Carbonylated Plasma Proteins As Potential Biomarkers of Obesity

Jul 10, 2014 - Department of Medicine, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, ... carbonylation also to obesity and type 2 diabetes m...
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Carbonylated Plasma Proteins As Potential Biomarkers of Obesity Induced Type 2 Diabetes Mellitus Ravi Chand Bollineni,†,‡ Maria Fedorova,†,‡ Matthias Blüher,‡,§ and Ralf Hoffmann*,†,‡ †

Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, ‡Center for Biotechnology and Biomedicine, and Department of Medicine, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany

§

S Supporting Information *

ABSTRACT: Protein carbonylation is a common nonenzymatic oxidative post-translational modification, which is often considered as biomarker of oxidative stress. Recent evidence links protein carbonylation also to obesity and type 2 diabetes mellitus (T2DM), though the protein targets of carbonylation in human plasma have not been identified. In this study, we profiled carbonylated proteins in plasma samples obtained from lean individuals and obese patients with or without T2DM. The plasma samples were digested with trypsin, carbonyl groups were derivatized with O-(biotinylcarbazoylmethyl)hydroxylamine, enriched by avidin affinity chromatography, and analyzed by RPC-MS/MS. Signals of potentially modified peptides were targeted in a second LC-MS/MS analysis to retrieve the peptide sequence and the modified residues. A total of 158 unique carbonylated proteins were identified, of which 52 were detected in plasma samples of all three groups. Interestingly, 36 carbonylated proteins were detected only in obese patients with T2DM, whereas 18 were detected in both nondiabetic groups. The carbonylated proteins originated mostly from liver, plasma, platelet, and endothelium. Functionally, they were mainly involved in cell adhesion, signaling, angiogenesis, and cytoskeletal remodeling. Among the identified carbonylated proteins were several candidates, such as VEGFR-2, MMP-1, argin, MKK4, and compliment C5, already connected before to diabetes, obesity and metabolic diseases. KEYWORDS: aldehyde reactive probe (ARP), LC−MS/MS, obesity, protein carbonylation, type 2 diabetes mellitus (T2DM)



INTRODUCTION Reactive oxygen species (ROS), such as superoxide anion (•O2−), hydrogen peroxide (H2O2), and hydroxyl radical (•OH), are generated by several enzymatic and nonenzymatic reaction pathways in organisms but also by exogenous factors.1,2 Overproduction and insufficient removal of ROS induces oxidative stress, which has been linked to several human disorders, such as heart failure, endothelial dysfunction, cardiovascular disorders, and brain degenerative impairments.3 Though the precise mechanisms remain to be disclosed, oxidative stress contributes to various diseases via oxidation of proteins, lipids, and DNA and thus affects many cellular functions. Recent data indicate that oxidative stress may trigger obesity, insulin resistance, and type 2 diabetes mellitus (T2DM) or at least contribute to disease progression.4−6 Clinical studies identified elevated systemic oxidative stress levels in obesity and even further enhanced ROS production during the development of insulin resistance.7,8 Adipose tissue dysfunction is a key mediator of insulin resistance9 with ROS being proposed as a likely source based on mouse models of obesity induced by high fat diet and in ob/ob mice.10 Studies on adipose tissue and adipocyte cell cultures provide a compelling link between insulin resistance, inflammation, and mitochondrial dysfunction © XXXX American Chemical Society

again connecting elevated ROS levels to disease onset. Moreover, obesity and insulin resistance down-regulate the main enzymes of glutathione metabolism, such as glutathione peroxidases 3 and 4, peroxiredoxine 3, and glutathione Stransferase (GST).9 GST isoform A4, the key antioxidant enzyme, can detoxify reactive aldehydes produced during lipid peroxidation. Its down-regulation triggers mitochondrial dysfunctions and thus might be connected to insulin resistance and T2DM.11,12 Additionally, aldehyde dehydrogenase (important for detoxification of reactive aldehydes) is downregulated in obesity and insulin resistance.13 Reactive aldehydes generated by lipid peroxidation, such as 4-hydroxynonenal (HNE) and 4-oxononenal (ONE), are present in epididymal white adipose tissue of ob/ob mice and mice living on high fat diets at 5- to 10-fold higher levels than in control animals. In subcutaneous white adipose tissue, however, aldehyde levels were elevated only slightly, indicating that different types of white adipose tissues are not equally affected by oxidative Special Issue: Proteomics of Human Diseases: Pathogenesis, Diagnosis, Prognosis, and Treatment Received: March 30, 2014

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stress.10 Elevated levels of 4-HNE were also detected in blood and muscle tissue of obese people.14−16 Nucleophilic amino acid residues in proteins can react with α,β-unsaturated aldehydes (e.g., HNE and ONE) via Michael addition, which is usually referred to as protein carbonylation or protein carbonyl formation. Alternatively, these amino groups can also react with the aldehyde functional group of the α,βunsaturated aldehydes via Schiff base formation, which does not retain the reactive carbonyl group in the side chain. Numerous reports indicate increased levels of protein carbonylation in adipose tissue during obesity, insulin resistance, and T2DM.9,17−19 Most studies have specifically focused on HNE and ONE protein adducts. Experimental data prove that HNE and lipid peroxidation products in general impair glucose stimulated insulin secretion in skeletal muscles and pancreatic β-cells.20−22 Several important molecular targets of HNE were identified, and carbonylation was connected to the loss of protein functions, for example for adipocyte fatty acid binding protein, mitochondrial F-ATP synthase, insulin receptor substrates-1 and 2, and complex I of the respiratory chain.23−25 It should be noted that “protein carbonylation” resembles not only Michael adducts formed by reactive α,β-unsaturated aldehydes, but also “reactive carbonyls” produced by direct protein oxidation (oxidation on Trp, Lys, Arg, Pro, and Thr), reaction with low- or high-molecular weight dicarbonyls (modifications of Lys, Arg, and Cys) generated during lipid peroxidation and glycoxidation, and oxidative degradation of Amadori products.3 High glucose levels characteristic for hyperglycemia in T2DM elevate the carbonylation levels. First, glucose can be oxidatively degraded to reactive dicarbonyls capable of modifying proteins at Lys-, Cys-, and Arg-residues. Second, glucose can react with Lys-residues to Amadori compounds that can be degraded by oxidation. Despite the large variety of protein carbonyls and their suggested relevance for different diseases, they were rarely studied in the context of obesity and T2DM. Protein carbonyl research requires high-throughput analysis by mass spectrometry to reveal simultaneously the modified positions in proteins, the carbonylation types, and the modification degree. The thorough analysis of protein carbonylation in blood plasma collected from lean individuals and obese patients with and without T2DM should provide new insights in the underlying pathological mechanisms (disease onset and progression) and thus provide a disease-related set of potentially diagnostic, predictive, and prognostic biomarkers. Given the importance of protein carbonylation in obesity-induced insulin resistance and T2DM, the objective of this study was the identification of carbonylated proteins in plasma of lean, obese, and obese T2DM individuals with the use of mass spectrometry.



hydroxylamine (aldehyde reactive probe, ARP), from Cayman chemical company (Michigan, U.S.A.). Pierce monomeric avidin agarose was purchased form Thermo Scientific (Perbio Science Deutschland GmbH, Bonn, Germany). Plasma Samples

Blood plasma was obtained from 5 lean individuals, 5 obese patients without T2DM and 5 obese patients with T2DM matched for age (44−46 years) and gender. Obese patients had a body mass index (BMI) ≥40 kg/m2 and the glycated hemoglobin (HbA1c) in T2DM patients was above 7.5%. In lean and obese individuals without diabetes, HbA1c was 9 Gpt/L, Creactive protein (CrP) > 5.0 mg/dL or clinical signs of infection, (2) undetectable antibodies against glutamic acid decarboxylase (GAD), (3) no clinical evidence of either cardiovascular or peripheral artery disease, (4) no thyroid dysfunction, (5) no alcohol or drug abuse, (6) no pregnancy. Tryptic digest

Each plasma sample was digested in triplicate and processed similarly in all consecutive steps. An aliquot of plasma (protein content of 0.1 mg) was diluted with ammonium bicarbonate (25 mmol/L) to a final protein concentration of 1 g/L. Sodium deoxycholate was added (1% w/v). Disulfide bridges were reduced with TCEP (5 mmol/L, 60 °C, 30 min), and the thiols were alkylated with iodoacetamide (10 mmol/L, 37 °C, 30 min, dark). Excess of iodoacetamide was quenched with dithiothreitol (10 mmol/L, 37 °C, 30 min). Proteins were digested by trypsin (50:1 enzyme to protein ratio, 25 mmol/L ammonium bicarbonate; 37 °C, 16 h). The digest was terminated by adding formic acid (0.5% v/v), and the precipitated sodium deoxycholate was removed by centrifugation. ARP Derivatization26

The tryptic digest (150 μL) was acidified with formic acid (1% v/v) and incubated with ARP (100 μL, 25 mmol/L in water) at room temperature (RT) for 2 h. Excess of ARP was removed by solid-phase extraction using Waters Oasis HLB 1 cc (10 mg) cartridges (Waters GmbH, Eschborn, Germany). The stationary phase was rinsed with methanol and equilibrated with water (1 mL each). The samples were loaded, the stationary phase was washed three times (1 mL, 0.1% formic acid in 5% aqueous acetonitrile), and the peptides were eluted (0.5 mL, 0.5% formic acid in 70% aqueous acetonitrile). The eluates were vacuum concentrated and stored at −80 °C.

MATERIALS AND METHODS

Chemicals

Sodium deoxycholate, tris-(2-carboxyethyl)phosphine (TCEP), hydrochloric acid, all sodium, potassium, and ammonium salts were purchased from Sigma-Aldrich GmbH (Steinheim, Germany). Acetonitrile (ULC-MS grade), formic acid, and methanol were obtained from Biosolve GmbH (Valkenswaard, Netherlands). Dithiothreitol (DTT) was purchased from Carl Roth GmbH & Co. KG (Karlsruhe, Germany), and iodoacetamide, from AppliChem GmbH (Darmstadt, Germany). Trypsin was obtained from Promega GmbH (Mannheim, Germany), and O-(biotinylcarbazoylmethyl)-

Affinity Enrichment

Samples were reconstituted in PBS (0.1 mL, 20 mmol/L NaH2PO4, 0.3 mol/L NaCl) and enriched by avidin affinity chromatography using a published protocol with slight modifications.27 Pierce monomeric avidin agarose (100 μL) was packed in mini-spin columns with Luer-lok adapter (Thermo Scientific, Bonn, Germany), and washed (1.5 mL, 10 mmol/L NaH2PO4, pH 7.4); irreversible binding sites were B

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blocked with D-biotin (300 μL, 2 mmol/L), and washed again (500 μL, 0.1 mmol/L glycine-HCl, pH 2.8). The stationary phase was equilibrated (2 mL PBS) and the sample loaded (0.1 mL). After an incubation time of 15 min, the stationary phase was washed with PBS (1 mL), phosphate buffer (10 mM NaH2PO4, 1 mL), a mixture of ammonium bicarbonate (50 mmol/L) and methanol (20% v/v; 2 mL), and water (1 mL) before the peptides were eluted (500 μL, 0.4% formic acid in 30% aqueous acetonitrile). Peptides were vacuum concentrated and stored at −80 °C. Shortly before MS analysis the samples were dissolved in 50 μL of 0.1% formic acid in 3% aqueous acetonitrile.

SuperHirn were exported to Prequips, and a retention time segmented inclusion list of m/z values was created for features found in at least two technical replicates of a given plasma sample. On average, 2000 features were detected in at least two technical replicates. On the basis of the retention times features were divided into 3-min long segments, with an overlap of 1 min between segments. The generated inclusion lists were directly exported (.csv file format) into the Xcalibur software (version 2.0.7) and further analyzed via targeted LC−MS/MS by activating the global mass list function. The LC and MS settings were similar as mentioned above, except that the dynamic exclusion was set to 120 s with a repeat count set of 30 s.

Mass Spectrometry

Database Search

A nano-Acquity UPLC (Waters GmbH, Eschborn, Germany) was coupled online to an LTQ Orbitrap XL ETD mass spectrometer equipped with a nano-ESI source (Thermo Fischer Scientific, Bremen, Germany). Eluent A was aqueous formic acid (0.1% v/v), and eluent B was formic acid (0.1% v/ v) in acetonitrile. Affinity enriched peptides (1.5 μL) were loaded onto the trap column (nanoAcquity symmetry C18, internal diameter 180 μm, length 20 mm, particle diameter 5 μm) at a flow rate of 10 μL/min. Peptides were separated on BEH 130 column (C18-phase, internal diameter 75 μm, length 100 mm, particle diameter 1.7 μm) with a flow rate of 0.4 μL/ min using several linear gradients from 3% to 9% (2.1 min), 9.9% (1.9 min), 17.1% (10 min), 18% (0.5 min); 20.7% (0.2 min), 22.5% (3.1 min), 25.6% (3 min), 30.6% (5 min), 37.8% (2.8 min), and finally to 81% eluent B (2 min). Together with an equilibration time of 12 min the samples were injected every 46 min. The transfer capillary temperature was set to 200 °C and the tube lens voltage to 120 V. An ion spray voltage of 1.5 kV was applied to a PicoTip online nano-ESI emitter (New Objective, Berlin, Germany). The precursor ion survey scans were acquired at an orbitrap (resolution of 60,000) for an m/zrange from 400 to 2000. The CID-tandem mass spectra (isolation width 2, activation Q 0.25, normalized collision energy 35%, activation time 30 ms) were recorded in the linear ion trap by data-dependent acquisition (DDA) for the top six most abundant ions in each survey scan with dynamic exclusion for 60 s using Xcalibur software (version 2.0.7).

The acquired tandem mass spectra were searched against the human plasma database (FASTA file created from the human plasma peptide atlas32) using Sequest search engine (Proteome Discoverer 1.1, Thermo Fischer). The setting allowed up to two missed cleavage sites and a mass tolerance of 10 ppm for precursor and 0.8 Da for product ion scans. As Proteome Discoverer allows only a maximum of six protein modifications per search, each sample was analyzed four times using carbamidomethylation on cysteine, oxidation of methionine, and four different variable modifications. The first search included carbonylated and ARP-derivatized lysine (mass shift of 312.08 m/z units), arginine (270.06 m/z units), threonine (311.10 m/z units), and proline (329.11 m/z units) as variable modifications. The second set consisted of ARP-derivatized malondialdehyde adducts (367.13 m/z units) at Lys- and Argresidues as well as acrolein (369.14 m/z units), pentenal (397.17 m/z units), and crotonaldehyde (383.16 m/z units) adducts on Cys-, His-, and Lys-residues as variable modifications. The third set contained ARP-derivatized alkenal adducts: HNE (469.23 m/z units), HHE (427.18 m/z units), ONE (467.22 m/z units), and OHE (425.17 m/z units) adducts at Cys-, His-, and Lys-residues. The fourth set focused on methyl glyoxal (385.14 m/z units) and glyoxal (371.12 m/z units) adducts at Arg-, Cys-, and Lys-residues as well as deoxyglucasone (457.16 m/z units) and glyoxal (353.11 m/z units) adducts on Arg- and Lys-residues. In this study only peptides with high and medium confidence (Xcorr value ≥1.5) ranked in position 1 in the database search were considered, if the corresponding protein was identified in at least two of the three technical replicates within each sample and at least three of the five biological samples within each group.

Trans-Proteomic Pipeline (TPP)

The raw files generated by LC−MS/MS were uploaded to the TPP software28,29 and converted to mzXML format.30 The mzXML files were searched against the human plasma database (FASTA file created from the human plasma peptide atlas31−33) using the X!Tandem plug-in for unmodified, carbamidomethylated, and methionine oxidized peptides. Identified peptides were further validated using Peptide Prophet34 and a single pepXML file containing high confidence identifications was generated for three technical replicates of each sample.



RESULTS

Design of Experimental Workflow

This study aimed at establishing a robust and reproducible MSbased bottom-up proteomics strategy for in-depth characterization of carbonylated proteins and their modification sites in blood plasma of nondiabetic obese patients (Ob) and obese patients with T2DM (Ob/T2DM). To improve both sensitivity and specificity, the reactive carbonyls were first derivatized with O-(biotinylcarbazoylmethyl) hydroxylamine, also known as aldehyde-reactive probe (ARP), and then enriched by avidin affinity chromatography. ARP labels both aldehydes and ketones very efficiently and fast at acidic conditions.26 Though the affinity enrichment strategy greatly reduced the sample complexity, the reliable identification of carbonylated peptides present at low quantities by data-dependent acquisition (DDA) still remained challenging due to the weak

Feature Detection, Alignment and Inclusion List

The three mzXML and one pepXML identification files resulting from the three LC−MS analyses of a given plasma sample were uploaded into SuperHirn. Feature detection alignment was performed using a retention time tolerance of 1 min, m/z tolerance of 10 ppm in MS and 30 ppm in MS/MS, and charge states from 2 to 5.35,36 All other parameters were used as defaults. Signals identified as unmodified peptide sequences were removed, and the remaining features were used to build an inclusion list with the inclusion list builder software implemented as plug-in in Prequips.37 All features detected by C

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Figure 1. (A) Experimental work flow used for in-depth profiling of carbonylated proteins in control, Ob, and Ob/T2DM plasma samples. Features of the LC−MS/MS (six most intense signals of DDA) present in at least two technical replicates were detected and aligned by SuperHirn, excluding all unmodified peptides for a further sequence analysis by targeted LC−MS/MS. (B) Strategy of a database search using Sequest. The data set resulting from the LC−MS/MS was searched against a database of plasma proteins created from the human plasma peptide atlas build 2012. A smaller database of 470 unique carbonylated proteins identified in at least three plasma samples was created, and the raw files were searched once more against this database. Proteins were considered as carbonylated, if identified in at least three biological and two technical replicates within each group.

signal intensities. Thus, we employed a targeted MS approach that utilized a timed inclusion list considering the retention times and precise m/z-values of all signals excluding signals already identified as unmodified peptides. Peptides containing only oxidized Met- or alkylated Cys-residues as modifications were also not further considered (Figure 1). All reaction conditions and analytical parameters were optimized for the identification of as many carbonylated sequences with high scores as possible and a desirably low background of noncarbonylated peptides. The final workflow integrated previously reported strategies and bioinformatic tools,29,35−37 which provided at least for this instrumental setup the best results: 1. Five plasma samples obtained from lean individuals, Ob and Ob/T2DM patients were split in three equal aliquots and analyzed in parallel, which provided three technical replicates for each of the 15 samples to judge the reproducibility. 2. Tryptic digests were enriched by streptavidin affinity chromatography and analyzed by LC−MS. Tandem mass spectra were acquired for the six most intense signals

with doubly or higher charge states of each MS survey scan in DDA-mode. 3. RAW files were converted to mzXML format and searched with the X!tandem plug-in for unmodified or only carbamidomethylated and methionine oxidized peptides using TransProteomic Pipeline (TPP). The confidence of the peptide identifications was validated by Peptide Prophet. Results were saved as pepXML file. 4. mzXML files were uploaded to SuperHirn software tool, which automatically extracts all peptide signals from the MS-scans and aligns detected MS signals across multiple LC−MS runs based on their m/z values, charge states and retention times (feature alignment). Identified peptides (step 3; pepXML files) were uploaded to SuperHirn and mapped to the mass spectrum. 5. All features annotated to unmodified or only carbamidomethylated and methionine oxidized peptides were deleted. The remaining features representing potentially modified peptides were used to create a timed inclusion list of precise m/z-values with Prequips using 3-min long segments based on the peptide retention times with an overlap of 1 min for consecutive segments. D

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Figure 2. ESI-LTQ-CID tandem mass spectra of ARP-derivatized carbonylated peptides with different modification types: (A) Arg-143 of proteinglutamine γ-glutamyltransferase was carbonylated to glutamic semialdehyde residue and (B) histidine residue of prolow-density lipoprotein receptorrelated protein 1 was carbonylated via Michael addition of 4-hydroxy nonenal. (C) Lys-206 of ATP-sensitive inward rectifier potassium channel 1 and (D) Cys-289 of human serum albumin were carbonylated by malondialdehyde and glyoxal, respectively.

the peptide eluting at 33.1 min (m/z 613.33, z = 3) was deduced mostly from almost complete y2- to y7- and b1- to b8ion series as residues 143 to 155 of protein-glutamine γglutamyltransferase K (Figure 2A). The mass shift of 270.09 m/ z-units relative to the unmodified sequence and the signals of the b-series identified residue 1 as glutamic semialdehyde derivatized with ARP, which resulted probably from direct oxidation of Arg-143 in the protein. The compounds with m/z 691.04 (z = 3) eluting at 31.4 min was identified as a tryptic peptide of the prolow-density lipoprotein receptor-related protein 1 (LRP1), also known as α-2-macroglobulin receptor (A2MR), apolipoprotein E receptor (APOER), or cluster of differentiation 91 (CD91). The signals corresponding to the displayed y-ions and the b14-ion (Figure 2B) clearly indicate that His-3941 was modified via Michael addition of the α,βunsaturated aldehyde 4-hydroxy nonenal. Similarly, Lys-206 of ATP-sensitive inward rectifier potassium channel 1 was confirmed as a malondialdehyde adduct (Figure 2C) and Cys-289 of serum albumin modified by glyoxal (Figure 2D). All identified carbonylated proteins including the modification sites and types are provided as Supporting Information (SI) (file 1).

6. The affinity enriched tryptic digests were reanalyzed by targeted LC−MS/MS using the generated inclusion list. 7. RAW files of the targeted LC−MS/MS were searched with Sequest against the human plasma database. A FASTA file containing potentially carbonylated proteins was generated by considering all proteins identified in at least three different biological replicates as being carbonylated at any residue. 8. The RAW files were searched with Sequest against this reduced FASTA database, which consisted of 420 proteins. 9. Finally, only proteins identified in at least two technical replicates of at least three plasma samples were annotated as carbonylated. Carbonylated Plasma Proteome

The CID tandem mass spectra of carbonylated peptides allowed the reliable sequence analysis by b- and y-ions series, the location of the carbonylated residue, and the type of modification indicating the reaction pathway that generated this modification site. The spectra interpretation shall be illustrated for four common carbonyl types (Figure 3). The sequence of E

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Figure 3. (A) Venn diagram showing the total number of carbonylated proteins and unique carbonylated proteins identified in control, Ob and Ob/ T2DM plasma samples. (B) Types of carbonyl modifications (direct oxidation products, low-molecular weight carbonyls and α,β-unsaturated aldehydes) used as variable modifications in the database searching to identify potential carbonylated proteins.

Overall the optimized bottom-up proteomics strategy identified 94, 88, and 114 carbonylated proteins in plasma samples obtained from lean, Ob, and Ob/T2DM persons corresponding to a total of 158 unique proteins (Figure 3A). Among them were 52 carbonylated proteins with 91 carbonylation sites present in all three groups, whereas 8−16 proteins were identified only in two of the three groups (Figure 3A). The 52 carbonylated proteins detected in all samples reflect probably the basal level of the carbonylated plasma proteome within the sensitivity range of the applied analytical platform, although the three plasma groups may differ in quantitative terms. Interestingly, 36 proteins were identified only in Ob/ T2DM plasma samples and 18 specifically in lean and Ob plasma samples (Figure 3A). Considering the reactions that produced these carbonylation sites, around 21% resulted from direct oxidation of the side chains by ROS, about 26% represented Michael adducts of α,βunsaturated aldehydes, and approximately 53% were reaction products of low-molecular weight (di)carbonyls generated by

lipid peroxidation and glycoxidation (Figure 3B). Most modifications were detected at Lys (53%) and Arg residues (21%), whereas Thr, Pro, Cys, and His residues contributed only 6−7%. The high number of modified Lys and Arg residues probably reflects their high content in human protein sequences and the fact that they are typically located on the surfaces of folded proteins. The 18 proteins identified to be carbonylated only in nondiabetic samples are present in plasma at different concentrations and possess different functions: proteins participating in extracellular matrix remodeling (e.g., A disintegrin and metalloproteinase with thrombospondin motifs 1) or cell adhesion/migration (e.g., tensin and thrombospondin type 1 domain-containing protein 7A) and signaling proteins, such as nuclear factor NF-κ-B p100 subunit and serine/ threonine protein kinase DCLK1. As the analysis relied on a targeted strategy, it is very likely that the carbonylated peptides were indeed below the detection limits in the digested plasma samples obtained from obese patients with T2DM. It is not F

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Figure 4. (A) Tissue specific expression of carbonylated proteins identified in control, Ob and Ob/T2DM plasma samples, annotated using the DAVID functional annotation tool and (B) functional classification of unique carbonylated proteins identified in Ob and Ob/T2DM plasma samples.

transduction, and RNA/DNA processing (Figure 4B), whereas only a few affect cytoskeletal remodeling, redox regulation, and energy metabolism (Table 1). The carbonylated proteins specific for Ob/T2DM contained a similar degree of proteins participating in RNA/DNA processing, cell junction/adhesion, and signaling, but a higher percentage of proteins responsible for cytoskeletal remodeling and energy metabolism (Figure 4B). Three Ob/T2DM specifically carbonylated cytoskeleton proteins bind to actin: protein-enabled homologue ENAH (involved in changing the cell polarity), Ras GTPase-activatinglike protein IQGAP1 (cross-linking of actin and microtubules), and filamin-B (responsible for cross-linking actin to membrane glycoproteins).38 Interestingly, the main motor proteins of intracellular vesicle transport by actin filaments and microtubules-based motility, myosin-9 and dynactin subunit 1, were also identified. A link between modification-affected cytoskeletal remodeling and cell adhesion was provided by spectrin α, integrin α-6 and filamin. Oxidative modifications of these proteins can disrupt the actin cortical rim in endothelial cells, leading to increased endothelial permeability, which accompanies chronic inflammation and macrophage infiltration.38 Remarkably, proteins participating in angiogenesis (four proteins), lipid metabolism (two), oxidoreductases (four), and DNA repair (six) were detected only in diabetic patients but not in obese or lean persons. These data support the hypothesis that oxidative stress accompanies the progression of T2DM. KEGG canonical pathway analysis by DAVID pointed to three carbonylated proteins specific for Ob/T2DM to be

clear, however, if these carbonylation sites were absent in the proteins or if the sequences were more heavily modified (other nonenzymatic modifications) and therefore missed in the analysis. In either case it most likely indicates T2DM-related mechanisms and thus might be of diagnostic or prognostic value, which should be studied on larger cohorts. Tissue Origin of the Carbonylated Proteins

All carbonylated proteins were analyzed for their expression in different tissues using the DAVID (Database for Annotation, Visualization and Integrated Discovery) functional annotation tool (Figure 4A). Carbonylated proteins identified in the plasma of lean individuals, Ob and Ob/T2DM patients were mainly annotated to four different tissue classes which include liver, plasma, platelet, and endothelium. The 94 carbonylated proteins identified in plasma samples of lean individuals were annotated to plasma (9%), platelet (18%), liver (33%), and endothelium (30%). The 88 proteins of the Ob group showed a very similar distribution: 13% from plasma, 14% from platelets, 30% from liver, and 34% from endothelium. The same was true for the 114 proteins of the Ob/T2DM group: 7% from plasma, 16% from platelet, 30% from liver, and 33% from endothelium. Functional Analysis of Carbonylated Proteins Specific for Ob and Ob/T2DM

As shown above, 36 and 18 proteins were specific for Ob/ T2DM and Ob plasma, respectively, and 10 additional proteins present in both obesity groups but absent in plasma from lean persons (Figure 3A). Functional GO analysis related most of the 18 proteins present in Ob plasma to cell adhesion, signal G

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Table 1. Functional Classification of Carbonylated Proteins Identified in Plasma Samples of Ob and Ob/T2DM Patients biological process

Uniprot

cell junction and adhesion

O00468 Q9P1Y5 O75976 P15924 Q96RW7 Q15149 P13611 P08253 Q5T4S7 P23229 P00451 O43184 Q06033 Q8N2S1

cytoskeleton remodeling

Q14203 O75369 P35579 Q8N8S7 P46940 Q13813

angiogenesis

P52179 P12882 O60241 P08253 P35579 P35968

signaling

Q99683 O00750 Q07954 P46940 Q8N3C0 Q9Y6R4 Q9Y4I1 P01031 P10721

energy metabolism

lipid metabolism

Q13423 P23368 P30038 Q9UHQ9 Q07954

oxidoreductase

Q13423 P23368 Q9UHQ9

protein

Ob/ T2DM

Ob

function

agrin calmodulin-regulated spectrinassociated protein 3 carboxypeptidase D desmoplakin hemicentin-1 plectin versican core protein 72 kDa type IV collagenase (MMP2) E3 ubiquitin-protein ligase UBR4 integrin alpha-6 coagulation factor VIII disintegrin and metalloproteinase domain-containing protein 12 interalpha-trypsin inhibitor heavy chain H3 latent-transforming growth factor beta-binding protein 4 dynactin subunit 1 filamin-B myosin-9 protein-enabled homologue ras GTPase-activating-like protein IQGAP1 spectrin alpha chain, nonerythrocytic 1 myomesin-1 myosin-1 brain-specific angiogenesis inhibitor 2 72 kDa type IV collagenase (MMP2) myosin-9 vascular endothelial growth factor receptor 2 mitogen-activated protein kinase kinase kinase 5 (ASK1) phosphatidylinositol 4-phosphate 3-kinase C2 prolow-density lipoprotein receptor-related protein 1 ras GTPase-activating-like protein IQGAP1 activating signal cointergrator 1 complex subunit 3 mitogen-activated protein kinase kinase kinase 4 unconventional myosin Va complement C5 mast/stem cell growth factor receptor Kit

+ +

major role in development of neuromuscular junction regulation of microtubule dynamics

+ + + + + +

biosynthesis of peptide hormones (insulin) and neuropeptides anchors intermediate filaments to desmosomes organization of hemidesmosomes associates with intermediate filaments, microtubules and microfilaments regulation of cell adhesion, migration and differentiation tissue remodeling, angiogenesis and remodeling of vasculature

NAD(P) transhydrogenase NAD-dependent malic enzyme delta-1-pyrroline-5-carboxylate dehydrogenase NADH-cytochrome b5 reductase 1

+ + +

prolow-density lipoprotein receptor-related protein 1 NAD(P) transhydrogenase NAD-dependent malic enzyme NADH-cytochrome b5 reductase 1

+

+ +

+ + + +

protein ubiquitination and degradation receptor of laminin in epithelial cells and platelets cofactor for factor IXa and participates in blood coagulation muscle development and cell−cell, cell-matrix interactions

+

protease inhibitor and a carrier of hyaluronan in serum

+

role in activity of TGFB1

+ + + + +

movement of organelles and vesicles along microtubules intracellular signaling by linking actin filaments to membrane glycoproteins cytokinesis and cytoskeleton reorganization F-actin outgrowth formation and migration actin cytoskeleton reorganization, cellular adhesion and regulation of cell cycle calcium dependent calmodulin interaction and movement of cytoskeleton

+ + + +

binds to titin, myosin and role in structural orientation of sarcomeres muscle contraction and motility. angiogenesis inhibition

+

tissue remodeling, angiogenesis and remodeling of vasculature

+ +

cytokinesis and cytoskeleton reorganization cell surface receptor for VEGF family and role in regulation of angiogenesis, vascular development apoptosis signal transduction pathway, immune response and MAP kinase signal transduction pathway phosphorylation of phosphatidylinositol and phosphatidylinositol 4phosphate endocytosis and phagocytosis of apoptotic cells

+ + + + +

+

actin cytoskeleton reorganization, cellular adhesion and regulation of cell cycle alkylated DNA repair and NF-kappa-B transactivation

+

+

activation of P38 and JNK MAOK pathways

+

+ + +

transport of vesicle to plasma membrane inflammation and smooth muscle contraction activation of AKT1 signaling, STAT family, RAS and

+

MAP kinases ROS detoxification and hydride transfer between NADH and NADP oxidative decarboxylation of malate proline degradation pathway

+

conversion of methemoglobin, cholesterol synthesis and elongation of fatty acids endocytosis and phagocytosis of apoptotic cells and cellular lipid homeostasis

+ + +

ROS detoxification and hydride transfer between NADH and NADP oxidative decarboxylation of malate conversion of methemoglobin, cholesterol synthesis and elongation of fatty acids

H

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Table 1. continued biological process

Uniprot P30038

RedOx

P00390

DNA repair

Q9Y4L1 Q8N3C0 P52701 P11388 P46100 P55072 Q9UIG0

RNA/DNA processing

P61221 O75691 O75643 P42704 Q9P2N5 P61247 Q8IVL1 Q6P158 Q14146

miscellaneous

O43681 O75694 Q5FWE3 Q8WUJ3 P05023 Q4VC31 Q5T1M5 Q5T5S1

Ob/ T2DM

Ob

delta-1-pyrroline-5-carboxylate dehydrogenase glutathione reductase, mitochondrial hypoxia up-regulated protein 1 activating signal cointergrator 1 complex subunit 3 DNA mismatch repair protein Msh6 DNA topoisomerase 2-alpha transcriptional regulator ATRX transitional endoplasmic reticulum ATPase tyrosine-protein kinase BAZ1B

+

+

proline degradation pathway

+

reduction of oxidized glutathione (GSSG) to GSH

+ +

cytoprotective role under hypoxic conditions alkylated DNA repair and NF-kappa-B transactivation

ATP-binding cassette subfamily E member 1 small subunit processome component 20 homologue U5 small nuclear ribonucleoprotein 200 kDa helicase leucine-rich PPR motif-containing protein RNA-binding protein 27 40S ribosomal protein S3a neuron navigator 2 putative ATP-dependent RNA helicase DHX57 unhealthy ribosome biogenesis protein 2 homologue ATPase ASNA1 nuclear pore complex protein Nup155 proline-rich transmembrane protein 3 protein KIAA1199 sodium/potassium-transporting ATPase subunit alpha 1 coiled-coil domain-containing protein 58 FK506-binding protein 15 uncharacterized coiled-coil domain-containing protein KIAA1984

protein

+

function

+

DNA mismatch repair via formation of hMutS alpha complex

+ + +

+

during transcription controls the topologic states of DNA regulation of gene expression by controlling its chromatin association degradation of polyubiquinated proteins by proteasome, vesicle transport and regulation of spindle disassembling and nuclear envelope formation tyrosine phosphorylation of H2AX, chromatin remodeling and transcription regulation inhibition of ribonuclease L activity, regulation of mRNA turnover

+

18S rRNA processing

+

pre-mRNA processing

+

+

+

RNA metabolism in nuclei and mitochondira

+

+ + + +

mRNA processing RNA binding role in cell growth, migration and neuronal development ATP-dependent helicase activity

+

unknown

+ +

transport of tail-anchored proteins, insulin signaling transport of proteins between nucleus and cytoplasm

+

transmembrane protein, function unknown

+ +

unknown exchange of Na and K ions across plasma membrane

+

unknown + +

unknown unknown

basically oxidize all classes of biomolecules, such as sugars, lipids, and proteins, which all can be used to monitor oxidative stress in global terms. In proteins, for example, cysteine, methionine, and to a lower content, tryptophan, proline, threonine, and other residues can be easily oxidized with only the initial oxidation states of cysteine being reversible within cells. All other oxidative modifications are irreversible and thus will seriously affect the protein functions including incorporation of new functionalities and autoantigens. As (reactive) protein carbonyls resemble a heterogeneous group of modifications, such as oxidized amino acid residues or reaction products of oxidized sugar and lipids, it represents an ideal class of potential biomarkers that should reflect many different nonenzymatic oxidative reaction pathways. Additionally, the formed aldehydes and ketones are relatively stable in the body and thus can accumulate over time, reflecting at least a short period of the disease. From an analytical perspective, these (reactive) carbonyl groups can be derivatized easily and

involved in the focal adhesion pathway. The vascular endothelial growth factor receptor 2 (VEGFR-2), for example, plays an essential role in the VEGF signaling cascade and in regulating angiogenesis and vascular development. Interestingly, VEGFR-2 levels are reduced in diabetes patients39 which might contribute to diabetic angiogenesis impairment. Moreover, the reduced protein levels appear to be mediated by methylgloyxal and connected to RAGE (receptor for advanced glycation end products) activation and autophagy, thus leading to endothelial angiogenesis.40 In the current study both methylglyoxal and malondialdehyde adducts of VEGFR-2 were detected specifically in Ob/T2DM plasma.



DISCUSSION It is well established that oxidative stress contributes to the onset and progression of obesity and diabetes via β-cell and mitochondrial dysfunctions,5,11,18 though both the diseaserelated molecular changes and the oxidative modifications are much more complex and only partially revealed. Oxidants can I

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after the proteins are released from a tissue, or if the underlying oxidations are affected similarly within organs and tissues. In the first case, the modifications would be general biomarkers representing the overall age and disease status of a person, but not directly linked to the origin of a certain disease. If this is correct, our data set should contain mostly carbonylation sites from abundant proteins with long half-life times in serum (e.g., serum albumin and immunoglobulins). The identification of only five carbonylation sites in serum albumin (Cys-269, Cys289, Cys-485, Lys-490, and Lys-565) and four in immunoglobulins (Thr-75, Pro-97, Arg-1458, and Thr-2490) clearly indicate that protein carbonylation occurs in blood only at a low level. It appears more likely that most detected carbonylation sites were formed in the tissues or organs wherein the proteins were originally expressed. If true, it would indicate that the carbonylation degree of at least a few modification sites are disease specific and not a result of rather unspecific body-wide oxidative stress conditions. The modification sites and carbonylation types might even point toward the mechanisms underlying a disease, providing early diagnostic and prognostic biomarkers and maybe even new disease prevention strategies. Thus, additional longitudinal studies on different disease cohorts are required to identify and quantify disease-related carbonylation sites. The low number of carbonylation sites identified in abundant plasma proteins versus low-abundant proteins was unexpected, especially as serum albumin has a relatively long half-life time. However, previous data indicate that the half-life time of in vitro oxidized/carbonylated serum albumin is relatively low,47,48 whereas it was also shown that carbonylated proteins circulate in blood for longer periods.49 Though further studies are required to link circulation times and carbonylation levels for serum proteins, the data indicate that carbonylation sites are mostly formed in the tissues or organs and only to a relatively low degree in blood. Protein carbonylation in general can induce cell death when the damaged proteins cannot be cleared by the proteasome and/or autophagy. Oxidative stress can induce apoptosis and necrosis in enothelial cells (ECs) releasing intracellular proteins into the blood. Both development and pathogenesis of T2DM is closely related to chronic inflammation, which is mediated by macrophage and neutrophil activity leading to elevated ROS levels produced by NADPH oxidase.50,51 Interestingly, we found an enrichment of platelet-derived proteins, as redox changes occur as a part of their normal activation process and recent publications correlate increased platelet proteins carbonylation to disease and general aging processes.52 Accumulation of carbonylated proteins might explain inflammation-derived apoptosis and necrosis resulting in elevated blood levels of carbonylated proteins. The majority of the carbonylated proteins identified within the plasma of Ob and Ob/T2DM subjects are involved in cell adhesion, cytoskeletal remodeling, signaling, and angiogenesis. For example, VEGFR-2, which plays an essential role in regulation of angiogenesis and vascular development, was carbonylated in plasma of Ob/T2DM patients. Although the influence of carbonylation on the activity and/or expression of VEGFR-2 is currently unknown, it may contribute to the recently established link between VEGFR-2 activity, obesity, diabetes, and nonalcoholic steatohepatitis.53 However, previous studies indicate that carbonylation affects insulin-like growth factor receptors IGF-1R and IGF-2R (among other growth factor receptors) and insulin-like growth factor binding proteins IGFBP-2 and IGFBP-3.54

quantitatively, providing specific enrichment (e.g., biotin) and sensitive detection techniques. For all these reasons, protein carbonylation is widely used to determine oxidative stress globally. More recently, there are also serious efforts to identify protein carbonylation sites by mass spectrometry as potential disease-specific biomarkers. Here, we combined several strategies developed in different laboratories including ours to identify first a large number of carbonylated proteins in human plasma and then relatively quantify these modification sites in a targeted approach to identify potentially disease-specific carbonylation sites. Though the biotin−avidin affinity enrichment strategies reduced the complexity significantly, unspecific binding of peptides to avidin via hydrophobic and electrostatic interactions still challenged the analysis, as indicated by more than 600 unmodified peptides detected in the plasma samples. This was especially evident for very abundant proteins, such as serum albumin, immunoglobulins, complement components, and lipoproteins. The classical DDA-strategy, which considers only the most intense signals for consecutive MS/MS, mostly misses carbonylated sequences present at low signal intensities and often provides unfavorable fragment ion spectra. This greatly reduces the reproducibility of the analysis with respect to the identified carbonylation sites in different serum samples, as experienced here when profiling initially the samples for different sample groups to identify as many modification sites as possible. Typically, 100 carbonylated peptides were identified on average with Xcorr > 1.5 in each analysis with only 10% identified in all three technical replicates. Feature detection and alignment tools in combination with targeted mass spectrometry provide access to weak signals as opposed to the DDA-based strategies. Here, we explored the benefits of elution time segmented inclusion list of m/z values for MS/MS sequencing using previously published bioinformatic tools for in-depth characterization of low-abundant carbonylated proteins. An average of 50% of the carbonylated proteins (52 proteins) being similar between the three groups (control, Ob, Ob/T2DM) clearly highlights the reproducibility of the analytical workflow. Moreover the data clearly indicate that the elution time segmented inclusion list allowed the identification of low-abundant carbonylated proteins and thus overcomes the background provided by abundant plasma proteins, such as albumin, immunoglobulins, complement components, and lipoproteins. These 52 proteins represent the basal protein carbonylation level and thus might be typical for midlife persons (44−46 years old donors), as protein carbonylation increases with age and several age-related disorders.41,42 It should be noticed, however, that these conclusions resulted mostly from global carbonylated protein levels obtained by ELISA and spectrophotometric assays for whole plasma samples. Information on the carbonylation type, the carbonylated proteins, and the modification sites are very limited.43,44 From the 52 proteins identified here only 14 mostly highly abundant plasma proteins had been reported in recent metastudies dealing with aging or age-related disorders, i.e. Q02218, P12259, P12111, P13671, P21333, Q6WRI0, O15230, P11047, P78559, P35580, Q9UHD8, P02768, P11277, and Q8WZ42.42,45,46 Carbonylated proteins identified in the plasma of control and obese patients with or without T2DM were mainly annotated to liver, plasma, platelet, and endothelium, closely resembling the plasma proteome composition. This raises the important question whether carbonylation occurs mostly in the blood, i.e. J

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only accomplished by an additional feature detection and alignment followed by targeted LC−MS/MS. Thus, the molecular identities of hundreds of proteins carbonylated by different mechanisms were revealed for the first time in human plasma, providing a solid base for probing them as biomarkers. Importantly, many of the detected proteins are connected to well-known complications of Ob and Ob/T2DM, such as endothelial dysfunction, cytoskeletal remodeling, and DNA damage. The set of potential biomarkers provides a valid base for a quantitative target study using larger cohorts of Ob and Ob/T2DM patients. Additionally, the set of 54 carbonylated proteins present in all samples allows studying how the carbonylation degree depends on age, gender, and living conditions.

Supporting the important role of increased oxidative stress in the pathophysiology of metabolic diseases (e.g., T2DM) and long-term macro- and microvascular diabetes complications, we identified proteins exclusively carbonylated in the T2DM. Such proteins include oxidoreductases, proteins of angiogenesis, lipid metabolism and DNA repair. Among the identified proteins, there are previously established candidates for the link between diabetes and impaired biosynthesis of peptide hormones such as insulin (e.g., carboxypeptidase D)55 premature atherosclerosis (e.g., MMP-2),56 the development of diabetic nephropathy (e.g., agrin),57 macular degeneration (e.g., hemicentin1),58 adipose tissue stress (e.g., mitogen-activated protein kinase kinase kinase 4),59 increased risk for colorectal cancer (e.g., Msh6).60 As another example, we found MMP-9 carbonylation only in T2DM. Since MMP-9 is involved in the regulation of glucose transporter-4 (GLUT-4) granule transport and fusion, MMP-9 protein modifications may significantly alter glucose uptake into insulin-sensitive tissues such as adipose tissue or liver.61 Moreover, our data set may help to better dissect proteins and protein modifications primarily associated with type 2 diabetes (e.g., MMP-9), obesity (e.g., complement C5), or both (e.g., MKK4). Recently, the complement C5 system has been demonstrated to play an important role in adipose tissue inflammation and the development of insulin resistance.62 Carbonylation of C5 could therefore contribute to adipose tissue dysfunction in obesity, but not necessarily to T2DM. We have previously shown that activation of the Ask1-MKK4-p38 stress signaling pathway may link increased immune cell infiltration into visceral adipose tissue with increased diabetes risk via deterioration of insulin resistance.59 On the basis of these findings and the significant carbonylation signal in our current analysis, we postulate that MKK4 protein modifications may contribute to an increased T2DM risk in patients with adipose tissue inflammation. Taken together, previously unrecognized protein modifications in these potential markers may contribute to the development of metabolic and/or cardiovascular diseases. Moreover, our data may stimulate further research to verify a potential role of proteins, which have not been linked before to obesity and diabetes (e.g., plectin, filamin-B, brain-specific angiogenesis inhibitor 2, versican core protein). Though it is speculative to link carbonylation of these proteins to the onset or progression of obesity and T2DM, the identified carbonylation sites will provide a solid base for further evaluating the biological implications in the context of both diseases. Thus, absolute or relative quantification of these carbonylated peptides/proteins, for example via targeted mass spectrometry, should be the next step to reveal their relevance. The lack of synthetic carbonylated peptide standards, however, limits accurate quantitative studies.



ASSOCIATED CONTENT

S Supporting Information *

File 1: List of all the carbonylated proteins, carbonylation sites and types of modifications found in the plasma of control and obese subjects without and with type 2 diabetes. File 2: Structures of all carbonyl modifications on Lys, Arg, Cys, His, Pro and Thr residues considered in this study. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*Tel.: 49 (0) 341 9731330. Fax: 49 (0) 341 9731339. E-mail: Hoff[email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS Financial support from the European Fund for Regional Structure Development (EFRE, European Union and Free State Saxony; 100146238 to M.F.), the “Bundesministerium für Bildung and Forschung” (BMBF; 03IP604 to R.H.), the Free State Saxony and the German Obesity Biobank (GOBB) (FKZ: 01GI1128 to M.B.) as well as a stipend to R.C.B. provided by Universität Leipzig are gratefully acknowledged.



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CONCLUSIONS The established analytical strategy and workflow allows a reproducible in-depth profiling of carbonylated proteins including the modification sites and carbonylation type. It should be noted that our approach aims at the identification of reactive carbonyl groups present in proteins, whereas consecutive reaction products without reactive carbonyls will not be detected, such as Schiff bases of α,β-unsaturated aldehydes. Though affinity enrichment reduced the sample complexity significantly, it does not considerably increase the sensitivity for low-abundant carbonylated proteins. This was K

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Journal of Proteome Research

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dx.doi.org/10.1021/pr500324y | J. Proteome Res. XXXX, XXX, XXX−XXX