Profiling Carbonylated Proteins in Human Plasma - Journal of

Profiling Carbonylated Proteins in Human Plasma Ashraf G. Madian and Fred E. Regnier* Department of Chemistry, Purdue University, West Lafayette, Indiana 47907 Received October 4, 2009

This study reports the first proteomic-based identification and characterization of oxidized proteins in human plasma. The study was conducted by isolating carbonylated proteins from the plasma of male subjects (age 32-36) with avidin affinity chromatography subsequent to biotinylation of carbonyl groups with biotin hydrazide and sodium cyanoborohydride reduction of the resulting Schiff’s bases. Avidin selected proteins were digested with trypsin, and the peptide fragments were separated by C18 reversed phase chromatography and identified and characterized by both electrospray ionization and matrix assisted laser desorption ionization mass spectrometry. Approximately 0.2% of the total protein in plasma was selected with this method. Sixty-five high, medium, and low abundance proteins were identified, the majority appearing in all subjects. An interesting feature of the oxidized proteins isolated was that in addition to carbonylation they often bore other types of oxidative modification. Twentyfour oxidative modifications were mapped in 14 proteins. Fifteen carbonylation sites carried on 7 proteins were detected. Methionine oxidation was the most frequent single type of oxidative modification followed by tryptophan oxidation. Apolipoprotein B-100 had 20 oxidative modifications, the largest number for any protein observed in this study. Among the organs contributing oxidized proteins to plasma, kidney, liver, and soft tissues were the most frequent donors. One of the more important outcomes of this work was that mass spectral analysis allowed differentiation between different biological mechanisms of oxidation in individual proteins. For the first time, oxidation products arising from direct ROS oxidation of amino acid side chains in proteins, formation of advanced glycation endproducts (AGEs) adducts, and formation of adducts with lipid peroxidation products were simultaneously recognized and assigned to specific sites in proteins. Keywords: protein carbonylation • oxidative stress • human plasma • advanced glycation endproducts • protein lipid peroxidation adducts

Introduction Blood proteins have been widely used in the assessment of human health,1,2 primarily in single protein assays. There is great interest today in the possibility that new proteomics methods will enable the identification and assessment of groups of blood proteins that change in either structure or concentration in association with disease progression and in the course of doing so provide disease specific molecular signatures that are more diagnostic than single proteins. One such case would be with oxidative stress diseases. However, as a prerequisite to the identification of disease markers it is necessary to understand the “normal” distribution of proteins in plasma. At present, there is almost no data on oxidized proteins in human blood. Oxidative stress (OS) is a phenomenon in which reactive oxygen species (ROS) accumulate in cells to such a level that proteins, DNA, RNA, and lipids are irreversibly damaged by oxidation.3 OS has been implicated in multiple ailments ranging from neurological diseases such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis to a variety * To whom correspondence should be addressed. E-mail: fregnier@ purdue.edu.

1330 Journal of Proteome Research 2010, 9, 1330–1343 Published on Web 02/01/2010

of diseases stretching from atherosclerosis, diabetes mellitus, chronic renal failure, and chronic lung disease, to cancer.4-7 Antioxidants and cellular catalysts such as catalase, selenium dependent glutathione, superoxide dismutase and thioredoxin hydroperoxidase are sufficiently abundant in most subjects that ROS (e.g., hydrogen peroxide, singlet oxygen, peroxynitrite and superoxide) are destroyed before causing irreversible protein oxidation.3 However, when the capacity of cells to destroy ROS is exceeded the system becomes “oxidatively stressed”. This can lead to protein carbonylation8 and oxidative injury to cells.5-7 Protein carbonylation is considered to be a universal indicator of oxidative stress, being a major form of protein oxidation.9 Carbonyl groups can be formed in proteins directly by the oxidation of amino acid side chains (on Pro, Arg, Lys, Thr, Glu, and Asp10) or oxidative cleavage of the polypeptide backbone by the R-amidation or diamide pathways. Proteins can also be carbonylated indirectly. One route is by Michael and/or Schiff base addition of advanced lipid peroxidation products such as 4-hydroxy-2-nonenal, 2-propenal and malondialdehyde to Cys, His, or Lys residues. Another mode of carbonylation is by the formation of advanced glycation end (AGE) products adducts.9 These adducts are then oxidized to the carbonyl level. AGE 10.1021/pr900890k

 2010 American Chemical Society

Profiling Carbonylated Proteins in Human Plasma formation is directly proportional to reducing sugar levels and is a major route of carbonylation in untreated diabetics. It appears that some proteins are more prone to carbonylation than others11 and that levels of protein oxidation vary between species. It has been reported that albumin and R1macroglobulin showed the highest level of age-related carbonylation in mouse plasma while albumin and transferrin were the major products in rat plasma and only albumin was carbonylated by this route in Rhesus monkey plasma.12 In another study, keratins were shown to be oxidized in rat plasma samples.13 Beyond these studies there are surprisingly few reports of plasma protein oxidation in vivo. It has been shown in vitro with Western blot immunoassays that fribrinogen, albumin, immunoglobulin and transferrin in human plasma can be oxidized with metal catalysis11 but clearly in vivo and in vitro oxidation is different. The paucity of data on oxidized proteins in plasma is perhaps due to the difficulty of finding oxidized proteins in complex matrices, the multiplicity of possible oxidative modifications, and low abundance.14 Recent development of methods that label carbonyl groups in oxidized proteins with biotin and then affinity select them from complex protein mixtures circumvents some of these problems.15-18 Through affinity selection, it is possible to greatly enrich oxidized proteins for mass spectral analysis, eliminate most unoxidized proteins except those in complexes with oxidized proteins, identify the sites of oxidation, and to a major extent identify the mechanism of oxidation. The objective of the work presented in this paper was to develop and apply methods for assessing human oxidative stress through carbonylation of the plasma proteome. Plasma samples taken from normal, unmedicated, nonsmoking male subjects from 32-36 years of age were immediately derivatized with biotin hydrazide. After reduction of Schiff bases and dialysis to remove excess biotin hydrazide, biotinylated proteins were selected with avidin affinity chromatography and the affinity selected fraction was trypsin digested. Peptide cleavage fragments were identified and sites of oxidative stress induced post-translation modifications (OSi∼PTM) located by analysis with a combination of reversed phase chromatography (RPC) and tandem mass spectrometry (MS/MS). MS/MS analyses were carried out in both the matrix assisted laser desorption and electrospray ionization modes.

Experimental Procedures Materials. Sodium cyanoborohydride Biotin hydrazide, ultralinked immobilized monomeric avidin, D-biotin, and SlideA-Lyzer dialysis cassettes were purchased from Pierce (Rockford, IL). Iodoacetamide, dithiothreitol (DTT), trypsin, Glycine, R-Cyano-4-hydroxy-cinnamic acid (CHCA), proteomics grade N-p-tosyl-phenylalanine chloromethyl ketone (TPCK)-treated trypsin, ammonium bicarbonate, guanidine, dithiothreitol, iodoacetamide acid (IAA), and L-cysteine were obtained from Sigma Chemical Co. (St. Louis, MO). Protease inhibitor cocktail was purchased from Roche Diagnostics (Indianapolis, IN). The ABI 4700 Proteomics Analyzer Calibration Mixture (4700 Cal Mix, bradykinin, angiotensin I, glu1-fibrinopeptide B, ACTH fragment 1-17, ACTH fragment 18-39, and ACTH fragment 7-38) were purchased from Applied Biosystems (ABI, Foster City, CA). Trifluoroacetic acid (TFA), and HPLC grade acetonitrile were purchased from Mallinckrodt Chemicals (Phillipsburg, NJ). Sodium phosphate, sodium chloride were purchased from Mallinckrodt (St. Louis, MO). Amicon Ultra-4 centrifugal filter devices were purchased from Millipore.

research articles Methods. Biotinylation of Normal Human Plasma. Blood was withdrawn from four drug free, nonsmoking male subjects (age 32-36 years). Samples were mixed with EDTA and protease inhibitor, centrifuged at 1500× g for 15 min, and the supernatant was removed. After centrifugation again at 2000× g for 15 min, 50 mM biotin hydrazide was added to a final concentration of 5 mM and the reaction was allowed to proceed at room temperature for another 2 h. Sodium cyanoborohydride was then added to a final concentration of 15 mM and the reaction was permitted to proceed at 0 °C for 1 h. Three sequential dialyses were performed in at least 200 volume equivalents of PBS buffer to remove any unreacted biotin hydrazide. Avidin Selection of Biotinylated Proteins. For the sake of identification, avidin selection was performed individually on the four samples. Characterization of oxidative stress induced post-translational modifications (OSi∼PTM) was achieved however by pooling the four samples and enriching modified proteins. An Agilent series 1100 (Agilent Technologies) HPLC was used for packing immobilized monomeric avidin into affinity columns as well as the purification of biotinylated proteins. Ultralinked immobilized monomeric avidin was selfpacked in a PEEK column (4.6 mm × 100 mm). After packing, the column was washed with 50 column volumes (CV) of PBS (0.15 M, pH 7.4) followed by 100 CV of 2 mM biotin to block any nonreversible biotin binding sites. This was followed by a 100 CV wash with regeneration buffer (0.1 M glycine, pH 2.5) and 100 CV of PBS re-equilibration phase. Bradford assays were used to estimate the concentration of the protein in the plasma samples. A total of 15 mg of plasma proteins was applied onto the affinity column with PBS mobile phase (0.15 M phosphate buffered saline, pH 7.4) at a flow rate of 0.5 mL/min. To remove any unbound proteins, the column was washed thoroughly with more than 60 CV loading buffer. The bound proteins were then eluted with regeneration buffer (0.1 M glycine, pH 2.5). The resulting chromatogram was monitored at 280 nm. Proteolysis. Affinity purified proteins were concentrated with an Amicon Ultra-4 centrifugal filter devices. The samples were further dried and reconstituted in 6 M guandinie HCl and 10 mM dithiotheritol. After 1 hr incubation at 70 °C, iodoacetamide (at a final concentration of 10 mM) was added to the reaction and allowed to incubate for 30 min at 4 °C. The sample was diluted 6-fold in ammonium bicarbonate (0.1 M, pH 8.0) and sequence grade trypsin (2%) was added. Proteolysis was allowed to proceed at 37 °C for at least 18 h after which the reaction was stopped by addition of tosyl lysine chloroketone (TLCK) (trypsin/TLCK ratio of 1:1 (w/w)). The resulting tryptic peptide mixture was analyzed with the MALDI-TOF/TOF and LTQ orbitrap XL instruments. LTQ Orbitrap-Based Identification and Characterization. The digested peptide mixtures resulting from trypsin proteolysis were separated on an Agilent 1100 HPLC system using a 75 µm × 120 mm C18 reversed phase chromatography (RPC) column packed with 5 µm C18 Magic beads. Peptide separations were achieved using a 60 min linear mobile phase gradient from 0.1% formic acid to 0.1% formic acid in acetonitrile at a flow rate of 0.3 µL/min. The electrospray ionization emitter tip was generated on the prepacked column with a laser puller (Model P-2000, Sutter Instrument Co.). The HPLC system was coupled directly to the LTQ Orbitrap hybrid mass spectrometer (Thermoelectron, San Jose, CA). The LTQ Orbitrap was equipped with a nanoelectrospray ion source (Proxeon Biosystems, Odense, Denmark). The MS was operated in the dataJournal of Proteome Research • Vol. 9, No. 3, 2010 1331

research articles dependent mode, in which a survey full scan MS spectrum (from m/z 300 to 1600) was acquired in the Orbitrap with a resolution of 60 000 at m/z 400. This was then followed by MS/ MS scans of the 3 most abundant ions with +2 to +3 charge states. Target ions already selected for MS/MS were dynamically excluded for 180s. The resulting fragment ions were recorded in the linear ion trap. Proteins are listed in Table 2 according to their Swiss-Prot entry names and accession numbers. MALDI-TOF/TOF-Based Protein Identification. The tryptic peptides fractions obtained from the digestion of the affinity purified proteins were desalted and fractionated using an Agilent 1100 Series HPLC (Agilent Technologies). A Pepmap C18 trap column and a Zorbax 300sB-C18, 3.5 µm, 100 µm i.d. RPC column of 15 cm length, (Agilent Technologies, Santa Clara, CA) were used. The RPC separation was achieved using a 40 min linear gradient from 98% solvent A/2% solvent B to 60% solvent A/40% solvent B at a flow rate of 800 nL/min. Solvent A was composed of 0.1% TFA in deionized water while solvent B was composed of 0.1% TFA in acetonitrile. Peptide fractions from the RPC column were mixed with MALDI matrix (Rcyano-4-hydroxycinnamic acid, 4 mg/mL in 60% ACN/0.1% TFA) continuously using a mixing tee at the end of the RPC column. An Applied Biosystems Inc. model 4800 MALDI-TOF/ TOF Proteomics Analyzer equipped with a 200 Hz Nd:Yag laser was used in the positive ion mode for the analysis of peptides spotted on a MALDI plate. The 4000 Explorer software furnished with the ABI 4800 controlled the automated acquisition of MS and MS/MS data. Protein identification based on acquired MS/MS spectra were carried out using Protein Pilot software 2.0 with the Pro Group algorithm for protein identification. A 95% confidence level of peptide identification was used as the minimum acceptance criterion. Proteins were identified based on the presence of at least two unmodified peptides from the same protein identified by the Pro Group algorithm at the 95% confidence level. Proteins identified in this analysis are listed in Table 3 according to their Swiss-Prot entry names and accession numbers. Mascot Database Searching. The MS data files (recorded with the Xcalibur software version 2.0.7, Thermo Fisher Scientific) obtained by LC/MS/MS analysis on the LTQ Orbitrap XL instrument were converted to dta files using the in house online LTQ_dta software. Minimum scans per group parameter were set to 1. The charge of the precursor ions was determined automatically by the software. Files were then sent to an inhouse MASCOT server (Version 2.2, Matrix Science19). The human taxonomy in the Swiss-Prot/Uniprot database was searched. Mascot has the limitation of allowing just nine modifications per search. Therefore, it was searched separately six times for each sample with a maximum of six modifications each time. The variable modifications were as follows. Search one targeted carbamidomethyl (C), oxidation (C,D,F,H,K,L,N, P,R,W,Y) and oxidation (M). The second focused on carbamidomethyl (C), dioxidation (C,F,M,P,R,W) and trioxidation (C). The third was directed to carbamidomethyl (C), biotinylated HNE (H,C,K), biotinylated glyoxal (K), biotinylated amadori (K), biotinylated 3-deoxyglucosone (K) and biotinylated methyl glyoxal. The fourth search targeted carbamidomethyl (C), biotinylated oxidized arginine, biotinylated oxidized lysine, biotinylated oxidized proline, and biotinylated oxidized threonine. The fifth search was directed toward carbamidomethyl (C), the oxidation of tryptophan to kynurenin, the oxidation of tryptophan to hydroxykynurenin, the oxidation of tryptophan 1332

Journal of Proteome Research • Vol. 9, No. 3, 2010

Madian and Regnier

Figure 1. Schematic illustration of the strategy used for the identification of oxidized proteins and their oxidation sites in normal human plasma. Samples were examined individually to facilitate protein identification. Those with oxidative modifications were then enriched by pooling the samples.

to oxolactone, the oxidation of proline to pyroglutamic acid and the oxidation of proline to pyrolidinone. In the sixth search, only carbamidomethyl (C) was searched. A decoy method was used to estimate the false discovery rate. The decoy for the four samples was generally less than 1.8%. Four missed cleavages were allowed. The precursor mass tolerance was set to 5 ppm and the fragment mass tolerance was set to 0.6 Da. Only proteins with at least two unmodified peptides were considered as correct matches. Proteins that were identified based on just one peptide or appeared in just one sample were rejected. Strict criteria were used to eliminate false positive identification of any of the modifications identified. For all modifications except biotinylation, the modification had to have an expectation value less than 0.05 and was manually validated in order to be considered as a correct match. The fragmentation of biotin however produces noise in the spectrum that increases the expectation value.20-22 Consequently, the biotinylated modifications were validated manually only. Annotating the Identified Proteins Based on Their Tissue of Origin. The human plasma atlas (version 4.0)23 was used to annotate the identified proteins according to their expression in the different tissues inside the body. The degree of their expression can be strong, medium or weak. Figure 4 shows a distribution of these proteins in the different tissues.

Results Selection of Donors. Oxidative stress is thought to be influenced by factors ranging from aging, gender, and diet to smoking, diseases, and medications. For this reason, strict criteria were used in blood donor selection. Donors were of the same gender (males), nonsmoking, had no diagnosed diseases, were not receiving medications, and were age matched (32-36 years of age). Analytical Strategy. The analytical protocol used in these studies (Figure 1) is a modified version of a method first described for the analysis of the yeast proteome.15 A fresh plasma sample (6 mL) was drawn from a donor and carbonyl groups biotinylated with biotin hydrazide and the resulting

research articles

Profiling Carbonylated Proteins in Human Plasma Schiff bases reduced. Samples thus derivatized were dialyzed to remove excess biotin hydrazide along with low molecular weight biotinylated species including small polypeptides. Following dialysis only high molecular weight species remained, most of which were proteins. Avidin affinity chromatography was then used to select and enrich carbonylated species for identification of oxidized proteins, sites of oxidation, and the particular types of oxidation involved. Oxidized proteins were identified in two ways. One was by RPC fractionation of the avidin selected fraction followed by proteolysis of RPC fractions and MS based identification with MALDI-MS/MS. A second approach was to tryptic digest the affinity selected fraction and proceed directly to proteolysis and MS based identification. Because the second method is simpler and provided similar numbers of identifications to the first, the second method was used in this work. Avidin selected proteins were digested with trypsin, the peptide cleavage fragments fractionated with a C18 RPC column, and the peptides from RPC identified by MALDIMS/MS (ABI 4800 plus) and then further characterized with an LTQ Orbitrap XL. Protein Pilot and Mascot were used for the analysis of the mass spectra respectively as described in the Methods. Sample Preparation. The object of the present study was to isolate carbonylated proteins from the plasma proteome. It is important to recognize that carbonyl groups react readily with free amine groups on proteins as they sit on the bench or are stored, forming Schiff’s bases24 and making them inaccessible to biotin hydrazide derivatization. This possibility is precluded by adding a large excess of biotin hydrazide to plasma samples within a few min of the time they are drawn. Once biotin hydrazide has been added samples can be frozen and stored indefinitely. Degradation of blood proteins by intrinsic endoproteases during collection and storage has been shown to be another complication.25 Addition of a protease inhibitor cocktail to fresh plasma inhibits these cysteine and serine proteases and preserves samples. Avidin Affinity Selection. An avidin affinity column was used in our procedure. Plasma samples were applied directly onto the affinity column without abundant protein removal. The column was used in processing approximately 50 samples over six months without significant loss of capacity based on the binding of standard biotinylated bovine serum albumin at periodic intervals. Although abundant protein removal is widely used in discovery proteomics studies, that approach was not used in this work for several reasons. One was concern that oxidized proteins could be associated noncovalently with abundant proteins and be removed. Recent studies in which 129 low abundance proteins were found to be associated with abundant proteins gives credibility to this abundant protein “sponge effect” hypothesis.2,26 A second was that peptides from abundant proteins did not interfere with oxidized protein identification. In fact, there was no relationship between the presence of peptides from abundant proteins and their concentration in affinity selected fractions. The third reason was that mathematical modeling has indicated that washing affinity columns with 15 or more column volumes of loading buffers elutes most proteins bound to columns with low affinity.27 Sixty column volumes of loading buffer were pumped through the affinity column after loading. During this washing step eluent absorbance returned to zero (Figure 2) as expected. Approximately 0.2% of the protein in plasma was captured by the avidin affinity column based on absorbance at 280 nm

Figure 2. Chromatogram from avidin affinity chromatography of a normal human plasma sample. A sample of the plasma (15 mg of total protein) was applied directly to a 4.6 × 100 mm2 column packed with Agarose to which avidin had been immobilized. The column was eluted initially with 0.15 M phosphate buffered saline, (pH 7.4) at 0.5 mL/min for 120 min then switched to a mobile phase containing 0.1 M glycine/HCl (pH 2.5) for an additional 40 min at the same flow rate. Absorbance was monitored at 280 nm. Table 1. Relative Amounts of the Affinity Purified Protein from Normal Human Plasma Samples Selected by Avidin Affinity Chromatography donor number

% affinity purified proteins (based on the 280 nm absorbance)

Donor number 1 Donor number 2 Donor number 3 Donor number 4 Average Standard deviation

0.199% 0.234% 0.141% 0.275% 0.212% 0.057

(Table 1). It should be noted that naturally biotinylated proteins, proteins naturally complexed with or cross-linked to the biotinylated proteins, and nonspecifically bound proteins are included in this affinity selected fraction as well.15 The relative standard deviation of peak areas among the four human plasma samples was 0.057%. Oxidized Protein Characterization. Based on the fact that MALDI-MS/MS and ESI-MS/MS used together generally leads to the identification of more proteins than either alone, both types of ionization were used in these studies.28 Analysis of affinity selected proteins from the four plasma samples generated an average of 7724 spectra in the ESI-MS mode (LTQ Orbitrap XL) as opposed to 601 spectra in the MALDI-MS mode (ABI 4800). An average of 65 proteins were identified based on 525 unique peptides identified by ESI-MS/MS and 225 by MALDI-MS/MS. Twenty-four proteins were identified by both MALDI-MS and the ESI-MS. Thirty-two proteins were identified by ESI-MS alone and 5 proteins were identified by MALDIMS only (Figure 3). The data showed good reproducibility. Fiftyeight proteins were identified in all the 4 donors, 2 proteins were identified in 3 donors only and 5 proteins were identified in 2 donors only. The list of proteins identified (Tables 2 and 3) includes highly abundant proteins (e.g., R-1-antitrypsin precursor, R-2-macroglobulin precursor, apolipoprotein A-I precursor, apolipprotein B-100 precursor), some of moderate abundance (e.g., hempexin and kininogen-1 precursor) and several low abundance proteins (e.g., tetranectin precursor and transthyretin precursor). Tissue origin of the proteins found in this study Journal of Proteome Research • Vol. 9, No. 3, 2010 1333

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Madian and Regnier Proteins with no detectable carbonyls were also seen. This could be because they were cross-linked or complexed with carbonylated proteins. It could also be the result of nonspecific binding to the avidin-agarose affinity system. Still another possibility is that two other unoxidized peptides from the proteins were not found as specified in our identification criteria. This strict evaluation criterion eliminated a large number of ambiguous carbonylation sites not confirmed by manual interpretation of the mass spectra.

Discussion Figure 3. Comparison between the number of oxidized proteins identified in the four donors’ plasma using ESI-MS, MALDI-MS, or both.

was obtained from the Human Protein Atlas (version 4.0). This is a database containing approximately five million immunohistochemical-based images produced using 6120 antibodies against 5067 protein coding human genes.23 Kidney (cells in the tubules), soft tissues, and liver (hepatocytes) contributed the largest numbers of proteins found in this study (Figure 4). Seventy-four oxidative stress-induced post-translational modifications (OSi∼PTM) were mapped to these proteins (Figure 5). Among the sites identified, oxidation of methionine to methionine sulphoxide appeared more frequently than any other OSi∼PTM. Twenty-two proteins with a methionine sulphoxide OSi∼PTM were mapped. The oxidation of tryptophan to dihydroxytryptophan, kynurenin, hydroxykynurenin, and hydroxytryptophan came second in frequency of oxidation. The high frequency of methionine oxidation agrees with literature reports that sulfur amino acids (methionine and cysteine) are more sensitive to oxidation by ROS than other amino acids.29 The fact that only one cysteine oxidation product was detected is not surprising for several reasons. One is that sulfenic acid reduction occurs naturally in biological systems. Another was that detection of sulfhydryl to disulfide oxidation was precluded in these studies by the reduction of disulfide bonds with dithiothreitol and iodoacetamide alkylation during the sample workup.30 It was also found that carbonyl groups generated directly by oxidation of threonine, arginine, lysine, and proline and those formed indirectly by the formation of Amadori, glyoxal, methylglyocal, 3-doxyglucosone, and 2-hydroxy nonenal adducts could be isolated with the same procedure. This means it is possible to simultaneously identify oxidation products involving direct ROS oxidation of amino acid side chains in proteins, oxidation of adducts from AGE product addition, and oxidation of adducts from lipid peroxidation products. Fourteen proteins were shown to have at least one oxidation site as seen in Table 4. Apolipoprotein B-100 in contrast was found to undergo 20 oxidative modifications; the largest number of OSi∼PTMs of any protein observed. Interestingly, this protein was carbonylated due to the formation of both glycation/AGEs (3-deoxyglucosone and methylglyoxal) and lipid peroxidation (2-hydroxy nonenal) adducts. Fifteen carbonylation sites carried on seven proteins were detected. These proteins are: alpha-2-HS-glycoprotein precursor (fetuin-A), antithrombin-III precursor (ATIII), apolipoprotein B-100 precursor (Apo B-100), apolipoprotein E precursor (Apo-E), C4b-binding protein alpha chain precursor, clusterin precursor and coagulation factor V precursor. 1334


The goal of this study was to isolate and characterize proteins in normal human plasma that were oxidized in vivo. Based on the fact that plasma samples were worked up and analyzed immediately after they were drawn, it is assumed that all the oxidation detected occurred in vivo. Even so, methionine is sufficiently labile it is possible a small amount of methionine oxidation occurred in vitro. This is of low probability because in vitro oxidation of proteins is generally associated with lengthy exposure to oxygen during storage or by some type of oxidative catalysis associated with metal contamination. Under metal catalysis ROS can be produced in vitro that oxidizes plasma proteins.11 In vivo oxidation of human plasma proteins has been most widely studied with colorimetric methods as in the cases of risk assessment with breast cancer31 and smoking.32 The problem with the coloroimetric methods is that the actual proteins being oxidized are not identified. The advance reported in these studies is that through the use of previously described methods for biotin labeling of carbonyl groups and avidin affinity chromatography selection of biotinylated species15 it is possible to isolate heavily oxidized proteins from the plasma proteome, identify the proteins involved, and characterize the sites and types of oxidation. Approximately 0.2% of the total protein in plasma was isolated by avidin affinity chromatography in these 32-36 year old male subjects. Eleven different amino acids were found to be involved in at least 23 different types of OSi∼PTM. Sixtyfive proteins were identified from the 4 male subjects. With this degree of complexity, it is possible that a peptide with an OSi∼PTM can be of very similar mass to an unmodified peptide, making it difficult to differentiate between them.33 Differentation was most specifically achieved by going to high mass accuracy instrumentation such as the LTQOribitrap XL mass analyzer with a mass accuracy approaching 1 ppm. The fact that large numbers of oxidized proteins were found in these normal male subjects means that irreversible oxidative stress with accompanying permanent alterations of protein structure is a normal part of metabolic processes. This is not a process that occurs only in the elderly or oxidative stress type of diseases. A major question is whether oxidized proteins found in normal individuals are those associated with aging and oxidative stress diseases but at a lower level. Stadtman reported that the presence of a conjugated metal in a protein increases the probability it will be carbonylated, presumably by internal metal catalysis.11 As expected, serotransferrin, ceruplasmin, hemoglobin, hemopexin, and fibrinogen were identified in our study. Fibrinogen is (i) more likely to be oxidized than serum albumin,11 (ii) the most susceptible protein to oxidation in plasma under intravenous administration of iron gluconate,34 (iii) one of the best

accessions

C1QC_HUMAN

C1QB_HUMAN

FA5_HUMAN

CLUS_HUMAN

CD5L_HUMAN

CADH5_HUMAN

C4BB_HUMAN

C4BP_HUMAN

APOH_HUMAN

APOM_HUMAN

APOE_HUMAN

APOD_HUMAN

APOC3_HUMAN

APOC1_HUMAN

APOB_HUMAN

APOA4_HUMAN

APOA2_HUMAN

APOA1_HUMAN

ANT3_HUMAN

FETUA_HUMAN

A2AP_HUMAN

A1AT_HUMAN

name

Alpha-1-antitrypsin precursor (Homo sapiens (Human) Alpha-2-antiplasmin precursor Homo sapiens (Human) Alpha-2-HS-glycoprotein precursor (Fetuin-A) -Homo sapiens (Human) Antithrombin-III precursor (ATIII) - Homo sapiens (Human) Apolipoprotein A-I precursor (Apo-AI) Homo sapiens (Human) Apolipoprotein A-II precursor (Apo-AII) (Homo sapiens (Human) Apolipoprotein A-IV precursor (Apo-AIV) (Homo sapiens (Human) Apolipoprotein B-100 precursor (Apo B-100) Homo sapiens (Human) Apolipoprotein C-I precursor (Apo-CI) Homo sapiens (Human) Apolipoprotein C-III precursor (Apo-CIII) Homo sapiens (Human) Apolipoprotein D precursor (Apo-D) Homo sapiens (Human) Apolipoprotein E precursor (Apo-E) - Homo sapiens (Human) Apolipoprotein M (Apo-M) Homo sapiens (Human) Beta-2-glycoprotein 1 precursor (Beta-2-glycoprotein I) (Apolipoprotein H) (Homo sapiens (Human) C4b-binding protein alpha chain precursor (C4bp) Homo sapiens (Human) C4b-binding protein beta chain precursor - Homo sapiens (Human) Cadherin-5 precursor (Vascular endothelial-cadherin) (VE-cadherin) Homo sapiens (Human) CD5 antigen-like precursor (SP-alpha) Homo sapiens (Human) Clusterin precursor Homo sapiens (Human) Coagulation factor V precursor Homo sapiens (Human) Complement C1q subcomponent subunit B precursor - Homo sapiens (Human) Complement C1q subcomponent subunit C precursor - Homo sapiens (Human)

donor 1

donor 2

donor 3

donor 4

16.3

20.7

0.7

20.3

20.7

ND

34.9

48.1

19.1

39.9

12.9

26.5

27.3

26.5

18.7

30.8

43

51.3

ND

ND

3.5

20.1

2

3

3

8

5

ND

6

24

3

6

4

6

2

4

63

10

4

17

ND

ND

1

7

307

279

29

462

297

ND

1014

8900

65

253

148

497

477

685

3123

527

691

1953

ND

ND

23

342

17

21

0.6

23

40

ND

26

58

22

26

27

19

27

13

21

27

59

60

13

14

13

25

3

4

2

6

11

ND

4

25

4

3

7

3

2

3

69

8

5

20

3

4

2

15

175

232

39

611

612

ND

563

9153

229

174

523

235

189

240

4342

678

1246

3570

214

218

128

353

ND

ND

4.1

22.9

ND

5.2

12.3

17.6

40.6

29.3

10.1

20.1

62.6

45.8

ND

10.6

28

60.7

7.8

22.9

11.4

53.1

ND

ND

4

3

ND

7

6

8

32

2

10

5

4

5

ND

22

3

6

14

7

12

39

ND

ND

181

729

ND

86

55

573

875

272

89

273

1419

891

ND

136

382

2835

75

506

106

1801

ND

ND

3.9

27

ND

5.2

12

12

33

33

9.5

20

30

27

0.6

25

25

52

16

32

7.9

49

ND

ND

8

8

ND

6

2

4

10

2

2

3

4

3

6

6

2

18

4

6

2

15

ND

ND

436

918

ND

63

114

419

594

191

117

351

669

642

33

360

373

2639

224

488

125

1203

number of unique number of unique number of unique number of unique sequence peptides assigned Mascot sequence peptides assigned Mascot sequence peptides assigned Mascot sequence peptides assigned Mascot coverage to the protein score coverage to the protein score coverage to the protein score coverage to the protein score

Table 2. List of Proteins Identified Using the LTQ Orbitrap XL

Profiling Carbonylated Proteins in Human Plasma

research articles

Journal of Proteome Research • Vol. 9, No. 3, 2010 1335

1336


K2C5_HUMAN

K22E_HUMAN

K2C1_HUMAN

K1C9_HUMAN

K1C10_HUMAN

MUC_HUMAN

LAC_HUMAN

KAC_HUMAN

IGHG4_HUMAN

IGHG3_HUMAN

IGHG2_HUMAN

IGHG1_HUMAN

IGHA1_HUMAN

HRG_HUMAN

HEMO_HUMAN

HBB_HUMAN

HBA_HUMAN

HPTR_HUMAN

FINC_HUMAN

FIBG_HUMAN

FIBB_HUMAN

FIBA_HUMAN

CFAH_HUMAN

CO4B_HUMAN

CO3_HUMAN

accessions

name

Complement C3 precursor- Homo sapiens (Human) Complement C4-B precursorHomo sapiens (Human) Complement factor H precursor (H factor 1) - Homo sapiens (Human) Fibrinogen alpha chain precursor Homo sapiens (Human) Fibrinogen beta chain precursor Homo sapiens (Human) Fibrinogen gamma chain precursor - Homo sapiens (Human) Fibronectin precursor (FN) Homo sapiens (Human) Haptoglobin-related protein precursor - Homo sapiens (Human) Hemoglobin subunit alpha Homo sapiens (Human) Hemoglobin subunit beta Homo sapiens (Human) Hemopexin precursor Homo sapiens (Human) Histidine-rich glycoprotein precursor (HPRG) - Homo sapiens (Human) Ig alpha-1 chain C region - Homo sapiens (Human) Ig gamma-1 chain C region Homo sapiens (Human) Ig gamma-2 chain C region Homo sapiens (Human) Ig gamma-3 chain C region Homo sapiens (Human) Ig gamma-4 chain C region Homo sapiens (Human) Ig kappa chain C region - Homo sapiens (Human) Ig lambda chain C regions Homo sapiens (Human) Ig mu chain C region - Homo sapiens (Human) Keratin, type I cytoskeletal 10 (Cytokeratin-10) - Homo sapiens (Human) Keratin, type I cytoskeletal 9 (Cytokeratin-9) - Homo sapiens (Human) Keratin, type II cytoskeletal 1 (Cytokeratin-1) Homo sapiens (Human) Keratin, type II cytoskeletal 2 epidermal (Cytokeratin-2e) (Homo sapiens (Human) Keratin, type II cytoskeletal 5 (Cytokeratin-5) Homo sapiens (Human)

Table 2. Continued donor 1

donor 2

donor 3

donor 4

ND

7.1

18.3

14

20.7

58.6

70.5

86.8

44.6

29.7

40.8

59.4

45.3

10.5

1.7

21.8

25.4

12.1

1.5

24.1

30.3

31.1

4.5

4.6

5.5

ND

3

8

4

6

17

4

7

3

5

7

15

8

2

3

3

3

3

3

5

11

16

3

4

6

ND

238

1062

349

463

6770

2437

7925

1459

655

1629

3676

2006

131

40

353

261

164

61

263

428

5885

146

571

1474

ND

5.6

11

17

28

63

71

82

44

29

41

58

48

5.7

6.3

22

15

16

3.5

37

42

27

7.1

4.6

15

ND

6

7

4

8

25

5

7

2

2

7

13

10

2

3

3

1

3

7

10

13

22

8

4

10

ND

404

689

412

577

7084

2016

6495

1373

1056

1985

4293

1732

91

105

254

59

100

262

782

782

6213

397

651

1083

5.9

25.4

28.7

34.8

35.6

36.3

46.7

86.8

31.8

22.8

41.1

50.9

47.3

20.8

18.4

6.8

48.6

14.1

ND

28

27.3

32.3

1.5

ND

5

3

2

1

6

4

4

3

4

2

4

5

4

2

26

6

8

2

1

ND

5

3

32

5

ND

6

298

1207

2608

1794

2523

1348

574

3469

937

681

1241

2693

1318

444

219

33

158

226

ND

342

387

9346

61

ND

608

9

25

27

41

40

31

47

91

37

20

42

52

48

21

17

16

42

14

ND

26

20

26

1.9

ND

2.6

2

10

14

15

19

8

3

9

2

2

8

13

9

6

4

2

2

1

ND

5

10

16

1

ND

0

411

1171

2994

1875

2575

507

842

2345

912

520

1191

2288

2094

465

251

95

143

314

ND

601

617

7145

141

ND

610


research articles Madian and Regnier

name

Kininogen-1 precursor (Alpha-2-thiol proteinase inhibitor) Homo sapiens (Human) LBP_HUMAN Lipopolysaccharide-binding protein precursor (LBP) - Homo sapiens (Human) LYSC_HUMAN Lysozyme C precursor (EC 3.2.1.17) Homo sapiens (Human) PHLD1_HUMAN Phosphatidylinositol-glycan-specific phospholipase D 1 precursor Homo sapiens (Human) PCOC1_HUMAN Procollagen C-endopeptidase enhancer 1 precursor Homo sapiens (Human) PRG4_HUMAN Proteoglycan-4 precursor (Lubricin) (Human) TRFE_HUMAN Serotransferrin precursor (Transferrin) (Homo sapiens (Human) ALBU_HUMAN Serum albumin precursor - Homo sapiens (Human) PON1_HUMAN Serum paraoxonase/arylesterase 1 - Homo sapiens (Human) PON3_HUMAN Serum paraoxonase/lactonase 3 Homo sapiens (Human) TETN_HUMAN Tetranectin precursor (TN) 4-binding protein) - Homo sapiens (Human) PROS_HUMAN Vitamin K-dependent protein S precursor - Homo sapiens (Human) VTNC_HUMAN Vitronectin precursor (Serum-spreading factor) Homo sapiens (Human) TTHY_HUMAN Transthyretin precursor Homo sapiens (Human)

KNG1_HUMAN

accessions

Table 2. Continued donor 1

donor 2

donor 3

donor 4

2

15

8

37.5

5

12

32.2

14.4

ND

ND

13

17

9.2

24.7

5

12.7

ND

5

8.8

ND

3

26.4

1

2

6

2.5

ND

ND

76

419

1189

ND

59

1493

784

ND

889

450

245

209

87

ND

ND

22

23

ND

2.5

46

38

8.5

9.7

23

10

39

10

7.5

ND

5

12

ND

9

10

14

5

20

5

4

4

3

4

ND

787

1348

ND

122

1730

1204

123

725

299

218

614

228

115

23.8

23

22.9

5.9

2.5

47.3

24.8

8

9.2

36.5

2.6

20.3

14.6

23.3

11

2

2

3

4

1

3

2

8

2

1

2

10

5

180

988

569

63

56

882

1446

229

608

729

82

288

724

861

15

20

23

23

2.5

53

25

6.3

11

28

8.7

20

15

21

1

6

12

4

1

10

12

3

11

7

7

3

5

10

130

1053

866

197

128

1246

1070

120

790

324

243

385

322

452



research articles


research articles

Madian and Regnier

Table 3. List of the Proteins Identified Using MALDI-MS/MS (ABI 4800 plus) donor 1

accessions

name

P01009|A1AT_HUMAN

Alpha-1-antitrypsin precursor Homo sapiens (Human) P01023|A2MG_HUMAN Alpha-2-macroglobulin precursor (Alpha-2-M) - Homo sapiens (Human) P01008|ANT3_HUMAN Antithrombin-III precursor (ATIII) Homo sapiens (Human) P02647|APOA1_HUMAN Apolipoprotein A-I precursor (Apo-AI) Homo sapiens (Human) P04114|APOB_HUMAN Apolipoprotein B-100 precursor (Apo B-100) Homo sapiens (Human) P02654|APOC1_HUMAN Apolipoprotein C-I precursor (Apo-CI) (ApoC-I) - Homo sapiens (Human) P02649|APOE_HUMAN Apolipoprotein E precursor (Apo-E) - Homo sapiens (Human) P04003|C4BP_HUMAN C4b-binding protein alpha chain precursor (C4bp) Homo sapiens (Human) P00450|CERU_HUMAN Ceruloplasmin precursorHomo sapiens (Human) P10909|CLUS_HUMAN Clusterin precursor Homo sapiens (Human) P00736|C1R_HUMAN Complement C1r subcomponent precursor Homo sapiens (Human) P01024|CO3_HUMAN Complement C3 precursor - Homo sapiens (Human) P0C0L5|CO4B_HUMAN; Complement C4-B precursor P0C0L4|CO4A_HUMAN Homo sapiens (Human) P02671|FIBA_HUMAN Fibrinogen alpha chain precursor Homo sapiens (Human) P02675|FIBB_HUMAN Fibrinogen beta chain precursor Homo sapiens (Human) P02679|FIBG_HUMAN Fibrinogen gamma chain precursor - Homo sapiens (Human) P02751|FINC_HUMAN Fibronectin precursor (FN) Homo sapiens (Human) P68871|HBB_HUMAN Hemoglobin subunit beta Homo sapiens (Human) P02790|HEMO_HUMAN Hemopexin precursor - Homo sapiens (Human) P01876|IGHA1_HUMAN Ig alpha-1 chain C region - Homo sapiens (Human) P01877|IGHA2_HUMAN; Ig alpha-2 chain C region - Homo P01876|IGHA1_HUMAN sapiens (Human); Homo sapiens (Human) P01857|IGHG1_HUMAN Ig gamma-1 chain C region - Homo sapiens (Human) P01859|IGHG2_HUMAN Ig gamma-2 chain C region - Homo sapiens (Human) P01861|IGHG4_HUMAN Ig gamma-4 chain C region - Homo sapiens (Human) P01842|LAC_HUMAN Ig lambda chain C regions - Homo sapiens (Human) P01871|MUC_HUMAN Ig mu chain C region - Homo sapiens (Human) P02787|TRFE_HUMAN Serotransferrin precursor (Transferrin) Homo sapiens (Human) P02768|ALBU_HUMAN Serum albumin precursor - Homo sapiens (Human) P27169|PON1_HUMAN Serum paraoxonase/arylesterase 1 Homo sapiens (Human) P07225|PROS_HUMAN Vitamin K-dependent protein S precursor - Homo sapiens (Human) P04004|VTNC_HUMAN Vitronectin precursor (Serum-spreading factor) Homo sapiens (Human)

11.72

1

2

3.349

1

2

8.005

4

8

17.3

9

18

23.71

3

6.24

13.15

3

6

44.57

5

10

52.06

6

11.7

10.96

3

7.73

7.364

1

2

24.1

3

4

13.25

2

4

27.44

2

4.37

44.48

2

5.36

21.27

4

9.61

26.97

6

12.9

5.446

2

4.44

10.99

2

4.08

21.38

6

14.9

21.38

4

11

17.73

3

6

23.55

6

10.8

17.14

7

14.7

21.89

8

19.7

12.67

3

6.98

18.86

3

6.88

40.88

23

44.4

41.8

23

50.4

58.04

20

55.7

63.75

27

66

55.63

12

26.3

64.9

15

30

33.91

22

52.6

47.49

47

103

51.7

4

7.82

27.21

2

5.22

22.94

5

10.9

37.66

9

17.5

ND

ND

ND

ND

ND

ND

10

3

6

7.941

2

3.52

39.7

6

12

44.24

5

10

36.2

5

10

32.82

4

8.02

ND

ND

ND

ND

ND

ND

14.29

1

2

14.29

1

2

22.69

6

12

19.16

6

12

ND

ND

ND

10.74

3

5.53

30.54

6

12.4

27.42

8

14.2

28.45

4

8

25.63

4

8

6.657

2

5.15

20.71

5

10

23.85

5

11.7

26.57

6

12.3

indicators of oxidative stress in coronary heart disease,35 and (iv) readily oxidized to the level that it causes atherosclerosis and thrombosis36-38 along with changes in blood rheology.36,39 At least in the case of atherosclerosis, some of the same metalloproteins associated with the disease state are also seen in normal male subjects, albeit not at the same concentration. Unsequestered metals play a major role in protein oxidation through the Fenton reaction as well, especially in 1338


donor 2

number of unique number of unique sequence peptides assigned ProteinPilot sequence peptides assigned ProteinPilot coverage to the protein score coverage to the protein score

diseases that reduce the concentration of proteins that chelate metals. Friedreich’s Ataxia is a classic case in which production of the mitochondrial protein frataxin declines with disease progression. The function of frataxin is to sequester Fe2+ in mitochrondria. As its concentration declines with disease progression, Fe2+ concentration increases in mitochondria and ROS production rises sharply as a result of the Fenton reaction, causing serious neurological problems similar to muscular dystrophy.

research articles

Profiling Carbonylated Proteins in Human Plasma Table 3. Continued donor 3

accessions P01009|A1AT_HUMAN

name

Alpha-1-antitrypsin precursor Homo sapiens (Human) P01023|A2MG_HUMAN Alpha-2-macroglobulin precursor (Alpha-2-M) - Homo sapiens (Human) P01008|ANT3_HUMAN Antithrombin-III precursor (ATIII) Homo sapiens (Human) P02647|APOA1_HUMAN Apolipoprotein A-I precursor (Apo-AI) Homo sapiens (Human) P04114|APOB_HUMAN Apolipoprotein B-100 precursor (Apo B-100) Homo sapiens (Human) P02654|APOC1_HUMAN Apolipoprotein C-I precursor (Apo-CI) (ApoC-I) - Homo sapiens (Human) P02649|APOE_HUMAN Apolipoprotein E precursor (Apo-E) - Homo sapiens (Human) P04003|C4BP_HUMAN C4b-binding protein alpha chain precursor (C4bp) Homo sapiens (Human) P00450|CERU_HUMAN Ceruloplasmin precursorHomo sapiens (Human) P10909|CLUS_HUMAN Clusterin precursor Homo sapiens (Human) P00736|C1R_HUMAN Complement C1r subcomponent precursor Homo sapiens (Human) P01024|CO3_HUMAN Complement C3 precursor - Homo sapiens (Human) P0C0L5|CO4B_HUMAN; Complement C4-B precursor P0C0L4|CO4A_HUMAN Homo sapiens (Human) P02671|FIBA_HUMAN Fibrinogen alpha chain precursor Homo sapiens (Human) P02675|FIBB_HUMAN Fibrinogen beta chain precursor Homo sapiens (Human) P02679|FIBG_HUMAN Fibrinogen gamma chain precursor - Homo sapiens (Human) P02751|FINC_HUMAN Fibronectin precursor (FN) Homo sapiens (Human) P68871|HBB_HUMAN Hemoglobin subunit beta Homo sapiens (Human) P02790|HEMO_HUMAN Hemopexin precursor - Homo sapiens (Human) P01876|IGHA1_HUMAN Ig alpha-1 chain C region - Homo sapiens (Human) P01877|IGHA2_HUMAN; Ig alpha-2 chain C region - Homo P01876|IGHA1_HUMAN sapiens (Human); Homo sapiens (Human) P01857|IGHG1_HUMAN Ig gamma-1 chain C region - Homo sapiens (Human) P01859|IGHG2_HUMAN Ig gamma-2 chain C region - Homo sapiens (Human) P01861|IGHG4_HUMAN Ig gamma-4 chain C region - Homo sapiens (Human) P01842|LAC_HUMAN Ig lambda chain C regions - Homo sapiens (Human) P01871|MUC_HUMAN Ig mu chain C region - Homo sapiens (Human) P02787|TRFE_HUMAN Serotransferrin precursor (Transferrin) Homo sapiens (Human) P02768|ALBU_HUMAN Serum albumin precursor - Homo sapiens (Human) P27169|PON1_HUMAN Serum paraoxonase/arylesterase 1 Homo sapiens (Human) P07225|PROS_HUMAN Vitamin K-dependent protein S precursor - Homo sapiens (Human) P04004|VTNC_HUMAN Vitronectin precursor (Serum-spreading factor) Homo sapiens (Human)

donor 4

number of unique number of unique sequence peptides assigned ProteinPilot sequence peptides assigned ProteinPilot coverage to the protein score coverage to the protein score 10.5

2

4

3.35

2

2

19.3

14

29.02

10

4

7.5

20.5

3

6

12.5

2

4

62.5

7

14.33

50.9

6

12

12.5

3

6.48

13

4

8

13.3

2

4

32.5

3

4

45.1

6

12

28.7

2

4

23.3

5

10.35

33.8

7

15

14.3

3

5.7

13.9

2

4

30.5

4

11.35

24.3

4

11

19.4

4

8.2

27.2

5

12

25.2

11

25.16

19.7

12

21

17.2

4

8.13

20.1

3

6.3

49.1

37

85.8

39.1

20

45

70.9

28

74.05

62.7

29

69

77.9

20

43.09

63.8

14

30

28

49

105.6

40.6

47

100

27.2

2

4

ND

ND

ND

29.9

4

9.22

32.5

7

13

20.4

2

4.16

33.4

5

9.7

ND

ND

ND

35.3

6

9.7

44.2

7

15.38

50

8

15

ND

ND

ND

38.7

9

17

37

5

12

47.7

6

15

33.3

2

4

14.3

6

2

39.9

7

15.53

27.3

5

11

14.6

2

4

21.5

2

4

32.8

13

27.75

48.4

10

20

21.7

4

6

35.2

5

10

7.84

3

6.33

16.1

3

6.4

26.8

10

21.44

27.6

7

16

A series of other variables contribute to the presence of oxidized proteins in the plasma proteome as well. One is the mechanism by which proteins from organs and cells enter the plasma proteome and the location at which oxidation occurs. There is no evidence that oxidized proteins enter plasma by excretion. But cells die and lyse. Chronic oxidative stress can be fatal to cells through the accumulation of protein oxidation products that trigger either apoptosis or necrosis. As cells die and lyse, soluble proteins are apparently released into the

circulatory system. Analysis of plasma containing these proteins in essence allows a partial autopsy of these recently lysed cells. This means that protein oxidation can occur in a biological compartment (organ or cells) other than plasma and be released into the plasma proteome. Looking for these oxidized proteins will be a powerful tool in the study of neurological diseases resulting in a high incidence of cell death. The location of proteins relative to sources of oxidative stress can also play a role in their tendency to be oxidized. The Journal of Proteome Research • Vol. 9, No. 3, 2010 1339

research articles

Madian and Regnier

Figure 4. Annotation of proteins identified according to their tissues of origin. Proteins were searched in the human protein atlas database and their degree of expression (low, medium, strong) was plotted against tissue of origin.

clearest case is membrane proteins. As noted above, fragments of fatty acids resulting from lipid peroxidation can add to lysine, cysteine, or histidine residues on proteins through a Michael addition mechanism. Integral membrane proteins and proteins in close proximity to membranes are the most likely to undergo this type of adduct formation. Proximity may also play a role 1340


in the oxidation of proteins in the circulatory system. The largest number of oxidative modifications in a plasma protein was found on apolipoprotein B-100. This protein plays a major role in oxidative modification of LDL and atherosclerosis.40 R-1Antitrypsin was also noted to be readily oxidized and is a component of LDL. This protein is another part of the complex

research articles


Figure 5. Total number of modifications identified and the corresponding oxidative modifications. Table 4. List of the Proteins Identified in the Pooled Sample with at Least One Oxidation Site along with Oxidation Sitesa protein description

Alpha-1-antitrypsin precursor - Homo sapiens (Human) Alpha-2-HS-glycoprotein precursor (Fetuin-A) Homo sapiens (Human) Antithrombin-III precursor (ATIII) - Homo sapiens (Human) Apolipoprotein A-I precursor (Apo-AI) - Homo sapiens (Human) Apolipoprotein A-II precursor (Apo-AII) - Homo sapiens (Human) Apolipoprotein B-100 precursor (Apo B-100) Homo sapiens (Human) Apolipoprotein C-I precursor (Apo-CI) (ApoC-I) - Homo sapiens (Human) Apolipoprotein C-III precursor (Apo-CIII) (ApoC-III) - Homo sapiens (Human) Apolipoprotein E precursor (Apo-E) - Homo sapiens (Human) C4b-binding protein alpha chain precursor (C4bp) - Homo sapiens (Human) Cadherin-5 precursor - Homo sapiens (Human) Clusterin precursor Homo sapiens (Human) Coagulation factor V precursor (Activated protein C cofactor) Homo sapiens (Human) Complement C1q subcomponent subunit B precursor - Homo sapiens (Human)

oxidative modifications 16

M398,16M382, 15F376 16 M321,15F320, 1T158, 3R159 23

K182 M110, 6W74, 6W96,9W96,16M136,9W74, L48, 2Y44

16 22

12

N73,

11

D97,15F95

16

M3280, 13P3281, 16M1266, 21K3229, 18K3234, 16M1881, 11D1880, M1189, 16M2526, 22L1060, 16M901, 16M2597, 16M812, 16M495, 16 M499, 18K305, 19K314, 16M2042,18K766,23K2147 7 W67, 6W67, 9W67, 16M64, 8W67, 15F68 16

7

W85, 6W85, 7W62, 8W62, 9W62, 10W62, 6W62, 6W74, 9W62,11D65, Y73, 9W74 14 P30 16 M249, 6W163, 11D243, 22L555, 4C468, 22L466, 5C468, 2Y470, 9 W163, 1T592, 1T419 16 M396 1 T93, 17K94 20 K2132 2

16

M147

a 1, Biotinylated oxidized threonine; 2, tyrosine hydroxylation; 3, biotinylated oxidized arginine; 4, cysteic acid (sulfonic acid); 5, sulfenic acid; 6, dihydroxytryptophan; 7, kynurenin; 8, hydroxykynurenin; 9, 2,4,5,6,7 hydroxylation of tryptophan; 10, oxolactone; 11, aspartic acid hydroxylation; 12, asparagines hydroxylation; 13, proline hydroxylation; 14, biotinylated oxidized proline; 15, phenylalanine hydroxylation; 16, methionine oxidation (sulphoxide); 17, biotinylated oxidized lysine; 18, biotinylation of amadori adduct; 19, biotinylation of 3 deoxyglucosone adduct; 20, biotinylation of glyoxal adduct; 21, biotinylation of methylglyoxal adduct; 22, hydroxy leucine; 23, biotinylation of 2- hydroxy nonenal adduct.

that accumulates on arterial walls and contributes to early stage atherosclerosis.41 This study shows that proteins associated with heart disease are being produced in high abundance in normal individuals in the 32-36 year age group.

Although oxidation of advanced glycation end (AGE) products has been widely reported in aging and various disease states, numerous instances of AGE oxidation were found in this study of normal individuals. In view of the fact that glycation Journal of Proteome Research • Vol. 9, No. 3, 2010 1341

research articles of a protein seems to have relatively little impact on biological activity, it is interesting that glycated proteins are being oxidized. The resulting carbonylated proteins would seem to be far more dangerous to cells than the glycated starting material. Protein glycation along with the subsequent AGE oxidation is an issue of major importance in diabetes. The analytical protocol described here for the analysis of AGE oxidation products may be of value in the study of OS in diabetes. Tracing oxidized proteins back to their tissue of origin using the Human Protein Atlas showed that kidney, liver and soft tissues contributed more proteins than other organs. This observation is supported by literature showing that high levels of oxidative stress occur in the liver42 and kidney.43 This may allow the identification of organs experiencing high oxidative stress through the analysis of blood.

Conclusions On the basis of the work presented in this paper, it can be concluded that approximately 0.2% of the protein in the plasma of normal, 32-36 year old human male subjects can be selected by avidin affinity chromatography after biotinylation of carbonyl groups with biotin hydrazide and that this protein fraction has a series of characteristic, oxidative stress related features. One feature is that roughly 25 different types of oxidative stress induced post-translational modifications (OSi∼PTMs) are present in normal plasma. Multiple forms of OSi∼PTM were observed with the same amino acid species in addition to the same OSi∼PTM occurring on multiple amino acids. A second feature is that more than half of the amino acid species used in protein synthesis were modified by some type of post-translational modification and that these modifications did not appear to have involved enzymatic catalysis. A third prominent feature was that a protein could be oxidized at multiple sites in an unrelated, nonstoichiometric pattern, thus creating multiple oxidized isoforms of a protein. A further conclusion is that some of the proteins reported to be a factor in several oxidative stress related diseases are present in the plasma of these normal male subjects. Whether these individuals will later develop these disease is unknown. If they do, oxidized proteins might be useful as early indicators of impending health problems. Finally, it is concluded that the probability a plasma protein will be oxidized depends on a series of variable. Among these variables are (i) structural features such as the presence of conjugated metals and the position of specific amino acids in the structure of the protein, (ii) the degree of oxidative stress in the compartment where the protein originated before being released into the circulatory system, (iii) the extent of AGE generation in cells and plasma, and (iv) the concentration of ROS at locations in the circulatory system such as with atherosclerosis. The fact that plasma proteins can be used to assess localized oxidative stress might make them useful as clinical diagnostic agents.

Acknowledgment. We gratefully acknowledge support of this work by the National Cancer Institute (grant number 1U24CA126480-01) and National Institute of Aging (grant number 5R01AG025362-02). We also thank Drs. Susan Fisher, Michael Alvarado, Penelope Drake and Amanda Romani for supplying us with the human plasma samples. Supporting Information Available: Supporting info1: a list of proteins and their corresponding peptides identified 1342


Madian and Regnier by the LTQ orbitrap XL; Supporting info2: a list of proteins and their corresponding peptides identified by the MALDI/TOF/ TOF (ABI 4800). This material is available free of charge via the Internet at http://pubs.acs.org.

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