoplasm where it can induce distinct physiological responses including proliferation, differentiation, and apoptosis.69,72 However, physiological targets for H2O2 must be close to the site of its generation, due to the

Lipid peroxidation is both an enzymatic and nonenzymatic process that yields multiple aldehyde and radical species with oxidative pathophysiological effects. It plays an amplifying role in the generation of radicals via oxygen-dependent propagation chain reactions; it can be initiated by either OH• or NO2•. Lipid peroxidation occurs when such free radicals withdrawn an electron from polyunsaturated fatty acids (PUFAs) (particularly sensitive to this reaction); this determines a chain reaction generating lipid peroxyl radicals (LO2•) and primary free radical intermediates and ultimately results in reactive aldehydes (Figure 4). Specifically, lipid peroxidation proceeds by three distinct mechanisms: (i) free radical-mediated oxidation; (ii) free radical-independent, nonenzymatic oxidation; (iii) enzymatic oxidation catalyzed by lipoxygenase, cyclooxygenase (COX), and cytochrome P450 family enzymes.63 Each oxidation mechanism yields specific products. Free radicalmediated lipid peroxidation proceeds by a chain mechanism, where one initiating free radical oxidizes many lipid molecules. Hydroxyl radicals (OH•) may be formed through the reaction of H2O2 and Fe2+ (Fenton reaction), from ONOO−, or by high energy irradiation. Alkoxyl radicals (RO•) may be formed during decomposition of hydroperoxides by metal ions, such as those present in heme and copper proteins (Haber−Weiss reaction).63 Metals may contribute in chain initiation directly as well.6 O2•− and NO• are produced from NOX, XO, and NOS enzymes. Neither of them is active enough per se to initiate lipid peroxidation directly, but they react quite rapidly at a diffusioncontrolled rate to give ONOO−, which may initiate lipid peroxidation chain reactions.50 Chain propagation is carried by LO2•, independently of the type of chain-initiating free radicals. The most important chain propagation step is the abstraction of bisallylic H from lipids by LO2• to give conjugated diene lipid hydroperoxide and new lipid radicals, which continues another chain reaction. Hydroperoxides are formed as the primary products in lipid peroxidation. Lipid hydroperoxides are not stable end-products of lipid peroxidation. They are also good substrates for several enzymes, such as GPx and phospholipases, and may generate a number of α,β-unsaturated aldehyde or ketone derivatives. These α,β-unsaturated carbonyl species readily react with proteins, DNA, and phospholipids to cause deleterious effects. Some aldehydes can also function as messengers that activate or inhibit signaling pathways under certain pathophysiological conditions.64,65 The most important biologically relevant ROS and RNS, a number of which are produced according to the simplified schemes reported in Figures 2B, 3 and 4, will be discussed in detail in the following sections. 2.1.1. Superoxide Anion. O2•− is produced by the addition of a single electron to ground state oxygen in a variety of intracellular locations. Its intracellular generation is mediated by enzymes, such as NADPH oxidases and XO, or nonenzymatically by redox-reactive compounds, such as the semiubiquinone compound of the mitochondrial ETC, or by auto-oxidation reactions. Main sites of O2•− production in the mitochondria are complexes I (NADH dehydrogenase) and III (ubiquinone oxoreductase) of the ETC.46 Approximately 1% of all oxygen consumption in mitochondria is diverted to the generation of O2•−. However, this percentage varies depending on the cell type and on the respiration steady state. Recent predictions estimate that, under physiological conditions, O2•− production is around 0.1% of the respiratory rate.66 If the ETC becomes uncoupled, a significant increase in O2•− production also occurs. Cellular signals can also stimulate O2•− generation G


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abundant presence and high catalytic rates of Prxs (located in peroxisomes, mitochondria, and the cytosol)73 and GPxs (localized in the cytosol and mitochondria).74 Prxs are primarily thought to control and regulate the intracellular accumulation of H2O2 and its function as second messenger, thus contributing to redox signaling.75 H2O2 is actually a poor oxidant and reacts mildly with [Fe− S] clusters (rate constant of 102−103 M−1 s−1), loosely with bound metals (103−104 M−1 s−1), and very slowly with GSH, free cysteine (2−20 M−1 s−1), and methionine residues (10−2 M−1 s−1).13,68 H2O2 does not react rapidly with protein thiols, but the reaction rate increases markedly when they are in the thiolate (RS−) form, the product of reaction being a sulfenate anion (RSO−). Its selective reactivity and diffusibility makes H2O2 fit for signaling. Thus, H2O2 reacts with many different cellular molecules and can activate a variety of signaling pathways. Collectively, these properties make H2O2 one of the most important signaling molecule within cells.72,76 H2O2 may be converted either into water by CAT, GPx, or Prx enzymes or, in the presence of reduced transition metal ions (Fe2+ or Cu+), into OH• by means of the Fenton reaction.77 OH• may also arise from electron exchange between O2•− and H2O2 via the Haber−Weiss reaction (Figure 1B). The high reactivity at nearly the rate of diffusion of OH• with almost any organic molecule and its extremely short physiological halflife (about 10−9 s) restrict its damage to a small distance from its origin (the diffusion distance for OH• is about the same diameter as a small protein). For this reason, organisms have not probably developed specific enzymatic systems for its detoxification. OH• can thus react with practically any biological molecules in the near vicinity; for example, it determines H abstraction from unsaturated fatty acids leaving behind carbon-centered radicals. The prevention of its production is the most efficient way to protect cells against its deleterious effects. The lack of specificity makes OH• useless as a second messenger, whereas it is clearly involved in pathological processes. 2.1.3. Nitric Oxide. NO• is primarily synthesized during the oxidative conversion of L-arginine to L-citrulline, which is catalyzed by three different mammalian types of NO synthases (NOSs) that are constitutive endothelial (eNOS/NOS3), neuronal (nNOS/NOS1), and inducible (iNOS/NOS2) enzymes, all of which requiring NADPH and O 2 as cosubstrates. A fourth mitochondrial NOS was also described.78 In addition, NO• can be produced by other redox enzymes, such as XO.79 NO• is a lipophilic diatomic gas with a relatively small Stoke’s radius and a biological half-life of 1−30 s;80 this, in combination with a neutral charge, facilitates its rapid diffusion within membranes. NO• crosses cell membranes without channel or receptor need, as readily as O2 and CO2. Because NO• is freely permeable to membranes, it will repeatedly diffuse in as well as out of a cell over the time span of a second.50 The presence of an unpaired electron in NO• supports its high reactivity with O2, O2•−, and transition metals in heme or cobalamine, with non-heme iron, or with reactive radicals, such as hydroperoxide radicals formed during lipid peroxidation.81 In contrast, NO• does not react with thiols or other nucleophiles directly but requires activation with O2•− to generate RNS, such as ONOO−, NO2•, and N2O3. Specifically, O2•− and NO• combine spontaneously to form ONOO− by a diffusion-limited reaction; no enzyme is required to catalyze this process. NO• is the only known biological molecule that reacts faster with O2•−

and is produced in high enough concentrations to outcompete endogenous SOD levels.51 Consequently, the kinetics and thermodynamics of this reaction make ONOO− formation inevitable in vivo.50 Actually, NO• is a weak oxidant and, by itself, is hardly capable of inducing oxidative stress. In the extracellular milieu, NO• reacts with O2 and H2O to form nitrate and nitrite anions. NO• is rapidly removed by its swift diffusion through tissues into red blood cells, where it is rapidly converted to nitrate by reaction with oxyhemoglobin.82 In various organisms, NO• is known to have many intra- and intercellular signaling functions under physiological conditions, such as autoimmunity, innate cell immune response to pathogens, vascular smooth muscle relaxation, neurotransmission (in mammals),50,83 defense signaling against microbial pathogens, regulation of growth, development and cell death, modulation of hormonal response, control of flowering or stomatal closure (in plants).84,85 In addition to important and beneficial physiological functions, NO• can also cause cellular damage through a phenomenon known as nitrosative stress. Substantial evidence indicates that NO• plays a key role in most common neurodegenerative diseases, including Parkinson’s disease (PD), Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), and ischemic brain injury (stroke).83 Although the mechanisms of NO•-mediated neurodegeneration remain uncertain, NO• has been shown to modify protein function by S-nitrosylation and nitration, both in animal models of specific human neurodegenerative diseases and in affected patients thereof.83 Moreover, NO• may reversibly inhibit enzymes with transition metals or with free radical intermediates in their catalytic cycle, such as CAT and cytochrome P450.50 2.1.4. Peroxynitrite. NO• can readily react (at a diffusioncontrolled rate) with O2•− to form the highly noxious and strong oxidizing agent ONOO−, which reacts with lipids, DNA, and proteins through direct oxidative reactions or via indirect, radical-mediated mechanisms.50 Peroxynitrite and its protonated form, ONOOH, can exert direct oxidative modifications through one- or two-electron oxidation processes, reacting with only a few chemical groups, which favors selective modification with key moieties in proteins, such as thiols, [Fe−S] centers, and zinc fingers. Peroxynitrite also exerts deleterious effects in biological systems by causing DNA oxidation and fragmentation, lipid peroxidation and nitration (in membranes, liposomes, and lipoproteins), nitration of protein aromatic amino acids, forming 3-nitrotyrosine (NO2-Tyr), and depletion of thiol groups in cells.50,77 The latter modification may alter redox signaling and play an important role in numerous cell signal trasduction pathways.86 Specifically, the direct secondorder reaction of ONOO− with thiols (particularly with RS− form) results in the formation of an intermediate sulfenic acid (RSOH), which then reacts with another thiol, forming a disulfide (RSSR).87 Thiols may also be oxidized by the radicals formed from peroxynitrite, generating RS•. Thiyl radicals may then react either with O2 promoting oxidative stress by propagating free radical reactions, or with NO• to form nitrosothiols. Oxidation of critical cysteines by ONOO− usually inactivates enzymes and proteins;50 in some instances, however, it may result in enzyme activation instead of inhibition, as demonstrated for some matrix metalloproteases.88 In addition to protein-bound thiols, ONOO− can directly oxidize lowmolecular-weight thiols, mainly GSH, which thereby serves as an efficient endogenous scavenger of ONOO− and plays a major role in the cellular defense against this RNS. Accordingly, H


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Figure 5. Formation of electrophilic prostaglandins and isoprostanes. Electrophilic eicosanoids can arise by nonenzymatic transformations of arachidonic acid or of its products. Some prostaglandins (PGs) synthesized via the COX pathway (left) can be spontaneously dehydrated giving rise to cyclopentenone PGs of the J series (derived from PGD2) or of the A series (derived from PGE2). Isoprostanes (IsoPs) (right) are formed by nonenzymatic free radical mediated oxidation of esterified arachidonic acid, which can give rise to A- or J-series products as well. The scheme depicts a very simplified view where only some of the possible products are shown. Adapted with permission from ref 1045. Copyright 2011 Elsevier.

the susceptibility of cells to ONOO− toxicity largely depends on the amount of intracellular GSH. The ability of ONOO− to nitrate tyrosine residues can either inhibit or activate signaling processes that depend on tyrosine phosphorylation.50 Peroxynitrite formation also reduces the bioavailability of both O2•− and NO•, which could also influence cell signaling events.6,50 About 15% of O2•− produced by mitochondria goes toward the formation of ONOO−, the remaining forming H2O2.83 Peroxynitrite is a transient species with a biological half-life of 10−20 ms. At physiological pH, ONOO− is in fast, dynamic equilibrium with its conjugated acid, ONOOH, having a pKa of 6.8, which may either rapidly undergo homolytic cleavage to yield the very reactive radicals NO2• and OH•89 or isomerize to nitrate radical (NO3•). Another key subcellular compartment where ONOO− is generated is the phagosome, during phagocytosis of invading microorganisms by neutrophils or macrophages through the assembly and activation of NOX2, which generates O2•− in the lumen of the vacuole. In immune cells previously exposed to cytokine stimulation, expression of iNOS will yield large levels of NO• that will diffuse, reach the phagosome, and subsequently react with O2•−, thus generating ONOO−; therefore, the latter molecule will act as a cytotoxic effector, reacting with the plasma membrane and intracellular components of the pathogen.90 Under biological conditions, ONOO− reacts rapidly with CO2 to form nitrosoperoxycarbonate (CO2−OONO−), which shortens its lifetime to 8.0; because of the reducing environment of the cytoplasm, they therefore remain essentially in the protonated state at physiological pH and are largely not sensitive to redox reactions. Conversely, redox-sensitive proteins have one or more specific cysteine residues localized in a basic environment, being flanked by basic amino acids within the tertiary or the quaternary structure, which lowers perceptibly their pKa values, causing them to be in the deprotonated, highly reactive RS− form at physiological pH. Additional factors eventually reducing cysteine pKa value are helix−dipole effects and hydrogen bonding with serine or histidine residues.160 Thus, the thiolate form renders proteins susceptible targets for a variety of reversible oxidative/nitrosative modifications, yielding cysteine derivatives containing the -SOH moiety, intramolecular (PSSP) or intermolecular (PSSP′) disulfide bonds, mixed disulfide bonds with low molecular mass nonprotein thiols (principally with GSH and free cysteine, PSSG and PSCys, respectively), or protein S-nitrosothiols (PSNO) (Figure 7).161−163 Once formed, -SOH can further undergo irreversible oxidation to -SO2H and -SO3H or additional reactions with other protein or nonprotein thiols to generate intra- or intermolecular disulfides (PSSP, PSSP′, PSSG, and PSCys).104,164 Indeed, -SNO can hydrolyze to form -SOH or react with a second thiol forming the disulfides mentioned above.105 Cysteine oxidation may protect protein catalytic residues from irreversible damage due to severe oxidative stress or act as a switching device in dynamic regulation of protein structure or

Figure 7. Oxidative modifications at protein thiols. Cysteine thiol group is susceptible to several oxidative modifications, generating cysteinyl radical (PS•, not shown) or sulfenic (PSOH) (reaction a), sulfinic (PSO2H) (reaction b), or sulfonic (PSO3H) (reaction c) acids (the latter being irreversible). Alternatively, oxidation can determine the formation of a disulfide bridge (cystine) between two adjacent sulfhydryl groups within different proteins (intermolecular cystine) (reaction d) or within the same protein (intramolecular cystine) (reaction d′), causing changes in protein aggregation and conformation. Reaction between protein thiols and low-molecular-mass thiols, such as GSH and free cysteine, can yield S-glutathionylated (reaction e) or S-cysteinylated (reaction f) proteins. Cysteines found in consensus motifs as adjacent to basic, acidic, or aromatic residues can serve as sites of S-nitrosylation, which can be mediated by NO•, Snitrosothiols, or several higher N-oxides (reaction g), or catalyzed by transition metals. Reprinted with permission from ref 102. Copyright 2009 Elsevier.

activity. In both cases, a key element of these processes is reversibility that is ensured in vivo by thiol−disulfide oxidoreductases, such as the Trx or Grx systems, which quickly restore the original redox state upon cell return into a nonstressing condition, guaranteeing the transient nature of N


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Figure 8. Protein S-glutathionylation and deglutathionylation. (A) Reactions for the formation of S-glutathionylated proteins (PSSG). Mixed disulfides can be formed in response to changes in the GSH/GSSG ratio, through thiol/disulfide exchange reactions between a protein sulfhydryl group (PSH) and GSSG (reaction A). Thiol/disulfide exchange reactions can also occur between a reduced protein thiol and PSSG. PSSG can also be formed following partial oxidation of PSH or the sulfhydryl group in GSH. This can occur by a radical reaction, by one electron S-oxidation of PSH or GSH to give the respective thyil radical, which further reacts with GSH or PSH to yield PSSG (reactions B and C), or from two electron Soxidation of PSH to sulfenic acid (PSOH), followed by reaction with GSH (reaction D). PSSG can also arise from reactions between GSH and a Snitrosylated protein (PSNO) (reaction E) and from reactions between PSH and S-nitrosothiols such as GSNO (reaction F), glutathione sulfenic acid (GSOH) (reaction G), or glutathione disulfide S-monoxide [GS(O)SG] (not shown). (B) Main mechanisms of protein S-glutathionylation and deglutathionylation. GSSG concentration can be sufficient to trigger protein S-glutathionylation by a SH/S−S exchange reaction between a PSH and GSSG (reaction a) in response to drastic changes in GSH/GSSG ratio (i.e., under conditions of severe oxidative stress); ROS-catalyzed generation of protein thiyl radicals (reaction b) or sulfenic acid intermediates (reaction c) can enable S-glutathionylation in the absence of GSSG accumulation. Thiol−disulfide exchange reactions can also occur between PSH and PSSG. Indeed, minor changes in the intracellular GSH/GSSG ratio are unlikely to lead to substantial S-glutathionylation of proteins according to thiol−disulfide exchange involving GSSG and PSH because of the redox potential of most protein cysteine residues. In addition, the GSSG export by most cells (as a protective mechanism against oxidative stress) drastically limits large shifts in the intracellular redox potential under normal physiological conditions. Therefore, although thiol−disulfide exchange can lead to PSSG formation under extreme conditions (i.e., high GSSG concentration),182 it is an unlikely mechanism in vivo. Thus, PSSG are formed following oneelectron oxidation of PSH (reaction b) or GSH (reaction b′) to the respective thyil radicals (PS• or GS•), which then react with GSH or PSH, respectively. The reaction of GS• with proteins to generate PSSG is catalyzed by glutaredoxin (Grx), an enzyme normally acting as a reductant. Sglutathionylation can also occur by two-electron oxidation of PSH to the sulfenic acid derivative (PSOH), which then reacts with GSH (reaction c). Furthermore, PSSG can arise from reactions between PSH and GSNO, GSOH, or GS(O)SG (reaction d) or from reaction between GSH and PSNO (reaction e). Biochemical and cellular studies support the GSNO-mediated protein S-glutathionylation as a feasible mechanism for PSSG formation in vivo. However, it is not well understood what protein properties or conditions would favor PSNO vs PSSG upon reaction of PSH with GSNO. Whatever the route of S-glutathionylation, the process can be reversed by means of reactions catalyzed by the thiol−disulfide oxidoreductases Grxs (reaction f). Reprinted with permission from ref 102. Copyright 2009 Elsevier.

the modification.101,104,164−168 Conversely, -SO2H and -SO3H derivatives are generally considered in vivo to be irreversible oxidation products, with the only exception of the catalytic cysteine in 2-Cys Prxs, which in vivo can be reduced back to −SH by sulfiredoxin or sestrin, protein-specific sulfinic acid reductases.169−171 Protein tyrosine phosphatases (PTPs) are proteotypic examples of enzymes containing a conserved, active site resident cysteine with a pKa value of 4.6−5.6, susceptible to reversible ROS/RNS-mediated functional modulation.44 Indeed, oxidation of this residue into a sulfenic acid derivative abolishes its nucleophilic properties, thereby inhibiting enzymatic activity,172,173 with important consequences on tyrosine dephosphorylation mechanisms associated with signal transduction pathways involved in cell metabolism, motility, proliferation, differentiation, and survival.44,161,174 Since sulfenic

acid-mediated PTP inactivation can be reversed by cellular thiols, ROS and RNS may be therefore considered as second messengers. As a protection against further irreversible oxidation, the sulfenic acid derivative may undergo secondary reactions with neighboring amide nitrogens or cysteines to yield sulfenamide, isothiazolidin-3-one, or disulfide derivatives, respectively.174−177 Formation of a S−S bond can occur with cysteine residues from the same or other polypeptide chains or from low molecular mass thiols (e.g., GSH), yielding various Sthiolated protein species. Once formed, disulfide bonds are susceptible to migration to other sites or other proteins through thiol−disulfide exchange reactions, often mediated by GSH and Trx or Grx system, thus regenerating the native cysteine within the protein active site. This general mechanism does not always imply functional inactivation, but it has also been reported for protein activation, as in the case of protein tyrosine kinases like O


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Src.103 Reversible cysteine conversion into sulfenic acid or disulfide(s) has also been described for different transcription factors,18 kinases,72,161 phosphatases,172−174,176 proteolytic enzymes, heat shock proteins, membrane receptors and many other enzymes,14 which have in common the occurrence of oxidation-susceptible, low-pKa cysteinyl residue(s) acting as a regulatory nanoswitch in dynamic modulation of the polypeptide structure or activity. These redox-regulated proteins may undergo reversible chemical changes in response to variations of the local redox potential or ROS/RNS levels, with important consequences on their activity and function;13−16,105 this phenomenon is at the basis of the tenet of redox sensing.14,44,102,104,105,164 Since some of these proteins are key enzymes in important cellular pathways, whole metabolic processes such as glycolysis, the tricarboxylic acid cycle, and the Calvin−Benson cycle become sensitive to environmental redox conditions and/or specific stimuli.14,17 On the other hand, cysteine residues are not equal in their ability to undergo reversible redox modifications, which provides the basis for reaction selectivity and specificity. In fact, it has been proposed that approximately 0.5% of all cysteine residues in the cellular proteome are subjected to continuous, reversible oxidation−reduction cycles; this redox cycling controls a number of essential mechanisms in eukaryotic cells.121 In this context, sulfenic acids are relatively unstable and have traditionally been viewed as intermediates toward formation of other oxidized derivatives. In recent years, however, stable -SOH moieties have been identified in a growing list of proteins, i.e. albumin, peroxidases, and Prxs,178,179 and have also been shown to a have a role in cell signaling.13,163,178 In this case, stability of the protein sulfenic acid moiety has been related to some structural parameters,180 such as the presence of nearby cysteine residues and steric accessibility to low molecular mass thiols,163 or cellular microenvironment. Finally, protein RS− is a powerful nucleophile that may irreversibly react with a large variety of electrophile compounds originating from either nonenzymatic or enzymatic peroxidation of lipids. Most of these important α,β-unsaturated aldehydes or ketones have been already reported in section 2.1.5 and in Figures 4 and 5. These RCS have been amply documented to alkylate sensitive protein thiols under oxidative stress conditions, generating carbonylated protein derivatives (see section 3.3 for details). Although progressive accumulation of these irreversible protein adducts alters normal cell functions and can ultimately lead to cell death, at low, noncytotoxic concentrations RCS may be regarded as stress signaling molecules regulating various cell functions, such as the cellular cytoprotective signaling pathways and apoptosis.63 3.1.1. Protein S-Glutathionylation. In mammalian cells, significant amounts of GSH can reversibly modify proteins through an oxidative modification that generates S-glutathionylated species (PSSG). In some cases, S-glutathionylation occurs on proteins under basal conditions (constitutive S-glutathionylation),181,182 for example, for β-actin183 and mitochondrial complex II.184 In other cases, it takes place mainly under oxidative stress conditions, such as ischemia/reperfusion, for example, for α-actin185 and GAPDH.186 Depending on ROS/ RNS nature, reversible protein S-glutathionylation may occur through different mechanisms (Figure 8).104,164,187,188 Also in this case, the most important determinant for S-glutathionylation is thiol reactivity, which depends on factors already reported in section 3.1.102,164 Proteins containing SeCys residues are also readily S-glutathionylated at neutral pH

because of their pKa value (lower than 6.0). Glutationylation can be reverted by Grx enzymes, which efficiently catalyze protein deglutathionylation.165,187 Other enzymes have been reported to present this activity, but their contribution to protein deglutathionylation is uncertain.187 Grxs may also serve as a catalyst of protein S-glutathionylation in vivo,189 although mechanicistic details in this case are not clear.164,188 Protein S-glutathionylation is a dynamic redox process thought to be involved in protection of catalytic cysteine residues from oxidative damage and functional regulation of exemplificative proteins that contribute to a wide range of cellular functions, such as modulation of cell shape (i.e., actin and tubulin),106,183 signaling (i.e., p21ras),190−192 ion transport and vascular tone (i.e., SERCA, sarcolemmal Na+−K+ pump, and ryanodine receptor type 1),193−196 metabolism (i.e., GAPDH),186 mitochondrial efficiency (i.e., mitochondrial complex II),184 and transcription (i.e., IKK β subunit),107 with significant effects on multiple physiological events.102,104,164,165,197 In addition, S-glutathionylation may affect specific protein modulators of apoptosis that play a fundamental role in embryonic development, tissue homeostasis, or some diseases triggered by a wide variety of stimuli, including activation of death receptors, environmental agents, and cytotoxic drugs.136 In particular, reversible S-glutathionylation has been shown to regulate some key signal transduction mediators, such as death receptor Fas,198 which is critical to cell survival/apoptosis.136 Thus, protein S-glutathionylation has been implicated in regulation of cellular homeostasis in some diseases, such as diabetes mellitus, hyperlipidemia, myocardial infarction, cardiac hypertrophy, and atherosclerosis (reviewed in refs 164 and 188). In many proteins, S-glutathionylation results in the activesite-directed inhibition of protein activity, as in the case of NFκB,199,200 actin,183,201,202 the inhibitory κB kinase (IKK) β subunit,107 and caspase-3.203 Likewise, there are many proteins where S-glutathionylation increases protein activity, for example, SERCA,190,193,194 mitochondrial complex II,184 and p21ras.190−192 Additionally, it has recently been revealed that Sglutathionylation may regulate oligomerization and activity of 2-Cys Prx I,204 a dual-function enzyme that exhibits peroxidase activity and a molecular chaperone function upon overoxidation of its catalytic cysteine. Prx I functional change is caused by a structural change from low-molecular-weight oligomers to highmolecular-weight complexes. S-Glutathionylation of a specific Prx I cysteine, localized at the putative dimer−dimer interface, promotes changes in protein quaternary structure from decamers to smaller oligomers (mainly dimers) and concomitantly inactivates its molecular chaperone activity.204 In order to consider S-glutathionylation as a regulatory functional redox reaction, some basic criteria should be met: (i) reversibility; (ii) specificity for selected cysteine residues within particular proteins; (iii) association with a specific physiological stimulus; (iv) direct effect on protein activity or related cell functions.165,188 Partial fulfilment of these criteria has been convincingly demonstrated for several proteins in different cell types.106,191,192,196 For example, S-glutathionylation has been shown to uncouple eNOS complex, switching it from NO• to O2•− generation.205 This process is induced by oxidative stress in both endothelial cells and intact vessels, and is reversible. SGlutathionylation of eNOS decreases NOS activity (with reduced production of NO•) and determines an increase in O2•− generation (primarily from the reductase subunit). Two highly conserved cysteines have been identified as SP


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dysfunction.137,213 Therefore, S-nitrosylation of parkin may contribute to neuronal cell injury, in part, by triggering accumulation of misfolded proteins. Similarly, studies on human post-mortem brains revealed an increased Prx2 Snitrosylation in subjects affected with PD.216 S-Nitrosylation of Prx2 prevents its reaction with peroxides and inhibits its enzymatic activity and protective function against oxidative stress. Furthermore, post-mortem brains from patients with PD or AD exhibited increased levels of S-nitrosylated PDI compared with normal-aged brains.138 PDI S-nitrosylation seems to prevent PDI-mediated ER stress reduction and allow protein misfolding, thus favoring neurotoxicity. However, it should be noted that, in many cases, it remains to be determined whether changes in protein S-nitrosylation are causal in the development of disease. Nevertheless, a number of drugs, including statins, deprenyl, rosiglitazone, and carmustine, seem to exert disease-ameliorative effects, at least in part, through modulation of S-nitrosylation.207

glutathionylation sites and found to be critical for redox regulation of eNOS function. In hypertensive vessels, eNOS Sglutathionylation is increased with impaired endotheliumdependent vasodilation that, conversely, is restored by thiolspecific reducing agents.205 Thus, S-glutathionylation of eNOS is a clear example of a pivotal switch providing redox regulation of cellular signaling, endothelial function, and vascular tone. 3.1.2. Protein S-Nitrosylation. S-Nitrosylation, the reversible addition of NO• to reactive protein thiols (or more properly RS−) yielding PSNO products, has emerged as an important reaction, which effectively acts as a reversible molecular switch influencing a number of enzymatic activities.83 Different are the putative mechanisms leading to S-nitrosylated adducts, among which the most probable in vivo seems to be the reversible transnitrosylation of protein thiols by GSNO.206−208 This nitrosothiol appears to be generated in vivo by GSH reaction with N2O3, which is initially formed following NO• addition to O2. Indeed, different studies have demonstrated a role for GSNO and GSNO reductase in PSNO homeostasis.207 Moreover, PDI and Trx1 have also been shown to present a transnitrosylase activity.209,210 Trx1 ability to transnitrosylate target proteins is regulated by a crucial stepwise oxidative and nitrosative modification of specific cysteines.210 Thus, S-nitrosylation of a broad functional spectrum of proteins represents the largest part of the ubiquitous influence of NO• on various cellular functions.105,137,138,211 S-Nitrosylation inhibits the activity of a number of essential proteins for cellular life, including PTPs44,105,212 and members of the large family of E3 ubiquitin ligases;137,213 it also activates H-, K-, and N-Ras activities,214 affects different protein−protein interactions, and may modulate protein location.105 In addition, S-nitrosylation has been demonstrated to regulate degradation processes, as for phosphatase and tensin homologue (PTEN), whose activity is critical for multiple functions in the central nervous system. S-Nitrosylation but not oxidation induces PTEN protein degradation via the ubiquitin−proteasome system.212 S-Nitrosylated protein is the predominant form of PTEN in human brain at early stages of (i.e., mild cognitive impairment) and conclamate AD, where PTEN level reduction has been reported. These findings demonstrate that NO•mediated redox modification is the mechanism regulating PTEN degradation and activation, which is distinguished from the H2O2-mediated PTEN oxidation, known only to inactivate the enzyme. Interestingly, protein S-nitrosylation and oxidation partially occur on overlapping residues.212 Protein S-nitrosylation exhibits a number of essential features that are typical aspects of a cellular signaling process: (i) temporal regulation on physiological time scale (i.e., stimuluscoupled and rapid); (ii) affecting selected consensus motifs that facilitate reaction, providing specificity; (iii) colocalization of target proteins with a source of NO•; (iv) reversibility by cellular reductants such as the Trx/TrxR system, PDI, and GSH.44,105 These features further emphasize the importance of protein S-nitrosylation in redox regulation of cellular signaling processes. Altered levels of several S-nitrosylated proteins, which thus show a functional impairment, have been associated with disorders of the human cardiovascular, muscular, and nervous systems.207,215 This led to the hypothesis that aberrant Snitrosylation of specific protein targets is directly implicated in the etiology and/or symptomatology of a number of human diseases.207,215 For example, S-nitrosylation of parkin alters its E3 ligase activity, thus inducing ubiquitin−proteasome system

3.2. Methionine Sulfoxidation

Methionine residues are highly susceptible to ROS/RNSmediated side chain S oxidation.217 Mild oxidizing conditions determine in vivo generation of methionine sulfoxide (MetO), which can be further oxidized to methionine sulfone (MetO2) under stronger oxidizing conditions. Conversion of methionine into homocysteic acid was also described in heavily oxidized protein samples.218 MetO exists as a mixture of S and R diastereomers. Methionine oxidation may determine an altered protein conformation or function due to the conversion of a hydrophobic residue (methionine) into hydrophilic ones (MetO and MetO2).219 To overcome these effects, MetO production is tightly regulated in vivo by ubiquitous sulfoxide reductases, which catalyze the Trx-dependent reduction of MetO into methionine. 220 This reaction requires the participation of Trx, TrxR, and NADPH. MetO diastereomers necessitate separate enzymes: MsrA and MsrB for Met-S-SO and Met-R-SO, respectively. Conversely, MetO2 cannot be reduced in vivo. Cyclic methionine oxidation/MetO reduction leads to consumption of ROS and thereby acts as a scavenging system to protect proteins from oxidative damage.221,222 This hypothesis is consistent with the observation that, unlike other modifications, MetO formation has little or no effect on protein susceptibility to proteolytic degradation.223,224 Oxidation of a few surface methionines has little effect on polypeptide conformation and function, but accumulation of methionine damage can cause significant structural changes in proteins, which may lose their function, aggregate, and become toxic to the cell.219,225 Several studies provided evidence that MsrA and MsrB activities decrease during aging and under disease conditions, such as AD.226,227 Indeed, numerous studies reported that MetO levels in proteins increase during aging and in certain diseases, in particular, the neurodegenerative ones.228 Therefore, MetO and MetO2 may represent useful biomarkers of redox changes during aging and diseases. Evidence for an additional role of certain methionines as oxidation sensors in the redox regulation of enzyme activity is accumulating, with particular emphasis for proteins involved in calcium transport229 and signaling.230,231 3.3. Protein Carbonylation

Protein carbonylation is an irreversible oxidative modification associated with ROS insult, which results in the introduction of a carbonyl group on the polypeptide chain. It is considered to be the most general type of protein oxidation, because the Q


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Figure 9. Selected mechanisms of protein carbonylation. (A) Protein carbonylation can occur by oxidative cleavage of the protein backbone by either the α-amidation pathway (reaction a) or oxidation of glutamyl side chains (reaction b), leading to the formation of polypeptide fragments in which the N-terminal amino acid is blocked by an α-ketoacyl derivative. (B) It can also occur by the direct oxidation of proline, arginine, lysine, and threonine side chains, yielding different carbonyl derivatives, such as 2-pyrrolidone (from proline), glutamic semialdehyde (from arginine and proline), α-aminoadipic semialdehyde (from lysine), and 2-amino-3-ketobutyric acid (from threonine).

Schiff bases.63 Finally, carbonyl groups may be introduced in proteins by oxidation of what has come to be known as advanced glycation end-products (AGEs) (Figure 11).236,237 These compounds, or their early intermediates, are formed by direct addition to proteins of reactive carbonyl derivatives (3deoxyglucosone, dideoxysones, methylglyoxal, and glyoxal), generated by oxidation of sugars, ascorbate, polyunsatured fatty acids, or Schiff base species resulting from the reaction of lysine residues with reducing sugars, or by direct oxidation of the Amadori adducts resulting from the latter species.232 Structures of some carbonylated oxidation products are reported in Table 3. Under specific conditions, some oxidation products may occur simultaneously. Protein carbonylation cannot be reversed by any established cell repair machinery and carbonylated proteins are generally removed by proteasome degradation activities.150,238 However, there is a limit to the cell capacities to process carbonylated proteins, particularly because the proteasome itself may be a

largest family of oxidized amino acid products is represented by carbonylated derivatives. Carbonyl groups may be introduced within the protein structure at different sites and by different mechanisms.232 The first involves oxidative cleavage of the protein backbone by either the α-amidation pathway (Figure 9A, reaction a) or oxidation of glutamyl side chains (Figure 9A, reaction b), leading to the formation of polypeptide fragments in which the N-terminal amino acid is blocked by an α-ketoacyl derivative.233 A second mechanism involves the direct oxidation of proline, arginine, lysine, and threonine side chains, yielding different carbonyl derivatives (Figure 9B). 233 A third mechanism acts indirectly through a Michael-type addition of RCS generated during lipid peroxidation, such as HNE, acrolein, malondialdehyde and others, to the nucleophilic side chain of cysteine, histidine, or lysine, leading to the formation of the so-called advanced lipoxidation end-products (ALEs) (Figure 10).234,235 Carbonyl groups of reactive aldehydes/ ketone may alternatively react with the amino groups to form R


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Figure 10. Cellular fates of α,β-unsaturated aldehydes from lipid peroxidation and resulting carbonylated proteins at cysteine, histidine, or lysine residues. ROS stimulate peroxidation of polyunsaturated fatty acids (PUFA), an oxidative event that is reversible through reduction by peroxiredoxin (PRX) and glutathione peroxidase (GPX) enzymes. The lipid hydroperoxides (PUFA-OOH) generated are unstable and lead to a variety of reactive aldehydes. The lipid peroxidation products generated include the α,β-unsaturated aldehydes HNE, ONE, HHE, 2-hexenal, crotonaldehyde, and acrolein as well as the dialdehydes glyoxal and malondialdehyde. GSTA4 catalyzes the conjugation of the highly reactive α,β-unsaturated aldehydes to glutathione, leading to their efflux from the cell by the glutathione conjugate transporter RLIP76. In addition, oxidation by aldehyde dehydrogenase or reduction by alcohol dehydrogenase, aldehyde reductase, or aldose reductase converts free aldehydes into less toxic molecules. The α,βunsaturated aldehydes that escape cellular metabolism serve as electrophiles in the covalent modification of proteins through nonenzymatic Michael addition. The resulting aliphatic carbonyl adducts on cysteine, histidine, or lysine residues may alter the activity of protein targets or cause them to become degraded by the proteasome.

target for oxidative inactivation,141,150,239 as recently demonstrated in patients with end-stage heart failure or hypertrophic cardiomyopathy.240 Thus, nondegraded carbonylated proteins may form toxic aggregates,27,28 as observed in most human neurodegenerative diseases,141,241,242 establishing a linkage between their elevated levels and the loss of cell viability. This correlation was ascertained for patients with multiple sclerosis who showed accumulated levels of carbonylated proteins, impairment of three 20S proteasome peptidase activities, and a failure of the 26S proteasome in their cerebral white and gray matter.243 However, degradation of carbonylated proteins is not completely efficient even when the proteasome is not oxidatively impaired; also in this case, low levels of damaged proteins may escape degradation and form high molecular mass aggregates, which accumulate with age.242,244 Accumulation of carbonylated proteins generally occurs in vivo during cell/organism aging and in vitro during cellular replicative senescence;150,245,246 similarly increased levels of carbonylated protein aggregates are observed in patients with age-related disorders, such as PD, AD, and ALS.23,27,241 Autophagic destruction of protein aggregates provides a last line of defense against these toxic species but evidently is not wholly efficient.247 Since protein carbonylation correlates well with oxidative damage,248 it has been considered a widespread marker of

severe oxidative stress under various pathophysiological conditions and of disease-associated protein dysfunction.27,122,141,249 For example, HNE-dependent modification at a conserved cysteine of the adipocyte and epithelial fatty acid-binding protein decreases protein affinity for fatty acids, and may contribute to obesity-linked insulin resistance.250 GAPDH, creatine kinase, and carbonic anhydrase are additional examples where protein enzymatic activity is attenuated by in vivo carbonylation.251,252 Although carbonylation most typically inactivates proteins, such modification can also indirectly result in a gain of function for certain metabolic signaling systems. A representative example is Keap1, the cytoplasmic inhibitor of the key transcription factor Nrf2 involved in the regulation of antioxidant-responsive-element-containing genes, which are activated in response to oxidative stress. HNE-mediated carbonylation of Keap1 disrupts the Keap1−Nrf2 complex and results in the translocation of Nrf2 to the nucleus, where it activates the expression of its target genes, thus increasing antioxidant defenses.253 Carbonylation impact on function is generally not limited to a specific protein activity but is diffused to various targets determining cell, tissue, and even whole organ dysfunction. For example, recent studies unveiled that several myofilament, mitochondrial, and cytosolic proteins are carbonylated inside skeletal muscle fibers in many animal models of muscle S


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Figure 11. A simplified overview of the Maillard reaction. Selected pathways are shown that link together Amadori derivatives with potential carbonyl sources (initial and intermediate species) and some linear and cross-linked advanced glycation end (AGE) products. Abbreviations are reported within the text.

dysfunction (e.g., ischemia/reperfusion injury and sepsis) and in humans with impaired skeletal muscle contractility, like patients with chronic obstructive pulmonary disease or with obstructive sleep apnea syndrome.254 In particular, many reports documented augmentation of RCS-derived carbonylation derivatives (i.e., HNE−protein adducts).251,254−256 In addition, a clear association between oxidative stress and depressed skeletal muscle contractility has been described in

several acute and chronic conditions. Accordingly, it has been hypothesized that oxidative stress-induced skeletal muscle injury, along with an associated depression of muscle contractile function, may be mediated, at least in part, through selective carbonylation of different intracellular proteins, including the structural myofilament proteins actin, myosin, and tropomyosin.254 T


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Figure 12. Proposed pathways of ROS- and RNS-induced modifications at tryptophan.

binding sites. Diffused occurrence of histidine and tryptophan residues in heme- and/or metal-coordination sites of proteins have determined a widespread detection of their modification products under conditions characterized by oxidative stress or related pathophysiological states. Examples of irreversible metal-catalyzed tryptophan and histidine oxidations have been reported for myoglobin,260,261 β-amyloid peptide,262 Cu,ZnSOD,263 prion protein,264 α-crystallin,265 and surfactant protein B.266 On the other hand, the occurrence of reactive and/or accessible histidine or tryptophan residues in proteins present in close proximity to general sources of ROS, as in the case of normally respiring tissues or biological tissues/body fluids exposed to γ or UV radiations, promotes their selective oxidative modification. These oxidation processes have been reported to occur under normal conditions in cardiac mitochondria,267 lens,265 foam cells,268 and plant thylakoid membrane,269 inducing structural or functional protein alterations that, whenever they are over-represented, are

Although aberrant modification and dysregulation of proteins have been the major focus of most protein carbonylation studies, it has been recently proposed that some RCS may play an active role in redox cell signaling.257,258 Since these authors have described a serotonin- or endothelin-1-mediated carbonylation of proteins from different tissues, which can be reverted by a decarbonylation process still unknown, and have surprisingly reported carbonylated adducts highly sensitive to reducing agent treatment before DNPH derivatization, similarly to sulfenic acid derivatives,259 further studies are needed to advance the knowledge of possible roles of protein carbonylation and, eventually, decarbonylation in cell signaling. 3.4. Tryptophan and Histidine Oxidation

Reduced metal ions bound to specific binding sites in proteins may react with oxidants to generate highly reactive ROS.233 Thus, resulting metal-catalyzed oxidative reactions are not highly selective processes, but rapidly occur at amino acid residues present in the close proximity of the macromolecule U


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concentrations (1−2 mM) of CO2 to quickly yield CO3•− and NO2•. Furthermore, whereas OH• is highly reactive and unselective, CO3•− can diffuse farther and react preferentially with various amino acids, such as tyrosine, tryptophan, and cysteine. It has to be underlined that, once formed, Tyr• can react in parallel with OH• or other vicinal tyrosine residues to yield di-OH-Phe (DOPA) and di-Tyr derivatives as modified end-products (Figure 13A).271,272 Other biologically relevant processes leading to NO2-Tyr formation include MPO or EPO/H2O2-assisted reactions, which predominate during conditions of chronic inflammation, such as atherosclerosis.273 Alternative pathways may involve oxidation of nitrite to NO2• by myoglobin and some cytochrome P450 enzymes. Thus, the wide spectrum of reactions promoting NO2-Tyr formation in biological conditions indicates that this derivative cannot be used simply as a footprint for ONOO−. Because of the complexity of cellular systems, protein nitration has to be more likely considered as the product of multiple NO2•-generating reactions that operate simultaneously. Selectivity of protein tyrosine nitration is due to different parameters, which include (i) cellular localization of the target protein and its proximity to the generation of nitration agents, (ii) local electrostatic environment sorrounding the target tyrosine residue, (iii) relative tyrosine accessibility on protein surface, and (iv) absence of steric hindrance factors.270,274 Conversely, protein abundance, total number of tyrosine residues, and primary sequence are not dominant factors in tyrosine nitration selectivity.270,274 Thus, nitration of protein tyrosine residues represents a dynamic and selective process, rather than a random, indiscriminate event; this conclusion is supported by numerous studies on pathological conditions associated with oxidative stress, where this modification is limited to certain cell types and to selective sites of injury.275 Formation of NO2-Tyr alters the protein structure and function as a result of the creation of a bulky, anionic adduct on tyrosine, which triggers local changes in polypeptide conformation and electrostatic environment. In fact, addition of the nitro moiety to tyrosine can lower the pKa of the phenolic -OH by 2−3 units (from ∼10.3 to ∼7.2 ± 1.2); this in turn imparts a net negative charge to half of the nitrated residues at physiological pH.89 These alterations give rise to many different functional changes, including gain of protein function. Thus, tyrosine nitration is generally associated with the reduction or loss of essential protein activities, including modulation of catalytic activity, cell signaling, and cytoskeletal organization,89,276 eventually leading to cellular dysfunction and disease progression.50 In this context, elevated NO2-Tyr levels have been observed for acute and chronic disease states (i.e., during vascular diseases) generally associated with oxidative stress. In this case, specific nitrated proteins have been demonstrated, whose functional changes (usually in a negative manner) may reflect disease progression.50,270,277 For example, actin nitration has been shown to reduce the dynamic assembly/disassembly process of protein filaments in the vasculature, kidney, and liver of patients affected with sickle cell disease. In addition to adversely affecting vascular function, selective nitration of tissutal actin tyrosine may lead to cell apoptotis and loss of organ function observed in this pathology.278 Similarly, augmented in vivo nitration of fibrinogen was demonstrated in the plasma of patients with clinically documented coronary artery syndrome, which independently predicts risk for this disease.279 In vivo nitration of fibrinogen increases the initial velocity of fibrin clot formation, alters clot architecture,

implicated in a variety of diseases, including cataract, atherosclerosis, several neurodegenerative ailments, and aging. In the case of either metal-catalyzed or direct oxidative reactions, hydroxytryptophan (OH-Trp), oxindolylalanine (OIA), N-formylkynurenine (NFK), dihydroxytryptophan (diOH-Trp), kynurenine (Kyn), and hydroxykynurenine (OHKyn) (Figure 12), or 2-oxo-histidine (OHis) and 4- or 5hydroxy-2-oxo-histidine (OH-OHis) have been widely ascertained as main tryptophan and histidine oxidation products, respectively, together with other amino acid derivatives. 3.5. Tyrosine and Tryptophan Nitration

A variety of reactive species and multiple reaction pathways concur to modify tyrosine in vivo yielding NO2-Tyr,270 whose presence is generally the result of a combined, simultaneous production of various RNS (Figure 13A). Most pathways

Figure 13. Proposed mechanism for ROS- and RNS-dependent formation of nitrotyrosine, dityrosine, and DOPA and molecular structure of tyrosine derivatives. (A) The free radical pathways of tyrosine nitration, hydroxylation, and cross-linking. (B) Molecular structure of halogenated tyrosine derivatives. R and MeO indicate the remaining protein portion and oxo-metal complexes, respectively.

involve a free radical biochemistry with carbonate radicals (CO3•) and/or oxo-metal complexes that oxidize tyrosine to a tyrosyl radical (Tyr•), followed by a very rapid, diffusioncontrolled reaction with NO2• yielding NO2-Tyr. Tyr• could also be produced by the reaction of tyrosine with OH• and NO2•, which are concomitantly generated as a result of the ONOO− decomposition; however, this reaction is slow in vivo and ONOO− more favorably react with high intracellular V


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crucial for immune response, excessive or misplaced generation can damage host tissues; the latter phenomena have been implicated in several human inflammatory diseases, such as arthritis, some types of cancer, heart disease, cystic fibrosis, and asthma, where high levels of chlorinated and/or brominated tyrosine have been detected.144,303,304

increases clot stiffness, and reduces the rate of clot lysis. Finally, apolipoprotein A1 (ApoA1) nitration and functional impairment, also accompanied by tyrosine chlorination, has been documented in plasma and HDL particles isolated from human atherosclerotic lesions from patients with cardiovascular diseases.280−282 In particular, 3-chlorotyrosine and NO2-Tyr levels were markedly higher in circulating HDL isolated from patients with coronary artery disease than that from healthy controls.280−282 It was demonstrated that ApoA1 oxidation impairs protein ability to promote cholesterol efflux from artery wall macrophages via the membrane-associated ATP-binding cassette transporter A1 transporter.280,281 However, finding of nitrated proteins in a given condition does not necessarily imply a direct pathogenic role, but at least it indicates an increased formation of ONOO−, along with other RNS, during the disease process. Protein tyrosine nitration has also been detected under normal physiological conditions,140,283 raising the issue of whether this modification may also perform a role in cellular signaling, possibly through cooperation with tyrosine phosphorylation.276 An indirect effect can be definitively claimed due to its ability to block tyrosine phosphorylation, thereby disrupting protein TyrP-mediated signaling events.284 Notwithstanding some evidence on signaling capacities of tyrosine nitration have been accumulated, experimental proof of the reversibility of this modification is still scarce, although a few reports have described a “denitrase activity”,285−287 thus hampering satisfaction of a fundamental criterion to consider it as a signaling process. Despite its relatively lower abundance in proteins with respect to tyrosine, tryptophan has also been shown to be a specific target of irreversible nitration upon in vitro exposure of purified polypeptides to nitrating agents,288 such as the peroxynitrite and peroxidase/H2O2/nitrite systems.289−294 1-, 4-, 5-, and 6-NO2-Trp were identified as reaction products,295,296 together with some oxidized tryptophan derivatives,297,298 with 6-NO2-Trp being the most abundant compound. This result depends on stability of 6-NO2-Trp under physiological conditions, whereas 1-NO-Trp and 1-NO2Trp decompose with half-lives of 1.5 and 18 h, respectively. Several tryptophan-nitrated proteins have also been identified when cells were treated in vitro with nitrating agents.299 Recently, this modification was demonstrated to occur in vivo in plant thylakoid membrane proteins following photooxidation.269 A related oxidized tryptophan derivative, namely, nitrohydroxytryptophan (NO2-OH-Trp), was also detected in the mitochondrial protein succinyl-CoA:3-oxoacid CoA transferase from rat heart.300,301 In most cases, these modifications were associated with protein functional changes.

4. PROTEOMIC ANALYSIS OF OXIDIZED, NITROSYLATED, AND NITRATED PROTEINS 4.1. Proteomic Methods

Proteomic analysis, providing scientists with a holistic approach to describe all polypeptide species present in a cellular extract or a biological fluid/tissue at a specific moment, is the ideal choice for revealing protein modifications due to oxidative/ nitrosative reactions. Thus, dedicated proteomic methodologies have been developed and used for the qualitative−quantitative representation of the protein targets related to a certain ROS/ RNS activity, to be eventually associated with a specific pathophysiological condition; these methods have been ascribed to the general term of redox proteomics. When dealing with redox PTMs, two major issues have to be taken in account: (i) as result of their relative stability, ROS/RNSinduced modifications cannot survive the purification steps needed to extract proteins from the biological sources; (ii) the dynamic nature of these modifications often affect only a small percentage of the protein, in a given time and in a particular cellular space. Both issues deeply influence the relative abundance of the modified proteins; thus, analytical methods aimed at the analysis of oxidative/nitrosative modifications have to be highly specific and sensitive and possibly provide quantitative information. The most widely used bioanalytical techniques in redox proteomics experiments are two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), which have originated several gel-based or gel-free methods for the characterization of each redox modification, as discussed in the following sections. Gel-based methods rely on the ability of 2-DE to efficiently separate whole proteomes extracted from biological tissues/ body fluids or complex protein mixtures on the basis of polypeptide isoelectric point (pI) and molecular weight (Mr).305−307 Despite the well-known limitations of this technique, e.g., underestimation of certain protein classes such as high-molecular-weight, basic, and very hydrophobic proteins, low dynamic range, limitations on the amount of sample loaded, and comigration of multiple proteins in the same spot,308,309 2-DE has been extensively and successfully applied for differential protein expression profiling, quantitative proteomics, and post-translational modification detection.307,310−312 2-DE can in fact separate thousands of proteins in a single experiment and can highlight the occurrence of posttranslationally modified proteins because they often appear as distinct rows of spots in the horizontal or vertical axis of the gel, due to the differences in their pI and Mr values resulting from the addition of certain modifications. Visualization of proteins is generally obtained by using nonspecific protein dyes, such as Coomassie blue or silver staining. There are also more selective staining techniques that utilize a residue-specific or a modification-specific reagent, for example, some iodoacetamide- or maleimide-based reagents, which specifically label reduced cysteine thiols.313,314 In conventional 2-DE, different protein samples are separated on diverse gels, which are stained and analyzed by picture

3.6. Tyrosine Halogenation

MPO-catalyzed reaction pathways (associated with HOCl production) have been demonstrated to generate 3-chlorotyrosine (Cl-Tyr) and 3,5-dichlorotyrosine (diCl-Tyr);302 similarly, MPO- and EPO-catalyzed reaction pathways yielding HOBr were proven to generate 3-bromotyrosine (Br-Tyr) and 3,5-dibromotyrosine (diBr-Tyr) (Figure 13B).302,303 HOCl and HOBr are formed by the reactions of H2O2 with Cl− or Br−. HOCl and HOBr are integral factors in mammalian host defense, being strong oxidants with potent antibacterial properties, and are produced in vivo by activated neutrophils, monocytes, and possibly macrophages (HOCl) and activated eosinophils (HOBr). Although hypohalous acid formation is W


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methodology minimizes intragel variations, thus enabling accurate quantitation of differences between samples, with an associated statistical significance that allows one to distinguish real biological changes from sample preparation deviations.318−321 Recently, 2D-DIGE has been specifically used in redox proteomics experiments;322 by designing new iodoacetamido cyanines for labeling cysteine thiol residues, the identification and quantification of S-nitrosylated proteins323 or the analysis of the thiol redox state of plasma proteins has been investigated.313 The ability of 2-DE to display thousands of proteins and to quantitate small changes in protein expression and modification has been exploited in parallel with immunoblotting for the detection of several redox PTMs (when a specific antibody is available). In this approach, the immunoblotting step is used to assign the proteins of interest on a reference 2D map, and a subsequent 2D gel is run to perform the identification and characterization of the proteins of interest (Figure 14). Examples of this strategy are studies that used Western blotting with anti-NO2-Tyr antibodies on 2-DE maps to identify several nitrated proteins in peripheral blood mononuclear cells from ALS patients and in mouse heart mitochondria, respectively.324,325 Similar strategies have also been widely used to detect protein carbonylation248 or S-glutathionylation326,327 adducts. Although gel-based methods allow the visualization and identification of proteins sensitive to redox modifications (detected either as differential spot migration or by selective antibodies), the identity of the protein and, most importantly, the localization of the modified residues have to be achieved by MS analysis. Continuous improvements in MS technology over the past decade have made this technique the primary method for the identification and characterization of proteins.328 Mass spectrometry in proteomics can be used to measure the mass of a peptide or a protein (single stage MS), but it can also provide structural information by fragmenting polypeptides (tandem MS or MS/MS), thus deriving direct information on the amino acid sequence and the nature and localization of the residues bearing PTMs.35,329−333 Since their introduction in the late 1980s, matrix-assisted laser desorption ionization (MALDI)334,335 and electrospray ionization (ESI)336 have been the most widely used soft ionization techniques for biomolecular analysis. Both ionization techniques have been coupled to a large choice of MS instrumentation. Nowadays, the most commonly used mass analyzers are ion trap (IT), linear ion trap (LIT) and time-offlight (TOF) devices, triple quadrupoles, TOF−TOF devices, quadrupole/TOF hybrids, LIT/Orbitrap hybrids, and LIT/ Fourier transform ion-cyclotron resonance (FT-ICR) hybrids, which show different sensitivity, resolution, mass accuracy, and ability to produce high-quality MS/MS spectra. This is one of the major achievements of the last generation instruments, such as Orbitrap or FT-ICR mass spectrometers, which have the capability to measure masses with very high resolution (up to 200 000 for the FT-ICR) and very high mass accuracy (up to 1 ppm).337,338 This allows approaching the elemental composition of peptides commonly measured in bottom-up proteomic experiments, and increases the confidence of protein and posttranslational modification identification. Improvements in peptide sequencing have also been recently achieved with the introduction of fragmentation techniques alternative to the most popular collision-induced dissociation (CID), which is typically used with the mass analyzers

comparison, using image analysis software able to depict densitometric spot variations between samples (Figure 14).

Figure 14. Proteomic analysis of oxidatively or nitrosatively stressed tissues/body fluids by 2-DE and specific immunodetection by Western blotting. Samples are separated on different 2D gels that are stained in parallel with derivatives/commercial antibodies specific for ROS/RNSmodified proteins and with nonspecific staining chemicals. Images are compared by dedicated analysis software. Selected spots from gels subjected to nonspecific staining are digested with a protease and further analyzed by MS or MS/MS procedures. Adapted with permission from ref 9. Copyright 2004 John Wiley and Sons.

Despite the robustness and the reproducibility that this technique has now achieved,315,316 comparative 2-DE image analysis is often extremely time-consuming. This limitation derives from the high degree of gel-to-gel variation in spot patterns, thus making two gels difficult to completely superimpose and finally to properly distinguish true biological changes from experimental variations, in particular when subtle PTMs are investigated. To limit this effect, several replicate gels have to be run for each sample to create electronic “average” gels, which can then be compared and matched with “average” gels from control samples to provide a reproducibility >95% of spot location, number, and volume. This tedious procedure has been overcome by the development of two-dimensional differential in gel electrophoresis (2D-DIGE) by Unlu and colleagues,317 who proposed to compare two or three protein samples simultaneously on the same gel. In 2D-DIGE, the proteins in each sample are covalently tagged with Nhydroxysuccinimide ester-modified cyanine fluorophores, the most popular being named Cy2, Cy3, and Cy5, which react with the ε amino group of lysine residues in proteins. These dyes have been designed to have no effect on the relative migration of proteins during electrophoresis; 2D-DIGE Cy dyes have in fact a similar molecular weight and possess a positive charge to replace the loss of the εNH2 group of lysine, but are spectrally resolvable (differing in their excitation and emission wavelengths). Differently labeled protein samples are mixed in equal amounts before the first dimension run so that all samples will experience the same electrophoretic separation. Proteins that are common to the samples appear as spots with a fixed ratio of fluorescent signals, whereas proteins that differ between the samples have different fluorescence ratios. This X


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spectrometer is set to detect only those peptides that produce this specific diagnostic ion and to preferentially select them for further fragmentation, thus allowing the identification of the components bearing that particular modification in a complex peptide mixture. This technique has been widely used for the identification of redox PTMs, such as tyrosine nitration,358 tryptophan halogenation and proline oxidation,359 carbonylation,360 tyrosine and tryptophan oxidation,361 and glycation.362 A similar experimental setting is based on the detection of specific neutral fragments deriving from the modified peptide. In this case, the mass spectrometer detects those peptides that produce a specific neutral loss (NL) and preferentially select them for further fragmentation. NL scanning is typically exploited to detect phosphorylated peptides, but it has also been used in redox proteomics for the analysis of S-nitrosylated peptides that, in CID fragmentation settings, lose the NO group, thus producing a characteristic NL of 30 Da.363 In any proteomic experiment aimed at the characterization of PTMs, it is crucial to gain information not only on the type and site of modification but also on the dynamics of site occupancy; therefore, assays capable of comprehensively assessing the dynamic changes in site-specific oxidation, nitrosylation, and nitration states are necessary to fully understand the contribution of the redox status to protein functioning. Because MS is not inherently a quantitative technique, being the ionization tendency of any single peptide a unique characteristics that depends on its physicochemical properties (size, charge, hydrophobicity), several strategies have been developed to achieve the accurate quantification of proteins and of their PTMs. Most quantitative proteomics methods rely on stable isotopic labeling of proteins or peptides. The approach is based on the idea that a stable isotope-labeled polypeptide is chemically identical to its native counterpart and behaves identically during chromatographic and/or MS analysis, but is distinguishable in a mass spectrometer owing only to a mass difference given by the corresponding isotopic content. The ratio of signal intensities for the isotope-labeled species provides an accurate measure of the relative abundance of peptides/proteins under different biological conditions. Strategies for isotope-based quantitative proteomics are distinguished into two groups, depending on whether the isotopic label is incorporated in vitro, during sample preparation, or in vivo by metabolic labeling (reviewed in refs 364−367). The most popular in vivo labeling method for proteomics studies is stable isotope labeling by amino acid in cell culture (SILAC), developed in 2000 by the Mann group.368 SILAC is based on the metabolic incorporation of stable isotopically labeled amino acids during protein synthesis in cells grown in special media, supplemented with either normal amino acids or counterparts labeled with heavy isotopes. The most commonly used amino acids are lysine and arginine, which are labeled with various combinations of [13C], [15N], and [2H] to generate tags with different mass shifts. Once labeled, the light and heavy cell populations (or protein extracts) are mixed in a 1:1 ratio and samples are processed together thereafter. Information on relative abundance of peptides coming from the light and the heavy labeled samples is achieved by MS, measuring the area under the peak of peptide pairs (Figure 15).369−372 In vitro labeling techniques are based on the introduction of an isotopic tag via either chemical or enzymatic reactions. One of the first in vitro labeling approaches used in proteomics

mentioned above. In particular, electron capture dissociation (ECD) has greatly increased the specificity of the analysis and the rate of protein identification when used in combination with CID.339−341 Another recently introduced fragmentation technique, named electron transfer dissociation (ETD), has been shown to produce extensive peptide sequence information that is often missing in conventional CID and proven to be especially useful for sequence analysis of post-translationally modified and highly basic peptides.342−344 Among other applications, ETD has been demonstrated to be very useful for studying oxidatively modified peptides and proteins, in particular for oxidative modifications affecting the side chains of sulfur-containing amino acids that show an impaired fragmentation during conventional CID.345,346 Moreover, when coupled with high-resolution mass spectrometer (Orbitrap and FT-ICR MS), ETD and ECD have been used in topdown proteomics approaches, where the intact protein gets fragmented to gain sequence information and post-translational modification characterization.347,348 In a bottom-up proteomic experiment aimed at the identification of PTMs, gel-separated proteins or proteins in solution are digested enzymatically with a sequence-specific protease (most commonly trypsin) and the derived peptide mixtures are analyzed by peptide mass fingerprinting (PMF) or by tandem MS. In the first approach, typically performed by MALDI-TOF MS, the measured masses of proteolytic peptides are matched with those derived from in silico digestion for each protein present within a sequence database.349−351 With this approach, identification of the occurring post-translational modification can be achieved by detection of a specific mass shift of the modified peptide. Unfortunately, some peptides can bear the same modification on different residues, or different modifications yielding the same mass shift can be present within one peptide, as for instance is the case of simple oxidation that can occur on cysteine or methionine, producing a mass increase of +16 Da. Thus, peptide mass fingerprinting alone is not sufficient to unambiguously localize the residue subjected to post-translational modification, and additional MS/MS experiments have to be performed in order to derive the peptide sequence and the identity of the modified amino acid.331,332 Developments in mass spectrometry and separation technologies have streamlined the analysis of entire proteomes in the so-called shotgun proteomic approach pioneered by the Yates group.352 In this procedure, a total protein extract is digested in solution, and the derived peptide mixture is separated via 2D (usually strong cation exchange and reversed phase) chromatography and analyzed by tandem MS. Further evolutions in prefractionation and separation techniques have made the analysis of complete proteome amenable to different gel-free experimental settings.353−356 Despite these promising results, when dealing with PTMs, one has to take into account that in most cases only a portion of the protein is modified, making more challenging the isolation and sequencing of the modified peptide. This limitation can be partially overcome by adopting specific strategies to trap the modified proteome only (see the following section for a comprehensive description of redox-specific trapping methods) or to detect, among all the peptides derived from the enzymatic digestion, only the ones bearing a specific modification. In the latter case, a particular MS experiment can be performed, called precursor (or parent) ion scanning (PIS).329,331,357,358 PIS requires a fragment ion highly characteristic for the modified amino acid; the mass Y


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is a tag with various masses (at least four masses, allowing fourplex experiments, but eight-plex implementations are also available) and is based on various combinations of isotopic elements. iTRAQ reagents with different tags are used to label peptides from different biological conditions. The balance group varies also in mass to ensure that the combined mass of the reporter and balance group remains constant. As such, peptides labeled with different isotopes are isobaric in MS, while the reporter groups are released during CID fragmentation, thus generating a low molecular mass reporter ion used for the relative quantification of the specific peptides and proteins from which it originates (Figure 16B). The iTRAQ technology was also adapted for the specific analysis and quantification of NO2-Tyr-containing proteins.383 In this case, all N-terminal groups in peptides generated by proteolytic digestion were blocked with acetic acid N-hydroxy succinimide, then a specific reduction of NO2-Tyr to NH2-Tyr was performed by Na2S2O4-based treatment; finally, all newly formed NH2 groups were labeled with amine-specific iTRAQ reagents, thus leading to the relative quantification of nitration sites. All the techniques described above belong to the so-called discovery proteomics approaches, where all the peptides brought to the mass spectrometer are fragmented and potentially identified. In the past few years, a new targeted proteomics approach has gained popularity for its peculiar feature to provide a reliable quantitative method for lower abundance proteins and protein PTMs as well as for the ability to discriminate highly homologous protein isoforms. Nowadays, this is the method of choice for validation of global proteomics data and biomarkers discovery.384−386 Targeted proteomics is based on a mass spectrometric technique called multiple reaction monitoring (MRM), also referred to as selected reaction monitoring (SRM). MRM, which was widely used in toxicology and pharmacokinetics studies, only recently has been introduced also in the proteomics field with the concept of the proteotypic peptide, an experimentally observable peptide that uniquely identifies a specific protein or protein isoform.387,388 MRM experiments are most predominantly performed on triple quadrupole mass spectrometers, which can follow multiple transitions characteristic for the studied peptide, thus concentrating only on the analysis of the peptide of interest within a complex peptide mixture. Transitions are designed such that the first mass analyzing quadrupole (Q1) is set to transmit only a narrow mass window around the desired parent ion and the third quadrupole (Q3) is set to transmit a narrow mass window around the desired fragment ion, which is produced by CID in the second quadrupole (Q2). The high specificity and sensitivity of MRM MS have made this technique highly suitable for the analysis of protein PTMs.36,389,390 By adding a stable isotope-labeled version of the peptide of interest, absolute quantitation can also be achieved.391 Both gel-based and gel-free approaches have been adopted in redox proteomics studies and specific protocols aimed at the identification of particular redox PTMs will be discussed in the dedicated paragraphs reported below.

Figure 15. SILAC workflow. Control and stimulated cells are grown in special media supplemented with lysine and arginine labeled with various combinations of [13C], [12C], [15N], [14N], [2H], and [1H] to generate tags with different mass shifts (light, medium, or heavy). After complete incorporation of stable isotopically labeled amino acids during protein synthesis, the light and heavy cell populations (or protein extracts) are mixed in a 1:1 ratio and samples are processed together thereafter. Information on relative abundance of peptides coming from the light and the heavy labeled samples is achieved by MS, measuring the area under the peak of each peptide pair.

studies was introduced by the Aebersold group a dozen of years ago373,374 and is based on the use of cysteine-specific isotopecoded affinity tags (ICAT). In ICAT, cysteine residues are specifically tagged with a reagent containing either eight or no [2H] atoms, as well as a biotin group that can be exploited for affinity purification of labeled peptides prior to MS analysis, where both identification and quantification of the labeled peptides is achieved. An interesting development of this technique is represent by the OxICAT method that couples ICAT with differential thiol labeling,375 thus allowing the precise quantification of oxidative thiol modifications (see subsequent sections for a detailed description). Alternatives to the ICAT reagent, but always specific for cysteine residues, are represented by deuterated acrylamide or deuterated 2-vinylpyridine. These tags have been used for differential analysis in 2-DE maps376−380 coupled to MS detection and can also lead to relative quantitation of redox-sensitive cysteines in shotgun analysis. Other tags have been designed to target different amino acid residues, and among them, the most successful and widely used technique is the isobaric tagging for relative and absolute quantification (iTRAQ).381,382 The iTRAQ reagent consists of a reporter group, a balance group, and a peptide reactive group. The reactive group specifically reacts with the N-terminus and εNH2 group of lysine residues in peptides. The reporter group

4.2. Analysis of Protein Adducts Resulting from ROS/RNS-Induced Modifications at Cysteine

Due to the chemical properties of the S-containing side chain, cysteine residues may undergo different redox reactions. This is biologically very relevant because most of the 214 000 cysteines Z


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Figure 16. Quantitative proteomic strategies based on chemical labeling. (A) ICAT tags consist of a sulfhydryl-reactive moiety connected to a linker region with [2H] or [13C] substitutions to make the tag heavy. Biotin is attached to allow affinity purification of labeled peptides. ICAT-tagged peptide purification prior to LC-MS reduces the sample complexity and facilitates protein quantitation, which is achieved by comparing the peak intensities of the heavy- and light-labeled peptides. (B) iTRAQ labeling is performed after enzymatic digestion of the extracted proteins. All the Ntermini and lysine-containing peptides are labeled, including peptides bearing PTMs. Each sample is labeled individually and can then be pooled. nLC-ESI-MS/MS using the latest HCD technology in OrbiTrap and Q-TOF MS or nLC off-line MALDI-TOF-TOF is then performed. Identical peptides derived from different samples have the same mass. These peptides share similar chromatographic properties, allowing both peptide identification and quantification to be derived from the same MS/MS spectrum. In MS/MS analysis, the signal intensity ratios of the reporter groups indicate the ratios of the peptides; they can be used to determine the relative amount of the protein from which they originated.

encoded in the human genome are functional,392 and their redox-induced modification may affect corresponding protein activities. Misbalance in cell homeostasis or site-specific production of certain ROS/RNS in response to various stimuli are most often sensed by proteins with redox-reactive cysteine residues,7,31,392 which may undergo above-mentioned reactions to generate PSOH, PSO2H, and PSO3H derivatives, intramolecular (PSSP) or intermolecular (PSSP′) disulfide bonds, mixed disulfide bonds with low-molecular-mass nonprotein thiols (principally PSSG and PSCys), or S-nitrosothiols (PSNO) (Figure 7).161−163,393,394 While reversible cysteine oxidations may play a regulatory role, the irreversible ones generally lead to loss of protein function or degradation of the damaged protein.44,132,395 Overall, cysteine oxidation is dynamically dependent on the concentration, location, and specificity of small-molecule oxidants/nitrosants balanced by various antioxidant enzymes. Therefore, development of proteomics tools enabling the identification of different cysteine oxidation sites, the characterization of their nature, and the quantification of their modification extent is an important step for determining the role that these oxidative events may play in modulating protein function.33,39,396−399

Depending on cysteine modification nature, identification and site localization of modified adducts generated after ROS/ RNS action may be achieved by a direct MS measurement on purified protein or peptide components, which reveal a specific mass shift of the cysteine-containing species. However, despite the continuous improvements in terms of accuracy and sensitivity in MS techniques for the analysis of PTMs,333,400,401 thiol modifications may be not easily identified either because of their low abundancy or because of their labile nature. Nevertheless, the occurrence of S-glutathionylated, Scysteinyl-glycinylated, S-cysteinylated, and intermolecular disulfide-containing proteins has been ascertained by direct MS analysis of the isolated species, revealing the corresponding adducts presenting a mass difference of +305, +176, and +119 Da, respectively, with respect to the unmodified counterpart, or having a mass of MP1 + MP2 − 2n, where MP1 and MP2 are the mass values of the proteins 1 and 2 (linked by S−S bonds), respectively, and n is the number of present disulfide bridges.402,403 As expected, the mass spectra of these species are totally affected by reductant treatment. On the other hand, intramolecular disulfide-containing proteins resulting from ROS/RNS activity present a limited AA


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mass variation with respect to unmodified species (Δm = −2 Da for each S−S bond), which make it required for additional measurements.402 To this purpose, a modification of the MS strategy conventionally used for the titration of free thiols in proteins has been applied for the detection of oxidized cysteines.404,405 By simple comparison of the molecular mass values of an intact protein in its native and stressed state before and after extensive alkylation with iodoacetamide under denaturing nonreducing conditions, the number of the cysteine residues involved in oxidative/nitrosative insult, and the nature of the modification can be inferred. In fact, cysteines involved in disulfides will not react with iodoacetamide (IAM), thus not generating the corresponding mass increase (Δm = +57 Da for each available SH group), easily detectable by MS measurements. By assumption of a comparable ionization tendency for all of the different species obtained following alkylation, this procedure can be successfully applied to evaluate the quantitative extent of the oxidative insult. For example, this approach has been used for the molecular characterization of the products generated from oxidative modification of bovine lens aldose reductase induced by Cu ions or intermediates of GSH turnover.404,405 Conclusive assignment of mixed disulfides to specific cysteine residues can be obtained by MS mapping experiments on peptide mixtures generated from carboxamidomethylated species following alkylation under denaturing nonreducing conditions. A careful evaluation of experimental conditions suitable to avoid scrambling phenomena during protein digestion is strongly recommended. Thus, identification of the modified residues has been obtained by nLC-ESI or MALDI-TOF MS mapping experiments, by detection of the peptides bearing a mass difference of +305 Da (Sglutathionylated), +176 Da (S-cysteinyl-glycinylated), and +119 Da (S-cysteinylated), and confirmed by MS/MS measurements.403,404 Similarly, cysteine pairing identification in species containing inter- or intramolecular disulfides has been derived by using the MS mapping and CID or ETD-based MS/MS approaches conventionally used for the assignment of disulfides in native polypeptide species,406−412 as in the case of ROS/RNS-induced formation of disulfides in Pax8 and OxyR transcription factors, VIA phospholipase A2, aldose reductase, mitochondrial complex II, glyoxalase 1, glutaredoxin, and various tyrosine phosphatases.404,405,413−420 Other cysteine oxidative modifications may not be easily identified by direct MS measurements either because of their labile nature (S-sulfenylation and S-nitrosylation) or because their mass shift coincides with other modifications (Ssulfinylation, S-sulfonylation and S-sulfation, +32, +48, and +80 Da, respectively). Furthermore, when applied to proteomic studies, most of the MS-based approaches mentioned above cannot be directly used to describe the ROS/RNS-induced protein modifications in a cell extract, biological tissue, or fluid on a holistic basis. To overcome these issues, several indirect methods have been developed during the years, which mainly rely on the differential labeling of the modified cysteine residues; they will be discussed in detail in the following sections. Typically, unmodified protein thiols are blocked with an irreversible alkylating reagent (IAM or N-ethylmaleimide, NEM); then, oxidized or S-nitrosylated cysteine residues are selectively reduced with specific reagents and labeled with a thiol-directed probe, which contains a detectable tag (a biotinylated, fluorescent, or isotopically-modified moiety present in the reagent). Depending on the proteomic approach used (gel-based or shotgun), proteins containing selectively

labeled cysteine residues are then identified by Western blot analysis, fluorescence detection, or nLC-ESI-MS/MS analysis, after their eventual proteolytic digestion. Such labeling procedures also allow for affinity-chromatography enrichment of the proteins or peptides of interest prior to their MS-based characterization, thus increasing the overall sensitivity of the analysis. 4.2.1. Analysis of Protein Adducts Resulting from Disulfide Bond Formation. Oxidation of cysteine residues to produce a disulfide bond is the first post-translational modification that occurs during the assembly/folding of the nascent polypeptide chain. For a long time, it has been thought that this modification is permanent, but it is now clear that incipient formation of (intra- and intermolecular) disulfide bonds involving redox-sensitive cysteine residues may be part of redox-sensing switch mechanisms, which are dynamically regulated and may modify protein structure or activity.421−423 In general, the subset of proteins that contain disulfide bonds or reactive cysteines susceptible to redox regulation has been defined as the “disulfide proteome”; it has been studied in almost all organisms, due to its relevance in regulation of almost all cellular processes.394 Several strategies have been developed during the years to select and recognize cysteinecontaining proteins according to their redox state (free thiol groups or disulfide bridges), whenever present in complex mixtures. The first method for global analysis of the disulfide proteome was originally developed to identify artificially disulfide-cross-linked ribosomal proteins and is based on the use of nonreducing/reducing “diagonal” 2D-PAGE.424 Briefly, protein extracts are separated in the first dimension on a normal SDS-PAGE under nonreducing, denaturing conditions; then, the whole gel lane containing the separated species is incubated with a reducing, denaturing buffer and placed horizontally on the second dimension-gel, where proteins are then separated under reducing conditions and finally stained (Figure 17). Depending on their cysteine redox state, proteins can be detected as spots placed into three different regions. Proteins not containing disulfides have the same electrophoretic mobility in both dimensions and therefore migrate along the gel diagonal. On the contrary, proteins containing intramolecular disulfides have a higher mobility (a more compact structure) in their oxidized form than their reduced counterparts, and therefore migrate slightly above the diagonal. Finally, proteins that are cross-linked by intermolecular S−S bridges migrate slower in the first dimension (higher molecular mass) than in the second one, and can therefore be identified below the diagonal.424,425 More recently, this strategy has been applied for identifying oxidative stress protein products in mammalian heart,426 cotranslational protein folding and disulfide bond formation427 and disulfide-bonded proteins in mammalian neuronal cells exposed to various oxidative stimuli or for isolating S−S bonded proteins from various cell compartments.428,429 The study of the disulfide proteome is complicated by the possibility of thiol−disulfide scrambling phenomena that may impair final results. In this context, recognition of the original protein cysteine redox state requires, as most important step, the rapid freezing of the cysteine redox state. This is usually obtained through the irreversible alkylation of all reduced cysteines by addition of an alkylating reagent to the protein extract immediately after cell lysis. The most straightforward method for disulfide proteome assignment relies on a differential alkylation of redox-sensitive cysteine residues by AB


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NEM).430,431 This strategy is suitable when dealing with simple protein mixtures or purified proteins. However, this approach does not distinguish between the nature of the disulfidecontaining species (S-glutathionylated, S-cysteinyl-glycinylated, S-cysteinylated, intra- and intermolecular disulfide-containing proteins). Moreover, eventual artifacts resulting from sulfenylated and S-nitrosylated proteins have to be considered, although with poor impact due to the poor relative concentration of these adducts. They can be eventually evaluated by performing additional experiments with milder reductants, such as sodium arsenite or ascorbate. To achieve a more general characterization of the cysteine redox state on a proteome scale, also performing differential studies after ROS/RNS insult, more sophisticated approaches are needed. Typically, these approaches are again based on preventive blocking of all available reduced cysteines with IAM or NEM, followed by selective reduction of all disulfidecontaining species with a reducing agent (such as dithiothreitol or the NADPH Trx system) and selective labeling of the newly formed thiols with a tagged alkylating agent, such as biotinconjugated iodoacetamide (BIAM), biotin-conjugated NEM (BNEM), iodoacetamidofluorescein (IAF), or monobromobimane (mBrB) derivatives, which allow Western blotting or fluorescence-based visualization of the protein spots bearing disulfide-involved cysteine residues after their 2-DE resolution, or eventual enrichment by affinity-chromatography (BIAM and BNEM) and their final MS-based identification (Figure 18).431−433 Reagents suitable for their application in intact organelles or cells have also been developed, as in the case of (4-iodobutyl)triphenylphosphonium, which accumulates in mitochondria and yields stable thioether adducts easily detectable by Western blotting.434 Different versions of these gel-based approaches have been developed and used to investigate ROS-sentitive mitochondrial proteins in alcoholic fatty liver of humans435 and rats,434,436 the effect of major enzyme sources of NADPH on yeast disulfide proteome,437 oxidant-sensitive proteins in Jurkat T lymphocytes,438 thioredoxin targets in plants439,440 and RNS-sentitive proteins in mitochondria from rat liver.441 A shotgun version of this approach has also been developed, which is based on thiol labeling with N-[6-(biotinamido)hexyl]-3-(2-pyridyldithio) propionamide (biotin-HPDP), which generates a disulfide-

Figure 17. Nonreducing/reducing “diagonal” 2D-PAGE for proteins containing redox-reactive cysteine residues. Proteins not having redoxactive cysteines run at the level of the diagonal across the gel. In contrast, proteins with an intermolecular disulfide bridge (homo- or heterodimers) migrate below the diagonal, whereas proteins with an intramolecular disulfide bridge run above the diagonal line.

using different alkylating agents. Briefly, the procedure consists in labeling all available reduced cysteines with IAM under denaturing nonreducing conditions, treating the resulting protein products with a specific reducing agent (dithiothreitol) under denaturing conditions, and then alkylating them with NEM. The protein mixture is then enzymatically digested and analyzed by nLC-ESI-MS/MS. Database search using as variable modifications for cysteine residues those from both alkylating agents (+57 and +125 Da for IAM and NEM, respectively) can distinguish peptides originally containing a reduced cysteine (alkylated with IAM) from those containing cysteine residues engaged in disulfide bridges (alkylated with

Figure 18. Differential thiol labeling for proteomic assignment of sensitive-cysteine-containing proteins. All available reduced cysteines are labeled with iodoacetamide (IAA) under denaturing nonreducing conditions. Resulting protein products are treated with a specific reducing agent under denaturing conditions and then alkylated by N-ethylmaleimide (NEM), NEM derivatives containing a biotin moiety (enabling selective purification on streptavidin beads) or NEM derivatives containing a fluorophore (enabling fluorescence-based visualization). AC


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development of this shotgun strategy was obtained with the OxICAT approach, which couples ICAT with differential thiol labeling.375 OxICAT allows the precise relative quantification of all oxidative thiol modifications in different proteins present in a single sample; it identifies the affected proteins also defining their redox-sensitive cysteines. It consists of several steps: (i) proteins are denatured to gain access to all reduced cysteines and labeled irreversibly with light ICAT; (ii) all reversible oxidative thiol modifications within the same sample are reduced by using tris(2-carboxyethyl)phosphine (TCEP) and subsequently modified with heavy ICAT; (iii) after trypsin digestion, the ICAT-labeled peptides are affinity-purified and analyzed by nLC MS/MS analysis, where both identification and relative quantification is achieved. Comparative analysis by OxICAT of a proteome under two experimental conditions determines relative quantitative changes of all oxidative thiol modifications in hundreds of proteins. It has to be underlined that the OxICAT method detects all reversibly oxidized cysteines (disulfides and S-glutathionylated, S-nitrosylated, and sulfenic acid derivatives), whereas higher irreversible oxidation states are not detected. Substitution of TCEP with specific reductants, such as glutaredoxin or ascorbate, will allow the use of OxICAT to specifically quantify S-glutathionylation or S-nitrosylation, respectively. 4.2.2. Analysis of Protein Adducts Resulting from SGlutathionylation. The first proteomic evidence that Sglutathionylation was quite a common post-translational modification affecting a large number of proteins came from the work of Fratelli and colleagues.450 In this study, authors radiolabeled cellular GSH with [35S], then exposed T cells to oxidants (diamide or H2O2), and performed nonreducing 2-DE, which was followed by detection of labeled proteins by phosphorimaging and their identification by MALDI-TOF PMF and nLC-ESI-MS/MS analysis. In this study, several proteins belonging to different functional classes were identified, and their regulation by S-glutathionylation was assessed; however, no localization of the reactive cysteines was achieved because no glutathionylated peptides were detected by mass spectrometry. N-Biotinylated analogues of reduced cysteine, GSH, or GSH ethyl ester have also been used in the study of protein Sglutathionylation.186,451,452 One problem with the use of these compounds in vitro or in cell culture systems was the difficulty to adequately replicate a pathophysiologically relevant oxidative stress. Many diseases with an oxidative component are modeled simply by treatment with metabolic inhibitors, chemical oxidants, or ROS-generating systems. Consequently, the (patho)physiological significance of changes in such systems was questionable, and it was difficult to be sure of the mechanism by which the oxidant manifested a measured functional effect. This is because the introduced oxidant likely reacts with many biomolecules, not solely with protein thiols to induce S-glutathionylation. On the other hand, antibody-based approaches for the detection and study of protein Sglutathionylation have also been used because of the ability of many laboratories to undertake analysis involving Western blotting, immunoprecipitation, and immunolocalization. Panspecific antibodies that detect S-glutathionylated proteins using these methodologies have been reported,192,202,453,454 but they often showed disadvantages.455 To overcome the abovementioned limitations, the use of biotinylated ozidized GSH (biotin-GSSG) was proposed.455 The treatment of cells with this compound replicates one specific consequence of oxidative

linked label on all cysteines that were originally involved in S−S bonds. Proteins are digested and biotinylated peptides are enriched on streptavidin beads, which then are eluted with βmercaptoethanol to generate material suitable for nLC-ESIMS/MS-based characterization.442 Detection of vicinal dithiols, which are likely to form intramolecular disulfide bridges because of their proximity, has been obtained by using the vicinal dithiol-specific reagent phenylarsine oxide (PAO).443 After reversible blocking of the vicinal dithiols with PAO, all other free thiols are alkylated by NEM treatment. PAO is then removed by reduction with 2,3dimercapto-1-propanesulfonic acid, and the newly exposed vicinal dithiols are labeled with BIAM, which serves both for Western blot detection and for affinity purification and enrichment of vicinal dithiol-containing proteins. In order to achieve a dynamic description of thiol susceptibility to redox regulation, more accurate quantitative gel-based methods have also been developed. The redox difference in gel electrophoresis (redox-DIGE) approach uses differential labeling of thiol-containing proteins under two experimental conditions (for example, before and after redox insult) with SH-reactive Cy3 and Cy5 maleimide dye reagents;444,445 Cy3- and Cy5-labeled proteins are then mixed and run on the same 2-DE gel. By measurement of changes in the relative fluorescence of the two tags within a single protein spot, it is possible to detect and quantify redox-sensitive thiols under two experimental conditions. A modified version of this technology based on iodoacetamide-substituted cyanines, C3NIASO3 and C5NIASO3, has also been developed and applied to monitor plasma protein thiol redox status.313 A preliminary MS-based method suitable to quantitatively monitor the redox status of cellular proteins in vivo upon redox stress has been originally proposed by Leichert and Jakob in 2004.446 This method is based on the sequential labeling with [12C]- and [14C]-labeled IAM of thiol-containing proteins before and after their treatment with reducing agents. High [14C]/[12C] ratios are predicted for cysteine-containing proteins with significant redox modifications, while low ratios occur for proteins whose thiol groups are not significantly modified in vivo. The limit of this approach relies on the absence of protein identification and modification site localization. To overcome these problems and perform quantitative analysis on a shotgun proteomic basis, the ICAT technology has been adapted to investigate the disulfide proteome changes.373,447 ICAT reagent is composed of three functional elements: a thiol-reactive group alkylating all reduced cysteines, an isotopically coded linker (existing in a light [12C]-labeled and a heavy [13C]-labeled form) that gives a mass shift of 9 Da used in MS to identify peptide pairs to be quantitated, and an affinity tag that allows specific enrichment of thiol-containing peptides. In this case, comparative quantitative analysis of the redox cysteine status has been performed by (i) direct ICAT labeling of proteins before (light-ICAT) and after (heavyICAT) exposure to a redox treatment, (ii) dithiotreitol-based reduction of protein samples followed by alkylation with IAM, (iii) combination of the labeled protein mixtures from both conditions and their concomitant digestion, (iv) avidinchromatography-based trapping of the labeled peptides, and (v) quantitation of the cysteine-containing peptides by nLCESI or MALDI-TOF-TOF MS/MS analysis (Figure 16A). This approach has been used, in combination with redox-DIGE or iTRAQ, to identify redox-sensitive proteins or thioredoxin target proteins in heart tissues, respectively.448,449 A further AD


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determination.468 Recently, two analytical methodologies for improved MS detection of sulfonic acid-containing peptides have been developed. The first one tries to compensate the poor intensity of signals for cysteine sulfonic acid-containing peptides in MALDI-TOF MS spectra. 469 Addition of diammonium hydrogencitrate (DAHC) to α-cyano-4-hydroxycinnamic acid highly improved detection of the peaks corresponding to the oxidized species, as well as when using 2,4,6-trihydroxyacetophenone and 2,6-dihydroxyacetophenone as matrices with DAHC. A matrix-free ionization procedure, based on desorption/ionization on porous silicon (DIOS) with DAHC was also developed. In the second method, a protocol for selective enrichment and identification of peptides containing cysteine sulfonic acid in tryptic protein digests was proposed; it is based on peptide capture using polyargininecoated nanodiamonds as high-affinity probes.470 This method was applied to selectively concentrate modified peptides from either a highly dilute solution or a complex peptide mixture, in which their abundance was as low as 0.02%. The polyargininecoated probes exhibited a higher affinity for peptides containing multiple sulfonic acids than peptides containing a single sulfonic acid group. The limit of the detection was ascertained in the femtomole range, with the MALDI-TOF mass spectrometer operating in the negative ion mode. On the other hand, proteomic analysis of cell extracts has also demonstrated that cysteine oxidation results in more acidic protein isoforms, which can be visualized as separated novel spots in 2-DE.464 Wagner and colleagues performed an extensive study on peroxiredoxins extracted from HeLa cells as model system, and by coupling 2-DE and MS, they were able to demonstrate that the most acidic spot contains a protein that is irreversibly oxidized at the active-site cysteine to the corresponding sulfinic or sulfonic acid derivative. 2-DE separation can thus represent a general method to distinguish differently oxidized forms of cysteine-containing proteins. On this basis, sulfinylated or sulfonylated derivatives of peroxiredoxin isoforms and transthyretin were identified as early biomarkers of oxidative stress in early ischemia/reperfusion injury during human orthotopic liver transplantation and in amniotic fluid of preeclamptic women.471,472 In the latter case, concomitant production of a protein isoform containing a cysteine to glycine oxidative modification was also observed.472,473 Although cysteine modification to sulfenic acid has been known for many years, only recently have specific tools become available for the comprehensive study and quantitation of the sulfenylated proteome. Indirect detection of sulfenylated proteins has been originally pursued based on a modified version of the biotin-switch (BS) approach.474,475 This method is based on arsenite-specific selective reduction of sulfenic acid in proteins under denaturing conditions. Liberated free thiols can then be labeled with a biotin−maleimide alkylating reagent that facilitates not only the detection of protein sulfenic acids on Western blots probed with streptavidin−horseradish peroxidase but also their affinity purification on streptavidin− agarose beads. As will be discussed in the following sections reporting other versions of the biotin-switch approach, indirect detection methods always need a complete blocking of all free thiols, usually obtained under denaturing conditions, before performing the subsequent selective reduction. On the other hand, the most widely used approach for direct analysis of sulfenic acid-containing proteins is based on the use of 5,5-dimethyl-1,3-cyclohexanedione (dimedone), which irre-

stress, that is, an increase in GSSG. Augmented biotin-GSSG concentration, as with unlabeled GSSG, promotes protein Sglutathionylation, and the biotin tag offers the advantage that it allows detection, purification and identification of the proteins that become modified using avidin-based procedures. Unless used on isolated proteins, however, all biotinylated reagents and antibodies mentioned above did not allow identification of the S-glutathionylation sites. To overcome this limitation, Cotgreave and colleagues proposed a method based on the specific tagging of deglutathionylated proteins with a cysteine-reactive biotin− maleimide reagent, which allowed the selective purification of the modified species through avidin binding.181,456 A mutant glutaredoxin was used to specifically deglutathionylate cellular proteins. Purified proteins were separated by 2-DE and identified by MS analysis. Over 40 novel glutathionylated proteins, both in stressed cells and in cells undergoing constitutive metabolism, were discovered. Alternatively, tagged proteins were digested with trypsin prior to avidin purification, which lead to the subsequent isolation of tagged peptides that were then eluted and directly characterized by nLC-ESI-MS/ MS. This second approach is much more suitable for the identification of the precise site of glutathionylation. In conclusion, application of these proteomics techniques has led to the identification of dozens of proteins as potentially regulated by reversible S-glutathionylation, either inhibited or activated (reviewed in ref 102), making this post-translational modification one of the most important players in several essential cellular pathways associated with apoptosis, protein folding, energy metabolism, calcium homeostasis and ion channel activity, regulation of cytoskeletal assembly, and signaling. 4.2.3. Analysis of Protein Adducts Resulting from Cysteine Oxidation. H2O2, hydroperoxides, HClO, or peroxynitrite can oxidize protein free thiol groups producing -SOH, -SO2H and SO3H derivatives. Although stable sulfenic acid adducts have been demonstrated to occur in a number of redox-sensitive proteins, often they have been considered as transient intermediates in the formation of more stable cysteine oxidation products, both under basal conditions and in response to several redox-active extrinsic compounds. In fact, depending on their polypeptide microenvironment and proximity to other thiols, sulfenic acid derivatives may undergo further oxidation reactions with nearby -SH groups to form disulfide bonds or more oxidized species, such as sulfinic or sulfonic acids.178,180,457−460 While sulfenylated species are labile, both in vivo and during sample preparation prior to MS analysis, sulfenic and sulfonic derivatives are more stable and can be detected with MALDITOF and ESI MS analysis, by measuring the occurrence of the corresponding adducts with Δm = +32 and +48 Da, respectively.170,461−464 These species are stable and insensitive to treatment with reducing agents. Identification of the modified cysteines can be obtained by MALDI or LC-ESI mass mapping experiments on protein digests, specifically revealing peptides bearing the corresponding mass increase, and confirmed by MS/MS analysis. This approach has been successfully used for characterization of sulfinic and sulfonic acid-containing matrix metalloprotease 9, chaperone GroEL, mitochondrial complex II, peroxiredoxin I, II, and III, actin, and various tyrosine phosphatases.177,211,416,418,420,462,464−467 Frequently, fragments containing cysteic acid are totally suppressed; this effect significantly facilitates peptide sequence AE


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Figure 19. Protein sulfenic acid-labeling reactions. Reaction of a protein sulfenic acid with 5-dimethyl-1,3-cyclohexanedione (dimedone) (reaction a), and with its deuterated form 2H6-dimedone (reaction b). Reaction of 2-iododimedone with free protein thiols (reaction c) is also shown. In cells, permeable azide-based and alkyne-based analogs of dimedone, such as DAz-1, DAz-2, DYn-1, and DYn-2, are used to trap and tag sulfenic acidmodified proteins (reaction d and e).

versibly modifies the -SOH moiety476−478 and does not react with free thiols or other oxidized cysteine forms (Figure 19, reaction a). During the years, several analogues of the dimedone reagent have been designed, which incorporate a fluorescent group or a biotin affinity tag in the cyclohexanedione moiety to enable either fluorescent detection or affinity purification of the modified proteins.476,479,480 These reagents can be used to label intact cells or proteins at the time of cellular lysis. In this latter case, it is essential also to add alkylating reagents and catalase directly into the lysis buffer in order to avoid artifactual formation of sulfenic acid derivatives.40 Recently, membrane-permeable azide-based and alkyne-based analogs of the dimedone, termed DAz-1, DAz-2, DYn-1, and DYn-2, have been developed by the Carroll’s group39,481−483 and have been used to trap and tag sulfenic acid-modified proteins directly within cells. These probes can be selectively coupled with alkyne or phosphine biotin tags, thus allowing the specific enrichment of sulfenylated proteins (Figure 19, reactions d and e). They have the remarkable

advantages to bypass reduction of sulfenic acids and to work in intact cells, thus avoiding potential disruption of cellular redox homeostasis. When used for the global analysis of sulfenylated proteins in a human tumor cell line, they enabled the identification of more than 175 new targets of oxidation above the already known sulfenic acid-modified proteins.483 A further improvement of this class of compounds is represented by the development of tagging agents that allow not only the identification of sulfenylated protein but also the quantification of the in vivo modification levels.484 These reagents consist in a deuterated form of dimedone (d6-dimedone), which reacts with sulfenylated cysteines, and in a 2-iododimedone derivative, which in turn reacts with free thiols (Figure 19, reactions b and c). Derivatization with this reagent couple leads to the formation of two identical peptides that will differ in the specific mass of their label, depending on the original redox state of the cysteine residue. After digestion of the labeled proteins, the extent of sulfenic acid formation can thus be determined by dividing the heavy-isotope labeled peak intensity AF


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Figure 20. The biotin-switch assay and its related proteomic procedures. S-Nitrosoproteins are selectively tagged and specifically purified. In the first step, free thiols (-SH) are blocked with the thiol-specific methylating reagent MMTS. During the second step and under conditions where disulfides (−SSR) are not reduced, S-nitrosothiols (−SNO) are selectively reduced by sodium ascorbate. In the last step, these newly formed thiols are reacted with specific reagent as biotin-HPDP, digestion sensitive histidine-tag reagent, fluorescence-based reagents, or thiol-beads. Modified proteins are then purified on avidin resin or nickel column, and then specifically visualized. After the enrichment or detection of S-nitrosylated species, selected proteins are digested with trypsin and identified by LC-MS/MS analysis.

quantify the level of nitrosylation in response to exogenous or endogenous stimuli. Direct detection of S-nitrosylation by MS is hampered by its low concentration at physiological levels and by its high lability; in fact, S−NO bonds are usually more easily fragmented than the peptide backbone giving rise to NO loss, expecially in MALDI ionization.489 This phenomenon is less relevant under milder conditions, such as in electrospray MS, where Snitrosylated proteins or peptides occur with a +29 Da mass shift with respect to the unmodified ones.461,490−493 Wang and collaborators reported a method for the direct detection of Snitrosylated peptides by quadrupole time-of-flight mass spectrometry (Q-TOF MS). Fine tuning of instrument parameters, which regulates ionization (cone and collision energy voltages), and adjusting buffer composition (1 mM EDTA and 0.1 mM neocuproine at neutral pH), which preserves the labile S−NO bond, were found to be necessary to directly detect S-nitrosylated sites.494 A NL MS-based method to identify peptides containing S−NO groups was first developed by Hao and colleagues.363 By using a triple quadrupole instrument, these authors studied the GSNOinduced nitrosylation of argininosuccinate synthetase. After protein tryptic digestion, they identified the unique peptide that was S-nitrosylated. In the experimental setup, the two quadrupole mass analysers were used to monitor the neutral loss of NO in the collision cell, where only the labile S−NO bond was fragmented under the mild collision energy conditions used. By using a similar strategy, Chen and coworkers identified the putative S-nitrosylation sites in a protein

by the sum of the heavy- and light-isotope labeled peak intensities in the mass spectrum. By combination of these different approaches, more than 200 proteins undergoing sulfenic acid modification have been identified so far; they are transcription factors, metabolic enzymes, proteostasis regulators, and cytoskeletal components, whose activity is modulated by a redox switch.39,479,483 4.2.4. Analysis of Protein Adducts Resulting from SNitrosylation. Protein S-nitrosylation is the reversible, covalent binding of nitric oxide to specific cysteine residues to form nitrosothiols.485 It widely occurs on proteins involved in virtually all classes of signaling pathways and thus functions as the prototype of redox thiol-dependent cellular signaling mechanisms. It may be finely tuned in vivo both in time and in space,166 and deeply affects protein activity, localization, and stability, thus regulating a large variety of pathophysiological cellular events, such as apoptosis, neurotransmission, cellular trafficking, and muscle contraction.486 S-Nitrosylation has also been implicated in many pathological conditions. Global detection of S-nitrosothiols in biological samples is generally performed by using chemiluminescent methods, which involve cleavage of the S−NO bond and measurement of free nitric oxide.487,488 These methods, although efficient and sensitive, lack of specificity, because they can only quantitate the total content of nitrosothiols in a sample; they cannot identify the Snitrosylated proteins or amino acids. These methodologies are generally used in the preliminary steps of a proteomic experiment to monitor the presence of nitrosothiols and to AG


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tyrosine phosphatase (PTP1B).495 In vitro nitrosylated PTP1B was digested in solution with trypsin and then subjected to LCMS scan. Only the signal pairs differing in mass by 30 amu were selected for MS/MS analysis, based on the feature that Snitrosylated peptides tend to lose the NO moiety. After the mass difference scan, five peptide pairs were selected for fragmentation, among which four ones were characterized by MS/MS analysis as carriers of a cysteine residue that became presumably S-nitrosylated in response to a biological stimulus. Up to now, these direct strategies have been mainly used with synthetic peptides or purified recombinant proteins nitrosylated in vitro, with the only exception being phytochelatins that are in vivo S-nitrosylated in Cd-stressed Arabidopsis thaliana cells,489,496 thus demonstrating that the direct detection of in vivo S-nitrosylation still represents a challenging task. In fact, nitrosothiols undergo photolytic degradation497,498 and are reduced by cytosolic reducing agents (ascorbate, glutathione) or reduced metals, such as Cu+.499,500 Thus, identification of S-nitrosylated cysteine sites has been generally obtained by using indirect strategies, with the biotin-switch technique being the method of choice for this kind of analysis. This very elegant protocol was introduced by Jaffrey and Snyder in 2001 and paved the path for the large scale proteomic studies of the S-nitrosylome.501 It is based on the conversion of the S-nitrosylated cysteines into biotinylated ones, which can then be efficiently purified prior to MS analysis. In the first step of the protocol, proteins are treated with a thiol-blocking agent, such as monomethyl thiosulfonate (MMTS), to chemically block all free cysteines. This blocking step is crucial because, if it is incomplete, it could lead to false positive identifications; therefore, it needs proper controls and has to be performed under denaturing conditions (in the presence of SDS at 50 °C) to gain access to all structurally masked free thiols. Following the blocking step, the S−NO bond is specifically reduced to a free thiol with sodium ascorbate, which does not affect disulfide bonds or other forms of oxidized cysteine. Free thiols are then reacted with a thiol-specific biotinylating agent, such as biotinHPDP, which results in a disulfide-linked label on all cysteines that were originally S-nitrosylated. Biotin labeling can be used either for Western blot detection or for affinity enrichment of the tagged proteins or peptides on avidin−agarose beads.501−504 MMTS was the blocking agent originally used, due to its high reaction rate with -SH groups. Since its reaction is reversible and may lead to false negative identifications, later on it was substituted with alkylating agents, such as NEM, which afford irreversible products.431 Besides the blocking step, also the ascorbate-assisted S−NO reduction has been debated for a long time. In fact, some studies have demonstrated that sodium ascorbate at low concentration is insufficient to completely reduce S-nitrosylated cysteine, while at high concentration it behaves as a nonspecific reagent.505−509 Thus, a combination of Cu2+ and sodium ascorbate at low concentration was proposed for efficient selective reduction.443,508,510,511 This combination generates Cu+ that reacts with S-nitrosylated cysteines to selectively reduce them. Despite all these concerns, it is now clear that, if performed with cautions (in the dark and by adding metal chelators such as neocuproine) and with proper control reactions, only Snitrosylated cysteines are specifically affected (for a detailed review, see ref 504). Modifications of this original protocol and the introduction of different labeling molecules have favored the evolution of the biotin-switch assay into several new methods (Figure 20), which improved the detection,

quantitation and modification site-assignment of S-nitrosylated proteins.398,512,513 The biotin-switch method has also been adapted to work with functional protein microarrays, which allow the immobilization of thousands of proteins including the low abundance ones. In fact, Foster and colleagues developed the first microarray-based proteomic screen of S-nitrosylated proteins, based on a modified BS assay, which uses an anti-biotin antibody and a fluorescently-labeled secondary antibody for detection of immobilized S-nitrosylated proteins.514 In this experimental setting, potential S-nitrosylation targets are immobilized via their GST-tag on microarrays; after treatment with different types of low molecular weight S-nitrosothiols (Snitrosoglutathione, S-nitrosocysteine, and S-nitroso-4-mercaptophenyl acetic acid), the biotin-switch assay is performed and nitrosylated proteins are detected. Another methodological option to study S-nitrosylation is represented by the so-called “fluorescence switch”, which was introduced by Tello and co-workers.515 This method has improved the sensitivity of the classic biotin-switch technology when coupled to 2-DE. In this case, labeling of the selectivelyreduced S-nitrosylated groups is performed with fluorescent maleimide, allowing fluorescent detection of labeled proteins within 2-DE gels. No pull-down enrichment of the Snitrosylated proteins is performed, so that the whole amount of the fluorescent protein is available for MS identification, thus enabling the use of a lower amount of starting material. In fact, it has to be noted that detection of S-nitrosylated proteins from a crude protein extract usually requires milligrams of starting material, because only a small percentage of the proteins is affected by S-nitrosylation. A similar approach, relying on a gelbased detection of modified proteins, has been proposed by Chouchani and colleagues, who coupled DIGE with specific detection of S-nitrosylated proteins (SNO−DIGE).323 In this modified version of the BS method, S-nitrosylated mitochondrial proteins were efficiently visualized and identified. Authors used a specific NO• donor (MitoSNO) produced by covalent linking of an S-nitrosothiol to the lipophilic triphenylphosphonium cation, which accumulates within mitochondria where it generates NO• and S-nitrosylated proteins. Protein extracts derived from rat mitochondria, treated or not with MitoSNO, were incubated with Cu2+ and sodium ascorbate to selectively reduce S-nitrosothiols, which were then labeled with thiolreactive Cy3 (for control) and Cy5 (for treated sample) fluorescent dyes, mixed together in a 1:1 ratio and finally resolved by 2-DE. Gel image analysis following fluorescence scanning enabled detection of mitochondrial S-nitrosylated proteins by measuring an increase in Cy5 fluorescence. Spots of interest were then trypsin-digested and identified by MS techniques. Parallel analysis by redox-DIGE enabled the authors to distinguish S-nitrosylated thiol proteins from those oxidized due to NO• metabolism. As also stated by the authors, gel-based BS approaches show some limitations since multiple proteins can be electrophorized within the same fluorescent spot, which makes more difficult the identification of the real Snitrosylated protein and the labeled cysteine-containing peptide within the digest. This bias was solved by introducing a modified version of the BS technology,516 where the biotin tag is substituted by a trypsin digestion-sensitive histidine tag. This particular tag contains a thiol-reactive group conjugated to an argininecontaining tripeptide that is linked to an exa-histidine tag (Figure 21). This tagging reagent irreversibly modifies the AH


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measurements. This detergent-free SNOSID method has been recently applied to the quantification of S-nitrosylated proteins by combining it with the SILAC procedure.520 RAW264.7 cells were grown in heavy and light SILAC media to achieve complete metabolic labeling of the entire proteome. After labeling, cells were treated to induce expression of the inducible nitric oxide synthase (iNOS), thus promoting endogenous protein S-nitrosylation. Treated cells were mixed with the control group and lysed, and proteins were extracted in detergent-free conditions to optimize LC-ESI-MS/MS detection and sensitivity.519 After biotin-switch, tryptic digestion, and purification, the peptides that contained both quantitative (relative intensity of the light and heavy peptide) and site-specific information were identified by mass spectrometry. Quantitative data on protein S-nitrosylation have also been obtained in another modified version of the BS procedure, known as the ICAT-switch method, where the ICAT reagent has been introduced in a shotgun procedure.521 In this approach, the light and heavy versions of the ICAT reagent have replaced the biotin-HPDP, and have been used to differentially label S-nitrosylated proteins in liver extracts from normal vs diabetic mice. After affinity purification, 58 Snitrosylated peptides have been identified by MS/MS analysis, among which 37 have changed their level of S-nitrosylation in diabetic mice, as determined by the relative intensity of the ICAT labeled peptide pairs. A modified version of the BS method combining the labeling and the pull-down steps, called SNO resin-assisted capture (SNO-RAC) approach, uses a thiol-reactive resin instead of the thiol-reactive biotin reagent.522 After blocking of free thiols and reduction with ascorbate of the S-nitrosylated sites, proteins are bound to the thiol-reactive resin. At this stage, the sample is trypsinized directly on resin beads and the resulting peptides are labeled with iTRAQ for quantitative proteomics measurements. SNO-RAC advantages reside in a reduced number of experimental steps, an increased detection of high-mass Snitrosylated proteins, and facilitation in MS identification and quantification of S-nitrosylated sites. Application of this method revealed that intracellular proteins may undergo rapid denitrosylation in vivo with varying rates of S−NO turnover among individual sites. It compensates most of the studies performed so far (based on above-mentioned BS method versions), which are referred to exogenously added or endogenously induced NO• donors, thus providing the identification of putative S-nitrosylation sites that are not necessarily those modified in vivo under physiological conditions. In these cases, different applications of the biotinswitch technology and its modifications led to the determination of the S-nitrosylated proteome in different cell lines and tissues.503,516,523−528 Few hundreds of S-nitrosylated proteins have been identified so far, making this post-translational modification biologically relevant for a number of cellular processes. It is now clear that not all cysteine residues and not all proteins with reduced cysteine residues can be modified, indicating that a specific sequence or structural feature determines S-nitrosylation biological specificity. As more and more S-nitrosylated proteins were identified, it became clear that recognition of the “nitrosylatable” cysteine does not reside into a linear sequence motif, although a strong trend for flanking acidic/basic residues was revealed,518 thus suggesting a role for a specific tertiary structure around the modifiable

Figure 21. Properties of the histidine tag specific for S-nitrosylated proteins. The histidine tag contains a thiol-reactive group conjugated to a GRAHHHHHHH peptide tag. This tag irreversibly alkylates newly formed thiols liberated by sodium ascorbate-dependent reduction of S-nitrosylated proteins. After trypsin digestion, the original S-nitrosylated cysteines specifically bear a tag that determines a mass shift (+271 Da) with respect to the reduced counterparts.

liberated cysteines after sodium ascorbate treatment and can be therefore detected by MS. In fact, it produces a specific mass shift (+271 Da) with respect to the original S-nitrosylated peptide that is maintained through all the purification steps, also strengthening MS localization of the modified cysteine. When this approach was applied to the analysis of neuronal cytosolic proteins extracted from cerebral cortex, the precise localization of 28 S-nitrosylated cysteine residues in 19 proteins was obtained. A simplified shotgun solution for direct assignment of modified amino acids in S-nitrosylated proteins from complex mixtures has been proposed by the development of the SNO site identification (SNOSID) method.517 In this case, the total protein mixture, after the free thiol blocking and the biotinHPDP labeling steps, is digested with trypsin before the pulldown step. Labeled peptides are then enriched and eluted from streptavidin beads by disulfide reduction. Eluted peptides are directly characterized by nLC-ESI-MS/MS analysis, thus conferring higher confidence to the results. The utility of this approach was initially demonstrated with the identification of 68 S-nitrosylated cysteines from GSNO-treated rat cerebellar proteins.517 The original SNOSID procedure was modified by Greco and co-workers, who used formic acid instead of a reducing agent to elute the biotinylated peptides from streptavidin−agarose beads.518 Thus, the HPDP-biotin moiety (as detected by a mass shift of +428 Da) remained on the eluted peptides and was used for precise S-nitrosylation site assignment. Increased specificity is fundamental for identification of the originally S-nitrosylated residues and for the exclusion of false positive results. This method was used to study the behavior of human aortic smooth muscle cells after exposure to S-nitrosylating agents. Eighteen proteins with very different functions were identified as S-nitrosylated, and peptide sequence analysis led to the identification of conserved motifs around the cysteine residues. Later on, the original SNOSID methodology was further modified, trying to improve the LC/MS performances and to reduce the amount of sample usually needed for analysis.519 Authors developed a detergent-free protocol by using urea as denaturing agent, which replaced ionic and nonionic detergents (SDS, Triton) known to be detrimental during LC/MS AI


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MetO, which can be further oxidized to MetO2 under stronger oxidizing conditions. Identification of oxidized methioninecontaining proteins has been generally achieved through both bottom-up and top-down proteomic experiments. In fact, MetO- and MetO2-containing polypeptide species are easily detected in the course of ESI or MALDI measurements, by the corresponding adducts having a mass shift of +16 and +32 Da, respectively, with respect to their unmodified counterpart.266,466,529−531 When MS techniques are coupled with a preliminary reversed phase LC separation, it has been observed that the oxidized products generally elute earlier than the unmodified ones.530,531 In general, oxidized peptides containing MetO or MetO2 have been properly detected by revealing the metastable ion loss in MALDI-TOF MS, or the characteristic loss of methanesulfenic acid and methanesulfonic acid (−64 and −80 Da for singly protonated ion, respectively) from the side chain of oxidized methionine derivatives during low-energy CID experiments.532−534 Modification assignment to a specific methionine residue has been always obtained by MS/MS experiments and database search routines accounting for the mass shifts associated with sulfoxide and sulfone moieties. In this context, a large number of examples are reported in literature for ROS- and RNS-modified proteins obtained from in-gel or in solution separations.300,361,466,467,535−540 LC-ESIMS/MS-based quantitative measurement of the modification extent was based on the evaluation of the relative area of oxidized peptides with respect to that of corresponding unmodified counterparts.540−548 All these studies definitively demonstrated that various in vitro and in vivo oxidative, nitrosative, haloxidative, lipoxidative, and glycoxidative conditions are able to induce MetO and MetO2 formation in proteins. In particular, these modifications were ascertained in brain proteins from patients with idiopathic Parkinson's and Alzheimer's disease,549,550 plasma proteins from patients with several inflammatory states,539,545 and HDL proteins from patients with coronary artery disease or type 1 diabetes.543,551 It has been hypothesized that methionine oxidation in polypeptides may also result from sample manipulation or reactions during their proteomic characterization.267,544 To address these points, reduced standard proteins resolved or not by preventive electrophoretic separation were subjected to quantitative mass mapping experiments after in-gel or in solution digestion.552 Several methionine residues were found highly converted into MetO following their in-gel proteomic analysis; their modification extent was reduced when in solution manipulation was performed. It was proposed that high voltages used during electrophoresis may cause the formation of O3 amounts leading to methionine oxidation. On the other hand, atmospheric O2 dissolved in basic solutions used for protein enzymatic digestion seems also to contribute, generating a background of MetO within samples. Furthermore, it was also demonstrated that gradual corrosion of stainless steel ESI emitters, under conditions of normal MS use, generates surface irregularities that promote electrochemical reactions associated with the spray process, finally determining artifactual MetO formation.553 These electrical discharge processes can be avoided by adding a redox buffer or (preferably) by repolishing the emitter, especially to a rounded geometry. To distinguish between artificial methionine oxidation deriving from sample handling and modification occurring in vivo, a dedicated procedure based on protein Nterminal acetylation, selective hydrolysis at methionine residues by CNBr, and specific labeling of the newly generated amino

cysteine residue. By using a 55 S-nitrosylated protein data set with established crystal or NMR structures, Marino and Gladyshev recently demonstrated that an acid−base motif better describes the modifiable cysteine residues.433 This motif implies the presence of a charged residue in close proximity to the S-nitrosylatable cysteine and the presence of another one with opposite charge within a larger region. Authors also suggested that this acid−base motif may be engaged in protein−protein interactions, thus contributing to trans-Snitrosylation reactions. In parallel, Doulias and collaborators used a much larger data set of endogenously S-nitrosylated proteins and defined a putative sequence and structure consensus for S-nitrosylation.527 In particular, they purified endogenously S-nitrosylated proteins from mouse liver by using organomercury reagents (either immobilized on agarose beads or as biotinylated derivative) that react directly, efficiently, and specifically with S-nitrosocysteines. After immobilization, the Snitrosylated proteins were digested; captured peptides were eluted with perfomic acid before LC-ESI-MS/MS analysis. This strategy led to the identification of 328 sites on about 200 proteins. Bioinformatics analysis of the identified S-nitrosocysteine proteome not only confirmed the occurrence of the modified acid−base motif already described433 but also highlighted that S-nitrosylation sites were over-represented in structural areas containing α-helices and under-represented in coils (compared with unmodified cysteine residues within the same proteins), and showed a preferential distribution in highly exposed areas on the molecular surface. 4.2.5. The OxMRM Method. Recently, a new methodology named oxidation multiple reaction monitoring (OxMRM) has been developed as a general procedure to assess cysteinespecific redox sensitivity of proteins.36 It combines protein purification, differential alkylation with stable isotopes, and MRM MS and can be applied in a targeted manner to virtually any cysteine or protein. This approach has been designed to overcome lack of sensitivitity and flexibility of the indirect methods described above. OxMRM is based on different experimental steps: (i) irreversible alkylation of reduced cysteines under denaturing conditions with a stable isotopelabeled tagging reagent (light form); (ii) reduction of oxidized cysteines followed by chemical tagging with the same stable isotope-labeled alkylating reagent (heavy form). Depending on the chemical reductant, several types of oxidation can be investigated: (i) sodium ascorbate is used for S-nitrosylated thiols; (ii) sodium arsenite selectively acts on sulfenic acids; (iii) strong reducing agents, such as DTT or TCEP, affect all reversible oxidative cysteine products. Differentially alkylated proteins of interest are then purified via specific antibodies and enzymatically digested, and the resulting peptides are subjected to LC-MRM MS analysis. Pitfalls of this method consist in the need for a specific antibody to purify the protein of interest and the previous knowledge of the peptide bearing the oxidized cysteine in order to design the proper MRM experiment. Despite these limitations, OxMRM benefits from the high sensitivity and reproducibility of the MRM MS approach and can be adapted to quantitate the percentage of various reversible cysteine oxidative states. 4.3. Analysis of Protein Adducts Resulting from ROS/RNS-Induced Modifications at Methionine

Methionine residues are highly susceptible to side chain oxidation exerted by most of the active ROS and RNS.217 Mild oxidizing conditions determine in vivo generation of AJ


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Figure 22. Combined fractional diagonal chromatography (COFRADIC) analysis of methionine sulfoxide-containing peptides. Peptides, as derived from protein(s) digestion with trypsin, are separated by reversed-phase LC, then subjected to a sorting reaction to reduce methionine sulfoxide (MetO) into methionine. A second, but identical, peptide chromatographic separation is then performed, and fractions are collected. MetOcontaining peptides, which have been converted into their more hydrophobic methionine-containing counterparts, shifted their elution times in the second chromatographic run to higher values, compared with oxidized species. Only peptides having different retention times in the two chromatographic runs are then analyzed and identified by MS and MS/MS, respectively.

groups with a Br-containing compound was proposed.554 This method allowed the unequivocal localization of oxidized methionine residues even in complex peptide mixtures but found a reduced application. In MetO- or MetO2-containing peptides, as for cysteinecontaining ones, side chain oxidation may hamper CID fragmentation, thus leading to a more difficult interpretation of resulting MS/MS spectra. This limitation was partially overcome by Srikanth and colleagues, who showed that ETD can be used to obtain extensive sequence information for peptides with oxidized cysteine, histidine, and methionine residues.345 Similarly, Guan and collaborators used a combination of ECD and CID coupled to FTICR mass spectrometry to solve this problem.533 In the latter case, MetO occurrence was easily detected in the low-energy CID spectrum, which showed the characteristic loss of methane sulfenic acid, while the position of oxidized methionine was localized by ECD, which was capable of providing extensive peptide backbone fragmentation without detaching the labile oxidized methionine side chain. In the past, a solid-phase isolation procedure for the selective enrichment of protein digests in MetO-containing peptides has also been proposed,555 but its application has been very limited. Very recently, polyclonal antibodies against oxidized methionine have been developed and used to recognize modified methionine in pure proteins or in mouse and yeast protein extracts by Western blot analysis.219,556,557 Even if they have not been even tested in 2-DE-based proteomic applications, one may speculate that these antibodies might be very useful to specifically enrich oxidized methionine-containing proteins or peptides, thus simplifying MS detection and assignment of this modification. Shotgun approaches based on 2D chromatographic separation followed by MS/MS-driven protein identification have been used to identify MetO-containing proteins in protein

extracts from various biological sources. In particular, the occurrence of MetO was ascertained in αA, αB, βA1, βB1, βB2, βB3, γB, γC, and γS crystallin isoforms from congenital cataract patients.544,558 Although a number of differences were found in cataract compared with normal lenses, no correlation with pathological outcome was derived, and tryptophan oxidation was preferred as proper biomarker of the disease. Very recently, a combined fractional diagonal chromatography (COFRADIC) approach was used to map in vivo oxidized methionine residues and to quantify their degree of oxidation.559 COFRADIC technology is based on two consecutive, identical peptide separations via reversed-phase LC coupled to MS/MS analysis.560−562 Between the first and the second chromatographic separation step, a chemical or enzymatic process (named sorting reaction) is performed to selectively affect a specific amino acid, thereby changing the chromatographic properties of the peptides holding this targeted residue. In this work, proteins extracted from SILAC-labeled Jurkat T cells after H2O2 treatment were trypsin-digested and subjected to COFRADIC analysis. The sorting reaction was performed with a mixture of MetO reductase A and B3, able to convert MetO-containing peptides into their more hydrophobic methionine-containing counterparts, which therefore selectively shifted their elution times in the second chromatographic run to higher values compared with oxidized species (Figure 22). Only peptides with altered column retention time were then collected for their subsequent MS/MS characterization. Thus, 2626 oxidation-sensitive methionine residues present in more than 1600 different proteins were identified, thereby representing the largest proteomic description of methionine oxidation in mammalian cells obtained so far. Bioinformatic analysis of the identified MetO-containing peptides highlighted a sequence motif, composed of polar amino acids with a noticeable enrichment for proline in heavily oxidized peptides, which favors AK


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Figure 23. Chemical labeling of carbonylated proteins with 2,4-dinitrophenylhydrazine (DNPH) (A), N′-aminooxymethylcarbonylhydrazino-dbiotin (ARP) (B), biotin hydrazide (BH) (C), and Girard’s P reagent (D). Additional reduction with NaCNBH3 is eventually performed in the case of BH- and Girard’s P reagent-adducted species to yield stable derivatives for further proteomic analysis.

methionine oxidation, and identified specific sequence features influencing the degree of methionine modification at individual sites.

or by the 2,4-dinitrophenylhydrazine (DNPH)-based colorimetric test.564 The latter procedure, based on the pioneering analysis of stable 2,4-dinitrophenylhydrazone (DNP) products by Levine and Stadtman, has become the most widely used method for the measurement of protein oxidation in purified components139 or extracts from various pathophysiological models122,565 (Figure 23A). Detection of carbonylated proteins has also been facilitated by the introduction of anti-DNP antibodies, which allow ELISA analysis according to quantitative standards.248,566 In parallel, the occurrence of specific carbonylated amino acids has been ascertained by their direct identification in biological fluids or protein hydrolysates, as generated from isolated polypeptide species or whole biological tissues/body fluids, through conventional LC analysis or dedicated MS-based procedures, after eventual precolumn derivatization. In particular, quantitation of carbonylated derivatives resulting from direct oxidation of proline, arginine, and lysine residues was obtained by GC-MS and LC-MS analysis.567,568 In fact, γglutamic semialdehyde (from proline and arginine) and α-

4.4. Analysis of Protein Carbonylation Products

Protein carbonylation occurs as a result of various irreversible oxidative modifications associated with ROS insult; carbonylated proteins are not repaired and are either removed by proteasome/lysosome activities or accumulate as damaged or unfolded species.232 In previous sections, we have already described that carbonyl groups may be introduced within the protein structure at different sites and by different mechanisms.35 Under specific conditions, different carbonylated amino acids may occur simultaneously. The analytical problem is in recognizing and differentiating among the various types of oxidative modifications.9 Because protein carbonyls have no distinguishing spectrophotometric absorbance or fluorescence properties, they have been initially assessed by measuring the incorporation of tritium following reduction with sodium [3H]-borohydride563 AL


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Figure 24. continued

AM


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Figure 24. Chemical structure of linear and cross-linked advanced glycosylation end-product (AGE) derivatives. (A) Linear and cross-linked AGEs from lysine modification. FL, Nε-fructosyl-lysine; PYR, pyrraline; CML, Nε-carboxymethyl-lysine; CEL, Nε-carboxyethyl-lysine; GALA, Nε-glycoloyllysine; AFGP, 1-alkyl-2-formyl-3,4-glycosyl-pyrrole; GLUCOLD, 1,3-bis-(5-amino-5-carboxypentyl)-4-(1′,2′,3′,4′-tetrahydroxybutyl)-3H-imidazolium salt; GOLD, 6-{1-[(5S)-5-ammonio-6-oxido-6-oxohexyl]imidazolium-3-yl}-L-norleucinate; MOLD, 6-{1-[(5S)-5-ammonio-6-oxido-6-oxohexyl]-4-methylimidazolium-3-yl}-L-norleucinate; DOLD, 6-{1-[(5S)-5-ammonio-6-oxido-6-oxohexyl]-4-[(2S,3R)-2,3,4-trihydroxybutyl]imidazolium-3yl}-L-norleucinate; lysine-hydroxy-triosidine, {1-(5-amino-5-carboxypentyl)-3-[(5-amino-5-carboxypentylamino)methyl]-5-hydroxypyridinium}; GOLA, N6-(2-{[(5S)-5-ammonio-6-oxido-6-oxohexyl]amino}-2-oxoethyl)-L-lysinate. (B) Linear AGEs from arginine modification. G-DH1 and G-DH2, glyoxal-derived dihydroxyimidazoline 1 and 2; G-H1, G-H2, and G-H3, glyoxal-derived hydroimidazolone 1, 2 and 3; MG-DH1 and MGDH2, methylglyoxal-derived dihydroxyimidazoline 1 and 2; MG-H1, MG-H2, and MG-H3, methylglyoxal-derived hydroimidazolone 1, 2, and 3; 3DG-DH1 and 3DG-DH2, 3-deoxyglucosone-derived dihydroxyimidazoline 1 and 2; 3DG-H1, 3DG-H2, and 3DG-H3, 3-deoxyglucosone-derived hydroimidazolone 1, 2, and 3; CMA, Nω-carboxymethyl-arginine; RPYR, argpyrimidine; THP, Nδ-(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6tetra-hydropyrimidine-2-yl)ornithine. (C) Cross-linked AGEs from combined lysine and arginine or lysine and histidine modification. PENT, pentosidine; glucosepane, 6-[2-{[(4S)-4-ammonio-5-oxido-5-oxopentyl]amino}-6,7-dihydroxy-6,7,8,8a-tetrahydroimidazo[4,5-b]-azepin-4(5H)-yl]L-norleucinate; GODIC, N6-(2-{[(4S)-4-ammonio-5-oxido-5-oxopentyl]amino}-3,5-dihydro-4H-imidazol-4-ylidene)-L-lysinate; MODIC, N6-(2{[(4S)-4-ammonio-5-oxido-5-oxopentyl]amino}-5-methyl-3,5-dihydro-4H-imidazol-4-ylidene)-L-lysinate; DOGDIC, N6-{2-{[(4S)-4-ammonio-5oxido-5-oxopentyl]amino}-5-[(2S,3R)-2,3,4-trihydroxybutyl]-3,5-dihydro-4H-imidazol-4-ylidene}-L-lysinate; arginine-hydroxy-triosidine, {2-(4amino-4-carboxybutylamino)-8-(5-amino-5-carboxypentyl)-6-hydroxy-3,4-dihydro-pyrido[2,3-d]pyrimidin-8-ium}; histidine-threosidine, 2-amino-5(3-((4-(2-amino-2-carboxyethyl)-1H-imidazol-1-yl)methyl)-4-(1,2-dihydroxyethyl)-2-formyl-1H-pyrrol-1-yl)pentatonic acid.

a reaction that proceeds via a Schiff base adduct formation. In this case, quantification of 3-(Nε-lysino)propan-1-ol, 1,3-di(Nεlysino)propane, and 2-ornithinyl-4-methyl(Nε-lysyl)1,3-imidazole also led to an evaluation of the cross-linked adducts.569,570 On the other hand, a GC-MS-based approach allowed accurate quantification of HNE−cysteine adducts in various biological samples.571 This procedure involves reduction of bound HNE to 1,4-[2H]dihydroxynonane ([2H]DHN) with NaB2H4, followed by treatment with Raney nickel (RN) to release [2H]DHN, which is then converted into a di-t-butyldimethyl (TBDMS) derivative. Under GC-MS operating conditions, this derivative undergoes a loss of O-TBDMS, resulting in an easily detectable ion. Also in this case, the use of deuterated internal standards led to more accurate quantitative measurements. Later on, the differential distribution of HNE adducts to sensitive amino acids present in blood proteins was revealed by a procedure derived from that reported above, which is based on the different reactivity of HNE-adducts at lysine and histidine residues to RN treatment, with respect to HNE− cysteine adducts.572 Recently, a further method was developed for the analysis of protein residues modified by reaction with different enals.573 Protein adducts were again stabilized by

aminoadipic semialdehyde (from lysine) products (Figure 9B) were detected by analyzing the corresponding 5-hydroxy-2aminovaleric acid (HAVA)- and 6-hydroxy-2-caproic acid (HACA)-derivatives obtained following protein reductive stabilization and extensive acid hydrolysis.567 In this case, amino acids were converted into their N,O-trifluoroacetyl methyl esters or N(O)-ethoxycarbonyl ethyl esters before chromatographic analysis. This approach allowed researchers both to perform selective ion monitoring (SIM) experiments for the quantification of trace quantities in biological samples and to use deuterated internal standards for analyte loss correction and precise quantitative measurements. On this basis, the quantification of HAVA and HACA was obtained for a series of model proteins and mammalian tissues under normal, aged, and pathological conditions.567,568 The extent of amino acid modification after RCS addition has also been evaluated with GC-MS analysis, by revealing the 3-(Nε-lysino)-4-hydroxynonal-1-ol generated in protein hydrolysates following reduction with NaBH4.569 The use of deuterated internal standards allowed more accurate measurements. This approach was also applied to the quantification of the modified lysine and arginine residues after MDA treatment, AN


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ammonio-6-oxido-6-oxohexyl]amino}-2-oxoethyl)-L-lysinate (GOLA); {1-(5-amino-5-carboxypentyl)-3-[(5-amino-5-carboxypentylamino)-methyl]-5-hydroxypyridinium} (lysine-hydroxytriosidine); {2-(4-amino-4-carboxybutylamino)-8-(5-amino-5carboxypentyl)-6-hydroxy-3,4-dihydro-pyrido[2,3-d]pyrimidin8-ium} (arginine-hydroxy-triosidine); 2-amino-5-(3-((4-(2amino-2-carboxyethyl)-1H-imidazol-1-yl)methyl)-4-(1,2-dihydroxyethyl)-2-formyl-1H-pyrrol-1-yl)pentatonic acid (histidinethreosidine); and glucose-lysine dimer cross-link 1,3-bis-(5amino-5-carboxypentyl)-4-(1′,2′,3′,4′-tetrahydroxybutyl)-3Himidazolium salt (GLUCOLD) derivatives were developed to this purpose (Figure 24).568,580−593 Some of these derivatives do not contain a carbonyl moiety but originate from modification of early carbonylated adducts. Also in this case, the synthesis of [2H]-, [13C]-, or [15N]-containing internal standards and the possibility to perform SIM experiments allowed for an accurate evaluation of trace quantities in plasma and lens proteins or other biological samples from patients under different pathophysiological states. Main drawbacks in the analytical studies mentioned above are the lack of information on the identity and concentration of individual modified proteins, together with the assignment of the modification sites. Obtaining this information is key to relating a specific amino acid modification to a biological mechanism, opening the possibility to establish a relationship between defined modified proteins and a pathophysiological condition. Thus, various proteomic methodologies have been developed to identify/characterize carbonylated proteins as purified from biological matrices or, directly, in biological tissues/fluids subjected to oxidative stress conditions. In vitro studies using chemicals that induce carbonylation were eventually performed to predict and further investigate the reactivity of proteins under real oxidative pathophysiological conditions. In the case of gel-based proteomic experiments, carbonylated proteins have been detected by reducing them with sodium [3H]-borohydride and separating the resulting labeled proteins with 1- or 2-DE; then, excised bands or spots have been analyzed for the amount of incorporated tritium by incubating each gel portion with 30% H2O2 and by measuring the released radioactivity.563 On the other hand, carbonylated protein samples derivatized with DNPH have been resolved by electrophoretic approaches, and carbonylation associated with specific bands or spots has been detected in Western blots by using anti-DNP antibodies (Figure 23A).248 By means of this second approach, a great number of proteomic studies on carbonylation have been published that used the commercially available Oxyblot kit (Chemicon/Millipore, Billerica, MA, USA) in the course of classical 1D or 2D gel-based experiments. Generally, DNPH-treated proteins are subjected to electrophoresis for Western blotting, while nontreated counterparts are resolved in parallel gels for protein identification by MALDI-TOF PMF or nLC-ESI-MS/MS procedures (Figure 14). Examples, in this context, are the differential proteomic studies on carbonylated proteins in liver, brain, temporal cortex, and olfactory bulb tissues or bronchoalveolar lavage fluid of control and aged rats;594−598 human plasma before and after physical exercise, limb amputation, or treatment with anticancer drugs;599−601 E. coli cells before and after nutrient starvation, aging, or exposure to UV radiation;602−605 fibroblasts, brain, and hippocampal tissues of control, AD, PD, and HD patients,241,549,606−611 related amnestic mild cognitive impairment subjects 612−614 or

reduction with NaBH4, hydrolyzed with HCl, derivatized with propylchloroformate and then subjected to LC-ESI-FT-ICR MS analysis. This approach was able to characterize in parallel Schiff base, Michael-type, pyridinium, 3-formyl-3,4-dehydropiperidino, and cross-linking adducts from the reaction of HNE, acrolein, crotonaldehyde (CRO), 2-pentenal, hexenealdehyde, and heptenaldehyde with lysine, histidine, and arginine. On the other hand, linoleic acid hydroperoxide-modified lysine derivatives, such as N ε (hexanoyl)Lys, N ε (azelayl)Lys, Nε(succinyl)Lys, and Nε(propanoyl)Lys, were also revealed and quantified in biological fluids by dedicated LC-ESI-MS/MS procedures using deuterated derivatives as internal standards.574 Similarly, various LC-ESI-MS-based procedures have also been developed for quantification of protein adducts resulting from the reaction of various IsoPs and PGs with cysteine, lysine, and histidine residues or IsoKs and LGs with lysines. Also in this case, carbonylated amino acids were directly identified in biological fluids or protein hydrolysates by GC-MS and LC-MS analysis, after eventual precolumn derivatization. In particular, IsoPs and PGs were demonstrated to provide Michael addition to proteins according to their α,β-unsaturated carbonyl reactivity.575,576 On the other hand, IsoK/LGs were shown to selectively form various derivatives that include imine, pyrrole, lactam, hydroxylactam, and cross-linked species.577−579 A number of lysine- and arginine-derived linear or crosslinked eventually carbonylated AGE derivatives (Figure 11) have also been detected by dedicated LC-, GC-MS- or LC-ESIMS-based procedures, when biological fluids or protein hydrolysates from samples previously subjected to extensive acid or enzymatic hydrolysis were analyzed. Thus, different methods for partial or concomitant detection of Nε-fructosyllysine (FL); pyrraline (PYR); Nδ-(5-hydro-4-imidazolon-2-yl)ornithine, 5-(2-amino-5-hydro-4-imidazolon-1-yl)norvaline and 5-(2-amino-4-hydro-5-imidazolon-1-yl)norvaline (glyoxal-derived hydroimidazolones G-H1, G-H2 and G-H3); Nδ-(5hydro-5-methyl-4-imidazolon-2-yl)-ornithine, 5-(2-amino-5hydro-5-methyl-4-imidazolon-1-yl)norvaline, and 5-(2-amino4-hydro-4-methyl-5-imidazolon-1-yl)norvaline (methylglyoxalderived hydroimidazolones MG-H1, MG-H2, and MG-H3); N δ -[5-hydro-5-(2,3,4-trihydroxybutyl)-4-imidazolon-2-yl]ornithine, 5-[2-amino-5-hydro-5-(2,3,4-trihydroxybutyl)-4-imidazolon-1-yl]norvaline, and 5-[2-amino-4-hydro-4-(2,3,4-trihydroxybutyl)-5-imidazolon-1-yl]norvaline (3-deoxyglucosonederived hydroimidazolones 3DG-H1, 3DG-H2, and 3DGH3); Nε-carboxymethyl-lysine (CML); Nε-carboxyethyl-lysine (CEL); Nω-carboxymethyl-arginine (CMA); pentosidine (PENT); argpyrimidine (RPYR); Nδ-(4-carboxy-4,6-dimethyl5,6-dihydroxy-1,4,5,6-tetra-hydropyrimidine-2-yl)ornithine (THP); 6-[2-{[(4S)-4-ammonio-5-oxido-5-oxopentyl]amino}6,7-dihydroxy-6,7,8,8a-tetrahydroimidazo[4,5-b]-azepin-4(5H)yl]-L-norleucinate (glucosepane); 6-{1-[(5S)-5-ammonio-6oxido-6-oxohexyl]imidazolium-3-yl}-L-norleucinate (GOLD); 6-{1-[(5S)-5-ammonio-6-oxido-6-oxohexyl]-4-methylimidazolium-3-yl}-L-norleucinate (MOLD); 6-{1-[(5S)-5-ammonio-6oxido-6-oxohexyl]-4-[(2S,3R)-2,3,4-trihydroxybutyl]imidazolium-3-yl}-L-norleucinate (DOLD); N6-(2-{[(4S)-4ammonio-5-oxido-5-oxopentyl]amino}-3,5-dihydro-4H-imidazol-4-ylidene)-L-lysinate (GODIC); N6-(2-{[(4S)-4-ammonio5-oxido-5-oxopentyl]amino}-5-methyl-3,5-dihydro-4H-imidazol-4-ylidene)-L-lysinate (MODIC); N6-{2-{[(4S)-4-ammonio-5-oxido-5-oxopentyl]amino}-5-[(2S,3R)-2,3,4-trihydroxybutyl]-3,5-di-hydro-4H-imidazol-4-ylidene}-L-lysinate (DOGDIC); N6-glycoloyl-lysine (GALA); N6-(2-{[(5S)-5AO


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correlated animal models;615−618 yeast cells before and after anoxic conditions, aging, or herbicide treatment;619−622 plasma or cerebrospinal fluid of control, AD, and Guillain-Barré syndrome patients;623−626 plant seeds before and during germination, early defense, and aging,627−629 fruits before and during senescence630 or leaves before and during the life cycle;631 plasma and adipocyte cells of animal models of diabetes and control;632,633 hepatocyte cells before and after treatment with ROS-inducing toxic or allergenic substances;634−636 thalamic tissues of animal models of absence seizure;637 bronchoalveolar lavage of control and patients with sarcoidosis and pulmonary fibrosis;638 gland, gill, and mantle tissues of clams and blue mussels unexposed or exposed to different pollutants;639−644 animal muscle tissues before and after acute oxidative stress;645,646 mitochondria of different tissues before and after ischemia-reperfusion conditions;647,648 Dictyostelium discoideum cells before and after exposure to Hg2+;649 diaphragm and muscle tissues of severe chronic obstructive pulmonary disease patients, controls, or related animal models;251,255,650 human cells unexposed or exposed to chemicals inducing apoptosis;651,652 saliva fluid of controls and patients with periodontal disease;653 proteasome preparations unexposed or exposed to ROS;654 and cerebellar astrocytes of a animal models of autoimmune encephalomyelitis.655 In all cases, analysis of carbonylated proteins was limited to their identification; no DNPH-derivatized peptides were detected in their digests, hampering modification site assignment. This was due to the restricted amount of carbonylated peptides generally present in a stained gel spot corresponding to an Oxyblot immunopositive one, with respect to their unmodified counterparts. Notwithstanding their widespread application, it was recently pointed out that use of commercial DNPH kits may be associated with an overestimation of protein carbonyl content,656 a diminished reagent labeling efficiency upon storage (due to a decrease in acidity of the derivatizing solution),657 and instability of certain DNPH-derivatized protein samples upon storage either under reducing conditions at 4 °C656 or at low pH values.658 Thus, caution should be exercised when commercial kits are used. Relevantly, it has been reported that DNPH is also reactive toward protein sulfenic acids in the absence of reducing reagents.259 A very recent comparative study on 2D redox proteome maps of mouse cerebellum realized by performing the DNPH derivatization step before or after electrophoresis demonstrated that, when the same approach is used for protein detection and identification, both methods are useful to identify carbonylated proteins.659 However, whereas pre-DNPH derivatized proteins were successfully analyzed, high background staining complicated the analysis when the DNPH derivatization was performed after transblotting. Differently from what was hypothesized before,35,606,632 comparative analysis of protein migration in 2-DE and of MS identification scores by using both methods demonstrated that no major differences are observed before and after DNPH treatment. Conversely, differences were detected in the protein patterns produced on the 2D gels; in fact, the different sample treatments led to differential areas of protein distribution enrichment. This precluded complete matching of 2D oxyblots of pretreated samples with the 2D Coomassie-stained gels of untreated samples, supporting the idea that these differences may be the consequence of a differential protein precipitation.

Other chemical probes containing a hydrazine-like moiety linked to a functionalized biotin or fluorescent molecule have been also used to visualize protein spots/bands in the course of gel-based proteomic investigations. In the first case, biotin− hydrazide (BH)660 and N′-aminooxymethylcarbonylhydrazinod-biotin (ARP)661 have been used to biotinylate carbonylated proteins before 2-DE, eventually capture them and detect them by Western blotting experiments without using secondary antibodies, thus rendering the recognition process more efficient and less subject to error (Figure 23B,C). In the case of biotin−hydrazide, derivatization has to be followed by a reduction step (with NaCNBH3) to yield the stable hydrazine derivatives, which then need protein precipitation and washing prior to further analysis.660,662−664 Conversely, the use of ARP is not associated with the removal of reagent’s excess, if the biotinylated samples are directly subjected to gel-based analysis,661,665 because the oxime bond formed between ARP and the carbonyl group is stable. Two visible-wavelength fluorescence probes have also been reported for proteomic analysis of carbonylated proteins, namely, fluorescein-5-thiosemicarbazide (FTC)666,667 and Alexa488 fluorescence hydroxylamine (FHA)668 (Figure 25).

Figure 25. Chemical structure of visible-wavelength fluorescent probes for gel-based proteomic analysis of carbonylated species. FTC, fluorescein-5-thiosemicarbazide; FHA, Alexa488 fluorescence hydroxylamine.

Both probes are commercially available and have been successfully used in 2D gel-based investigations.667,668 These probes offer at least two advantages in gel-based analysis of carbonylated proteins. First, there are no Western blot experiments to be carried out, so the whole process, from gel running to image documenting, can be completed in a much shorter time. Second, the same gel can be used for both protein staining and carbonyl imaging, which reduces errors associated with gel spot identification and excision for subsequent MS analysis. This is in contrast to DNPH- or biotin-based 2D gel analysis of carbonylated proteins, whereby gels are used for total protein staining and Western blot membranes are used for imaging the carbonylated proteins. Also in this case, gel-based analysis of carbonylated proteins by derivatization with these fluorescent molecules was limited to their identification by different MS procedures. In addition to general drawbacks mentioned before, 2-DEbased proteomic analysis suffers from some specific limitations that clarify why the identified, carbonylated proteins are generally limited to abundant and soluble proteins: (i) the limited protein load on gel, associated with low carbonylation levels on a given protein, and the low abundance of some modified proteins; (ii) the incomplete recovery of carbonylated peptides from a gel digest and/or a LC column during subsequent analysis; (iii) the concomitant occurrence of AP


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Figure 26. Gel-based and gel-free proteomic approaches for enrichment, visualization, quantification, characterization, and modification site assignment of carbonylated species. Reagents and analytical procedural steps used for each approach are shown (in white squares), together with the resulting information (in gray squares). Optional steps are indicated with dotted white quares.

subjected to oxidative stress, challenged with 2-nitropropane or EtOH-containing diet.675−680 Differently from what was reported for selective staining of carbonylated proteins in combined 2-DE-Western blotting, when ARP is used in affinity capture experiments (see below), further steps on labeled proteins are indeed required to completely remove the ARP excess, such as buffer exchange using centrifugal filters or TCA precipitation and washing.665,681 Either for biotin−hydrazide or ARP, affinity purification of carbonylated species was obtained by using different kinds of avidin/streptavidin columns (Figure 26). Compared with shotgun approaches (see below), the advantage of this strategy is that fractionation is occurring at the protein level and all tryptic peptides generated from a certain protein(s) will be found in the same fraction, being contemporaneously suitable for MS analysis and thus facilitating their identification. However, since a number of unmodified peptides are also present in the sample subjected to MS measurements, characterization of the modification site or type may be eventually hampered with respect to proteomic strategies that analyze only modified peptides. Nevertheless, a number of modified amino acid residues were also assigned in these studies.250,674−678 Recently, a strategy for the identification of carbonylated proteins from complex protein mixtures that combines biotin− hydrazide labeling of intact carbonylated proteins, avidinaffinity chromatography, multiplexed iTRAQ reagent stable

unmodified proteins within a stained gel spot corresponding to an immunopositive one on the blotting membrane, which may determine the presence of false positive results, generally concluding the stringent need for MS/MS confirmation for the final assignment of a protein carbonylation. To overcome the above-mentioned 2-DE limitations and since modified proteins may be present at low concentrations with respect to unmodified counterparts, multidimensional separation approaches have been introduced, which are generally based on a combination of affinity-dependent capture of intact carbonylated proteins and their further resolution by reversed phase-LC or SDS-PAGE, followed by a MS-driven protein identification of their digests. For the first purification step, either anti-DNP antibodies or hydrazine-like reagents were used in developing enrichment methods for modified components (Figure 26). However, anti-DNP antibodies found a limited application in enrichment of DNPH-derivatized peptides,669,670 because they always implied the unpleasant occurrence of immunoglobulins within immunoprecipitates, whenever they are not being covalently immobilized.648,671 On the other hand, biotin−hydrazide and ARP were highly effective in selectively isolating intact carbonylated proteins from different sources. The first reagent was successfully used for the purification and identification of naturally carbonylated species from brain, adipose tissue, and plasma of mammals,250,672−674 from yeast cells or other mammalian tissues AQ


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pyrrolidone, pyrrolidinone, or pyroglutamic acid (from proline), in proteins subjected to a different degree of oxidative insult. They verified that the main carbonylated amino acids were specific lysine, cysteine, arginine, proline, histidine, and tyrosine residues; oxidative damage preferentially affected subdomains exposed on protein surface and near S−S bridges. Similar experiments performed with oxidized RNase A, βlactoglobulin or lysozyme demonstrated the same pattern of mass shifts for semialdehyde derivatives, together with the occurrence of a satellite signal at −18 Da with respect to that of Aasa, when this carbonylated residue occurred at the peptide Nterminus.686−688 The latter phenomenon was ascribed to the formation of a cyclic peptide intermediate. On the other hand, the occurrence of Arg469 to Gsa modification was recently assigned in Hsp70-1, among the mammalian hippocampus proteins that are differentially carbonylated after ischemiareperfusion events; this result was obtained following 2D Oxyblot experiments and direct MALDI-TOF-TOF analysis of Coomassie-stained gel spot digests.689 This study is one of the few examples available in literature where assignment of semialdehyde derivatives was obtained by direct MS analysis of 2-DE-resolved proteins. An alternative MS method for selective recognition of Gsa-, Aasa-, and Akb-containing peptides exploits the ability of multistep tandem mass spectrometry (MSn) to deliver bidimensional selection via PIS and MS3 analysis.688 Prior to MS analysis, proteins are digested and carbonylated peptides are selectively labeled with dansyl-hydrazide, which determines a mass shift of +247 Da with respect to the underivatized carbonylated species. Modified protein digests are then analyzed in combined MS2 and MS3 scan mode, to specifically select the dansylated species, taking advantage of the dansylspecific fragmentation pathways. A significant increase in signal/noise ratio and in sensitivity of analysis was obtained by application of this procedure, due to the reduction of duty cycle of the mass spectrometer. This method ensured the unambiguous localization of carbonylated lysine residues in RNase A and the identification of RNase A carbonylated peptides spiked into an E. coli proteome tryptic digest. Very recently, Hoffmann and colleagues have presented a combined strategy to identify semialdehyde-containing sites in proteins by a bottom-up approach, which is based on protein digest derivatization with DNPH.690,691 Resulting DNP derivatives show a mass increase of +180 Da with respect to nonderivatized carbonylated species. In a first approach, DNPlabeled peptides are separated by hydrophilic interaction chromatography (HILIC) and then are analyzed by MALDITOF MS with DNPH as the reactive matrix, which favors ionization of modified species.690 The mass list generated for each HILIC fraction, representing mostly DNP-modified peptides, is then used for a subsequent nLC-ESI-OrbitrapMS/MS analysis. This comprehensive two-dimensional HILIC × nLC-ESI-Orbitrap-MS/MS strategy was exemplified for tryptic digests of native bovine serum albumin (BSA) and βlactoglobulin (LG), and their in vitro oxidized versions, i.e. oxBSA and oxLG. In total, 3 carbonylation sites were identified in native LG, 9 in native BSA, 11 in oxLG, and 32 in oxBSA. In a second approach, the same authors verified that DNPHlabeled carbonylated peptides show a favorable ionization behavior in negative ion mode ESI, providing a sensitive detection method.691 Regular peptides were mostly discriminated under these conditions. Among the fragmentation techniques tested for the negatively charged ions, PQD

isotope labeling, and analysis using pulsed Q dissociation (PQD) operation on an LTQ linear ion trap mass spectrometer was reported.682,683 This strategy provided the ability to distinguish biotin hydrazide-labeled, avidin purified, carbonylated proteins from noncarbonylated background proteins with affinity for the avidin column, as derived from a control sample. By applying this strategy to the identification of crudely enriched rat skeletal muscle mitochondrial protein isolates, authors identified mitochondrial proteins susceptible to carbonylation in a muscle type (slow- vs fast-twitch)-dependent and age-dependent manner from rat skeletal muscle.683 Fasttwitch muscle contained twice as many carbonylated mitochondrial proteins than did slow-twitch muscle, with 22 proteins showing significant changes in carbonylation state with age. Other oxidative modifications (e.g., S-nitrosylation, hydroxylation) were also identified on many of the carbonylated proteins, thus providing further evidence of the susceptibility of these proteins to oxidative damage. These results were obtained on the basis of the ascertained quantitative reproducibility of the avidin-affinity enrichment method. Depending on the various mechanisms generating different carbonylated amino acid derivatives, specific dedicated proteomic methodologies for their analysis will be reported in the following sections. 4.4.1. Analysis of Protein Adducts Resulting from Oxidation at Lysine, Arginine, Threonine, and Proline. Whenever oxidation of proline/arginine to γ-glutamic semialdehyde (Gsa), lysine to α-aminoadipic semialdehyde (Aasa), and threonine to 2-amino-3-oxo-butanoic acid (Akb) has been ascertained (Figure 9B),567,568 it is important to have definitive information on the nature of the modified protein(s), together with the assignment of the modification site(s). In addition to the methods reported in the previous section to visualize and identify carbonylation protein targets by gel-based and gel-free proteomic experiments, direct ESI- or MALDI-based MS detection of modified amino acids in intact polypeptides or their digests has been always encouraged for the final assignment of oxidative modifications.9 Thus, Gsa-, Aasa-, and Akb-containing proteins or peptides were tentatively assigned through direct MS measurements of polypeptide mixtures, by detecting the corresponding adducts showing a mass shift of −43 Da (arginine to Gsa), +16 Da (proline to Gsa), −1 Da (lysine to Aasa), and −2 Da (threonine to Akb) with respect to unmodified counterparts. However, modification assignment simply based on mass increase is not certain, since other amino acids can bear the same chemical groups or even other modifications can be present on polypeptide yielding the same mass shift. Thus, MS/MS experiments were used to properly assign the modified amino acids within the protein sequence. By using serum albumin and glycated insulin as model proteins oxidized under metal-catalyzed in vitro conditions, Domingues and co-workers obtained a direct site assignment of arginine to Gsa and lysine to Aasa modifications through bottom-up experiments, which were performed by using a combined nLC/off-line MALDI-TOF-TOF approach.684,685 Definitive localization of semialdehyde derivatives was obtained on the basis of the mass shifts in the corresponding MS and MS/MS spectra. Authors identified these carbonylated derivatives, together with other oxidized amino acids (from methionine, histidine, tyrosine, cysteine, proline, phenylalanine, valine, leucine, glycine, or tryptophan), α-aminoadipic acid (from lysine), aspartate or asparagine (from histidine), and AR


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provided three diagnostic ions at m/z values 152.0, 163.1, and 179.0, specific for DNPH-modified peptides. These marker ions were successfully applied to detect model carbonylated peptides (30 pg) in a spiked tryptic digest of a whole HeLa cell protein extract (150 ng). A major disadvantage of the latter approach is the lack of sequence information provided in negative ion mode. Therefore, the data obtained from negative ion mode ESI-MS will only provide a list of m/z values that most likely represent carbonylated peptides. Thus, the peptide sequences and the carbonylation sites have to be retrieved from a second LC-ESI-MS/MS experiment in positive ion mode, which specifically analyzes the peptides presumed to be carbonylated based on the first analysis in negative ion mode. Both approaches mentioned above were verified as extendible also to pyruvic acid- or HNE-modified lysine or histidine residues, as well as peptides containing pyroglutamic acid (from proline oxidation). These recent studies have reintroduced the possibility of using DNPH for derivatization of carbonylated peptides before MS analysis, but still leave open the debate on the efficacy of this reagent in gel-free proteomic studies. In fact, DNPH was originally introduced as reagent and reactive matrix for the characterization of carbonylated protein species in the course of MALDI-TOF peptide mapping experiments.669,692,693 However, it was verified that DNP−peptide derivatives, when separated from the DNPH excess, dissociate in solution to an extent that depends on time and pH.694 It was suggested that simple derivatization with DNPH is unsuitable for determining peptide-based carbonyls using a protocol where the oxidized protein is derivatized with DNPH prior to proteolysis. The proteolysis step in solution and diluted acid conditions typically used for nLC-ESI-MS/MS analysis should be apparently sufficient to cause significant reaction reversibility. This factor explains the limited success of other authors to find DNPHlabeled peptide carbonyls generated during protein oxidation in the absence of using a reductive step stabilizing 2,4dinitrophenylhydrazones.695−699 Thus, a careful evaluation of all these aspects have to be further considered, also in light of the availability of immobilized anti-DNP antibodies for selective trapping of DNP−peptide derivatives before MS analysis.669 On the other hand, it has been reported before that biotin− hydrazide is highly effective in selectively enriching carbonylated proteins from different sources in the course of gel-free proteomic studies.674 Purification of intact carbonylated proteins, their NaCNBH3-based reduction, SDS-PAGE separation, and trypsinolysis, followed by nLC-ESI-MS/MS analysis of peptide digests further demonstrated the effectiveness of this reagent also in direct assignment of metal-catalyzed oxidation sites. In this case, arginine to Gsa-, proline to Gsa-, lysine to Aasa-, and threonine to Akb-containing peptides were identified and characterized by detecting the corresponding adducts showing a mass shift of +199, +258, +241, and 240 Da with respect to unmodified counterparts, respectively, and directly assigning the modified amino acids in the corresponding MS/ MS spectra (Figure 27A). Examples in this context are the studies of modified residues in serum albumin, peroxisomal malate synthase, and catalase subjected to in vitro modifications by ascorbic acid/FeCl3 system, H2O2, or HClO treatment,678,700 which were characterized by nLC-ESI-IT-MS/MS or nLC-off line MALDI-TOF-MS experiments, or in carbonylated proteins from rat and human plasma,673,674 rat liver,675 or yeast cells,676,677 which were characterized by nLCESI-Q-TOF-MS/MS, nLC-ESI-Orbitrap-MS/MS, or nLC-off

Figure 27. MS/MS analysis of carbonylated peptides from the enzymatic digest of metal-oxidized proteins further labeled with biotin−hydrazide (A), Girard’s P (B), and N′-aminooxymethylcarbonylhydrazino-d-biotin (C) reagent. (A) Assignment of modification sites in albumin subjected to metal-induced carbonylation and further reduction with NaCNBH3 to yield stable derivatives. ESI−MS/MS analysis of the [M + 2H]2+ ion at m/z 1087.1, which was assigned to the aminoadipic semialdehyde-containing peptide 89−105. Dominant fragment ions, yn (n = 8−15), b8, and b10, arise from the biotin-labeling AS


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To circumvent these difficulties, the same authors later reported a new strategy for labeling protein carbonyls, which uses light and heavy isotope coded GPR, allowing confident identification of carbonylated proteins via a multistep LC/MS and MALDI-MS/MS data filtering process.702 This strategy was used for the identification of heavily oxidized transferrin peptides and resulted in the identification of 13 distinct modified species.701,702 The competency of the method was validated in a complex mixture using oxidized transferrin spiked in a yeast lysate as well as analyzing oxidized yeast proteins. Twenty-five percent of the peptides identified in a pure oxidized sample of transferrin were successfully identified from the complex mixture. On the other hand, analysis of yeast proteome stressed with H2O2 resulted in the identification of 41 carbonylated peptides from 36 distinct proteins. Differential isotope coding of model peptides at different concentrations followed by mixing at different ratios was used to establish the linear dynamic range for quantification of carbonylated peptides using light and heavy forms of GPR. The possibility of using a shotgun approach for the identification and characterization of proteins containing αaminoadipic and γ-glutamic semialdehyde moieties was recently assayed by Maier and colleagues (Figure 26).703 They used a targeted approach, which combines protein labeling with ARP,665 trypsinolysis, ARP-modified peptide enrichment, and nLC-ESI-Q-TOF-MS/MS or nLC-off line MALDI-TOF-TOFMS/MS analysis for the site-specific identification. In this case, Gsa-, Aasa-, and Akb-containing peptides were identified and characterized by detecting the corresponding adducts presenting a mass shift of +270, +329, +312, and +311 Da with respect to unmodified arginine, proline, lysine, and threonine, respectively, also assigning the modified amino acids in the corresponding MS/MS spectra (Figure 27C). Examination of the tandem mass spectra of the ARP-labeled semialdehydecontaining peptides revealed some common features aiding in their manual interpretation but, on the other hand, also complicating the automated interpretation of the spectra by using database searching algorithms. The first of these features is the neutral loss of the ARP moiety from the y-ion in which the ARP-labeled residue is the N-terminal residue. This results in the respective y-ion appearing at −331 m/z from its expected value. In some cases, NL is not complete, and the respective yion also appears at the expected m/z, including the mass of the ARP tag. The other two features commonly observed in the tandem mass spectra of ARP-labeled peptides are the fragment ions present at m/z 227 and m/z 332, which originate from the ARP tag. This shotgun approach was successfully applied to the analysis of in vitro oxidized glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and native cardiac mitochondrial proteins. The analysis of GAPDH resulted in the site-specific identification of two Aasa and four Gsa residues. Computational evaluation of the identified sites in GAPDH indicated that these sites are located in flexible regions, with high solvent accessibility values and in proximity to metal ion binding sites. The targeted proteomic analysis of semialdehydic modifications in cardiac mitochondria yielded nine Aasa modification sites, which were unambiguously assigned to distinct lysine residues in four proteins. ARP was successfully used also during shotgun analysis of carbonylated proteins resulting from reaction with lipooxidation end products (see below).665 Concomitant isolation, identification, and quantification of Gsa-, Aasa-, and Akb-containing peptides was also achieved by using ((S)-2-(4-(2-aminooxy)-acetamido)-benzyl)-1,4,7,10-tet-

Figure 27. continued at Lys97, as also demonstrated by the mass difference between y9 and y8, equal to a molecular mass of 128 (lysine) + 241 (biotin) = 369 Da. (B) MS/MS spectrum of the peptide KCSTSSLLEACTF from transferrin derivatized with the Girard’s P reagent and acquired by using a MALDI-TOF-TOF instrument. All y ions and some b ions are assigned. The difference between y2 and y1 indicates the loss of a reagent-tagged threonine residue. (C) Tandem mass spectrum of the N′-aminooxymethylcarbonylhydrazino-d-biotin (ARP)-labeled aminoadipic semialdehyde-containing peptide 61−72 from rat ADP/ATP translocase 1. ESI−MS/MS analysis of the [M + 2H]2+ ion at m/z 910.5 yields a complete yn-ion series. Fragment ions marked with an asterisk retained the ARP moiety during CID analysis and allowed the unequivocal localization of the modified residue at position 63. The fragment ion peak at m/z 1278.72 represents the y10-ion after loss of the ARP moiety associated with the proposed formation of an azacyclohexane moiety. Fragment ions specific for the ARP tag are observable at m/z 227.1 (ARP-F1) and m/z 332.1 ([ARP+H]+). Adapted with permission from refs 700, 701, and 703. Copyright 2006 and 2010 Springer and 2006 American Chemical Society.

line MALDI-TOF-TOF-MS/MS measurements. In most cases, other oxidized amino acids (from histidine, tyrosine, or tryptophan), hydroxylated amino acids (from histidine or tryptophan), and aspartate or asparagine (from histidine) were simultaneously detected. Oxidation sites were predominantly found at lysine, arginine, proline, histidine, threonine, and methionine residues. Generally, authors preferred not to use avidin/streptavidin trapping of biotinylated products at peptide level, but rather for selective purification of carbonylated proteins (Figure 26). They keep away from using the biotin− hydrazide chemistry in shotgun approaches to avoid obtaining modified peptide fragments that are very large and/or bear too many modifications for easy identification.677 This is because lysine and arginine are major targets of oxidation and after their modification they will not be hydrolyzed by trypsin. In addition to using biotin−hydrazide, Regnier and colleagues also developed a method for labeling and enriching Gsa-, Aasa-, and Akb-containing peptides that utilizes the Girard’s P reagent (GPR) (Figure 23D).701 Also in this case, derivatization has to be followed by a reduction step (with NaCNBH3) to yield the stable hydrazine derivatives. Girard’s Pderivatized peptides were then identified and characterized by detecting the corresponding adducts having a Δm of +134 Da with respect to unmodified carbonylated species, directly assigning the modified amino acids in the corresponding MS/ MS spectra (Figure 27B). Structural features of GPR critical to its analytical efficacy are the presence of a quaternary amine group that enhances MS ionization efficiency and a hydrazide group that reacts with carbonyl groups in proteins. The solubilizing properties of this reagent become especially important for oxidized proteins since they can be of reduced solubility due to cross-linking, denaturation, and backbone cleavage.233 Presence of the quaternary amine also allows enrichment of tagged peptides by strong cation exchange chromatography. Even though GPR has several important advantages over other labeling strategies for detection of carbonylated proteins, it still suffers from inherent problems associated with complex PTMs such as those related to the oxidative damage, which targets around 13 amino acid side chains to produce more than 30 new structures. This multiplicity problem enormously increases the probability of false positives during database searches of the MS/MS spectra. AT


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Figure 28. Oxidation-specific element-coded affinity tag (O-ECAT) for quantization of metal-induced carbonylation sites in proteins. (A) Chemical structure of the ((S)-2-(4-(2-aminooxy)-acetamido)-benzyl)-1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid reagent. (B) A representative portion of the two-dimensional spectrum showing m/z vs retention time for an affinity-purified Fe-EDTA/ascorbate oxidized human serum albumin sample tagged with TbAOD and HoAOD. (C) An enlargement of the two-dimensional spectrum shown in panel B. (D) FTMS spectrum showing a doubly charged mass pair. The peaks shown here correspond to the peptide TPVSDRVTK with the arginine oxidized and tagged with TbAOD and HoAOD. Adapted with permission from ref 704. Copyright 2006 American Chemical Society.

raazacyclododecane-N,N′,N″,N‴-tetraacetic acid, which ensured the development of a method based on oxidationdependent element coded affinity tags (O-ECAT) (Figure 28). After derivatization of carbonyl groups present in a protein sample, the introduced O-ECAT moiety was used to chelate a rare earth metal such as Tb (158.9 Da) or Ho (164.9 Da).704 Treating different protein samples with different rare earth metals permitted differential coding of samples. Native and oxidized serum albumin samples were allowed to react with this reagent and, after coding and mixing, were digested with trypsin. Coded peptide fragments were then selected with an immunosorbent column targeting the derivatizing agent. Trapped peptides were then analyzed by nLC-FT-ICR-MS/ MS for concomitant protein identification and a measurement of the modification extent. By using Tb−O-ECAT, the observed mass shift for oxidized lysine, arginine, proline, and threonine was +719, +677, +736, and +718 Da, respectively. Relative intensities of the tagged peptides from the two samples were used to determine the degree of oxidation, which is independent of the modified amino acid.

Carbonyl derivatives formed by direct metal-catalyzed oxidation attacks on the side chain of lysine, arginine, proline, and threonine residues were experimentally evaluated in vitro on BSA and on 23 proteins from E. coli.705 The presence of a carbonylated site rendered the neighboring carbonylatable site more prone to modification. Most carbonylated sites were present within hot spots of carbonylation. These observations led authors to suggest rules for identifying sites more prone to carbonylation. They used these rules to design an in silico model, allowing an effective and accurate prediction of sites and of proteins more prone to carbonylation in the E. coli proteome. Authors observed that proteins evolve to either selectively maintain or lose predicted hot spots of carbonylation depending on their biological function. Because this predictive model also allows efficient detection of carbonylated proteins in B. subtilis, they believed that it may be extended to foresee metal-catalyzed oxidation attacks in all organisms. 4.4.2. Analysis of Adducts Resulting from Protein Reaction with Lipooxidation End-Products. Phospholipid peroxidation occurs continuously in mammals and can be increased by oxidant challenges. Reduction of the initially AU


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In vitro studies with acrolein, HNE, ONE, HHE, CRO, NONE, MDA, 15-deoxy-Δ12,14-PGJ2, Δ12-PGJ2, PGJ2, PGA1, PGA2, and 8-iso-PGA1 added to isolated proteins were initially performed to simulate and assign cysteine, lysine, and histidine reactivity under normal and pathophysiological conditions. Thus, the occurrence of modified amino acids was ascertained by MS measurements, detecting the corresponding protein or peptide adducts having a mass increase of +56, +156, +154, +114, +70, +140, +54, +316, +334, +334, +336, +334, and +336 Da, respectively, with respect to the unmodified counterparts (Figure 29A and 30B).360,736,737 In some cases, preventive reduction with borohydride derivatives was performed to stabilize adducts before protein digestion, generating an additional mass shift of +2 Da per carbonyl/ aldimine group in the corresponding modified species.360,718,724,732,737−744 Generally, modification assignment simply based on MH+ signal mass increase during peptide mapping experiments was considered as not definitive; thus, MS/MS experiments were performed to properly assign the modified amino acid(s) within the peptide sequence, commonly providing evidence for covalent modification in a Michael addition-type reaction (Figure 29 and 30). To this purpose, bottom-up studies based on nLC-ESI-MS/MS or nLC-off line MALDI-TOF-MS/MS analyses were carried out for the characterization of HNE-,250,268,360,663,712,715,716,718,738−741,743−753 ONE-, 360,718,737,745,754 acrolein-, 360,742,755−759 HHE-, 360 CRO-,360,737,758 NONE-,360 and prostaglandin-modified proteins.732,733,736,760−764 Sometimes these investigations detected multiple reactive amino acids on protein structure. No significant differences in modification site assignment efficacy were observed depending on the ioniziation (ESI or MALDI) technique used. This was due to the general stability of the analyzed adducts during spectral analysis with both techniques. Whenever measured, reaction modification kinetics were analyzed following nLC-ESI-MS/MS experiments by comparing the relative area of modified peptides with respect to that of the corresponding unmodified counterparts or by performing SRM using stable isotope tagging with different reagents.663,737,741,743,744 Formation of minor Schiff base adduct at lysine residues was also observed. Concomitant reactivity of acrolein also at arginine residues was occasionally proven by detecting the corresponding cyclic adducts, which showed a mass increase of +54 Da in the corresponding MS and MS/MS spectra.737,758 In some cases, modification site assignment was obtained for peptide samples recovered after digestion of proteins extracted from biological tissues or fluids subjected to oxidative stress conditions, which were previously resolved by mono- and bidimensional electrophoresis.663 In the case of Michael adducts resulting from HNE reaction (with Δm = +156), their general tendency to cyclize yielding hemiacetal derivatives that do not show mass changes was evident (Figure 31).658 MS analysis of HNE-modified species also demonstrated the concomitant occurrence of minor Schiff base- or in-source dehydrated Michael-adducts (with Δm = +138) and related cyclized 2-pentylpyrrole species (with Δm = +120) (Figure 31).663,740,741,747,750 Their assignment within the protein sequence was obtained by MS/MS experiments; ECDbased fragmentation experiments were also used to this purpose (Figure 29C).750 Since racemic HNE preferentially reacts with cysteine residues to form stable Michael addition adducts having three chiral centers, stereochemical configuration of these products was also investigated in redox-regulated proteins

formed fatty acyl hydroperoxides by one-electron reductants generates alkoxyl radicals that decompose to a plethora of products, some of which contain reactive functional groups, such as epoxides and aldehydes (Figures 4 and 5).706 We previously mentioned HNE, ONE, HHE, acrolein, OHacrolein, MDA, CRO, OCT, NONE, OCTA, ONEA, HODA, DODE/KODA, OUEA, EKODE, DHKODE, KODDE, HKODE, and various IsoPs, IsoKs, LGs, and PGs as significant examples of reactive carbonyl products. Additional reactive species are glyoxal (GO), methylglyoxal (MGO), and glycolaldehyde, which are oxidation products of both lipids and sugars (treated in the following section). All these molecules can react with proteins, generating lipid-derived adducts that are detectable in mammalian tissues and can alter the properties of the target proteins (Figure 10).234,235,707,708 The contribution of these electrophiles to pathological events, such as neurodegeneration, pain, inflammation, and cellular aging, has largely been defined through the study of individual protein targets.707,708 On the other hand, a major challenge in studying protein damage by lipid electrophiles is the sheer diversity of products generated. These investigations, which have been initially performed in vitro by adding reactive carbonyl products to isolated proteins or whole cells, were realized to simulate/investigate cysteine, lysine, and histidine protein reactivity under real pathophysiological conditions. To identify protein targets of various lipoxidation products, gel-based proteomic procedures were developed, taking advantage of the availability of anti-HNE, anti-acrolein [together with related anti-Nε-(3-methylpyridinium)-lysine (MP-Lys) and anti-Nε-(3-formyl-3,4-dehydropiperidino)-lysine (FDP-Lys)], anti-ONE, anti-MDA, and anti-carboxyethylpyrrole (CEP) antibodies, or the biotinylated 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2)/HRP−streptavidin and biotinylated PGA1/HRP− streptavidin reagent combinations, which were used in Western blot experiments to probe modified proteins resolved by monoand bidimensional electrophoresis. These reagents were often used in parallel with Oxyblot Western blotting procedures. Thus, protein spots, specifically recognized by parallel Western blotting experiments, were sampled from Coomassie-stained preparative gels for subsequent in-gel digestion and further identified by MALDI-TOF peptide mass fingerprinting or nLCESI-MS/MS (Figure 14). Examples, in this context, are the differential proteomic studies on (i) HNE-modified proteins in the brain from control and AD patients709,710 and related amnestic mild cognitive impairment subjects,711 retina tissues before and after photooxidative stress,712−714 liver tissues of rats fed with high fat/ethanol diet and control,715−718 20S proteasome,719 and limb and heart muscle tissues of cachectic rats and control;720 (ii) acrolein-modified proteins in myocytes superfused or not with reactive carbonyl products,721 lysates of HT-29 cell treated or not with molecules inducing apoptosis/ cytoskeleton disorganization,722 plasma of patients with brain infarction and control,723 human atherosclerotic lesions,724 and mouse hepatocytes;725 (iii) ONE-modified proteins in liver tissues of rats fed with high fat/ethanol diet and control;718 (iv) MDA-modified proteins in limb and heart muscle tissues of cachectic rats and control,720 frontal cortex brain tissues of Lewy body disease patients and control;726 (v) CEP-modified proteins in plasma of control and patients with age-related macular degeneration;727 and (vi) cyclopentenone prostaglandin-modified proteins in mesangial, HeLa, NIH-3T3, SH-SY5Y cells, or liver mitochondria and mesenteric vessels.728−735 AV


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Figure 29. continued peptides (Δm = +156 Da) were detected in the spectrum of HNEtreated hBAT. (B) ESI-LIT-CID-MS/MS spectrum of the [M + 3H]3+ ion at m/z 625.688, which was assigned to the HNE-modified hBAT chymotryptic peptide 271−284. Sites of HNE modification were identified from a series of differentially Michael adduct-modified fragment ions. Fragment ions uniquely localize two possible sites of HNE Michael adduction, to His271 and His280, and eliminate His275 as a modified site. (C) ESI-LIT-ECD-FT−MS/MS spectrum of the hBAT peptide 335−346. The spectrum contains a rich series of c- and z-ions. The c2 ion has a m/z 364.234; the expected value for the corresponding unmodified ion is m/z 226.130. The difference is 138.104, the expected value for a Schiff base adduct, or a dehydrated Michael adduct. All the other cn ions (n > 2) (labeled with +) were increased by the same amount. Adapted with permission from refs 663 and 750. Copyright 2008 the American Society for Biochemistry and Molecular Biology and the Biochemical Society, respectively.

(Figure 31). By using the pyridylamination method along with MS to characterize R and S stereoisomers, Uchida and colleagues demonstrated the residue-dependent stereoselective formation of the HNE−cysteine adducts in proteins.765 In most cases, HNE Michael-adducted species were easily assigned in nLC-ESI-MS/MS experiments on protein digests by using NL scanning for m/z 156, 78, and 52, corresponding to the loss of a neutral reduced HNE moiety from singly, doubly, and triply charged precursors, respectively (Figure 32).739 This procedure was of limited analytical utility in the case of HNEmodified histidine-containing peptides, but proved particularly suited for HNE-modified peptides at cysteine residues, since their typical MS/MS fragmentation pattern is characterized by a retro-Michael reaction. Thus, data-dependent and NL-driven MS3 acquisition was reported for the identification of HNE adducts by MS-based proteomics (Figure 32).766,767 In addition to abundant Michael adducts, this approach was also used to enhance detection of Schiff base derivatives.768 However, the limitation associated with the NL-driven MS3 acquisition approach resides in the ambiguity for a correct assignment of the HNE modification site when more than one candidate site is present, because MS3 is triggered on the neutral loss ion. To overcome this problem, Prokai and co-workers introduced a NL-triggered ECD-MS/MS strategy for the characterization of HNE-modification sites in peptides (Figure 32).769 With this method performed using a hybrid LIT-FT-ICR mass spectrometer, ECD in the FT-ICR unit of the instrument is initiated on precursor ions of peptides showing the neutral loss of 156 Da (corresponding to a HNE molecule) in the prescan acquired via collision-induced dissociation MS/MS in the linear ion trap. In addition to manifold advantages associated with the ECD method of backbone fragmentation, including extensive sequence fragments, ECD tends to retain the HNE group during MS/MS of the precursor ion, facilitating the correct localization of the modification site. By study of acrolein-conjugated proteins, it was evident that cysteine, lysine, and histidine residues may react with this reagent forming Michael adducts (Δm = +56). For lysine residues, Schiff base, MP-Lys, and FDP-Lys adducts were also observed, as revealed by the presence of the corresponding peptide species showing a mass increase of +38, +76, and +94 Da, respectively (Figure 33A).268,723,724,742,770,771 The latter two derivatives result from the progressive addition of multiple acrolein molecules to the same modified amino acid through different mechanisms.772 The occurrence of both species was

Figure 29. MS and MS/MS analysis of peptide digests from human bile acid CoA:amino acid N-acyltransferase (hBAT) adducted with 4hydroxy-2-nonenal (HNE). (A) MALDI-TOF MS peptide mass fingerprints of native (top) and HNE-modified hBAT (bottom) following chymotrypsin digestion. Several hBAT chymotryptic peptides were identified in the spectrum of the unmodified protein; additional signals tentatively associated with Michael adducted AW


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Figure 30. MALDI-TOF MS and MS/MS analysis of cyclopentenone prostaglandin adducts on H-Ras. (A) Structures of some PGs. (B) Spectra obtained following incubation of each compound (50 μM) with protein (5 μM) for 1 h. The arrows indicate the position where the PGJ2−H-Ras adduct occurs in the spectrum. MALDI-TOF-TOF MS/MS analysis of the modified peptides with m/z 984.8 and 1997.2 from tryptic digests of PGA1- (C) and Δ12-PGJ2-treated H-Ras (D), respectively. The sequence of the modified peptides is given above the spectra. Asterisks denote the ions for which peaks compatible with the addition of the respective PG are observed. Adapted with permission from ref 736. Copyright 2007 American Chemical Society.

Figure 31. Reactivity of HNE toward cysteine, lysine, and histidine residues. Michael adduct formation with the amino acids mentioned above or Schiff base formation pathway with lysine are shown, together with dehydration reactions described within the text. Reduction with NaBH4 is optionally used to generate stable adducts. Asterisks indicate chiral carbons.

identification of in vivo adducts of acrolein with protein residues. In the case of the adducts resulting from ONE reaction with cysteine, histidine, and lysine, it was evident that different derivatives (with Δm = +154) occur depending on the nature of the modified amino acid. Sayre and co-workers demonstated that the long-lived ONE-derived adduct to lysine is actually a 4ketoamide rather than a Michael adduct (with no apparent changes in mass value).774 A number of advanced reaction products originating from ONE Michael adduction to cysteine were also identified by dedicated MS procedures.775 They were the major 2-cyclopentenone derivatives, i.e. 2-(acetylamino)-3[(3-butyl-4-oxocyclopent-2-en-1-yl)sulfanyl]propionic acid and 2-(acetylamino)-3-[(4-butyl-5-oxocyclopent-3-en-1-yl)sulfanyl]propionic acid (both with Δm = +136 Da), which might be generated through the base-catalyzed cyclization of the C-2 or the C-3 Michael addition products (with Δm = +154 Da), respectively, and the minor thiomorpholine derivative, i.e. 4-acetyl-5-hydroxyl-6-(2-oxoheptyl)thiomorpholine-3-carbox-

proven by nLC-ESI-MS/MS experiments, and whenever assigned to ApoA1, apolipoprotein B100 (ApoB100), or albumin residues, it was shown to colocalize with atherosclerotic lesions, eventually impairing cholesterol removal from artery wall cells (Figure 33), or to be associated with infarctual brain conditions, respectively.268,723,724 On the other hand, acrolein−cysteine adducts (showing a Δm = +56 Da) do not seem stable at physiological pH and temperature; other derivatives generally appear (Δm = +38 Da), which are formed from the adducts mentioned above via an intramolecular reaction involving a Schiff base compound.759,773 Formation of the corresponding lysine and histidine derivatives is much slower and generally requires higher acrolein concentration. Collectively, these data demonstrate that acrolein reacts avidly with cysteine, histidine, and lysine residues and that the apparent loss of protein−acrolein Michael adducts (with Δm = +56 Da) over time may be related to the appearance of additional species. These findings may be important in AX


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Figure 32. continued neutral loss-driven MS3 (A) versus neutral loss-driven electron capture dissociation (B) approaches. (C) CID-MS/MS spectrum of the (M + 2H)2+ ion at m/z 904.03 assigned to the ATP synthase subunit β tryptic peptide 49−63, as obtained from data-dependent acquisition. (D) MS/MS/MS spectrum of the neutral loss ion at m/z 826.02, as obtained by the NL-MS3 method. (E) ECD MS/MS spectrum of the (M + 2H)2+ ion at m/z 904.03, as obtained by NL-ECD-MS/MS analysis. An asterisk denotes the HNE-modified amino acid. Adapted with permission from ref 769. Copyright 2009 American Chemical Society.

ylic acid (Δm = +154 Da), originating from the C-2 Michael addition product. Also in this case, apparent loss of protein− ONE Michael cysteine adduct (with Δm = +154 Da) over time was related to the appearance of these additional species, with important consequences on identification of in vivo ONE adducts in proteins. In addition to the modifications mentioned above, acrolein, ONE, and CRO were also proven to generate a number of protein cross-linking products.360,658,749,771,776 To assign their presence to specific amino acids and to establish their structural characteristics, adduct stability was generally increased by reduction with borohydride derivatives; the resulting products were digested with a proteolytic enzyme and peptide digests were then subjected to MALDI-TOF-MS and nLC-ESI-MS/ MS analysis. In the case of acrolein-modified proteins, intrapeptide cross-linked species were characterized by a mass increase of +38 and +40 Da for nonreduced and reduced species, respectively (Figure 34).771,777 On the other hand, inter-peptide P1−peptide P2 cross-linked species were characterized by a mass value of MP1 + MP2 + 38 and MP1 + MP2 + 40 Da for nonreduced and reduced species, respectively, where MP1 and MP2 correspond to the mass of peptide P1 and that of peptide P2.771 Nature of the characterized cross-linked products indicated that the acrolein-induced cross-links were accompanied by two consecutive reactions, Michael addition and Schiff base formation. The main carbonylated products resulting from MDA reaction were generally S- or N-propenal derivatives (Δm = +54), which are formed via Michael adduction followed by dehydration reaction, or dihydropyridine (DHP) derivatives (Δm = +134) (Figure 35A).360,751,752,778 ELISA analysis of these separated species demonstrated that available anti-MDA antibodies generally recognize DHP-type adducts.778 Relative measurements on quantitative site modification were performed by isotope dilution with reconstructed ion chromatograms of precursor and product peptides, using the [15N]labeled protein as internal standard.751 On the other hand, MDA was also proven to generate lysine-based cross-linking products (Figure 35A).751 To assign their presence to specific amino acids and to establish their structural characteristics, proteins were digested and corresponding digests were subjected to nLC-ESI-MS/MS analysis (Figure 35B,C). Intrapeptide cross-linked species showed a mass increase of +36 Da; conversely, inter-peptide P1−peptide P2 cross-linked species were characterized by a mass value of MP1 + MP2 + 36, where MP1 and MP2 correspond to the mass of peptide P1 and peptide P2, respectively.771 The nature of the 1-amino-3iminopropene cross-linked products indicated that the MDAinduced cross-links were accompanied by three consecutive reactions: (i) Michael addition to lysine; (ii) dehydration of the

Figure 32. Improved methods for structural characterization of HNEmodified peptides by liquid chromatography−tandem mass spectrometry using data-dependent acquisition procedures. Flowcharts of AY


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Figure 33. Mass spectrometric characterization of linear acrolein adducts. (A) Reaction pathways for the formation of Nε-(3-methylpyridinium)lysine (MP-Lys) and Nε-(3-formyl-3,4-dehydropiperidino)lysine (FDP-Lys) products. (B) Modified peptide identified during LC-ESI-MS/MS analysis of an endoprotease GluC digest of apolipoprotein A1 (ApoA1) (25 μM) exposed to acrolein (500 μM) for 24 h, at 37 °C. MS/MS analysis demonstrated that peptide 224−235 ([M + H]+ at m/z 1432.6) contains Lys226 as modified amino acid having a mass increase of +76 Da. (C) Conversion of Lys226 to MP-Lys is associated with a decrease in the ability of ApoA1 to remove cholesterol from cells by the ABCA1 pathway. [3H]-Cholesterol-labeled ABCA1-transfected BHK cells were incubated for 2 h with native or acrolein-modified ApoA1 (5 μg/mL); [3H]-cholesterol efflux to the acceptor apolipoprotein was then measured. MP-Lys adduct at Lys226 was detected and quantified by LC-ESI-MS analysis. (D−F) Immunohistochemical colocalization of ApoA1 and acrolein adducts in human atherosclerotic lesions. Photomicrographs of adjacent sections of a human coronary atherosclerotic plaque demonstrating Movat’s pentachrome stain (D), immunostaining for ApoA1 (E), and acrolein−lysine adducts (F). With the Movat’s stain, cells are stained red, elastin is stained black, glycosaminoglycans are stained blue, and collagen is stained yellow. Positive immunohistochemical staining is indicated by a red immunoreaction product (E, F). There is a widespread colocalization of acrolein epitopes with extracellular ApoA1 (comparison of E and F). Adapted with permission from ref 724. Copyright 2005 the American Society for Biochemistry and Molecular Biology.

resulting propenal derivative; (iii) Schiff base formation with another lysine residue. Levels of these MDA−protein adducts were elevated in HDL isolated from human atherosclerotic lesions, suggesting that lipid peroxidation might render HDL dysfunctional in vivo.751 These authors showed that MDA damages ApoA1 by a pathway that generates lysine adducts at specific sites in the protein; such damage may facilitate the formation of macrophage foam cells by impairing cholesterol efflux via the ABCA1 pathway. Other cross-linking products resulting from concomitant reaction of MDA with lysine and arginine residues were also identified.570

On the other hand, peptide mapping experiments on 15deoxy-Δ12,14-PGJ2-modified GSH and proteins following reduction with NaBH4 and further proteolytic digestion were used to identify modified amino acids, eventually selecting diagnostic fragments for additional ion mapping searching studies.732 From these spectrometric analyses, it was concluded that the fragment ion at m/z 301, arising from the dehydrated ion of the modified species, is a typical product ion of 15-deoxyΔ12,14-PGJ2 adducted to cysteine-containing peptides and, hence, can be used as the diagnostic ion in the ion mapping analysis. This approach was successfully used to identify AZ


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Figure 34. continued MS/MS spectrum of the [M + H]+ ion at m/z 797.4, which was associated with the peptide 1−6 having a Δm = +40 Da. (C) MS/MS spectrum of the [M + H]+ ion at m/z 797.4, which was associated with the peptide 1−6 bearing an intramolecular cross-link between Phe1 and His5 (Δm = +40 Da). (D) MS/MS spectrum of the [M + 2H]2+ ion at m/z 1110.5, which was associated with the peptide 1−6 crosslinked to the peptide 19−30 through an acrolein-induced inter-peptide bridge linking His5 and Lys29 (Δm = +40 Da). Asterisks denote modification and crosslinking sites. Acrolein-induced modification on Phe1 occurred at protein N-terminus; Cys19 was present as cysteic acid derivative. Adapted with permission from ref 771. Copyright 2007 Informa Healthcare.

Cys374 as modification site in 15-deoxy-Δ12,14-PGJ2-treated actin. Conversely, IsoK/LGs react remarkably rapidly with the lysine residues of proteins to form adducts that include imine, pyrrole, lactam, hydroxylactam, and cross-linked species.579 In vitro studies with IsoK added to isolated cytochrome c revealed a protein adduct showing a mass increase of +298 Da with respect to unmodified counterpart. This component corresponds to the lysine−anhydropyrrole species (Figure 36).779 MS/MS experiments were performed to properly assign the modified residues within the peptide sequence. To this purpose, a top-down approach was used, which identified modified amino acids at protein N-terminus. Nowadays, no other studies are available in this field. In order to have widespread information on the wide array of carbonylated adducts resulting from protein modification with lipoxidation products of linoleic acid (LA), a deuterated analogue (d5-LA) was synthesized and used for in vitro protein labeling and peptide mapping experiments, thus facilitating their global detection and characterization by MS procedures (Figure 37).780 In fact, LA generates unsaturated hydroperoxides, which undergo further oxidative evolution resulting in a mixture of electrophiles, including epoxyketooctadecenoic acid and dienones with intact C-18 chains as well as oxidative cleavage products, such as HNE, ONE, OCT, HODA, KODA, and OUEA. Reduction by NaBH4 served to trap protein reversible adducts and to quantify the number of reducible functional groups in each compound. This study, which originally mimicked the distribution of reactive lipid peroxidation products generated by a continuous low level flux of ROS present in vivo under conditions of oxidative stress, confirmed that many irreversibly formed adducts previously identified following exposure of model proteins to pure electrophilic modifiers are also generated during in situ oxidation of proteins. These derivatives included HNE−, HODA−, OUEA−, EKODE−, ONEA−, KODDE−, HKODE−, DHKODE−, and OCT−histidine Michael adducts (having a mass shift of +156, +228, +198, +310, +170, +294, +312, +328, and +126 Da, respectively), ONE− and DODE− lysine 4-ketoamides (having a mass shift of +154 and +226 Da, respectively), Nε-hexanoyl-lysine (having a mass shift of +98 Da), ONE− and DODE−lysine pyrrolinones (having a mass shift of +136 and +208 Da, respectively), and cysteine/ histidine−ONE− and cysteine/histidine−DODE−lysine pyrrole cross-linked products (characterized by a mass value of MP1 + MP2 + 118 and MP1 + MP2 + 190 Da, respectively, where MP1 and MP2 correspond to the mass of the cross-linked peptides P1 and P2) (Figure 38). However, reversibly formed

Figure 34. Mass spectrometric characterization of cross-linked acrolein adducts. (A) Reaction pathways determining the formation of the crosslinking adduct at lysine and histidine residues through the aldimine-type or the propanal-type intermediates. LC-ESI-MS/MS analysis of an endoprotease AspN digest of insulin subjected to modification with acrolein and further reduction with NaCNBH3. (B) BA


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Figure 35. Mass spectrometric characterization of malondialdehyde linear and cross-linked adducts at lysine residues. (A) Chemical structure of the N-propenal-Lys, dihydropyridine (DHP), and cross-linked Lys-1-amino-3-iminopropene-Lys derivatives. Tandem mass spectrometric analysis of the adduction products in apolipoprotein A1, after digestion with endoprotease Glu-C. (B) MS/MS spectrum of the [M + H]+ ion at m/z 1555.8, which was assigned to the N-propenal-Lys118 adduct of the peptide 114−125. (C) MS/MS spectrum of [M + H]+ ion at m/z 792.5, which was assigned to the intramolecular cross-linked Lys206−1-amino-3-iminopropene-Lys208 adduct of the peptide 206−212. Adapted with permission from ref 751. Copyright 2010 the American Society for Biochemistry and Molecular Biology.

displayed a statistically significant increase in adduction with increasing HNE exposure concentration. Authors further identified 18 biotin−hydrazide-modified, HNE-adducted peptides by specific capture using anti-biotin antibody and analysis by high-resolution nLC-ESI-MS/MS. A subset of the identified HNE targets was validated with a streptavidin capture and immunoblotting approach. An analogous strategy, which performed trapping at protein level, was similarly used to identify cyclopentenone prostaglandin-modified proteins after treatment of various cell types/tissues with biotinylated 15deoxy-Δ12,14-PGJ2 or biotinylated PGA1.662,728−730,732−735,761−763,782,783 On the other hand, a biotin-modified linoleoylglycerylphosphatidylcholine probe (PLPBSO) was synthesized and used to investigate typical linoleate oxidation products and trap covalent adducts with intact proteins.784 Supplementation of human plasma with PLPBSO followed by free radical oxidation resulted in covalent adduction of PLPBSO to plasma proteins, which were stabilized by NaCNBH3 reduction, isolated with streptavidin, resolved by SDS-PAGE, trypsinolyzed, and identified by nLC-ESI-MS/MS (Figure 39A).785 ApoA1 phospholipid adduct sites were mapped by MS/MS analysis of tryptic peptides following mild base hydrolysis to release esterified phospholipid adducts (hampering analyte ionization). Several carboxylated adducts formed from phospholipidesterified KODA, HODA, 7-oxoheptanoic acid, 8-oxooctanoic acid, and 9-oxononanoic acid were identified (Figure 39B−E);

Figure 36. Chemical structure of the lysyl-anhydropyrrole.

adducts, such as the HNE−lysine Schiff base, were not present at detectable levels. The isotopic labeling allowed less commonly identified “mirror-image” adducts derived from the carboxy terminus of LA to be identified. A novel 2-octenoic acid−histidine Michael adduct (having a mass shift of +142 Da) was also discovered. Reactive electrophiles generated by lipid peroxidation are thought to contribute to various oxidative stress-related pathologies by covalently modifying proteins and affecting critical protein functions; the difficulties in capturing and analyzing the relatively small fraction of modified proteins have complicated the identification of the protein targets of lipid electrophiles. Similarly to what was reported for metal-catalyzed oxidized species, biotin−hydrazide has also been used for trapping and characterizing intact protein components modified by various lipoxidation products. In particular, it was originally utilized to isolate intact modified proteins after treatment of RKO cells with various HNE concentrations.781 HNE−Michael adducted proteins were captured with streptavidin, resolved by SDS-PAGE, trypsinolyzed, and identified by nLC-ESI-MS/MS. Out of the proteins identified, almost 400 BB


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Figure 37. continued HIRL by d0-ONE (H), d5-ONE (I), or DODE (L). Adapted with permission from ref 780. Copyright 2009 American Chemical Society.

they showed a mass shift of +226/210, +228, +128, +158/142, and +156 Da, respectively, with respect to unmodified counterparts. Free radical oxidations of isolated HDL also generated adducts with HNE and other noncarboxylated electrophiles, but these were only sporadically identified in the PLPBSO-adducted ApoA1, suggesting a low stoichiometry of modification in the phospholipid-adducted protein. Very recently, the click chemistry was used to biotin-label intact HNE-modified proteins, thus allowing their trapping on streptavidin beads (Figure 40).786 Click chemistry describes the 1,3-dipolar cycloaddition reaction between azide (or alkyne) labeled probes to conjugate alkyne (or azide) labeled reporter tags. For proteomic purposes, the probe used was an analogue of HNE, modified at the terminal carbon with an azide (azidoHNE) or substituted at the ω and ω-1 positions with an alkyne (alkynyl-HNE) (Figure 40A). The attachment of either an azide or alkyne tag to HNE is a subtle change, so that HNE, azido-HNE, and alkynyl-HNE exhibited comparable cytotoxicity and abilities to stimulate gene expression. Liebler and coworkers used click chemistry, streptavidin-based enrichment, and nLC-ESI-MS/MS to compile large data sets of protein targets of azido-HNE or alkynyl-HNE in the human colon cancer RKO cell line. Later on, the same authors labeled proteins modified with alkynyl-HNE by using a click reagent that bears azido and biotin groups separated by a photocleavable linker (Figure 40B).787 Proteins modified in this way were affinity purified on streptavidin beads. Photolysis of the beads with a low-intensity UV light then released bound biotinylated proteins, i.e. proteins modified by alkynyl-HNE, which were further identified by nLC-ESI-MS/MS. Conversely, three shotgun approaches were developed for the simultaneous identification and characterization of HNEadducted proteins (Figure 26). The first approach combines protein labeling with ARP, trypsinolysis, enrichment of ARPmodified peptides, and nLC-ESI-MS/MS or nLC-off line MALDI-TOF-TOF-MS/MS analysis for site-specific identification.665,681 In this case, HNE-Michael adducted peptides are identified by detecting the corresponding species presenting a mass shift of +469 Da with respect to unmodified counterparts; these components are further characterized by assigning the modified amino acids in the corresponding MS/MS spectra (Figure 41). Similarly to what was reported previously for aldehyde-containing ARP-labeled peptides, MS/MS spectral inspection of the ARP-labeled HNE-Michael adducted peptides revealed the occurrence of (i) the diagnostic (partial) NL fragment lacking the ARP and HNE−ARP moiety (at −331 and −469 Da, respectively), (ii) the fragment ions at m/z 227 and 332 originating from the ARP tag, and (iii) the Arp−HNEmodified histidine, cysteine, and lysine immonium ions (mainly observed in MALDI-TOF-TOF-MS/MS) (Figure 41A−C). Conducting peptide mass measurements with the highest possible accuracy is of particular relevance for the ARP-labeling strategy, because shotgun identification is based on the correct assignment of a single peptide adduct. This approach was successfully applied to the characterization of the protein targets and sites of HNE modification in human monocytic THP-1 cells after exogenous exposure to this lipoxidation product. Sixteen proteins were unequivocally identified as

Figure 37. Mass spectrometric characterization of protein modifications generated by the products of nonenzymatic oxidation of linoleic acid (LA). Deuterium-labeled linoleic acid (d5-LA) was synthesized to facilitate the detection and characterization of the protein modifications by mass spectrometry. Reduction by NaBH4 served to trap reversible LA-derived protein adducts and to quantify the number of reducible functional groups in each adduct. (A) Total ion current of the chymotryptic digest of modified β-LG by LA (d0/d5 = 1:1) peroxidation products and selected ion chromatogram of the peptide KIDALNENKVL modified by d0-ONE (B), d5-ONE (C), or DODE (D) through 4-ketoamide formation. Part of the left figure was enlarged and shown on the right. Tandem mass spectra of the modified peptide KIDALNENKVL at Lys91 by d0-ONE (E), d5-ONE (F), or DODE (G) through 4-ketoamide formation. Tandem mass spectra of the peptide KIDALNENKVL cross-linked to the peptide BC


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Figure 38. Protein adducts modified by linoleic acid peroxidation products. Structures were elaborated on the basis of the proteomic data also reported in Figure 37. Reprinted with permission from ref 780. Copyright 2009 American Chemical Society.

of these proteins to HNE modification was demonstrated by analyzing HNE-treated yeast cell cultures with a complementary biotin−hydrazide enrichment strategy. Coupling solidphase enrichment approach with combined NL-triggered CID and ECD-MS/MS strategies facilitated the detection of HNEmodified peptides from low-abundance proteins subjected to substoichiometric carbonylation during oxidative stress.769,789 To improve proteomic analysis of HNE-modified peptides, Griffin and co-workers then developed a method based on partial [18O]-labeling of reactive carbonyl modifications, which produces a unique isotope signature in mass spectra of carbonylated peptides and enables their detection without reliance on matching MS2 spectra to a peptide sequence.790 Key to this method are optimized experimental measures for eliminating trypsin-catalyzed incorporation of [18O] at peptide C-termini, and for stabilizing the incorporated [18O] within the carbonyl modification to prevent its loss during LC separation. By applying this method to a rat muscle homogenate treated with HNE, authors demonstrated its compatibility with solidphase hydrazide chemistry. Additionally, they demonstrated the value of [18O] isotope signatures for confirming HNE-modified peptide sequences matched via sequence database searching, and identifying modified peptides missed by MS2 and/or sequence database searching. By combining [18O]-labeling method with a customized automated software script, authors systematically evaluated the efficiency of MS2 and sequence database searching for identifying HNE-modified peptides.

targets of HNE adduction, and 18 sites of HNE modification at cysteine and histidine residues were assigned. Very recently, ARP was also used for simultaneous shotgun identification of in vivo endogenous protein targets of acrolein, OH-acrolein, CRO, HHE, HNE, and ONE modification in cardiac mitochondria (Figure 41D−F).788 This study was performed by detecting the corresponding species presenting a mass shift of +369, +387, +383, +427, +469, and +467 Da, respectively, with respect to unmodified counterparts; these components were further characterized by assigning the modified amino acids in the corresponding MS/MS spectra. Combined nLC-ESI-MS/MS and nLC-off line MALDI-TOF-TOF-MS/MS analysis was used for this purpose and allowed identification of 39 unique lipoxidation sites on 27 proteins. Several of the target sites were modified by multiple 2-alkenal products. This study emphasized the wide potential of this reagent in shotgun characterization of carbonylated proteins. The second shotgun approach uses a solid-phase capture and release strategy, based on reversible hydrazide chemistry, to enrich HNE-modified peptides from whole protein digests (Figure 42); protein and modification site identifications are obtained by their LC-ESI-MS/MS analysis.767 To maximize the detection of fragment ions diagnostic of HNE modification, both NL-dependent acquisition of MS3 spectra and PQD operation mode are employed. When the solid-phase hydrazide enrichment strategy was applied to a yeast lysate treated with HNE, 125 distinct amino acid sites of HNE modification were mapped on 67 proteins. The endogenous susceptibility of many BD


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Figure 39. Characterization of proteins adducted by lipid peroxidation products in plasma with the biotinylated phospholipid probe PLPBSO. (A) Structure of PLPBSO and flowchart showing its application to the identification of protein targets of phospholipid electrophiles. (B, C) MS/MS spectrum of the tryptic peptide 161−171 from apolipoprotein A1 modified at His162 with KODA and HNE, respectively. (D) MS/MS spectrum of the tryptic peptide 132−140 from the same protein modified with 7-oxoheptanoic acid at His135. (E) MS/MS spectrum of the tryptic peptide 178− 188 from the same protein modified with 8-oxooctanoic acid at Lys182. Adapted with permission from ref 785. Copyright 2008 American Chemical Society.

purified on streptavidin beads. Photolysis of the beads with a low-intensity UV light released bound biotinylated peptides, i.e. peptides modified by alkynyl-HNE, which were further characterized by nLC-ESI-MS/MS, thus permitting identification of the adduction site (Figure 40C,D). Identification of 30 separate peptides from human serum albumin by peptide catch

The third shotgun approach used the click chemistry mentioned above, where labeled proteins modified with alkynyl-HNE were reacted with a click reagent that bears azido and biotin groups separated by a photocleavable linker (Figure 40B).787 Whole protein extracts modified in this way were digested with trypsin and resulting peptides were affinity BE


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Figure 40. Methods for adduct capture and analysis using “click” chemistry-compatible HNE analogues. (A) Chemical structure of 4hydroxynonenal (HNE) and the alkynyl analogue (aHNE), together with the reactivity of the latter toward cysteine, lysine, and histidine residues. (B) Flowchart for ligation of biotin to protein adducts of aHNE by “click” cycloaddition and photorelease of modified protein from biotin reagent. (C) Two flowcharts for isolation of adducted peptides and adducted proteins by “click” biotinylation, trypic digestion, and photorelease of peptides. (D) MS/MS spectrum of plasma apolipoprotein A1 peptide 161−171 modified with aHNE and (E) MS/MS spectrum of plasma apolipoprotein A1 peptide 189−195 modified with aHNE, as detected by applying the peptide catch and photorelease method to a human plasma sample. Adapted with permission from ref 787. Copyright 2009 the American Society for Biochemistry and Molecular Biology.

on a model protein or mitochondrial protein preparations that were modified in vitro with HNE. Efficacy and potential of the HICAT strategy for concomitant identification, characterization, and quantification of different in vivo oxylipid−protein conjugates was demonstrated on cardiac mitochondrial protein preparations. 4.4.3. Analysis of Protein Glycooxidation End-Products. The term protein glycoxidation generally indicates a number of reactions yielding the nonenzymatic attachment of reducing sugars/sugar derivatives to a polypeptide species.592,792 These processes generally affect protein lysine and arginine residues and are not available to nonreducing oligosaccharide derivatives, where aldehyde or ketone groups have been converted to ketal and acetal groups of glycosidic

and photorelease revealed 18 alkynyl-HNE adduction sites on the protein. Protein catch and photorelease showed that both HSA and ApoA1 in human plasma undergo significant modification by HNE. Recently, an isotopically coded affinity probe was also developed and evaluated for the characterization and quantification of proteins adducted by 2-alkenals derived from lipid peroxidation.791 This novel aldehyde-reactive, hydrazide-functionalized, isotope-coded affinity tag (HICAT) (Figure 43) was found to be effective for the selective isolation, detection, and quantification of Michael-type adducts of 2alkenals on proteins using a combination of affinity isolation, nLC, and MALDI-TOF-TOF-MS/MS. Chemical and mass spectrometric properties of the new probe were demonstrated BF


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Figure 41. Shotgun proteomic analysis of lipid peroxidation adducts according to the N′-aminooxymethylcarbonylhydrazino-d-biotin (ARP)-based procedure. (A, B) Tandem mass spectrum of the peptide 282−296 from malate dehydrogenase, which was found with the ARP−acrolein modification at Cys285, during analysis of endogenous protein targets of electrophilic 2-alkenals in rat cardiac mitochondria. (C) Tandem mass spectrum of the ARP−HHE modified version of the same peptide. (D) Pie chart displaying the mitochondrial proteins and amino acid sites identified with modifications by lipid peroxidation-derived 2-alkenals. (E) Pie chart illustrating the percentage breakdown of the types of residues identified with Michael-type additions of lipid peroxidation-derived 2-alkenals. (F) Pie chart indicating the percentages of the different types of lipid peroxidation-derived 2-alkenals identified. Adapted with permission from ref 788. Copyright 2011 Elsevier.

moieties. In the physiological setting, one of the most important sugars participating in glycation of mammalian metabolism is glucose, and some of the most important sugar derivatives are reactive dicarbonyl metabolites, such as glyoxal (GO) and methylglyoxal (MGO). Glycoxidation of proteins occurs by a complex series of sequential and parallel reactions called collectively the Maillard reaction (Figure 11), which have been originally summarized in the Hodge scheme.793 Glucose (or another mono- or oligosaccharide) reacts with protein amino groups to initially form a glycosylamine, which then dehydrates to yield a Schiff base. The Schiff base undergoes an Amadori rearrangement to form Nε-fructosyl-lysine (Figure 11). Glycosylamine, Schiff base, and Nε-fructosyl-lysine derivatives are considered to be early stage glycation adducts. In later stage reactions, Nεfructosyl-lysine degrades to many stable end-stage adducts called advanced glycoxidation end-products (AGEs). Incon-

sistencies in the application of this classification and nomenclature have recently arisen because glucose may also degrade when unattached to protein to form reactive αoxoaldehydes. α-Oxoaldehydes are potent glycoxidating agents and react with proteins to form AGEs directly. The Schiff base adduct may also degrade via non-Amadori rearrangement reaction pathways to α-oxoaldehydes, which also leads to the formation of AGEs (Figure 11). The latter are also formed by the direct modification of proteins by α-oxoaldehydes produced by the degradation of glycolytic intermediates and lipid peroxidation. Important α-oxoaldehyde or dicarbonyl glycating agents are GO, MGO, and 3-deoxyglucosone (3-DG). Therefore, AGEs may be formed in glycation by glucose in pre- and post-Amadori product reactions, and indeed in processes where an Amadori product is not a precursor. In conclusion, AGEs may be formed in both the early and late stages of glycation processes (Figure 11). Molecular structures BG


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Figure 42. Shotgun proteomic analysis of lipid peroxidation adducts according to the solid-phase hydrazide (SPH) enrichment procedure. (A) Scheme for SPH enrichment and analysis of HNE-modified peptides within complex mixtures. (B) left, Base peak chromatogram from NL-MS3 datadependent acquisition showing a complex mixture of mouse brain tryptic peptides spiked with three HNE-modified peptide standards, DRVYIH*PF, LVLEVAQH*LGESTVR, and IVYGH*LDDPANQEIER, where asterisk indicates the modified residue. right, Full-scan FTICR spectrum displaying the doubly protonated ion of DRVYIH*PF peptide at m/z 601.84, but other coeluting peptides present in the complex mixture of mouse brain tryptic digest, as well. (C) Base peak chromatogram showing the HNE-modified peptide standards enriched by SPH enrichment (left), along with the full-scan FTICR spectrum revealing only peptide DRVYIH*PF after its release from the SPH beads by 10% formic acid (right). Adapted with permission from refs 767 and 769. Copyright 2007 and 2009 American Chemical Society. BH


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Figure 43. Structure of the hydrazide-functionalized isotope-coded affinity tag (HICAT) reagent.

of Nε-fructosyl-lysine and some common AGEs are given in Figure 24 (for abbreviations, please refer to section 4.4). The contribution of protein glycoxidation to various nephropathies, retinopathies, osteopathies, and neuropathies also related to cellular aging, atherosclerosis, and diabetes has largely been defined through the detection of different lysineand arginine-derived linear or cross-linked AGE derivatives in biological fluids or protein hydrolysates from sample tissues.581,583,585−587,589−592 These studies demonstrated that a major challenge in studying protein damage by glycoxidation is the sheer diversity of the products generated. However, these investigations lack information on the identity of individual modified proteins, together with the assignment of the modification sites. Obtaining this information is key to relating specific protein modifications to biological mechanisms or pathophysiological conditions. Thus, various proteomic methodologies have been developed to identify and characterize glycoxidation in proteins as purified from biological matrices or, directly, in biological tissues/fluids subjected to oxidative stress conditions. In vitro studies using chemicals inducing glycoxidation in isolated proteins or whole cells were initially performed to predict and further investigate the reactivity of proteins under real oxidative pathophysiological conditions. However, it has to be recognized that proteomic approaches in this field have been less developed than for other oxidative modifications, probably as a result of the number of glycoxidation derivatives present in the modified proteins and of their complex structure. Since no antibodies exist that selectively recognize protein early glycoxidation end-products with respect to natural glycoproteins in the course of gel-based proteomic experiments, detection of Schiff base and further glycated species has been generally performed on isolated components through ESI or MALDI MS procedures, by revealing the corresponding components presenting a mass shift of +162 (glucose and fructose) or +324 Da (lactose).236,686,687,794−811 In some cases, modification was associated with pathological conditions.794,797,800,802,806,808,812 Glycation assignment to specific lysine residues was obtained by peptide mass mapping experiments by either nLC-ESI or nLC-off line MALDI-TOF MS procedures.686,687,794−796,798,801,803−805,807,809−816 Sometimes, preventive reduction with borohydride derivatives was performed to stabilize adducts before protein digestion, thus generating an additional mass increase of 2 Da in the corresponding modified peptides. Generally, MS/MS experiments were realized to definitively assign the modified amino acid(s) within the peptide sequence.807,809,811−815,817 However, CID experiments on glycated peptides revealed that sequence-informative b and y ions resulting from peptide backbone cleavage were reduced within MS/MS spectra,

hampering sequence-derived peptide identification (Figure 44A). In the case of glucosylated peptides, the most abundant

Figure 44. Comparison of the spectra obtained for glycated peptides under CID- and ETD-MS/MS conditions. Tandem mass spectra of the [M + 3H]+3 ion at m/z 499.6, which was assigned to the glycated tryptic peptide LVDkFLEDVKK from α-1-antitrypsin precursor; “k” represents Amadori modification of lysine glucose. Spectra obtained under CID and ETD fragmentation modes are shown in panels A and B, respectively. Insert in panel A is the zoom in view of the ions between m/z 445.5 and 487.4. Identified c and z ions were labeled above and below the sequence in panel B. Reprinted with permission from ref 820. Copyright 2007 American Chemical Society.

ions corresponded to the NL of H2O, 2H2O, 3H2O, 4H2O, and 3H2O + HCHO, as well as of the Amadori adduct C6H10O5; the species showing NL of 3H2O + HCHO was identified as the furylium ion (−84 Da).811,812,818−821 In the case of lactosylated peptides, the most abundant ions were associated with the cleavage of the glycosidic bond, with retention of the glycosidic O atom by the species carrying the reducing end, BI


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plus neutral losses of 2−3 H2O molecules to generate the corresponding (C6H12O6 + 2H2O) furylium ion (−216 Da); neutral losses of H2O, 2H2O, and C6H12O6 + 2H2O + HCHO were also observed.822,823 NL pattern of various H2O molecules and formation of furylium ions were generally chargeindependent for either glucosylated and lactosylated peptides. Neutral losses of 3H2O and 3H2O + HCHO (for glucosylated peptides) and of 2H2O + C6H12O6 (for lactosylated peptides) were used in NL scanning, NL-triggered MS3, and multistage activation experiments for glycoxidized peptide recognition and modification site assignment.821,822 During NL-triggered MS3 experiments on a peptide digest from serum albumin treated in vitro with D-glucose, MS3 scans triggered by neutral losses of 3H2O or 3H2O + HCHO produced similar results in terms of glycated peptide identifications. However, neutral losses of 3H2O resulted in significantly more glycated peptide assignments during multistage activation experiments. Overall, this study demonstrated that the multistage activation approach produces more glycated peptide identifications, while the NLtriggered MS3 procedure results in much higher specificity. To overcome difficulties observed under CID conditions, ETD-based approaches were developed in parallel for the analysis of peptide digests from glycoxidized proteins (Figure 44B).819,820,823−827 In this case, neither furylium ions nor ions corresponding to NL of H2O molecules from the glycoxidation site were observed, clearly demonstrating that the Amadori product is stable under ETD conditions and the bond cleavage is less dependent on peptide composition and side chain modification. The abundance and the almost completeness of the c- and z-type ion series observed, regardless of whether the modification site was located, greatly facilitated peptide sequencing and final modification site assignment. Using supplemental collisional activation after electron transfer increased the number of modified peptides identified.823,824,827 Since boronic acid has been demonstrated to form reversible covalent esters with 1,2 and 1,3 diols in aqueous media, intact early glycoxidation end-product-containing proteins or their digests have been selectively purified by affinity chromatography using phenylboronate-functionalized resins (Figure 45A).820,828,829 Different magnetic beads or chromatographic phases are commercially available for this purpose. These resins were initially tested on model proteins glycated in the course of in vitro experiments.812,820 Later on, they found a widespread application in the course of shotgun proteomic studies on various biological fluids.820,823−825,827 Their use for enriching glycoxidized peptides from whole protein digests, followed by nLC-ESI-MS/MS analysis of the trapped species according to the CID and ETD procedures mentioned above allowed identification of modified proteins present even at lowabundance levels in plasma and milk, also assigning modification sites. Very recently, this approach was used for the comprehensive proteomic identification of glycated proteins in control and diabetic human plasma and erythrocytes (Figure 45B).826 By using immunodepletion, enrichment, and fractionation strategies, Metz and co-workers identified 7749 unique glycated peptides, corresponding to 3742 unique glycated proteins. Semiquantitative comparisons showed that glycation levels of a number of proteins were significantly increased in diabetes and that erythrocyte proteins were more extensively glycated than plasma proteins. A glycation motif analysis revealed that some amino acids were favored more than others in the protein primary structures in the vicinity of the glycation sites in both sample types. Data on glycated proteins

Figure 45. Boronic acid-based proteomic analysis of glycated proteomes. (A) The equilibria between boronic acid-functionalized resin and cis-diol-containing compounds, showing affinity attachment, elution, and resin regeneration. (B) Flowchart depicting exemplificative glycated peptide identification from blood plasma and erythrocyte membranes of individuals with normal glucose tolerance, impaired glucose tolerance, and type 2 diabetes mellitus. Pooled samples from each patient group were processed and analyzed individually by ETDMS/MS analysis as reported in Figure 44. Adapted with permission from refs 820 and 825. Copyright 2007 and 2008 American Chemical Society.

provided a foundation for potential identification of novel markers for diabetes, hyperglycemia, and diabetic complications in future studies. On the other hand, gel-based proteomic procedures have been developed to identify protein glycoxidation end-products, taking advantage of the availability of monoclonal and polyclonal anti-CML, anti-CEL, anti-3DG-H, anti-PENT, antiPYR, anti-RPYR, or general anti-AGE antibodies, which were used in Western blot experiments to probe polypeptide species resolved by 1D and 2D electrophoresis (Figure 14). These reagents were often used in parallel with Oxyblot and anti-HNE Western blotting procedures (Figure 26). Thus, protein bands or spots specifically recognized by parallel Western blotting experiments were sampled from Coomassie-stained preparative gels for subsequent in-gel digestion and further identified by MALDI-TOF peptide mass fingerprinting or nLC-ESI-MS/MS procedures. Examples, in this context, are the differential proteomic studies on (i) AGE-containing proteins in blood lymphocytes, liver mitochondria, skeletal muscle, and fibroblasts during aging,246,830−832 (ii) RPYR- and AGE-containing proteins in yeast cells treated or not with MGO or dihydroxyacetone,833,834 (iii) RPYR-containing proteins in brain of a mouse model for trait anxiety,835 (iv) CML-, CEL-, and AGE-containing proteins in brain of control, AD, and Pick disease patients,836,837 (v) RPYR-, PENT-, and AGE-containing proteins in urine and kidney of patients with dialysis-related amyloidosis and control,838,839 (vi) CEL- and AGE-containing proteins in lens of control, nondiabetic, and diabetic cataractous BJ


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patients,840 and (vii) CML-, CEL-, PENT-, 3DG-H-, PYR-, RPYR-, and AGE-containing proteins in heart and kidney tissues of patients with diabetes and control or related mouse models.841−843 In most cases, further MS analysis of modified spots or bands allowed protein identification; rarely, AGEs were observed in the corresponding peptide digests.833,843 In order to identify lysine and arginine residues involved in the generation of AGEs, various in vitro studies were performed either by adding GO, MGO, or 3-DG to isolated proteins or to intact cells to be further resolved for its polypeptide components or by investigating the oxidative degradation of glycated proteins, which were obtained by preventive ad hoc modification procedures. Modified proteins were then subjected to bottom-up MS-based experiments, where the occurrence of CML, CEL, PYR, and 1-alkyl-2-formyl-3,4glycosyl-pyrrole (AFGP) or of G-H1, MG-H1 A, 3DG-H1 A, 3DG-H1 B, RPYR, and THP was ascertained by detecting the corresponding peptide adducts having a mass increase of +58, +72, +108, and +270 Da with respect to unmodified lysinecontaining homologues, or +40, +54, +144, +142, +80, and +144 Da with respect to unmodified arginine-containing homologues, respectively. Dihydroxyimidazolidine derivatives (G-DH, MG-DH, and 3DG-DH) were also observed at arginine residues, as deduced by the corresponding species showing a mass shift of +58, +72, and +162 Da, respectively. MS/MS experiments were often performed to assign the modified amino acid(s) within the peptide sequence (Figure 46). To this purpose, nLC-ESI-MS/MS or nLC-off line MALDI-TOF-MS/MS procedures were carried out for the characterization of GO-,360,805,844,845

MGO-,360,810,816,833,846−851 D-glucose-,805,807,809,810,845,852−855 807 D-fructose-, lactose-,686,687 ribose-,845 and 3DG-modified846 proteins. In some cases, modification site assignment was obtained for peptide samples derived from digestion of proteins recovered from cells/biological fluids associated with real pathophysiological conditions, which were previously resolved by mono- and bidimensional electrophoresis.804,816,833,843,848,851,856−858 A careful evaluation of the protein functional properties/structural characteristics following AGE formation was eventually performed by dedicated enzymatic assays or molecular modeling simulations. Since some modifications may generate similar mass shifts for the adducted peptides, variations in source voltage and collision energy to obtain the dehydration and decarboxylation products of the THP-containing species and dehydration of the MG-DHcontaining species were used for their identification in the course of ESI-MS experiments.850 Whenever measured, modification percentage values were derived from nLC-ESI-MS/MS experiments by evaluating the total current ion area of the modified peptides with respect to that of the modified peptides plus that of the corresponding unmodified counterparts or by isotope dilution with reconstructed ion chromatograms of precursor and product peptides, using the [15N]-labeled protein as internal standard.751,805,807,810,844,848−850 On the other hand, MALDI-TOF MS peptide mapping experiments were also used to quantitatively evaluate site-specific AGE generation in model proteins; original experiments performed by integrating peak areas of unmodified and modified peptides nowadays have been replaced by more accurate [16O]/[18O]-labeling approaches.855 In the case of MGO-induced modifications, for example, dosedependent and time-course experiments on model proteins demonstrated that MG-H1 A and MG-DH adducts were the major products detected at minimal reagent to protein ratios.850 At higher MGO doses, RPYR and THP adducts were also detected, with lower levels of CEL, MOLD, and MODIC. Among these products, MG-H1 A appears to be the major AGE on plasma or tissue proteins and is increased in patients with diabetes and renal disease, or in animal models of these illnesses. Mechanistic considerations on the reactivity of various aldehydic and diketonic dicarbonyl compounds, and pathways yielding various AGE products were elaborated on the basis of dedicated ESI-MS-based studies on modified amino acids.859,860 In addition to the linear modifications mentioned above, glycoxidized proteins were also proven to contain a number of AGE cross-linking products, which were characterized by bottom-up proteomic analysis. Dedicated studies on RNase A treated with D-glucose, β2-microglobulin from chronic hemodialysis patients with amyloidosis, or type 2 ryanodine receptor calcium-release channel, myosin heavy chains and sarco(endo)plasmic reticulum calcium-ATPase from heart of a rat model of type 1 diabetes are well documented examples in this context.804,814,816,843,857,858 To assign modifications to specific amino acids and to establish their structural characteristics, proteins were digested with a proteolytic enzyme and peptide digests were then subjected to MALDI-TOF-MS or nLC-ESI-MS/MS analysis. These experiments demonstrated that inter-peptide P1−peptide P2 cross-linked species were characterized by a mass value of MP1 + MP2 + 58, MP1 + MP2 + 252, MP1 + MP2 + 126, and MP1 + MP2 + 108 Da for pentosidine, crossline, 3-deoxyglucosone-derived imidazolium cross-link (DODIC), and glucosepane-containing species, respectively, where MP1 and MP2 correspond to the mass

Figure 46. Mass spectrometric characterization of methylglyoxal adducts at arginine residues. (A) Reaction scheme showing formation of dihydroxyimidazolidine (MG-DH1) and hydroxyimidazolone (MGH1) products. (B) Product-ion spectra of the (M + 2H)2+ ions of the methylglyoxal-modified peptide 91−99 from the α chain of human hemoglobin. A summary of the observed fragment ions is shown above the spectrum. R indicates the methylglyoxal-modified arginine residue. Adapted with permission from ref 849. Copyright 2006 American Chemical Society. BK


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Figure 47. Linear and cross-linked AGE products in sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) from a rat model of type 1 diabetes. (A) Representative analysis of carbonyl adducts on SERCA2a isolated from control (C) and streptozotocin (STZ)-induced diabetic (D) rats. Western blotting of membrane vesicle material obtained by using anti-argpyrimidine, -AGEs, -3-deoxyglycosone/imidazolone, -CML, -carboxyethyllysine, -pentosidine, -pyralline, and -actin antibodies. (B) Alignment of a MALDI-TOF mass spectral region obtained following trypsin digestion of SERCA2a from control (top), STZ-induced diabetic (middle), and insulin-treated STZ-induced diabetic (bottom) rats. An MH+ signal at m/z 2485.12 was present in diabetic samples but not in the others. (C) ESI-Q-TOF tandem mass spectrum obtained after fragmentation of the peptide component at m/z 2485.12 (panel B), suggesting its nature as a pentosidine cross-linking adduct between Lys460 in peptide 452−464 (within the SERCA2a nucleotide domain) and Arg636 in peptide 629−637 (within the SERCA2a phosphorylation domain). Reprinted with permission from ref 843. Copyright 2011 American Diabetes Association.

by a dihydroxyimidazolidine structure and a peptide mass shift of +350 Da with respect to the unmodified counterpart. This adduct occurs at conserved modification sites in collagen and presents structural analogies with arginoline and hydroxylysyl pyridinoline cross-linking derivatives, also present in cartilage. In addition, it was also demonstrated that cysteine residues may behave as targets of glycoxidation and that low molecular mass thiols may act as protective agents.863 In fact, incubation of model proteins with GO resulted in protein cross-link formation, with concurrent loss of thiol groups and formation of S-carboxymethylcysteine, a product of GO adduction to cysteine residues. Further MS experiments demonstrated the formation of thiol-glyoxalamine species to generate 2(alkylthio)acetamide cross-links.864 A range of low molecular mass thiols were shown to inhibit dicarbonyl adduction to and cross-linking of the thiol-free proteins, consistent with these thiols being alternative (sacrificial) targets of glycoxidation. Some of these thiols seemed more efficient modulators of glycoxidation than established glycoxidation inhibitors such as aminoguanidine.

values of unmodified peptides P1 and P2 (Figure 47). In parallel, intrapeptide cross-linked species were also observed (Figure 48). In all cases, recognition of cross-linked peptides was facilitated by using dedicated algorithms. Conversely, other cross-linking adducts were observed during nLC-ESI-FT-ICR-MS analysis of glycoxidized human serum albumin.861 On the basis of accurate mass measurements and known protein sequence, it was proposed that inter-peptide P1−peptide P2 cross-linked species showing a mass value of MP1 + MP2 + 126, MP1 + MP2 + 48, MP1 + MP2 + 22, and MP1 + MP2 + 24 Da occurred in protein digests, where MP1 and MP2 again correspond to the mass values of unmodified peptide P1 and P2. These species were associated with the P1-(C6H6O3)P2, P1-(C4)-P2, P1-(C2H2)-P2, and P1-(C2)-P2 cross-linked adducts, respectively, Furthermore, glycoxidation reactions at specific lysine residues in collagen, followed by its ketoamine (Amadori) rearrangement, oxidation, and final addition of arginine residues was demonstrated to generate cross-linking adducts in mature articular cartilage.862 The resulting derivative was characterized BL


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Figure 48. continued the presence of intramolecular glucosepane moiety cross-linking Lys41 and Arg39. The inset shows the corresponding singly charged peptide with m/z 2896.4 and its m + 6 signal at 2902.4 present when glycation was obtained with a 1:1 [13C]/[12C] glucose mixture. (C) Tandem mass spectrum of the [M + 3H]+3 ion at m/z 972.3, which was assigned to the peptide 36−61 containing an intramolecular modification by DODIC cross-linking residues Lys41 and Arg39. Adapted with permission from ref 814. Copyright 2008 American Chemical Society.

4.5. Analysis of Protein Adducts Resulting from ROS/RNS-Induced Modifications at Tyrosine

Reaction of various radical or nonradical RNS, ROS, and hypohalogenous acids with protein tyrosine residues has been proven to yield NO2-Tyr, Cl-Tyr, diCl-Tyr, Br-Tyr, diBr-Tyr, di-Tyr, di-OH-Phe (DOPA), and DOPA-quinone derivatives as modified end-products (Figure 13 and 49). Unproblematic detection of most of these covalent adducts is dependent on their general stability, even at conditions yielding polypeptide backbone hydrolysis, which made some derivatives useful biomarkers of ROS/RNS insult.272,865 Initial studies on tyrosine modifications were based on GC- or LC-MS determination of amino acids after precolumn derivatization, which accurately quantified basal levels of Cl-Tyr, diCl-Tyr, Br-Tyr, diBr-Tyr, NO2-Tyr, di-Tyr, and non-natural o-Tyr and m-Tyr isomers (derived from phenylalanine oxidation) in biological fluids/ tissues or protein hydrolysates and ascertained the occurrence of increased levels of these compounds in organisms exposed to oxidative/nitrosative stress or disease conditions.271,272,866−869 Compared with UV- or fluorescence-based LC counterparts, these MS approaches provided structural information and allowed SIM experiments for quantification of trace quantities as well as the use of stable isotope dilution procedures for precise and accurate measurements.272,865,870−875 For example, analysis of human bronchoalveolar lavage proteins for Br-Tyr, Cl-Tyr, and NO2-Tyr content showed levels of 1093, 161, and 480 μmol, respectively, of each analyte per mole of tyrosine residues in asthmatic patients, whereas the levels in the controls were in the 13−65 μM range.876 Also in ApoA1 from patients with cardiovascular disease, levels of NO2-Tyr and Cl-Tyr were of the same magnitude.282 These data showed that between 0.00001% and 0.001% of protein tyrosine residues can be found modified, a phenomenon that raises serious analytical problems for the detection of these modifications directly within protein structures.154 Moreover, methods reported above for analysis of modified amino acids involve chemical derivatization steps that, in principle, can inherently decrease the detection sensitivity. Thus, extreme caution must be exerted any time that absolute quantitation of tyrosine modifications is needed. A major drawback of the analysis of free tyrosine derivatives is the resulting lack of information on the identity and concentration of individual modified proteins; obtaining sitespecific information is key to relating specific tyrosine modification to biological mechanisms, and also opens the possibility to establish a relationship between defined modified proteins and a pathological condition. Thus, various gel-based and gel-free proteomic methodologies have been developed to ascertain tyrosine modifications in proteins as preventively purified or from biological tissues/fluids subjected to oxidative/ nitrosative stress conditions.41,877 In vitro studies with ROS/ RNS, such as tetranitromethane, peroxynitrite, HClO, and

Figure 48. Linear and cross-linked AGE products in glycoxidized bovine RNase A. (A) Tandem mass spectrum of the [M + 2H]+2 ion at m/z 1339.0, which was assigned to the glycated tryptic peptide 40−61 (Δm = +160) (oxidized Amadori). Every b ion greater than b2 contained this increment, while every y ion up to y20 was consistent with the unmodified peptide sequence, strongly implicating the modification at Lys41 by an oxidized Amadori product. (B) Tandem mass spectrum of the [M + 2H]+2 ion at m/z 1449.4, which was assigned to the glycated tryptic peptide 36−61. The presence of ions b4 and y23, both containing the glucosepane mass increment, suggested BM


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Figure 49. Proposed pathways for ROS- and RNS-dependent formation of dityrosine, nitrotyrosine, DOPA, DOPA-quinone, TOPA, and TOPAquinone.

tissue before and during gland remodeling after lactation,912 spinal cord tissues of animal model of familial ALS,913 lung tissues of allergen-induced animal model of asthma,142 kidney tissues of control and spontaneously hypertensive animals,914 heart mitochondria of animal models of diabetes,297,325,915 islet of Langerhans/β-cells or adipocytes exposed to glucose,916,917 cells treated or not with diesel exhaust particles.918 To control for nonspecific antibody binding, parallel experiments using Na2S2O4-mediated reduction of nitro- to aminotyrosine on membrane have been used before Western blotting;902 however, reduction of protein spots on the membrane is often incomplete and may result in weak signals even after treatment.286,882,919 The dithionite-based reduction of nitrotyrosine is not free of byproducts, such as aminotyrosine Osulfate (5−10% yield);920 as an alternative, a quantitative reduction based on protein treatment with dithiothreitol in combination with heme-containing proteins, at 100 °C was proposed.921 Although 2-DE has been used successfully to recognize nitrated proteins, it suffers from some limitations that have been already reported before for other redox PTMs, generally determining the stringent need for MS confirmation for a final assignment of protein nitration.882,899,915,922 Some of these drawbacks were tentatively faced by analyzing nitroproteomes with alternative 2D separation techniques, such as solution IEF plus SDS-PAGE899,923 or LC plus SDS-PAGE882,924,925 that, although providing less resolution than 2-DE, have allowed a higher sample load in the first dimension926 and are more suitable for hydrophobic proteins. These alternative procedures always involved detection of nitrated proteins by Western blotting and did not therefore provide significant improvements in the localization of the modified residues. To overcome some of the limitations reported for gel-based analyses, multidimensional chromatographic separations (based on a combination of ion-exchange and reversed phase LC of digested protein mixtures) followed by MS/MS-driven protein identification have also been used for nitroproteome studies.882,922,927 Since nitroproteins are quite rare and so elusive, anti-NO2Tyr antibodies have also been used in the development of

HBrO added to isolated proteins, were performed to simulate and investigate tyrosine reactivity under pathophysiological conditions.41,361,877−879 Most of the reported studies have focused on tyrosine nitration, taking advantage of the commercial availability of anti-NO2-Tyr antibodies,880−884 among which the clone 1A6 monoclonal one has been widely used in Western blot experiments to probe proteins resolved by 1D and 2D electrophoretic or chromatographic procedures, according to its high specificity and sensitivity (Figure 14). Conversely, antiCl-Tyr, -Br-Tyr, and -di-Tyr antibodies have been prepared for histological applications,879,885−887 but their use in proteomic studies has not been reported so far. Proteins containing selected tyrosine redox PTMs have also been isolated taking advantage of their specific absorbance at 360 nm under acid conditions (pH < 5) (for NO2-Tyr)888,889 or fluorescence at 410 nm (excitation 315 nm) (for di-Tyr)890,891 during chromatographic separations, and aberrant migration in SDSPAGE under reducing conditions (for di-Tyr).890,892 To date, the most commonly used unbiased approach for identifying NO2-Tyr-containing proteins has been 2-DE, wherein protein spots, which are specifically recognized by parallel Western blotting experiments with an anti-NO2-Tyr antibody, are cut from preparative gels for subsequent in-gel digestion and MS-based analysis (Figure 14).881 About 40 studies have been published until now using this approach, which allowed identification of nitroproteins by MALDI-TOF MS peptide mass fingerprinting or nLC-ESI-MS/ MS.287,645,654,893−896 Examples, in this context, are the differential proteomic studies on nitroproteins from skeletal muscle, cardiac, and brain tissues of aged and control rats,897−899 lung and liver tissues or mast cells before and after inflammatory challenge,900,901 retina tissues before and after light exposure,902 brain tissues of control and AD patients or related animal models,25,903−905 injured brain tissues of control and antioxidant-treated animals,906 lung tissues of control and cancer patients,907 mitochondria of different tissues during ischemia-reperfusion conditions,140,908 control and pathogen challenged plasma or plant tissues,909−911 mammary BN


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Figure 50. Comparative MALDI and ESI analysis of a tryptic digest from nitrated bovine serum albumin. (A) Reflectron positive-ion MALDI-TOF spectrum of the whole tryptic digest. (Inset) Expanded spectral region where signals associated with the nitrotyrosine-containing peptide 137−143 were observed, including photodecomposition (nitroso, nitrene, and aminotyrosine) products. (B) ESI-IT-MS/MS spectrum of the [M + 3H]+3 ion at m/z 495.69, which was associated with the nitropeptide 336−347. (C) ESI-IT-MS/MS spectrum of the [M + 2H]+2 ion at m/z 486.93, which was associated with the nitropeptide 137−143. Adapted with permission from ref 878. Copyright 2008 Elsevier.

enrichment methods. Thus, various studies have reported on immunoprecipitation experiments to successfully isolate intact NO2-Tyr-containing proteins.278,899,919,928 Covalently immobilized antibodies (now commercially available) were used to

circumvent the unpleasant occurrence of immunoglobulin heavy and light chains within immunoprecipitates. However, some researchers have reported that immunoprecipitation of nitroproteins may result in insufficient amounts for further BO


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N2 ion laser (337 nm) (Figure 50A). The effect of the matrix nature, laser shots, laser power, and peptide concentration on the formation of PDC products was evaluated in dedicated studies.358,935,936 While this phenomenon produces a unique fingerprint facilitating nitropeptide assignment, it also compromises the sensitivity of modification detection (about 60− 70%), since it spreads the MH+ signal over different species. This may result in the impossibility to detect nitropeptides in complex peptide maps,358,934,937−939 also having in mind that NO2-Tyr is a low-abundance protein modification.154 On this basis, only a few studies reporting MALDI-TOF characterization of nitropeptides from gel-resolved proteins have been published,278,891,905,913,915 and also poor fragment information by MALDI-MS/MS or PSD spectral analysis have been reported so far.893,929,934 Reduction of nitropeptides to aminotyrosine-containing species (showing defined molecular ions upon MALDI) can avoid PDC problems and can be used to readily discern NH2-Tyr-containing peptides in a background of non-nitrated peptides.920,934 This reaction was achieved quantitatively by boiling analytes with dithiothreitol and heme, to avoid the generation of aminotyrosine O-sulfate byproducts (Δm = +80), otherwise occurring with dithionite.921 An alternative approach consists in analyzing NO2-Tyrcontaining peptides with IR-MALDI (emission at 2.94 μm, exciting water as the matrix), which generally leads to less PDC than UV-MALDI.937 Very recently, efficient and selective photodecomposition at 355 nm was observed for NO2-Tyr, NO-Tyr, and N-Tyr adducts generated by UV-MALDI of NO2Tyr-containing peptides.940 Product ion spectra obtained by postsource PDC at this wavelength contained useful information on the amino acid sequence. The spectra for nitropeptides obtained with 355 nm irradiation inside the ion source (MALDI/in-source PDC) displayed characteristic triplet patterns due to photodecomposition of the above ions. For peptides displaying prominent signal in a MALDI mass map of a tryptic mixture, in-source PDC allowed positive identification of their tyrosine-nitrated forms. It was possible to identify such nitropeptides (10 fmol) when spiked within a tryptic digest (100 fmol). In contrast, ES ionization does not show PDC phenomena and allows for a complete estimate of protein nitration by detecting stable peptide species with the expected mass shift (Δm = +45) (Figure 50B,C).41,881,882 When coupled online with a LC device, a selective detection of nitrated species can be concomitantly obtained by measuring absorbance at 360 nm.888,933 Due to the low amounts of nitrated proteins usually recovered by 2-DE, nLC has been generally used to increase sensitivity during MS analysis.933 On this basis, most of the nitroproteome studies published so far have used ESI sources for the characterization of nitrated proteins/peptides. Unequivocal assignment of NO2-Tyr in various proteins has been determined by CID experiments.269,278,287,293,297,300,325,358,878,892,893,898, 899,902,908,912,916,924,926,927,941−963 However, Stevens and others showed that data from mass analyzers of low mass accuracy, used in automated MS/MS-based peptide identification mode and with a large mass tolerance for precursor ion selection, may lead to the incorrect assignment of tyrosine nitration.964,965 This problem has been associated with the occurrence of (almost) isobaric peptides generated by a combination of odd events (e.g., carbamoylation due to the presence of urea together with deamidation, or His-Ala in place of NO2-Tyr within the sequence) or of short (modified) peptides having

electrophoretic and/or MS analysis, a common problem with identification of low-abundance, post-translationally modified proteins.897,899 On the other hand, Zhan and Desiderio incorporated an anti-NO2-Tyr antibody into a chromatographic format and dubbed it a nitrotyrosine affinity column, which enabled the purification of nine nitrated proteins from a human pituitary adenoma tissue, plus their interacting partners.929 Direct digestion of eluted species and subsequent MS/MS analysis of resulting peptides identified specific NO2-Tyr residues in each protein; hundreds of MS/MS scans were accumulated to improve signal/noise of b- and y-ions, which were further manually interpreted. They sought to increase sensitivity by allowing the sample to incubate overnight with the immunoaffinity resin and to enhance selectivity by using a stringent washing procedure. A similar approach was recently used to identify nitroproteins in human ex-smoker bronchoalveolar lavage fluid.930 Based on the pKa difference between the aromatic amine of NH2-Tyr (pKa of 4.75) and other aliphatic amines present in proteins (pKa of 8.0−10.5), Nikov and colleagues tentatively isolated nitroproteins by converting nitrointo aminotyrosine with Na2S2O4, selectively modified aminotyrosine with sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (sulfo-NHS-SS-biotin), at pH 5, and affinity captured the reaction products on an immobilized streptavidin column.931 In agreement with previous reports describing a certain reactivity of aliphatic amines with NHS esters at pH 5, some investigators observed that this method is not selective enough to enrich NO2-Tyr-containing proteins from complex samples.41,920,932 In contrast, no enrichment procedures have been developed for tyrosine halogenation or oxidation products so far. Since 2-DE and LC-based methodologies for detection of tyrosine-modified proteins may suffer from contamination drawbacks leading to the occurrence of false positive results, the direct MS detection of nitrated, oxidized, and halogenated tyrosine adducts in intact proteins or their digests by ESI- or MALDI-based techniques has been required for the final assignment of protein modifications.9 Thus, DOPA-quinone-, DOPA-, NO2-Tyr-, Cl-Tyr-, Br-Tyr-, and di-Tyr-containing proteins and peptides have been tentatively assigned by direct MS measurements of polypeptide mixtures, by detecting the corresponding adducts having a mass shift of +14, +16, +45, +34, and +78 Da or cross-linked species, respectively, eventually bearing the typical halogen isotopic distribution. However, modification assignment simply based on a mass increase measurement is not certain, since other amino acids (tryptophan, methionine and cysteine) can bear the same chemical groups or even other modification(s) can be present on polypeptide yielding the same Δm. Thus, peptide mass fingerprinting alone may be not sufficient, and additional MS/ MS experiments were often used to localize the modified tyrosine within the protein sequence.9,882,922,933 In the case of NO2-Tyr-containing polypeptides, when comparative analysis was performed by UV-MALDI- and ESIMS, it was evident that the first ionization technique does not correspond to the optimal choice for sensitive detection of modification, as it induces a prompt fragmentation involving the NO2 group.358,878,934 This phenomenon has been associated with a series of photodecomposition (PDC) reactions yielding NO-Tyr (Δm = +29), NH2-Tyr (Δm = +15), and N-Tyr (Δm = +13) adducts, which depend on the close vicinity of the absorbance maximum of NO2-Tyr under acidic conditions (∼360 nm) to the emission wavelength of the BP


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Figure 51. Comparative reaction diagram showing the enrichment procedures for nitrotyrosine-containing peptides according to different shotgun proteomic approaches. Procedures based on N-succinimidyl S-acetylthioacetate (scheme a), pyridine-2-carboxaldehyde (scheme b), biotinyl Nhydroxysuccinimide ester (NHS-biotin) (scheme c), N-succinimidyl 3-(perfluorobutyl)propionate (scheme d), and solid-phase active ester reagent (SPAER) (scheme e) are shown. Chemical structure of the original nitrotyrosine-containing peptide and the tagged products for final nLC-ESI-MS/ MS or MALDI-TOF-TOF MS/MS analysis are shown in the boxed portions.

have to be recognized without the mass shift; (vii) correct precursor ion charge state (QSTAR Elite only), (viii) correct monoisotopic precursor mass (QSTAR Elite only); (ix) precursor mass accuracy 6; v) one or more fragment ions in the MS/MS spectrum that include the modified tyrosine have to be recognized with the resulting mass shift due to the NO2-Tyr modification; (vi) one or more fragment ions in the MS/MS spectrum that do not include the modified tyrosine BQ


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Figure 52. Top-down mass analysis of nitrated lysozyme. ECD (A, B), IRMPD (C), and CID (D) MS/MS analysis of bis-nitrated lysozyme. Nitrated tyrosine residues are circled in the fragment summaries. Reprinted with permission from ref 975. Copyright 2010 American Chemical Society.

spiked into an E. coli proteome tryptic digest; later on, this approach was applied to the analysis of plasma nitroproteome of kidney disease patients.968 To enhance detection of nitrated proteins, facing eventual problems related to physicochemical properties hampering their further electrophoretic or chromatographic separation, enrichment procedures have also been developed in the context of a ESI-based shotgun proteomic strategy, thus labeling, trapping, and analyzing nitrated peptides directly from whole protein digests (Figure 51). Zhang and colleagues originally described a procedure for chemical labeling and enrichment of nitrotyrosine-containing peptides based on: (i) acetylation of

primary amines with acetic anhydride; (ii) reduction of nitrotyrosine with Na2S2O4; (iii) acylation of aminotyrosine with N-succinimidyl S-acetylthioacetate (SATA); (iv) deprotection of S-acetyl on SATA to yield thiol-containing peptides to be further trapped on thiopropyl sepharose, alkylated with iodoacetamide, and analyzed by LC-ESI-LIT-MS/MS (Figure 51, scheme a).932 Authors identified 150 nitration sites on 102 unique proteins from an in vitro nitrated tissue homogenate from mouse brain. Two years later, Lee and co-workers published a modification of this approach, where steps iii and iv were replaced by the reaction of aminotyrosine-containing peptides with pyridine-2-carboxaldehyde to generate the BR


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multiple losses of small neutral species including OH•, H2O, and NH3. Loss of ammonia appeared to be the result of noncovalent interactions between the NO2 group and protonated lysine side chains. A comparative evaluation of ECD versus CID and infrared multiphoton dissociation (IRMPD) for top-down MS/MS analysis of multiply charged intact nitrated protein ions of different proteins was performed in a commercial FT-ICR mass spectrometer (Figure 52). CID and IRMPD produced more cleavages in the vicinity of the nitration sites than ECD. However, the total number of ECD fragments was greater than those from CID or IRMPD, and many ECD fragments contained the site(s) of nitration. Authors concluded that ECD can be used in the top-down analysis of nitrated proteins, but the precise localization of the nitration sites may require either of the “slow-heating” methods. LC-based quantification of nitropeptides in protein digests has been reported by various authors, taking advantage of their selective absorbance at 360 nm.286,882,888 They generally underlined the limit of this method when analyzing low amounts of proteins recoverable from 2-DE spots. To increase sensitivity during quantitative analysis of protein tyrosine nitration, a fluorogenic tagging procedure of NO2-Tyrcontaining peptides and proteins with 4-(aminomethyl)benzenesulfonic acid (ABS) was recently proposed.976 It includes the initial dithionite-based reduction of NO2-Tyr to NH2-Tyr residues, which are then reacted with ABS in the presence of K3Fe(CN)6, at pH 9.0, to form highly fluorescent 2-phenylbenzoxazole products. They incorporate one or two ABS molecules per nitrotyrosine residue and have a fluorescence excitation and emission maxima at 360 and 490 nm and a mass shift of +179/196 or +366 Da with respect to tyrosine, respectively. LC analysis with fluorescence detection of digests subjected to derivatization with ABS was used for quantitative analysis of in vitro tyrosine nitration in a model protein or a protein mixture from C2C12 cells exposed to peroxynitrite. LC-ESI-MS/MS analysis of ABS-tagged peptides demonstrated that the fluorescent derivatives undergo efficient backbone fragmentations, allowing for sequence-specific characterization of protein tyrosine nitration during proteomic studies. Fluorogenic tagging with ABS also allowed visualization of nitroproteins in LC and gel-based protein separations. On the other hand, dedicated methods based only on MS measurements have also been proposed to quantitatively evaluate protein nitration in the course of proteomic investigations. Initial approaches were based on the use of a reference peptide from the protein of interest, which was used as the internal standard to assay relative nitropeptide level changes;977 this strategy showed a good precision (relative SD < 10%) and a limit of detection of 5 fmol. A related method based on the measurement of the relative area of nitropeptides and of corresponding unmodified counterparts from LC-ESIMS/MS analysis was also proposed for evaluation of the nitration extent in isolated proteins;978 this approach was also introduced in a MRM MS procedure to sensitively quantitate nitrotyrosine levels of specific α-synuclein tyrosine residues in an inducible transgenic cellular model of PD.979 Very recently, a quantitative LC-ESI-MS/MS-based proteomic evaluation of in vivo nitrotyrosine levels in A. thaliana thylakoid membrane proteins after photooxidative exposition was performed by measuring the ratio of the area of nitropeptides versus that of corresponding unmodified counterparts.269 Exposure of plants to light stress resulted in an increased level of tyrosine nitration

corresponding Schiff base products, which were further reduced with Na(CN)BH3 to yield the bis-pyridinylated-tyrosine derivatives. These compounds were then purified on Ni2+nitrilotriacetic acid functionalized magnetic agarose beads for further MS analysis (Figure 51, scheme b).969 Authors were able to detect spiked nitropeptides from nitrated albumin into a HeLa cell whole protein digest. A related chemical approach was also published by Abello and colleagues; it involves: (i) the quantitative reduction of nitrotyrosines with dithiothreitol/ heme at 100 °C; (ii)the biotinylation of resulting aminotyrosine residues; (iii) the enrichment of biotinylated peptides by avidinbased affinity chromatography and their final analysis by LCESI-IT-MS/MS (Figure 51, scheme c).970 In this case, authors were able to detect tyrosine-nitrated angiotensin II in a tryptic digest of albumin. On the other hand, Nuriel and colleagues proposed another approach based on reductive dimethylation of primary amines within peptides followed by reduction of nitrotyrosines with Na2S2O4, biotinylation of aminotyrosine products with sulfo-NHS-SS-biotin, their streptavidin-based trapping, and MS analysis.971 However, no results based on the use of this method have been reported at present. Recently, a novel nitropeptide-enrichment procedure was proposed, which allowed the identification of 28 unique nitrated peptides in 28 proteins from an in vivo nitrated Huh7 human hepatoma cell line lysate.972 This method is based on: (i) acetylation of primary amines with sulfosuccinimidyl-2-acetate; (ii) reduction of nitrotyrosines with Na2S2O4; (iii) perfluorination of aminotyrosines with N-succinimidyl 3-(perfluorobutyl)propionate; (iv) trapping of fluorinated carbon-tagged peptides on fluorinated carbon-linked silica beads and subsequent LC-ESILIT-MS/MS analysis (Figure 51, scheme d). Very recently, Prokai and co-workers proposed a simple and highly specific method to enrich nitropeptides by a chemoprecipitation procedure involving only two straightforward chemical modifications of the nitrated species prior to capturing (Figure 51, scheme e).973 Specifically, capping of the aliphatic amines in the peptides was done first by reductive methylation to preserve the charge state of peptides for ESI-MS analysis, followed by reduction of NO2-Tyr to the corresponding NH2-Tyr derivative. These peptides were then immobilized on a strategically designed solid-phase active ester reagent (SPAER), while other peptides carrying no free amino groups were washed away. Tagged peptide derivatives were easily released from the beads by acid-catalyzed hydrolysis at room temperature. This approach was applied to the analysis of synthetic nitropeptides and to the identification of several in vitro nitrated human plasma proteins. Authors demonstrated that converting the NO2 group to the small 4-formylbenzoylamido tag does not significantly alter fragmentation properties upon CID, compared with those of the native nitropeptides. At the same time, this derivatization actually improves ECD due to the conversion of the electron-predator nitro group into this novel tag. The proteomic approaches reported above always involve chemical derivatization steps that, although increasing nitroprotein selectivity, can inherently decrease its detection sensitivity; this point has to be considered when endogenous NO2-Tyr is studied. ECD MS was recently also applied to the analysis of NO2Tyr-containing peptides and proteins.974,975 Authors showed that nitration severely inhibits the production of ECD sequence fragments in doubly charged peptides, while some singly charged sequence fragments occur for the triply charged species. ECD of the nitrated peptides is characterized by BS


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Figure 53. Reaction diagram showing quantification of nitrotyrosine-containing peptides according to the 1-(6-methylnicotinoyloxy)succinimide and the iTRAQ-based strategies. The chemical structures of the corresponding amine-reactive and iTRAQ reagents are shown in the boxed portions at the end of the reaction pathways a and b, respectively.

adapted to nitrotyrosine analysis to quantify relative modification extents (Figure 53, scheme b).383 This method was based on blocking all primary amine groups after proteolytic digestion with acetic acid N-hydroxy succinimide, followed by Na2S2O4-based reduction of NO2-Tyr to NH2-Tyr residues, which were further labeled with amine-specific iTRAQ reagents. This procedure allowed the identification and relative quantification of nitropeptides from in vitro nitrated BSA spiked into an E. coli protein mixture or from in vitro nitrated milk proteins; a significant increase in sensitivity was obtained by using nitro-specific PIS. Differently from nitration, halogenation of tyrosine residues in proteins has been ascertained in the course of MS studies, where either ESI- or MALDI-based instruments were used without any specific limitation; thus, Cl-Tyr-, di-Cl-Tyr, BrTyr-, and di-Br-Tyr adducts in intact proteins or their peptide digests were detected by measuring the corresponding mass shift of +34, +68, +78, and +156 Da, respectively. MALDI-TOF MS measurements did not reveal additional signals for these modified species, except for the typical pattern of signals related to the isotopic distribution of mono- and dihalogenated derivatives. Thus, peptide mapping experiments on isolated proteins ascertained the occurrence of Cl- and di-Cl-Tyr residues in purified ApoA1 or histone proteins following treatment with HClO,983,984 which were further identified by PSD experiments. By assumption of identical ionization properties for the non-, mono-, and dimodified species, the integration of corresponding peak areas allowed evaluation of the relative modification percentages, depending on the experimental conditions.984 A similar MALDI-TOF-based approach was also used to assign specific Br- and di-Br-Tyr

in proteins of the photosystem II reaction center and the oxygen-evolving complex, compared with low light conditions. In contrast, the level of tryptophan nitration strongly decreased for all light-harvesting proteins of the photosystem II under the same conditions. Recent developments in MS reagents have led to improvements in protein quantification techniques, such as those based on ICAT980 and iTRAQ,381 which have also been adapted to evaluate protein nitration. Initially, [2H]-containing phenylisothiocyanate was used for stable isotope labeling of dithionitereduced nitrotyrosines, which reacted quantitatively at pH 3 without interference of aliphatic amines.981 Photochemically mediated cyclization of the obtained tyrosyl-3-thiourea resulted in a 2-anilino-benzoxazole derivative (yield: 80%), a process requiring the presence of the neighboring phenolic hydroxyl group, thus increasing the overall labeling selectivity. Relative ESI-MS quantitation was based on the unique mass increase due to the labeling with light and heavy phenylisothiocyanate (+116 and +121 Da, respectively, with respect to nitrotyrosine). Recently, Tsumoto and colleagues have proposed a method to identify nitropeptides and to quantify protein nitration levels in different biological samples that is based on a combination of chemical derivatization and MALDI-TOF MS.982 Their strategy includes: (i) the protection of both N-terminal and side chain amines by acetylation with [13C0]/[13C4]- or [2H0]/[2H6]acetic anhydride; (ii) the reduction of NO2-Tyr to NH2-Tyr with sodium hydrosulfite; (iii) the derivatization of aminotyrosine with 1-(6-methyl[2H0/2H3]nicotinoyloxy)-succinimide (Figure 53, scheme a). However, the utility of this method was only demonstrated with NO2-Tyr-containing angiotensin II and albumin as model compounds. Also the iTRAQ technology was BT


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Figure 54. Identification of the major site of tyrosine chlorination and nitration in human apolipoprotein A1 exposed to MPO-H2O2-NaCl and MPO-H2O2-NaNO2. MS/MS analysis of the (A) unmodified ([M + 2H]+2 ion at m/z 416.4), (B) chlorinated ([M + 2H]+2 at m/z 433.4), and (C) nitrated ([M + 2H]+2 at m/z 438.9) peptides from apolipoprotein A1. Lipid-free or lipid-associated apolipoprotein A1 (5 μM) was exposed to H2O2 (125 μM) in the MPO-chloride or the MPO-nitrite system for 60 min, at 37 °C. After the reaction was terminated by methionine addition, protein was digested with trypsin, and resulting peptides were analyzed by LC-ESI-MS/MS. Further experiments demonstrated a strong linear association between the extent of Tyr192 chlorination and the loss of ABCA1 transport activity. Reprinted with permission from ref 147. Copyright 2011 Elsevier.

the first examples of a quantitative peptide mapping investigation applied to the global analysis of in vitro and in vivo myeloperoxidase-catalyzed reactions affecting ABCA1dependent cholesterol efflux in patients with atherosclerotic lesions.536,985 In fact, selective or concomitant detection of nitrated and/or chlorinated peptides in products obtained under different experimental conditions specifically demonstrated that only chlorination markedly impairs cholesterol transport. A similar approach has been recently used to discriminate oxidative modifications in von Willebrand factor by neutrophil oxidants that inhibit its cleavage by ADAMTS13, with possible consequences in several inflammatory states,

residues in albumin following treatment with the myeloperoxidase−H2O2 system, in the presence of chloride, bromide, and nitrite.292 In this case, the application of pure Br isotopes was used to demonstrate the incorporation of the halogen to specific peptides, as ascertained by the specific mass shift of the corresponding signals within the MS profile. Similarly, LC-ESI-MS/MS approaches have been used for the assignment of chlorination sites in ApoA1 and histone proteins.984−986 Also in this case, detection of Cl-Tyrcontaining peptides was achieved by specifically revealing species presenting a Δm = +34 Da, which were further assigned by MS/MS analysis (Figure 54). The studies on ApoA1 were BU


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including sepsis and acute respiratory distress syndrome.539 In this case, parallel analysis of tyrosine chlorination and methionine oxidation sites was performed. The occurrence of DOPA, DOPA-quinone, and di-Tyrcontaining species (Figure 49) in proteins has been generally investigated by ESI or MALDI-TOF-TOF MS/MS analysis of LC-resolved digests, by detecting the corresponding adducts having a mass difference of +16 or +14 or cross-linked species, respectively.933 Since other amino acids can be modified by ROS or RNS with concomitant generation of identical peptide mass shifts, the direct localization of DOPA and DOPAquinone residues within the protein sequence, as ascertained by MS/MS experiments, has been generally preferred to avoid false positive results. On the other hand, identification of crosslinked peptides containing di-Tyr has been generally obtained by searching MS/MS data with dedicated algorithms.987 Thus, purified model proteins were initially investigated in in vitro studies and proven to include different DOPA residues following their exposure to various oxidizing conditions; examples, in this context, are the modifications induced on BSA and glycated insulin by the FeCl2/H2O2 system,684,685 ApoB100 after treatment of low-density lipoprotein with the FeSO4/H2O2 system,988 pertussis toxoid vaccine by H2O2 treatment of Bordetella pertussis cells,989 and myoglobin by O2•−.990 In the latter case, LC-ESI-MS/MS analysis of the protein digest also highlighted the occurrence of the superoxide product at Tyr151. Enhanced high mass-resolving power and mass-measurement accuracy proved very effective in curtailing erroneous identifications. Later on, direct assignment of DOPA to different tyrosine residues within proteins present in A. thaliana thylakoid membrane or sheep wool was obtained by quasi-proteomic approaches applied to biological samples subjected to photooxidation.269,991 The former investigation identified 16 tyrosine residues oxidized to DOPA in plant photosystems I and II, cytochrome b6f and ATP synthase. In addition to three DOPA residues assigned to three sheep intermediate filament proteins (IFPs), the latter study identified additional cross-linked photooxidation products bearing di-Tyr and hydroxylated diTyr on high glycine-tyrosine protein and IFPs. Very recently, the same authors have demonstrated the concomitant presence of trihydroxy-phenylalanine (TOPA)- and TOPA-quinonecontaining adducts (with Δm = +32 and +30 Da with respect to tyrosine, respectively) during analysis of photooxidation products of model peptides (Figure 49).992 Di-Tyr cross-linked species were also observed during LC-MS analysis of the products resulting from peroxynitrite/CO2 and H2O2 reaction with oxyhemoglobin or lactoperoxidase, respectively.890,993 Recently, a MS-based method has been proposed for simultaneous identification of DOPA- and Cl-Tyr-containing peptides in a digest from nine proteins exposed to HClO treatment (Figure 55).361 To this purpose, PIS for fragment ions of DOPA and chlorotyrosine was investigated as a labelfree MS approach to map specific oxidized and halogenated tyrosine residues. By use of a double quadrupole-LIT mass spectrometer, PIS was combined with detection of MS3 fragment ions from the immonium ions and collisionally activated decomposition peptide sequencing to achieve selectivity for the modifications. For DOPA, the immonium ion at m/z 152.1 fragmented to yield diagnostic ions at m/z 135.1 and 107.1 (Figure 55A); the Cl-Tyr immonium ion at m/ z 170.1 gave diagnostic ions at m/z 153.1, 134.1, and 125.1

Figure 55. Precursor ion scanning, detection of MS3 fragment ions from the immonium ions, and collisionally activated decomposition peptide sequencing for selective recognition of oxidized tyrosine residues in protein digests. (A) MS3 spectrum of DOPA (m/z 198.1), showing diagnostic fragment ions of the immonium ion at m/z 135.1 and 107.1. (B) MS3 spectrum of 3-chlorotyrosine (m/z 216.1), showing diagnostic fragment ions of the chlorotyrosine immonium ion at m/z 125.1, 134.1, and 153.1. (C) Precursors of m/z 170.1 from nLC-ESI-MS/MS of HOCl-modified lysozyme tryptic peptides. (D) MS3 spectrum of the precursor of m/z 170.1 at m/z 680.6 selected in the first information-dependent acquisition criteria. (E) MS/MS sequencing of the 3-chlorotyrosine-containing peptide at m/z 680.6. Adapted with permission from ref 361. Copyright 2009 John Wiley and Sons.

(Figure 55B). This method demonstrated its potential for application to complex biological samples. Very recently, the first proteome survey of endogenous DOPA and DOPA-quinone site-specific modifications was obtained in mouse brain and heart tissue samples by using a shotgun approach based on 2D-LC coupled to LTQ Orbitrap MS/MS analysis (Figure 56).933 Results from LC-ESI-LIT-MS/ MS analyses included 50 and 14 DOPA-modified tyrosine sites identified in brain and heart proteins, respectively, whereas only a few nitrotyrosine-containing peptides were detectable, thus BV


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linked with mitochondrially-derived oxidative stress and may serve as sensitive markers for disease pathologies. The generation of tyrosine radicals following oxidative insult of heme-containing proteins has been detected by trapping these species with specific spin-trapping (ST) agents and measuring the addition of the modifying moiety to the intact molecules by direct ESI or MALDI analysis.260,890,994 Depending on the ST compound used, a mass increase of +344 (for 3,5-dibromo-4-nitrosobenzenesulfonic acid), +113 (for 5,5dimethyl-1-pyrroline N-oxide), or +72 Da (for 2-methyl-2nitrosopropane) was observed for the corresponding adducts. Investigation on the nature of the trapped derivatives obtained under different experimental conditions permitted one to elucidate the interaction mechanism of different ROS or RNS with the heme group.260,995 Also in this case, identification of tyrosine residues subjected to ST agent addition was obtained by ESI- or MALDI-MS experiments on protein digests and confirmed by CID or PSD analysis of the modified peptides.890,994 On the other hand, potential mechanisms for superoxide-mediated oxidation of tyrosine were also investigated by LC-ESI-MS/MS analysis of model peptides exposed to a peroxidase/O2•− system or activated neutrophils.996,997 Authors demonstrated that the rate constant for the reaction of tyrosine radicals with O2•− is higher than that for dimerization, but the efficiency of O2•− addition to peptides depends on the position of the tyrosine residue. When tyrosine was N-terminal, the major products were hydroperoxides that had undergone cyclization through conjugate addition of the terminal amine. With non-N-terminal tyrosine, electron transfer from O2•− to the peptide radical prevailed. Peptides also containing methionine revealed a novel and efficient intramolecular oxygen transfer mechanism from an initial tyrosine hydroperoxide to give a dioxygenated derivative with one oxygen on the tyrosine and the other forming methionine sulfoxide. Notwithstanding the quantitative data indicating even slightly higher levels of DOPA, Cl-Tyr, and Br-Tyr than of NO2-Tyr in various biological tissues/body fluids under certain pathophysiological conditions,282,876 nitration has been the most investigated tyrosine modification, as deduced from the number of studies published so far. In contrast, no proteomic studies on Cl-Tyr, Br-Tyr, and di-Tyr-containing proteins from various biological matrices generated as a result of in vitro and in vivo modifications have been reported to date, and no dedicated approaches have been developed for global detection of Cl-Tyr, Br-Tyr, DOPA, and di-Tyr in proteomes. These observations give an idea of the wide unexplored scenarios that are still open to investigation in this field of redox proteomics. On the other hand, the historical development of the studies on NO2-Tyr shows that most of the data on nitrated proteins derived from gel-based approaches, where immunopositive spots in 2-DE gels were further identified by peptide mass fingerprint analysis.41,877 In some cases, confirmation on nitrated proteins and information on modification sites were further obtained by standard MS/MS analysis. Different proteomic studies on tyrosine nitroproteome have shown that only certain proteins are nitrated in specific tissues;275 for example, a correlation was found between βamyloid deposition and nitration of a number of proteins from brain hippocampus of patients affected with AD.25 Moreover, identification of nitrated tyrosine residues permitted development and elaboration of predictive structure-based models on factors influencing protein modification or group-based predictive systems (GPS) to recognize putative modification

Figure 56. Shotgun analysis of DOPA- and DOPA-quinone-modified proteins from mouse heart. MS/MS spectra of unmodified (top), DOPA-modified (middle), and DOPA-quinone-modified (bottom) peptides 133−146 from hemoglobin subunit β are shown for comparison. Y*, DOPA-modified tyrosine; Y#, DOPA-quinonemodified tyrosine. Reprinted with permission from ref 933. Copyright 2010 the American Society for Biochemistry and Molecular Biology.

suggesting the much higher abundance for DOPA modification compared with tyrosine nitration. Moreover, 20 and 12 DOPAquinone-modified peptides were also observed in brain and heart proteins, respectively; nearly one-fourth of these peptides were also observed with DOPA modification at the same sites. For both tissues, these modifications were preferentially found in mitochondrial proteins with metal-binding properties, consistent with metal-catalyzed OH• formation from mitochondrial O2•− and H2O2. These modifications also link to a number of mitochondrially-associated and other signaling pathways. In fact, many of the modification sites were common sites of previously reported tyrosine phosphorylation, suggesting potential disruption of signaling pathways. Collectively, these results suggest that DOPA and DOPA-quinone modifications BW


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sites.998,999 These algorithms demonstrated a promising accuracy of 76.51%, a sensitivity of 50.09%, and a specificity of 80.18% from the leave-one-out validations. As an example application, authors predicted potential NO2-Tyr sites for hundreds of nitrated substrates that had been experimentally detected in small-scale or large-scale studies, even though the actual nitration sites had not yet been determined. Through a statistical functional comparison with NO-dependent reversible S-nitrosylation, they evaluated that tyrosine nitration generally affects proteins involved in transcription, translation, and protein stability, while S-nitrosylation concerns components involved in ion transport, glycolysis, and multicellular organismal development. Frequently, information on modified tyrosine residues was deduced by in vitro studies, where isolated proteins or even biological tissues or body fluids were exposed to chemical reagents to predict potential polypeptide reactivity in vivo and to obtain standard modified peptides for the screening of biological samples. In some instances, in vitro and in vivo studies have produced quite different results.41,877 These distinct patterns of reactivity were associated with: (i) the nature and concentration of the reactive species, which may differ from that acting in specific cell environments; (ii) the target proteins, which may exist in vivo in complexes with other molecular species thus affecting their reactivity; (iii) specific isoforms of modified proteins, which may be eliminated in vivo through dedicated processes; (iv) unpredictable specific differences between in vitro and in vivo conditions. The low levels of protein nitration occurring in vivo prompted the development of non-gel-based approaches based on direct MS/MS analysis of nitropeptides from whole protein digests, which have provided a more comprehensive overview of the extent of nitration in a proteome at a higher throughput than gel-based methods.922,927 The above-mentioned difficulties in obtaining a true identification of nitrated tyrosine residues by using mass analyzers of low mass accuracy have raised the issue of a careful re-evaluation of the nitrated proteins reported so far. To do so, it is essential to develop more effective and selective labeling or enrichment strategies or both, because the abundance of false positives will otherwise make the discovery of truly in vivo nitrated proteins cumbersome.

Oxidative modification of tryptophan to Kyn and NFK/diOH-Trp has been often reported for proteins separated by gel electrophoresis, which were then subjected to peptide mass fingerprinting or MS/MS experiments. It has always been unclear whether Kyn and NFK/di-OH-Trp may represent exclusively post-translational events or sample handling artifacts generated during electrophoresis and in-gel proteolytic digestion. To address this problem, several authors characterized by LC-ESI-MS/MS analysis some proteins that are not normally expected to undergo oxidative modifications, with and without the use of electrophoretic separation.552,1000 Several tryptophan residues were found extensively modified to NFK/ di-OH-Trp and Kyn following SDS-PAGE separation and in-gel digestion. In this case, even hydroxy-N-formyl kynurenine (OH-NFK) (Δm = +48 Da) was observed. In contrast, the same residues were observed as unmodified or poorly modified upon in solution digestion. Authors supposed that high voltages used for SDS-PAGE may cause the formation of small amounts of O3 in the electrophoretic cell, which in turn may initiate a reaction cascade leading to tryptophan oxidation products. These results indicate that tryptophan oxidation may occur as an artifact in proteins separated by SDS-PAGE, and their presence should be carefully interpreted, especially when gel electrophoretic separation methods are used. 4.6.1. Analysis of Protein Adducts Resulting from Tryptophan Oxidation. Tryptophan oxidation has been known for decades, since the inactivation of lysozyme by oxidants modifying a critical tryptophan residue was reported;1001 these early reports relied on the identification of tryptophan modification products only by their characteristic electronic absorbance spectra. Later on, the first complete MS characterization of proteins from bovine lens, e.g. α-crystallin isoforms, presenting oxidized tryptophan residues after exposition to oxidative Fenton insult was described.265 The occurrence of OH-Trp/OIA, NFK/di-OH-Trp, Kyn, and OHKyn (Figure 12) in reaction products from αA- and αBcrystallin was ascertained through direct ESI-MS measurements, by detecting the corresponding adducts having the mass shifts mentioned above. The assignment of the modified residues was obtained by MALDI mass mapping experiments combined with LC-ESI-MS/MS analysis of the oxidized peptides, following protein in solution digestion. These derivatives were proved stable to MS analysis conditions and provided clear MS/MS spectra to assign modification to specific amino acids. On the other hand, myoglobin mutants treated with mchloroperbenzoic acid showed the heme-assisted oxidation of tryptophan to yield 2,6-dihydro-2,6-dioxoindole- and 2,6dihydro-2-imino-6-oxoindole-containing derivatives, as deduced by a combined MALDI-mass mapping, PSD analysis, and [1H]/ [13C] NMR spectroscopy approach. In this case, oxidized peptides from in solution digested myoglobin showed a selective mass difference of +30 Da compared with the unmodified one.261 It was proposed that formation of a cyclic derivative from the 2,6-dihydro-2,6-dioxoindole moiety, followed by its hydrolysis, might be responsible for generation of the 2,6-dihydro-2-imino-6-oxoindole group observed. Later on, the oxidation of a critical tryptophan residue in the chloroplast photosystem II protein CP43 was reported by a dedicated LC-ESI-MS/MS study, providing the first example of a selective functional modification in vivo.1002 In addition to Kyn and OH-Trp/OIA, authors demonstrated the presence of a dihydro-hydroxytryptophan-containing peptide (Δm = +18

4.6. Analysis of Protein Adducts Resulting from ROS/RNS-Induced Modifications at Tryptophan and Histidine

In vitro studies with ROS and RNS, such as the FeCl2/H2O2 system, H2O2, HClO, peroxynitrite, and the peroxidase/H2O2/ nitrite system, added to isolated proteins, were initially performed to simulate and investigate tryptophan and histidine reactivity under pathophysiological conditions. The absence of any specific antibody for OH-Trp/OIA, NFK/di-OH-Trp, Kyn, OH-Kyn, OHis, OH-OHis, and nitrohydroxyytryptophan (NO2-OH-Trp) generally limited these studies to pure MSbased researches. In this case, the occurrence of these tryptophan and histidine derivatives was ascertained by detecting the corresponding adducts having a mass increase of +16, +32, +4, +20, +16, +32, and +61 Da, respectively. Modification assignments simply based on MH+ signal mass increase during peptide mapping experiments were considered as not definitive and MS/MS analysis was always judged necessary to properly assign the modified amino acid(s) within the peptide sequence. BX


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solution or in-gel after SDS-PAGE separation. In some cases, a quantitative evaluation of the oxidation extent under different experimental conditions was provided; this was based on the measurement of the relative mass signal intensities, by assuming an identical ionization tendency for unmodified and modified peptides.467,992,1006 Detection of modified peptides at tryptophan residues after treatment of (partially) purified proteins with oxidizing agents has been facilitated by the recent introduction of a strategy that uses two dedicated softwares to visualize and analyze the LCMS data as 2D images, where the y-axis represents the retention time in the LC separation and the x-axis represents the m/z values from the mass spectrometer.540 The intensity of the images corresponds to the MS signal intensities. To evaluate differences in the LC-MS data, each oxidized sample is compared with a control sample. The 2D images are displayed in a transparency mode on top of each other to facilitate the comparison of the two data sets. This approach was used to determine oxidized hemoglobin and albumin products in blood samples treated in vitro with persulfates, and in nasal lavage fluid samples from hypersensitive subjects challenged in vivo with the same reagents, such as hairdressers, who are frequently exposed to bleaching powder containing persulfates. In both cases, OH-Trp/OIA- and NFK/di-OH-Trp-containing peptides were revealed and relatively quantified by using LC-ESI-MS/ MS in SIM mode. The oxidation degree before and after challenge increased after exposure for peptides with oxidized tryptophan. Recently, a MS-based method has been proposed for simultaneous identification of 2-OH-Trp- and 5-OH-Trpcontaining peptides in an in-gel digest from nine proteins exposed to HClO treatment.361 To this purpose, PIS for fragment ions of these tryptophan derivatives was investigated in a label-free MS approach to map these specific oxidized residues in mixture of peptides also containing Cl-Tyr and DOPA. By use of a double quadrupole linear ion trap mass spectrometer, PIS was combined with detection of MS3 fragment ions from the immonium ions and collisionally activated decomposition peptide sequencing to achieve selectivity for the modifications. For 2-OH-Trp, the immonium ion at m/z 175.1 fragmented to yield diagnostic ions at m/z 158.1 and 130.1, and the 5-OH-Trp immonium ion at m/z 175.1 gave diagnostic ions at m/z 158.1, 148.1, and 146.1 (Figure 57). This method demonstrated its potential for application to complex biological samples. Assignment of in vivo oxidized proteins at tryptophan residues was also obtained during proteomic analysis of mammalian tissues and body fluids. For example, the concomitant presence of OH-Trp/OIA, NFK/di-OH-Trp, Kyn, OH-Kyn, and OH-NFK was demonstrated in different isoforms of Mn-SOD purified from human medulloblastoma cells;1007 on the other hand, different tryptophan residues in mitochondrial complex I subunit NDUFA10 variants from Wistar rat brain were converted into OH-Trp/OIA and NFK/ di-OH-Trp derivatives.538 In both cases, proteins were resolved by gel electrophoresis before MS analysis. Moreover, a 2-DEbased proteomic approach was used to identify biomarkers of carcinogenesis in the serum of patients with hepatocellular carcinoma.535 Four isoforms of ApoA1 with different pI values were identified in diseased and control samples; specific enrichment of the most acidic ApoA1 isoform occurred in hepatitis B virus patients who developed hepatocarcinoma. A comparative analysis by LC-ESI-MS/MS analysis of the four

Da) in protein digests, probably formed during the oxidative cleavage of the indole ring to give Kyn. These derivatives were either observed in the case of samples that were purified by SDS-PAGE and of samples not subjected to electrophoresis. Taken together, these results imply that CP43 redox PTMs are an intrinsic property of the photosystem II and are not induced by handling procedures. The essential role of tryptophan oxidation to Kyn in vivo was confirmed for secreted protein MopE from Methylococcus capsulatus.1003 Also in this case, kynurenine occurrence was proven by MALDI mass mapping and LC-ESI-MS/MS experiments on protein digested in solution. Solving the crystallographic structure demonstrated that Kyn forms the Cu-binding site of the protein, together with other histidine residues. Functional studies on a heterologously expressed form of MopE demonstrated that the absence of Kyn paralleled the lack of binding to Cu ion. In addition to OH-Trp/OIA, NFK/di-OH-Trp, Kyn, and OH-Kyn, another product resulting from tryptophan oxidation was observed in wool proteins exposed to photooxidation, i.e. dihydro-kynurenine derivative (Δm = −14 Da).991 These tryptophan peptide derivatives were detected within the tryptic digest of photoyellowed wool by monitoring its HPLC profile at 400 nm. This quasi-proteomic approach revealed the occurence of these components in both type I (two sites) and type II (three sites) intermediate filament proteins and in the inner root sheath associated protein IRSA1 (one site), which were not subjected to SDS-PAGE. In addition to OHTrp/OIA, NFK/di-OH-Trp, Kyn, and OH-Kyn, the same authors have recently demonstrated the concomitant presence of OH-NFK (Δm = +48 Da), dihydroxyformylkynurenine (Δm = +64 Da), tryptophandione/dihydrodioxoindole (Δm = +30 Da), β-unsaturated 2,4-bis-tryptophandione (Δm = +28 Da), hydroxy-bis-tryptophandione (Δm = +44 Da), and tetrahydroβ-carboline (Δm = +12 Da) containing adducts during analysis of photooxidation products of model peptides (Figure 12).992,1004 These results confirmed previous observations on the role of tryptophan derivatives in protecting and filtering UV radiation. The hydroxyformylkynurenine, dihydroxyformylkynurenine, dihydro-hydroxytryptophan, dihydro-kynurenine, tetrahydro-βcarboline, and various tryptophandione derivatives mentioned above, which may result from very strong oxidative insult, were generally not reported in other studies that, conversely, demonstrated the wide occurrence of OH-Trp/OIA, NFK/diOH-Trp, Kyn, and OH-Kyn as main tryptophan oxidation species. Examples, in this context, are the modification products obtained from surfactant protein B during exposure of pulmonary lipid surfactant extracts to the FeCl2/H2O2 system or HClO,266 myoglobin treated with HClO,537 ApoB100 during CuSO4-mediated oxidation of low-density lipoprotein,268 cytochrome c treated with singlet oxygen,1005 parathyroid hormone treated with FeCl2/H2O2 system,547 albumin treated with FeCl2/H2O2 system,684 the preparation products of the pertussis toxoid vaccine by H2O2 treatment of B. pertussis cells,989 and actin or troponin I subjected to short-term X-ray irradiation.467,1006 In the latter case, the additional occurrence of OH-NFK and hydroxytryptophan oxolactone (Δm = +14) was described, together with an uncharacterized modified tryptophan derivative (Δm = +46 Da). These studies were generally performed by a combination of MALDI-TOF-MS and LC-ESI-MS/MS techniques, which assigned the nature of each modified tryptophan residue and its oxidation product(s). Depending on the study, protein digestion was performed in BY


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confirmed previous observations on the role of tryptophan derivatives in protecting and filtering UV radiation. Later on, a similar approach was used to comparatively evaluate on a quantitative basis the oxidative events occurring in urea-soluble proteins from the nucleus of two young, two aged, and two early stage nuclear cataract lenses following trypsin digestion.544 The presence of OH-Trp at Trp9 and Trp60 in αB, at Trp96, Trp99, and Trp198 in βA3, at Trp16 in βA4, at Trp123, Trp126, Trp192, and Trp215 in βB1, at Trp150 in βB2, and at Trp72 and Trp162 in γS was demonstrated. A number of differences were found in cataract compared with normal lenses, most notably an increase in the number of oxidized tryptophan residues. Semiquantitative analysis revealed a tentative trend toward increased levels of tryptophan oxidation with age; an increase in the extent of modification was also apparent in cataract lenses compared with aged normal lenses. These findings suggest that tryptophan oxidation may be involved in nuclear cataract development. Recently, a proteomic analysis of A. thaliana thylakoid membrane proteins after photooxidative exposure was performed by a combined SDS-PAGE/LC-ESI-MS/MS approach.269 Authors identified 49, 35, 4, 2, and 10 tryptophan residues modified into OH-Trp/OIA, NFK/di-OH-Trp, hydroxytryptophan oxolactone, OH-Kyn, and Kyn derivatives, respectively (Figure 58). Quantitative analysis based on measurement of the relative area of oxidized peptides and that of corresponding unmodified counterparts demonstrated that tryptophan oxidation products occurred mostly in photosystem II and to a lower extent in photosystem I. Exposure of plants to light stress resulted in an increased level of tryptophan oxidation in proteins of the photosystem II reaction center and the oxygen-evolving complex compared with low light conditions. Based on these data, authors proposed that oxidative modifications of proteins by ROS might represent an important regulatory mechanism of protein turnover under light stress conditions, especially for the photosystem II and its antenna proteins. Very recently, a polyclonal antibody for detection of NFK in Western blotting experiments has been developed.1008 The efficacy of this product in selectively recognizing NFKcontaining polypeptides was confirmed through SDS-PAGE analysis of proteins with and without tryptophan residues, which were subjected to photooxidation or radical-mediated oxidation. The sensitivity of this approach was validated through MS-based detection of NFK in photooxidized myoglobin and in carbonate radical-oxidized human SOD1. This antibody has a promising role in 2-DE-based applications for proteomic studies. When myeloperoxidase-mediated oxidative events involve proteins containing the Trp-Gly dipeptide within their sequence, as in the case of matrilysin 7, a novel tryptophan oxidation product was also detected.1009,1010 Authors named it WG-4, as the result of the mass shift (Δm = −4 Da) they measured in the course of peptide mapping experiments with respect to the unmodified counterpart (Figure 59). In fact, initial chlorination of the indole ring of tryptophan generates the corresponding 3-chloroindolenine, which in turn rearranges to yield a previously unknown cyclic indole-amide species, in which tryptophan cross-links to the main chain N atom of the adjacent glycine residue to form an aromatic six-membered ring. This derivative was proven to occur by a combined approach based on LC-ESI-MS/MS, UV/visible spectroscopy, H-D exchange, and NMR spectroscopy. WG-4 was shown to

Figure 57. MS3 spectra of hydroxytryptophan derivatives. (A) MS3 spectrum of 5-hydroxytryptophan (m/z 175.1) showing diagnostic ions at m/z 158.1, 148.1, and 146.1. (B) MS3 spectrum of 2hydroxytryptophan (m/z 175.1), showing diagnostic ions at m/z 158.1 and 130.1. Adapted with permission from ref 361. Copyright 2009 John Wiley and Sons.

ApoA1 isoform digests identified specific oxidation of Trp50 and Trp108 to NFK, and Met112 to MetO in this up-regulated isoform. Similar results were obtained for a murine model of hepatocarcinogenesis, in which the mice are deficient in hepatic AdoMet synthesis (MAT1A−/−). Although it is not clear at present whether the occurrence of these modifications has a causal role or simply reflects secondary epiphenomena, this selectively oxidized ApoA1 isoform may be considered as a pathological hallmark that may help us to understand the molecular pathogenesis of hepatocarcinoma. The distribution of NFK was investigated throughout the human heart mitochondrial proteome by using a combined approach based on MALDI-TOF MS and LC-ESI-MS/MS analysis of trypsin digested bands from SDS-PAGE of isolated complex components and whole mitochondria preparations.267 This study demonstrated that NFK is associated with a distinct subset of proteins, including an over-representation of complex I subunits as well as of complex V subunits and enzymes involved in redox metabolism. No relationship was observed between this tryptophan modification and methionine oxidation, a known artifact of sample handling. Because the mitochondria were isolated from normal human heart tissue and not exposed to any artificially induced oxidative stress, authors suggested that the susceptible tryptophan residues in these proteins are “hot spots” for oxidation in close proximity to a source of ROS in respiring mitochondria. A shotgun approach based on 2D (ion-exchange and reversed phase) chromatographic separation of protein digests followed by MS/MS-driven protein identification was also used to analyze the lens tissue proteome from a congenital cataract patient.558 This analysis demonstrated that oxidative modifications at methionine, tyrosine, and tryptophan residues (Δm = +16) were the most frequent PTMs present in lens proteins. In particular, the occurrence of OH-Trp was ascertained in αB, βA4, βB1, βB2, γB, and γC isoforms, with respect to the total ones (11 in number) identified in this tissue. These results BZ


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Figure 58. Oxidized and nitrated products of the LHCB1 protein from Arabidopsis thaliana thylakoid membranes as identified by LC-ESI-MS/MS after protein SDS-PAGE separation. CID spectra of the tryptic peptides 44−55 (A−C) and 126−133 (D−F) modified at tryptophan and tyrosine residues are shown. Peptide sequences and modification sites were identified by the series of b and y ions and are shown schematically at the top. Major b and y ions are labeled within the spectra. Oxolactone-, kynurenine-, and hydroxykynurenine-containing peptides are reported in panels A, B, E, and D, respectively; nitrotyrosine- and nitrotryptophan-containing peptides are reported in panels C and F. Ions at m/z 632 (A), 626 (B), 493 (D), and 485 (E) correspond to the loss of H2O from the corresponding precursor ions; ion at m/z 647 (D) comprises nitrosotyrosine. Reprinted with permission from ref 269. Copyright 2011 John Wiley and Sons.

Figure 59. Reaction pathway for the production of WG-4 in Trp-Gly-containing proteins.

CA


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Figure 60. Presence of nitrohydroxytryptophan in rat succinyl-CoA:3-ketoacid coenzyme A transferase and identification of Trp372 as the site of modification. The MS/MS spectrum for modified tryptic peptide 366−377 ([M + 2H]2+ ion at m/z 713.3), together with amino acid analysis, proved the nature and site of redox modification. W* indicates nitrohydroxytryptophan. Reprinted with permission from ref 1064. Copyright 2010 Elsevier.

unmodified tryptophan counterpart.300,301 In this case, comparative MALDI-TOF analysis with some standard oxidized or nitrated tryptophan derivatives also demonstrated the occurrence of NO-OH-Trp (Δm = +45) and 3N-OH-Trp (Δm = +29) adducts following photodecomposition of the nitrohydroxytryptophan species. In contrast, ES ionization of NO2-Trp-containing peptides does not present PDC phenomena and, when coupled online with a LC device, allows for a simultaneous assignment of the nitrated tryptophan residue(s) and an evaluation of the modification extent.300 Due to the low amounts of nitrated proteins, nLC has been generally used to increase sensitivity during MS analysis. Accordingly, a positive assignment of nitrated tryptophan residues in modified proteins after their in vitro exposure to nitrating agents was mostly achieved when LC-ESI-MS/MS analysis of the corresponding digests was performed. Examples, in this context, are the modification of Trp155 in frataxin by peroxynitrite,294 Trp15 in human hemoglobin β chain treated with the H2O2/nitrite system,293 Trp32 in human Cu,Zn-SOD treated with the peroxynitriteCO2 or MPO/H2O2/nitrite systems,298 Trp121 in human acidic fibroblast growth factor treated with peroxynitrite,944 and of various tryptophan residues in ribonuclease and lysozyme treated with peroxynitrite and MPO.1012 A LC-ESI-MS/MS-based approach was used for the assignment of the NO2-OH-Trp residue at position 372 in mitochondrial protein succinyl-CoA:3-oxoacid CoA transferase (SCOT) from rat heart; this enzyme is involved in the breakdown of ketone bodies in the extrahepatic tissues.300,301 The specific biochemical properties of this novel amino acid were characterized by a combination of LC electrochemical detection and simultaneous ESI-MS/MS analysis (Figure 60). Nonmodified SCOT decreases progressively with age; conversely, modified SCOT accumulates in vivo with age. Protein modification was associated with an increased enzyme activity.301 Authors hypothesized that increases in tryptophan modification of SCOT and in its catalytic activity constitute a plausible mechanism for the age-related metabolic shift toward enhanced ketone body consumption, as an alternative source of energy supply in the heart. Recently, a polyclonal antibody for detection of nitrotryptophan in Western blotting experiments has been

kink and stiffen the peptide backbone, eventually hindering the interaction of the substrate with the catalytic pocket of matrix metalloprotease 7. Conversely, a completely different pattern of oxidized tryptophan derivatives was observed when proteins were subjected to oxidative modifications by singlet oxygen.1011 Model peptides and proteins were photolyzed in the presence of Rose Bengal and O2 and digested with Pronase, and the resulting derivatives were identified by LC-ESI-MS/MS analysis. Seven major tryptophan photoproducts were characterized in peptides, including trans- and cis-3α-hydroperoxypyrroloindoles (Δm = +32 Da), trans- and cis-3α-hydroxypyrroloindoles (Δm = +16 Da), trans- and cis-3α-dihydroxypyrroloindoles (Δm = +32 Da), and NFK, consistent with singlet oxygen-mediated reactions. The hydroperoxides decomposed rapidly at elevated temperatures and in the presence of reductants to the corresponding alcohols. Most of these compounds were detected on proteins, providing in this case direct evidence for peroxide formation. Conversely, cytochrome c was modified by singlet oxygen at Trp59, generating a peptide adduct bearing a mass shift of +16 Da at this position, probably corresponding to OH-Trp/OIA, as deduced by LC-ESI-MS/ MS analysis of the corresponding tryptic digest.1005 4.6.2. Analysis of Protein Adducts Resulting from Tryptophan Nitration. The occurrence of NO2-Trp upon in vitro exposure of purified proteins to nitrating agents was initially ascertained through direct MS measurements, by detecting the corresponding adducts having a mass shift of +45 Da with respect to the unmodified counterparts.290,291,296 When peptide mapping experiments were performed by UVMALDI-TOF-MS to localize modified tryptophan residues in modified albumin and creatine kinase isoenzymes, however, it was evident that this ionization technique yields PDC of the nitro group to the NO-Trp adduct (Δm = +29 Da),290,292 as already observed during analysis of NO2-Tyr-containing peptides.358,934 The close vicinity of the absorbance maximum of NO2-Trp to the emission wavelength of the N2 ion laser also in this case compromises the detection sensitivity but also produces a unique MS fingerprint facilitating nitropeptide recognition. A similar phenomenon was also observed during the analysis of (NO2-OH-Trp)-containing peptides, which showed the expected mass shift of +61 Da with respect to the CB


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developed.1013 This antibody was immunoreactive with 6NO2Trp-containing Cu,Zn-SOD but not with unmodified proteins and 3NO2-Tyr-containing Mn-SOD. When the nitro group of 6NO2-Trp was reduced by sodium hydrosulfite to form 6NH2Trp no antibody recognition was observed. Later on, authors used this polyclonal antibody for the visualization of NO2-Trpcontaining proteins in PC12 cells treated in vitro with peroxynitrite, which were resolved by 1D and 2D gel-based proteomic analysis.299 LC-ESI-MS/MS analysis of the excised spots or bands, subjected to in-gel trypsinolysis, identified several nitrotryptophan-containing peptides, which were derived from 6 proteins, also assigning 10 modified tryptophan residues. On this basis, this anti-6NO2-Trp antibody seems to have a promising role in 2-DE-based applications for proteomic studies and could also be tested for immunoprecipitation and enrichment experiments of NO2-Trp-containing proteins. Direct assignment of NO2-Trp-containing proteins in A. thaliana thylakoid membrane after plant photooxidative exposition was performed by a combined SDS-PAGE/LCESI-MS/MS proteomic approach.269 Authors identified 12 Trpmodified residues present in 5 proteins (Figure 58). A quantitative evaluation of their in vivo levels was obtained by measuring the ratio of the area of nitropeptides vs that of corresponding unmodified counterparts. Nitration was found to occur in proteins from two photosynthetic complexes. Exposure of plants to light stress resulted in an increased level of tryptophan nitration in proteins of the photosystem II reaction center and the oxygen-evolving complex, compared with low light conditions. In contrast, the level of tyrosine nitration strongly decreased for all light-harvesting proteins of the photosystem II under the same conditions. 4.6.3. Analysis of Protein Adducts Resulting from Tryptophan Halogenation. HBrO and HClO produced by activated neutrophils, monocytes, eosinophils, and macrophages have been demonstrated to halogenate and oxidize proteins, thus yielding also the formation of 6-bromotryptophan (Br-Trp) and 6-chlorotryptophan (Cl-Trp).303,1014,1015 In addition to OH-Pro, some bioactive peptides from sea sponges, annelids, and mollusks also contain 6-Br-Trp, which is probably generated by a still unknown oxidative post-translational modification.1016 As a result of its general stability to the different ionization techniques, the occurrence of this derivative was demonstrated by either MALDI-TOF or LC-ESI MS/MS analysis of the corresponding peptide digests, by using CIDbased approaches.1016 Br contributed to a distinctive isotopic distribution in all fragments that contain Br-Trp. Conversely, ECD fragmentation resulted in the loss of bromine and return to the normal isotopic distribution. Searching for these molecular ion mass characteristics allowed screening for novel bioactive peptides using LC-ESI MS/MS analysis.1017 Br-Trp-containing peptides were also detected by a dedicated PIS procedure, which uses quadrupole TOF mass spectrometers with high-resolution and high-accuracy fragment ion selection (within Δm/z = ±0.04).359 The immonium ions of Br-Trp and OH-Pro were described as two fragment ions characteristic for tryptophan-brominated and proline-hydroxylated peptides, respectively. The “reporter” ion of trytophanbrominated peptides was highly mass deficient due to the presence of bromine, thereby allowing the selective detection of these species and the distinction from other dipeptidic a-, b-, and y-fragment ions by high-resolution, high-accuracy precursor ion scanning. This strategy also enabled the differentiation between precursors giving rise to the oxygen-containing

immonium ion of hydroxyproline and precursors of the immonium ions of near-ubiquitous leucine/isoleucine. Both immonium ions have the same nominal mass of 86 Da, but the exact masses differ by less than 0.04 Da. High-resolution, highaccuracy PIS enabled the identification of proline-hydroxylated and tryptophan-brominated species and the selective analysis of species carrying these modifications in a highly complex mixture. 4.6.4. Analysis of Protein Adducts Resulting from Histidine Oxidation. Histidine modifications as a result of metal-catalyzed or other ROS-dependent oxidative insults on proteins have been revealed by detecting the corresponding 2oxo-histidine (OHis) and 4- or 5-hydroxy-2-oxo-histidine (OHOHis) derivatives as main reaction products.264,1018 Although the occurrence of OHis in protein hydrolysates, as established by conventional amino acid analysis procedures, was a preliminary indication of the oxidative modification affecting histidine residues,264,1018 the presence of oxidized histidine derivatives was ascertained by direct ESI or MALDI analysis of intact molecules or their peptide digests, which detected the corresponding adducts having a mass increase of +16 and +32 Da, respectively.262−264 Definitive identification of modified amino acid(s) within the peptide sequence was achieved by MS/MS experiments. Due to the general stability of the OHis and OH-OHis derivatives to experimental conditions used during either MALDI-TOF or LC-ESI MS/MS-based peptide mapping experiments, MS/MS spectra were generated by using both ionization techniques. Thus, the concomitant occurrence of OHis at His44, His46, and His118 and OH-OHis at His46 was proven to occur in Cu,Zn-SOD after addition of H2O2;263 similarly, His6, His13, and His14 were demonstrated as being converted into OHis when β-amyloid was treated with the CuCl2/ascorbate system;262 analogously, His375, His569, His1113, His1864, and His3281 in ApoB100 were oxidized to OHis when low-density lipoprotein preparations were treated with CuSO4.268 On the other hand, the progressive increase of OHis at His66, His125, and His248 was shown in ascorbate-treated Mn2+-protein phosphatase 1γ after metalexchange with Cu2+,1019 and at various histidine residues in BSA after treatment with the FeCl2/H2O2 system for different times.684 In most cases, the selective detection of OHis and OH-OHis at positions corresponding to histidine residues involved in metal-binding suggested the combined use of in vitro treatment with oxidizing reagents and MS analysis as a tool for the assignment of the metal-binding sites in proteins. This was confirmed by selective localization of other oxidized amino acids corresponding to residues known as being located in close proximity to the metal. Quantitative analysis based on measurement of the relative area of oxidized peptides and of corresponding unmodified counterparts was used to highlight the histidine residues more susceptible to oxidative insult.684,1019 2-Oxo-histidine or 4- or 5-hydroxy-2-oxo-histidine derivatives were also proven to occur in various proteins as a result of their in vivo oxidation. Examples, in this context, are the determination of the presence of OHis at: (i) His15 in native silicatein beta from the marine sponge Petrosia f iciformis;1020 (ii) His2245, His2253, and His3960 in native ApoB100 from low-density lipoprotein preparations;268 (iii) His30 and His31 in Mn-SOD from human medulloblastoma cells.1007 In the latter case, the additional occurrence of an unknown adduct (showing a mass shift of +14 Da), probably associated with an intermediate of histidine oxidative modification, was also CC


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demonstrated to occur at His30 and His31 by MALDI-TOFTOF MS analysis. Indeed, the concomitant presence of OHis and OH-OHis derivatives at His149 and at His149 and His322, respectively, was proven in mitochondrial complex I subunit NDUFA10 variants from Wistar rat brain by a combined MALDI-TOF-MS/LC-ESI-MS/MS approach.538 In most of the cases reported above, proteins were resolved by gel electrophoresis before MS analysis. Oxidative modification to amino acid side chain can change the dissociation pathways of peptide ions, but these variations are most commonly observed when cysteine and methionine residues are oxidized. Bridgewater and colleagues described the very noticeable effect that oxidation of histidine residues can have on the dissociation patterns of peptide ions containing this residue.1021 They showed that a common product ion spectral feature of doubly charged tryptic peptides is the enhanced cleavage at the C-terminal side of histidine residues. This preferential cleavage arises due to the unique acid/base character of the imidazole side chain, which initiates cleavage of a proximal peptide bond for ions in which the number of protons does not exceed the number of basic residues. Authors demonstrated that this enhanced cleavage is eliminated when histidine is oxidized to OHis derivative, because the proton affinity and nucleophilicity of the imidazole side chain are lowered. Furthermore, they found that oxidation of histidine to OHis can cause the misassignment of oxidized residues when more than one oxidized isomer is simultaneously subjected to MS/MS analysis. These spectral misinterpretations can be generally avoided by using multiple stages of MS/MS or via specifically-optimized LC separation conditions. When these approaches are not accessible or do not work, N-terminal derivatization with sulfobenzoic acid can avoid the problem of mistakenly assigning oxidized residues. Later on, the same authors demonstrated that these misassignments can be more readily and easily avoided by using ETD MS to dissociate the oxidized peptides (Figure 61).1022 They observed that the relative insensitivity of ETD to side-chain chemistry allows the extent of histidine oxidative modification to be determined readily for peptide isomers having more than one site of oxidation, including tyrosine, phenylalanine, and tryptophan. These results, along with previous studies of oxidized peptides, suggest that ETD is probably a better technique than CID for obtaining correct sequence and modification information for oxidized peptides. This approach was used to identify the Cu2+coordinating residues in the prion protein (PrP) under different stoichiometric loadings of copper by a combined approach based on metal-catalyzed oxidation with the H2O2/ascorbate system and the MS/MS-driven assignment of the modified residues in the resulting oxidized products.1023 ETD analysis of the different oxidized PrP products demonstrated that coordination of Cu2+ by multiple histidine imidazoles is found at 1:1 and 1:2 PrP/Cu2+ ratios. Notably, there appeared to be four to seven isomers of this multiple histidine coordination mode in the 1:1 complex. Authors clearly showed that His96 is the dominant Cu2+-binding ligand, because in every isomer His96 is bound to Cu2+. The individual octarepeat binding sites begin to fill at ratios of 1:3 PrP/Cu2+, with no clear preference for the order in which they load with Cu2+, although the His77 octarepeat appears to saturate last. A shotgun proteomic approach based on 2D chromatographic separation followed by MS/MS-driven protein identifications was also used to comparatively evaluate, on a quantitative basis, the oxidative events affecting histidine

Figure 61. Efficacy of ETD analysis in oxidation site assignment at histidine residues. (A) CID of singly oxidized myelin proteolipid protein (MPP) peptide 139−151 (M + O + 3H) 3+ after simultaneously subjecting the isomeric oxidized forms to MS analysis without prior LC separation. Spectrum suggests a number of oxidation sites, with difficulty in making definitive assignments. (B) The UV trace of the three oxidized isomers of the singly oxidized MPP peptide after LC separation. The His1, Trp6, and His9 labels correspond to peptides that are oxidized at these residues, as proven by independent MS/MS analysis of each isomer (data not shown). (C) ETD of singly oxidized MPP peptide (M + O + 3H)3+ after simultaneously subjecting the isomeric oxidized forms to MS analysis without prior separation. The asterisks indicate the product ions that are oxidized. Unlike the CID spectrum, the ETD spectrum correctly identifies the three oxidation sites and provides the correct oxidation percentages for each residue (data not shown). Reprinted with permission from ref 1022. Copyright 2009 John Wiley and Sons.

residues present in urea-soluble proteins from the nucleus of two young, two aged, and two early stage nuclear cataract lenses following trypsin digestion.544 Only three different histidine residues were positively identified as oxidized to OHis, with little variation seen among the three studied groups. Oxidized histidine residues were identified in the N-terminal portion of αB-crystallin and the C-terminal portion of βA3-crystallin. A completely different pattern of oxidized histidine derivatives was observed when proteins were subjected to CD


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oxidative modifications by singlet oxygen.1005 In the case of cytochrome c, combined MALDI-TOF-MS and LC-ESI-MS/ MS analysis of the protein tryptic digest demonstrated the presence of different peptide adducts at His26 and His33, which presented a mass shift of −22, +14, +26, +32, and +34 Da relative to the unmodified counterparts. The second and the fourth species were associated with the 2-imidazolone and the hydrated imidazolone derivatives, respectively.1024,1025 The LCMS2 spectrum of the peptide identified as the −22 Da adduct was consistent with a multiple oxidation of the histidine residue to asparagine. Ozonolysis of histidine results initially in a +48 Da adduct identified by MS 2 as 2-amino-4-oxo-4-(3formylureido)butanoic acid followed by decomposition to asparagine, the −22 Da adduct.1026,1027 No information was derived for the adducts showing a mass shift of +26 and +34 Da.

residues located in the sirtuin 1 binding pocket for NAD+ and substrate, whose redox modification causes a loss of protein functionality.1033 Redox proteomic techniques, developed to trap and detect PSOH, PSNO, PSSG, and the total reversibly oxidized thiol pool on a holistic basis,31,510,513 have allowed the global identification of hundreds of redox-regulated proteins in cells and organisms, collectively called “the redoxome” (Figure 62).31,33,36,38−40,396,397,446,455,480,481,483,484,1034,1035 Exhaustive

5. CONCLUDING REMARKS AND FUTURE PERSPECTIVES In the past few years, integrated redox proteomics methodologies have contributed in a significant way to provide a better understanding of the oxidative and nitrosative PTMs occurring in prokaryotes and eukaryotes during their life cycle or eventually associated with the development of pathological states.9,29,31,32,36,37,39,375,559,710,907,965,1028,1029 In particular, progress in this proteomic field has advanced our knowledge of regulatory mechanisms that involve reversible oxidation of proteins at cysteine and methionine residues, leading to a better understanding of oxidative biochemistry under physiological conditions. On the other hand, a number of irreversible redox PTMs, including nitration, some oxidations, halogenation, and carbonylation, as detected by dedicated redox proteomics approaches, have been associated with specific stressing conditions, highly affecting protein activity and, ultimately, cell functionality. Thus, studies describing global changes in nitrated or carbonylated proteins from organisms subjected to environmental (by metals, organic compounds, photooxidation) or physiological (by starvation) challenges have been performed as a result of the recent technological developments in this field.269,489,496,602,622,991,992 A significant number of proteins whose activity is regulated by reversible redox modifications, especially at cysteine residues, have been identified by redox proteomics techniques. In some cases, redox regulation was demonstrated to involve active site residues essential for enzyme functionality. In this context, PTPs are a prototypic example of signal transducers,76,105,172,176,392,1030,1031 whose catalytic cysteine is the major site for oxidation by H2O2 and of S-nitrosylation by RNS, causing protein inactivation.172−174 Quantitative redox proteomic approaches were recently used to identify and quantify redox PTMs in PTPs expressed in human, mouse, and rat cell lines and tissues or in response to stimulation of growth factor receptors.44,1032 By applying these novel quantitative methods, authors found that normal and malignant cells display distinctive patterns of PTP expression and redox modifications. In other cases, reversible redox modification involves cysteine moieties, known as redox-sensing thiols, that are not essential for but affect activity indirectly, by modulating protein structural properties. A recently-investigated prototype of this class of molecules is sirtuin 1, a redox-sensitive class III histone deacetylase, whose activity is decreased when its cysteine residues are oxidized or alkylated.753 Cysteines do not play a role in enzymatic catalysis; however, there are key cysteine

Figure 62. Venn diagram representing the overlap between cysteineredoxosomes and cysteine-redox/electrophile-responsive proteomes in a specific organism. Reaction characteristics (reversible/irreversible) are reported. Each proteomic group (shown in different color) is defined by the number of cysteine residues susceptible to a specific reaction (also shown). This graph describes the possible subproteome overlaps and the resulting differential modulation of metabolic pathways or physiological functions. A similar representation can be used for other amino acids susceptible to different redox reactions.

detection of redox-regulated proteins is a challenging task, since 214 000 cysteines are present in about 20 000 proteins encoded in the human genome. The “redoxome”, also referred to as the “reactive cysteine proteome”, has undoubtedly provided convincing evidence that thiol-based redox switches exist in many proteins to modulate activity and function in response to ROS/RNS challenge. This subproteome is partially different from what is known as the “disulfide proteome”,394 which conversely defines the set of proteins containing disulfides susceptible to reduction by oxidoreductases, such as Trxs and Grxs. In this context, the thiol oxidation status of hundreds of different proteins during physiological and redox stressing conditions was recently quantified in vivo in a single experiment by using dedicated quantitative redox proteomic techniques, which were applied to exponentially growing yeast cells.375,1035 This study revealed that the majority of the identified cysteines are reduced in vivo, confirming that yeast maintain a reducing environment during exponential growth. Authors also found a substantial number (∼15%) of cytosolic and mitochondrial proteins partially oxidized (25−60%) during exponential growth. The latter group included TrxR1, a central player to maintain redox homeostasis. These partially oxidized proteins contain thiols having redox potentials close to the physiological one, which allows fine-tuning of physiological pathways by small alterations in the cellular redox environment. This proteotypic study also provided experimental evidence that the ability of protein thiols to react to changing ROS levels is likely governed by both thermodynamic and kinetic parameters, making prediction of thiol modifications challenging and de novo identification of peroxide sensitive protein thiols indispensable. For an optimal interpretation of these holistic studies, however, redox proteomics data have to be integrated in global proteome networks by dedicated bioinformatic procedures in order to provide punctual pictures of the redox-sensitive pathways existing in the cell,31 highCE


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regulators. 15d-PGJ2 was demonstrated to modify critical cysteines in Keap1, the negative regulator of transcription factor Nrf2. This modification alters Keap1 interaction with Nrf2, leading to stabilization and nuclear accumulation of this transcription factor, determining the activation of promoters containing antioxidant response elements.1047,1048 Redox proteomics has also been increasingly applied to investigate animal and plant diseases; for example, it has been demonstrated that NO• plays a fundamental role in the plant hypersensitive disease resistance response. To understand the mechanisms by which this RNS exerts its action, various gelbased redox proteomics approaches were originally applied to identify protein targets subjected to oxidative or nitrosative modification under this condition. In particular, S-nitrosylated and nitrated proteins were characterized in A. thaliana undergoing hypersensitive disease resistance response.911,1049 Taken together, these studies suggest that defense response in A. thaliana well correlates with redox modification of proteins involved in photosynthesis, glycolysis, nitrate assimilation, and antioxidant response.911,1049 Although further investigations are required to understand the whole functional significance of the modifications ascertained, these studies have contributed to improve the knowledge of plant defense responses and the mechanisms mediating plant−pathogen interaction, disclosing future applications in the field of agricultural biotechnologies. Pioneering redox proteomic studies have also identified specific modified protein targets in various human diseases related to oxidative/nitrosative stress, such as neurodegenerative disorders, diabetes mellitus, and ocular or cardiovascular pathologies, providing mechanistic information on their development and insights into the pathways involved in their pathogenesis as well as into downstream functional consequences.325,588,915,1050 Moreover, a number of studies have also been performed to reveal distinctive redox modification patterns related to tissue aging.246,300,301,594−598,830−832,897−899,1051 For example, the identification of nitrated proteins and the characterization of the functional consequences of ROS/RNS activity on various enzymes from human specimens or animal models of human disease have provided a greater depth of knowledge into the molecular mechanisms associated with neurodegenerative and cardiovascular diseases or with chronic pathologies related to airway inflammation, such as asthma.142,277,282,903,986 In the first context, Butterfield and co-workers used gel-based redox proteomics to identify protein targets of oxidation in the brain of subjects affected with AD.606,607 Indeed, they originally demonstrated increases in protein tyrosine nitration, carbonylation, and S-glutathionylation in AD, mild cognitive impairment, or animal models of AD, PD, HD, and ALS.25,326,549,606,607,610,614,710,903,1052 Thus, redox proteomics led to the identification of a number of brain proteins that regulate glucose metabolism as being oxidatively modified in AD, consistent with results from positron emission tomography studies showing a decrease of glucose use in the brain of these patients.25,326,606,607,610,710,903 Other research groups used proteomics and redox proteomics approaches to identify brain proteins bearing oxidative modifications in subjects affected with idiopathic AD or PD.549,550,836 In particular, ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), a neuronal deubiquitinating enzyme whose mutation has been linked to an early onset familial PD, was found to be down-regulated in idiopathic AD and PD brains; similarly, DJ-1, a peptidase protecting neurons against redox stress and cell death, whose

lighting eventual protein nodes whose modification, at as little as one single amino acid, can control a whole metabolic route. Bioinformatics is also essential to define essential key elements known as being involved in signal transduction mechanisms, such as kinases, phosphatases or their respective targets, whose modification can highly affect important signaling processes essential for cell life cycle. The latter issue is very important for cell biology studies, since it directly unveils and assigns to specific proteins the interplay existing between ROS/RNSdependent (mainly H2O2- or NO•-dependent) PTMs and other modifications known as being involved in signaling processes, e.g. phosphorylation and dephosphorylation. Very recently, pioneering proteomics studies linking together redox PTMs and phosphorylation have been performed in plants1036,1037 and mammals,933,1032,1038,1039 highlighting some degree of interplay between redox- and phospho-signaling,44 as also proven by competitive modification of the same amino acid residues.933 In this context, the increasingly recognized role of protein S-nitrosylation in a wide range of signaling pathways observed in various pathophysiological states found a technological recognition in the number of novel redox proteomics procedures that were developed and applied for the study of the “S-nitrosoproteome”.486,501−527 In spite of the large number of SNO-proteins now identified (∼1000), the observed specificity of S-nitrosylation in terms of target proteins and specific cysteines within modified proteins is incompletely understood. Recent developments in redox proteomics789,1040−1042 have also allowed the identification of a subset of cellular proteins, collectively termed “the redox/electrophile-responsive proteome”,707,708,736,781,1041,1043,1044 which are involved in phenotypic adaptation to indirect oxidation processes. This specific proteome generally includes key cell regulators, such as Keap1, Ras, p53, Trx, and NF-κB, that, though widely varying in cellular localization and functionality, have cysteine residues with distinct nucleophilic character (pKa ≤ 5), which are readily and irreversibly modified by electrophilic species, for example, α,β-unsaturated aldehydes and PGs, often produced by lipid peroxidation under inflammatory conditions. Irreversible adduction at these thiols is generally a type of indirect carbonylation,122 often resulting in (at least partial) protein inactivation. The “redox/electrophile-responsive proteome” partly coincides with the “redoxome” but includes a subset of thiol-containing proteins that are selectively adducted by electrophilic species (Figure 62).708,730,1041,1045 Depending on the electrophile, a wide range of biological activities, cellular processes, and stress signaling pathways are affected.1046 Different unique cysteine residues may be modified on the same protein as a result of the electrophile nature; moreover, various electrophilic species may selectively target only one member of a closely related protein family.736,1045 For example, 15d-PGJ2 binds selectively to H-Ras over the closely related NRas and K-Ras, the reason for this selectivity relying on cysteine content of their hypervariable domains.736,1045 Thus, protein modification by specific RCS may display both inter- and intramolecular selectivity. In this context, redox proteomics allowed to establish a relationship between the antiinflammatory effect of different PGs and the covalent modification of critical cysteine residues in proteins involved in the modulation of inflammation, such as transcription factors NF-κB and AP-1.728,1041 These studies also proved for 15dPGJ2 a high degree of specificity for modification of protein cysteine residues in signaling proteins and transcription factor CF


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RNS and chronic inflammation, with consequent effects on proinflammatory, proapoptotic, and profibrotic pathways, no massive studies on oxidation, nitrosation, and nitration protein targets have been performed so far. Conversely, various redox proteomics studies on animal models of kidney disease, such as the spontaneously hypertensive rat, were carried out to explore oxidative/nitrosative stress in hypertensive kidney;1054 in particular, differentially nitrated,877,914 sulfenylated,475 or carbonylated proteins1055 were identified. A recent study on human plasma nitroproteome reports the first investigation on chronic and end-stage kidney disease patients.968 Plasma proteins from both patient groups showed a higher burden of protein nitration than that of healthy individuals, but main modification targets appeared to be the same in all subjects. Kidney proteins are particularly well-represented in plasma proteome,674 and appearance of proteins in urine is an early indicator of chronic kidney diseases, thus making urine a key sample source for kidney proteomics,1056,1057 overcoming limitations of invasive tissue monitoring. These considerations suggest that urine redox proteomics in the near future will likely be especially informative for detection of oxidation, nitrosation, and nitration protein targets in kidney pathologies. In this case, preliminary studies will be necessary to determine basal levels of oxidized proteins in this human body fluid, similarly to what has already been done for plasma.674,723,724 Examples mentioned above underline the overwhelming and urgent need to identify and easily measure specific biomarkers of oxidative damage in humans, which are related to a certain disease.135,1058−1060 However, it is not clear at present whether the occurrence of specific oxidative modifications on certain protein targets identified by holistic redox proteomic approaches has a causal role in pathogenesis or simply reflects secondary epiphenomena, and whether such redox PTMs have to be considered as pathological hallmarks helping to follow disease onset and/or progression. In this context, only a few biomarkers of oxidative stress/damage have been validated in controlled clinical studies and are commonly used.29,124,132,1029 Conversely, a number of oxidized and nitrated proteins related to inflammation processes have been detected in human tissues/body fluids.539,545,900,901 At present, it is practically not clear whether some oxidative stress-induced protein modifications may have a real potential in clinical trials as specific biomarkers of oxidative stress and, more importantly, may have diagnostic (particularly early and possibly presymptomatic) or therapeutic uses. Probably, a panel of complementary redox biomarkers will give more significant information regarding a disease,275,1061,1062 to be integrated with expression biomarkers, as derived by classical quantitative proteomic studies. Although it is likely that some future results will ultimately show that changes in ROS/RNS concentration and oxidative protein damage in some situations are merely epiphenomena, it is also likely that future research development in redox proteomics will be able to provide the biomedical research community with new tools to assess the significance of oxidative protein modifications and, indirectly, of ROS and RNS, in biological systems. Thus, current research progress in redox proteomics technologies suggests that many more biomarkers are likely to be developed and clearly validated in the near future. Availability of new high-speed and high-resolution MS platforms, combined with 2D nLC systems and dedicated affinity trapping procedures, will make feasible high sample throughput and accurate identification of protein oxidative PTMs. In this context, novel low-abundant redox modifications,

homozygous gene deletion is linked to recessively transmitted PD, was observed to be over-represented. Authors demonstrated that UCH-L1 and DJ-1 are the major targets of oxidative damage in AD and PD brains, where they are extensively modified by carbonylation, methionine sulfoxidation, and sulfonylation compared with age-matched controls.549,550 All together, these results provided strong evidence supporting a direct link between oxidation, damage to the neuronal ubiquitination/deubiquitination machinery, abnormal DJ-1 antioxidant function, and pathogenesis and progression of sporadic AD and PD. In parallel, various redox proteomics investigations have been performed to understand the oxidative modifications associated with cataract formation and progression in affected patients;544,588 in particular oxidative modifications at methionine, tryptophan, and histidine residues were monitored in lens proteins. Semiquantitative analyses showed increased levels of tryptophan oxidation paralleling with disease, suggesting its involvement in cataract development. On the other hand, redox proteomic analysis of plasma and atherosclerotic lesions from subjects affected with coronary artery disease revealed a significant tyrosine chlorination, carbonylation, and methionine sulfoxidation in ApoA1 compared with controls.536,551,724,751,985 ApoA1, the major protein of HDL, is involved in cholesterol mobilization from oxidized LDL and macrophages of the arterial wall during reverse cholesterol transport; it also reduces lipid peroxides by using methionine residues as reductants. These redox modifications were proven to affect protein functionality. High levels of oxidized methionine residues were also found in ApoA1 from type 1 diabetic patients,543 in agreement with increased levels of lipid peroxidation products in plasma during diabetes mellitus. Because coronary artery disease is the major cause of morbidity and mortality in type 1 diabetic patients, ApoA1-HDL sulfoxidation may contribute to the development and/or progression of atherosclerosis and cardiovascular diseases; therefore, monitoring of ApoA1 sulfoxidation may prove useful during therapeutic treatment of these patients to assess its efficacy. Redox proteomics methods have also been applied to investigate other diseases associated with lipid metabolism dysfunctions and related mitochondrial uncoupling; animal models of disease or cultured human cell lines were used to provide insights into disease pathogenesis and/or progression. In particular, animal models of alcohol-exposed fatty liver disease have been studied to identify abnormally oxidized cysteine-containing 434−436,1053 and carbonylated715−718 proteins, whose modification was related to their abnormal function, and eventual cellular dysfunction. Similarly, redox proteomics analysis of adipocytes, islets of Langerhans, and pancreas β-cells have revealed that alterations in the extent of tyrosine nitration and nitroproteome profiles tightly correlated with changes in glucose concentration.916,917 These studies delineated for the first time the potential pathophysiological impact of glucose-triggered redox PTMs on these cells, suggesting that protein nitration may be an important factor in insulin resistance, obesity, and the pathogenesis of diabetes. In this context, massive redox proteomics studies were also performed to identify glycated, carbonylated, and S-nitrosylated proteins in biological fluids of diabetic patients or tissues of related animal models.521,826,840−843 Notwithstanding the widespread occurrence of kidney pathologies, whose onset seems to be linked to a progressive loss of redox homeostasis, possible overproduction of ROS/ CG


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Leader first and then as Head of Unit. In 2010, she was appointed also as Scientific Director of the Proteomics Facility in the same Institute. Her main research interests are focused on the development and application of mass spectrometric methods in the field of biological and biomedical proteomics, with particular emphasis on quantitative proteomics and analysis of post-translational modifications. She has published more than 120 papers in international peer reviewed journals in these fields.

such as cysteine conversion into thiosulfonate, cysteine-SO2SH, dehydroalanine, and serine, have recently been identified in Prx6 and various mitochondrial proteins.1063 Thus, redox proteomics is now offering a technologically feasible route to more detailed characterization of complex PTM patterns in human pathologies, with potential for insight to disease onset (including novel early biomarkers), understanding biochemical mechanisms, fingerprinting of variations within patient populations and assessing new diagnostic strategies. In conclusion, redox proteomics promises a significant contribution in the field of cellular biology and biomedical research, especially in the elucidation of ROS/RNS-dependent irreversible modifications ultimately leading to cell dysfunction and death as well as of reversible redox PTMs affecting metabolic pathways/signaling mechanisms and associated with the development of human pathologies. It will also promote the discovery of novel diagnostic biomarkers enabling diagnosis of various oxidative stress-related diseases. In this perspective, we believe that redox proteomics will not only contribute to broaden our knowledge concerning potential biomarkers for disease diagnosis but will also provide insights into damaged metabolic networks and will identify novel therapeutic targets for modulation of disease progression. Indeed, it will plausibly play a pivotal role also in the design and testing of new drugs against those pathologies related to altered redox homeostasis. Several technological limitations still remain unsolved; however, these approaches and future developments will play an important role toward understanding the interplay between oxidative stress and important biochemical processes in health and disease.

Isabella Dalle-Donne received her Biological Sciences degree in 1989 and her Ph.D. in Cellular and Molecular Biology in 1994 from the University of Milan, Italy, followed by a Postdoctoral Fellowship in Cellular and Molecular Biology in 1995−1997 at the same university. She served as a Research Technician (1998−2003) and as Assistant Professor (2003−2010) at the Department of Biology of the University of Milan, Italy. In 2010, she became an Associate Professor of General and Cellular Biology at the Department of Biology, University of Milan, Italy, and in 2012 she joined the Department of Biosciences at the same university. She has authored over 90 peerreviewed scientific papers and coedited the book “Redox Proteomics: From Protein Modifications to Cellular Dysfunction and Diseases”. During her doctoral and postdoctoral training, she studied the effect of the anti-cancer drug doxorubicin and metal ions on cytoskeletal proteins. Since the second half of the 1990s, her research interests largely focus on protein oxidative modifications as well as on oxidative changes in the glutathione redox system, at the molecular, cellular, and tissue level under various oxidative conditions, including some oxidative stress-associated human conditions such as diabetes mellitus and aging.

AUTHOR INFORMATION Corresponding Author

*Mailing address: Proteomics & Mass Spectrometry Laboratory, ISPAAM National Research Council via Argine 1085, 80147 Naples, Italy. E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies

Angela Bachi received her B.Sc. in Pharmaceutical Chemistry and Technology from the University of Genoa, Italy (1990). She got a second B.Sc. in Pharmacy from the same university (1991) and a Ph.D. in Pharmacology at the Mario Negri Institute for Pharmacological Research, Milan, Italy (1996). She worked as postdoctoral associate at the EMBL, Heidelberg, Germany (1997−1999), where she completed her training on biological mass spectrometry. Since 2000, she has worked at the San Raffaele Scientific Institute in Milan, Italy, as Group

Andrea Scaloni received his B.Sc. in Chemistry (1987) and his Ph.D. in Chemical Sciences (1992) at the University of Rome La Sapienza, Italy. After a research experience at the Rockefeller University, New York (1992−1994), he joined the Italian National Research Council as Researcher (1994). As Research Director (from 2007), he currently CH


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heads the Proteomics & Mass Spectrometry Laboratory, ISPAAM, Italian National Research Council, Naples. His main research interests are focused on the development and application of advanced methods for the qualitative and quantitative characterization of eukaryotic and prokaryotic proteomes. Several investigations on clinical isolates defined a differential expression and/or an altered structure of different proteins to be associated with a specific pathophysiological condition or pharmacological treatment. Differential proteomic studies were also performed on organisms with agro-industrial interest. His activity has also focused on (redox) post-translational modifications in polypeptides from various sources. Finally, he developed massspectrometry-based strategies for functional proteomic studies. He has published more than 220 papers in international peer reviewed journals and books in these fields.

ACKNOWLEDGMENTS Authors thank Aldo Milzani, Ciro Orsini, Giuseppina De Simone, Angela Cattaneo and Vittoria Matafora for technical assistance during manuscript preparation. This work was partly supported by funds from Italian MIUR (Grants PRIN2008_CCPKRP_002 and FIRB2008_RBNE08YFN3_003) and from Italian MEF (Innovazione e sviluppo del Mezzogiorno-Conoscenze Integrate per Sostenibilità ed Innovazione del Made in Italy Agroalimentare-Legge n.191/2009) to A.S. REFERENCES (1) Zhang, Y.; Du, Y.; Le, W.; Wang, K.; Kieffer, N.; Zhang, J. Antioxid. Redox Signaling 2011, 15, 2867. (2) Sies, H. Oxidative stress: Introductory remarks. In Oxidative Stress; Sies, H., Ed.; Academic Press: London, 1985; pp 1−8. (3) Sies, H.; Jones, D. Oxidative stress. In Encyclopedia of Stress, 2nd Ed., Fink, G., Ed.; Academic Press: New York, 2007; Vol. 3, pp 45−48. (4) Valko, M.; Morris, H.; Cronin, M. T. Curr. Med. Chem. 2005, 12, 1161. (5) Valko, M.; Rhodes, C. J.; Moncol, J.; Izakovic, M.; Mazur, M. Chem.-Biol. Interact. 2006, 160, 1. (6) Halliwell, B.; Gutteridge, J. Free Radicals in Biology and Medicine; Oxford University Press: Oxford, U.K., 2007; pp 1−851. (7) Valko, M.; Leibfritz, D.; Moncol, J.; Cronin, M. T.; Mazur, M.; Telser, J. Int. J. Biochem. Cell Biol. 2007, 39, 44. (8) Jomova, K.; Vondrakova, D.; Lawson, M.; Valko, M. Mol. Cell. Biochem. 2010, 345, 91. (9) Dalle-Donne, I.; Scaloni, A.; Giustarini, D.; Cavarra, E.; Tell, G.; Lungarella, G.; Colombo, R.; Rossi, R.; Milzani, A. Mass Spectrom. Rev. 2005, 24, 55. (10) Barber, S. C.; Shaw, P. J. Free Radical Biol. Med. 2010, 48, 629. (11) Jomova, K.; Valko, M. Toxicology 2011, 283, 65. (12) Salmon, A. B.; Richardson, A.; Pérez, V. I. Free Radical Biol. Med. 2010, 48, 642. (13) D’Autreaux, B.; Toledano, M. B. Nat. Rev. Mol. Cell Biol. 2007, 8, 813. (14) Brandes, N.; Schmitt, S.; Jakob, U. Antioxid. Redox Signaling 2009, 11, 997. (15) Paulsen, C. E.; Carroll, K. S. ACS Chem. Biol. 2010, 5, 47. (16) Klomsiri, C.; Karplus, P. A.; Poole, L. B. Antioxid. Redox Signaling 2011, 14, 1065. (17) Antelmann, H.; Helmann, J. D. Antioxid. Redox Signaling 2011, 14, 1049. (18) Wood, M. J.; Storz, G.; Tjandra, N. Nature 2004, 430, 917. (19) Dansen, T. B.; Smits, L. M.; van Triest, M. H.; de Keizer, P. L.; van Leenen, D.; Koerkamp, M. G.; Szypowska, A.; Meppelink, A.; Brenkman, A. B.; Yodoi, J.; Holstege, F. C.; Burgering, B. M. Nat. Chem. Biol. 2009, 5, 664. (20) Lee, J. W.; Helmann, J. D. Nature 2006, 440, 363. (21) Ding, H.; Demple, B. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 8445. CI


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Chemical Reviews

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