ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 305, No. 2, September, pp. 595-599, 1999 Mass Spectrometric Identification of Modifications to Human Serum Albumin Treated with Hydrogen Peroxide Jeffrey W. Finch,’ Rosalie K. Crouch,* Daniel R. Knapp,t and Kevin L. Scheyt® Departments of *Cell and Molecular Pharmacology and "Ophthalmology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina 29425 Received April 12, 1993 Oxidized amino acid residues in human serum albumin ‘exposed to hydrogen peroxide have been identified in tryptic peptides using liquid secondary ion mass spec- trometry. Sites of oxidation identified include Cys34, ‘Met123, Met298, Met446, and Met548. The extent of oxidation varied with location in the protein sequence, suggesting a relationship between oxidation and protein three-dimensional structure. The data presented here for human serum albumin demonstrate the utility of mass spectrometry in studying protein alterations. This type of information may be helpful in assessing the ability of proteins to act as antioxidants in biological systems which are subject to oxidant stress as in cases of inflammation and in the aging process. < 1003 Academie Pres, ne Albumin has been proposed to serve as an antioxidant in biological systems subject to attack of oxygen radicals from species such as hydrogen peroxide (H,0;) (1). Al- bumin has been shown to reduce oxidative damage in cul: tured porcine aortic endothelial cells and lung fibroblasts (2) and to inhibit lipid autoxidation (3) and protect against inactivation of 1-antiproteinase by hydrogen peroxide (4) in serum. Plasma level concentrations of albumin have been shown to be oxidized by reactants such as H,0, gen- erated from the xanthine/xanthine oxidase system (5), suggesting that levels of albumin in vivo are appropriate for its role as an antioxidant. In the rat and human cornea, albumin has been shown to be oxidized by H,0, and a role for albumin as an antioxidant was proposed for this system as well (6). The latter finding is of clinical im- portance concerning the widespread use of contact lens disinfectant solutions which contain 3% H.0, (7). Pro- teolytic susceptibility of albumin has been demonstrated * Current address: JBOL USA, Inc., 11 Dearborn Ra., Peabody, MA, 01960, * To whom correspondence should be addressed. 003-9861 /88 $5.00 Copyright 19 by Academic Press, Inc. All rights of reproduction in any form reserved, to increase upon exposure to H,O», and increased turnover may be a generalized biological response to oxidative pro- tein damage (8, 9). Other studies have suggested that pro- tein oxidation and the ability to scavenge oxygen radicals may play important roles in the aging process (10). In assessing antioxidant activity of albumin, Radi et al, (11) studied the reactivity of the free thiol group on bovine serum albumin (BSA)’ with HO. and peroxyni- trite anion, using 5,5°-dithiobis(2-nitrobenzoic acid) (DTNB) and absorption spectroscopy to quantitate free thiol content. A 44% loss in DTNB-reactive BSA sulf- hydryls was observed under mild oxidizing conditions (0.6 mM HO», 20 min incubation) but they were recoverable by arsenite reduction, suggesting the formation of bio- logically reversible sulfenic acid (RSOH). However, xan- thine oxidase-derived 0; and H,0, caused nonrecoverable oxidation of BSA thiols for incubation periods longer than 20 min, indicating the formation of higher, biologically irreversible thiol oxidation states RSO,H (sulfinic acid) and RSO;H (sulfonic acid). ‘The purpose of this work was to confirm that Cys34 of human serum albumin (HSA) is oxidized by H,02, to identify other sites of oxidation, and to assess the extent of oxidation for these sites. Mass spectrometric (MS) analysis of tryptic peptides from oxidized HSA was used to locate each modified residue and estimate the extent of oxidation. The observed protein oxidation provides further evidence that HSA can indeed act as a sacrificial antioxidant. Furthermore, this study demonstrates the utility of mass spectrometry for identifying amino acid modifications in large proteins. * Abbreviations used: HSA, human serum albumin; BSA, bovine serum albumin; LSIMS, liquid secondary ion mass spectrometry; ESI, elec: trospray ionization; DTNB, 8,5'-dithiobia(2-nitrobenzoic acid); DTT. dithiothreitol; Tris, eris(hydroxymethyl)aminomethane: EDTA, ethyl enediaminetetraacetic acid; ‘TRA, trifluoracetic acid: CNBr, cyanogen bromide, 598« * 24919 cal ag id ie wee . 7 PREOHVK a Cosmin Te we te ae ay oe alo de cin oe . =, fomsznt ane ane MLVLIAEROVLOOC Preonwe Coyne ait Re, Abundance soo 10b0 1550 bo dbp 2000 300 acon we . 1. LSIMS data of HPLC fractions which contained « peptide corresponding to sequence 21-41, which has a free eysteine at position 54, (a) Fraction isolated {com tryptie digest of native HSA, retention time = 61.5 min. (b) Fraction isolated from tryptic digest of HSA treated with 1.5% hydrogen peroxide, retention time = 60.5 min, MATERIALS AND METHODS Fatty acid-free HSA was purchased from Sigma Chemical Co. (St Louis, MO) and used without further purification. H,O, solutions (1.5% in water) were made fresh daily from a 30% solution (Sigma) and stored in the dark at 4°C. Sequence grade trypsin was obtained from Boehringer ‘Mannheim (Indianapolis, IN). lodoucetic acid (Sigma) was recrystallized from hexane and stored in the dark at -15°C. The peptide TPTPTQFLC was synthesized by the MUSC Protein Chemiatry Facility and used 10 test the experimental protocol. All other chemicals and solvents were ‘obtained from commercial sources and used without further purification, Reaction of synthetic test peptide with H,0,. The test peptide (50 nmol), purified by reversed-phase HPLC, was incubated in @ 15% H:0, solution (80 yl) at 37°C for 30 min. The reaction produets were purified by HPLC and analyzed by liquid secondary ion mass spectrometry (LSIMS). The fraction containing the oxidized peptide was incubated at 87°C in NH,HCO, buffer (0.1 M, pH 8.0) containing DTT (0.08 ) for 4 h and the reaction mixture was analyzed again using HPLC and LSIMS. Preparation of oxidized HSA. HSA (30 nmol) was disolved in 1.5% 1,0, solution (500 ul) and incubated at 37°C for 4 h. The H,O, was then removed by diluting the sample with NHyHCO, baffer (14 mal, 0.1 M, pH 80) and using a Centriprep 30 concentrator tube (Amicon Di- FINCH BT AL, vision, W. R. Grace Co-Conn., Beverly, MA) for ultrafiltration. Ultra: filtration was accomplished by centrifuging the concentrator tube at 2500 rpm for 20 min, After the dilution/centrifugation process was re- peated three times, the solution containing the oxidized HSA was con- centrated to 0.5 ml by centrifugetion at 2500 rpm for 12 min. After Iyophilizing the sample, 5% acetic acid (200 pl) was added to remove the bicarbonate. The aaimple was then lyophilized once again in prep: aration for enzymatic digestion ‘Tryptic digestion. Native or oxidized HSA was diseolved in 200 yl of denaturing solvent (6M guanidine HCI, 0.3 M Tris HCI, 2 mM EDTA, pH 80), purged with argon, and incubated for 2 h at 37°C. The protein ‘was reduced with DTT (4 mg, 16 h) and carboxymethylated with io doacetic acid (2.5 mg, 1h at 0°C). Mereaptoethanol (2 pl) was added to the solution and the protein was desalted by diluting with NHJHCO, Dutfer (0.1 M, pH 8.0) and repeating the ultrafiltration procedure outlined above. The protein was digested with trypsin (wubstrate/enzyme ratio (of 100/1, w/w) in NHJHCOs butter (0.1 M, pH 8.0) for 14h at 37°C and the digestion was terminated by freezing and lyophilizing the solution. ‘The bicarbonate was removed by adding 5% acetic acid (200 ul) and |yophilizing the solution. Prior to HPLC analysis, the digest was recon stituted in 5% acetic acid (200 ul) and sonicated for 16 min. HPLC. Digents of native and oxidized HSA were separated by re- versed-phase HPLC using s 4.6 x 250-mm Vydac! Mg Peri win oidaed Mat ox Ppt win oxo Oye time (i) FIG. 2. Comparison of reversed-phase HPLC of tryptic digests from (a) native HSA and (b) HSA exposed to 1.5% H,0; for 4h in the region of 44-70 min. M, peptide in an HPLC fraction containing a Met; C, peptide containing Cys34,OXIDATION OF HUMAN SERUM ALBUMIN 597 TABLE 1 ‘Summary of Data from HPLC and MS Analysis of Tryptie Peptides Containing Oxidation Sites Retention HSA Peptide ‘ime Extent of digest sequence® (nin) MH® (m/s)? oxidation’ Cys34 Native al eis 2491.3 Oxidized 21-41 os, 24912 21-ttox 605 24813 1 Mews? Native 52.98 a7 1487 Osidized = . Not detected Meti23 Native 115-136 515 2651.1 Oxidized 115-136 515 2651.5 (w) 115-1360" 2667.4 (3) 2 Met208 Native va7-a13 32 Oxidized 287.313 2975.1 (w) 281-3130x 2991.2 (8) Mevi29 Native 24-397 1780.1 «w Oxidized = = Not detected Menai, Native 445-466, o 267 Oxidized 445-468, 6 2676.2 (w) 445-4660% 585 2602.4 Metsas 546-557 B15 1342.7 (3) 546-557 ~ 546-5570, a 1358.6 (@) 5 + ox—peptde containing an oxidized Cys or Met residue, * s-atrong ion intensity, w—weak ion intensity. Extent of oxidation of residue rated on a scale of 1 to 5 (1 lowest, 5—highest), based on the relative (M + H)' ion intensities of the unmodified ‘and oxidized peptides from HSA treated with HO, “Detected by electrospray MS, but not by LSIMS. CA) with 214-nm detection, The column was held at 100% solvent A (H,0 + 0.1% TRA) for 5 min and then ramped with a linear gredient to 70% solvent B (CH,CN + 0.084% TFA) at 90 min using a flow rate of 0.5 ml/min. Fractions were collected, lyophilized, and stored frozen nays, prior to MS Mase spectrometry. LSIMS analysis was carried out with a JEOL. HX110/HX110 four sector tandem mass spectrometer (JEOL Ltd. ‘Tokyo, Japan). An LSIMS cesium ion gun providing 15:keV Cs ions ‘was used for sample jonization, with MSI operating with a resolution 10 9000. Lyophilized HPLC fractions were sonicated in 12% acetic acid (15) prior to MS analysis, and 1 ofthis solution was mixed on the probe tip with a liquid matrix (1 l) of I:t (v/v) glyeerol/nitrobenzyl Alcohol. Electrospray ionization MS was carried out using a Nermag 'R30-10 triple quadrupole (Delsi Inc., Houston, TX) equipped with a ‘custom electrospray interface (12). Samples were dissolved in a solution of 50% methanol/44% H,0/6% acetic acid prior to ESI MS analysis, ‘Manual edman degradation. A modified procedure of Tarr (13) was used to obtain partial sequence information of tryptic peptides. A solution ‘of 50% aqueous pyridine (10 ul) was added to the lyophilized peptide, lowed by the addition of 5% phenvlisothioeyanate in pyridine (20 sl. the solution was incubated at 37°C for S min, the sample was lyophilized, and TPA (25 gl) was added. After incubating the mixture for 5 min, the sample was lyophilized and then reconstituted in 10% ‘TRA (5-1 sl) LSIMS analysis was earried out after each evele. RESULTS To test the experimental protocol of reducing oxidized protein with DTT prior to enzymatic digestion, a syn- thetic model peptide, TPTPTQFLC, was oxidized with HO, and subsequently treated with DTT. The native peptide, purified by HPLC, had a retention time (R,) of 37 min and MS analysis gave an (M + H)* ion signal at m/z 1007.3," which matched the calculated mass of the peptide. Treatment for 30 min with 1.5% H,0, yielded a new HPLC peak (R, = 33 min) with only a very small peak at 37 min. The new peak had an (M + H)* signal at m/z 1055.3, indicating oxidation of Cys to cysteic acid (Am = 48) within 30 min. The location of the oxidized residue was further confirmed by the tandem mass spec- trometry (data not shown). Treating the oxidized peptide with an excess of DTT for 4 h did not alter the cysteic acid moiety; therefore it seemed likely that oxidized HSA could be reduced and carboxymethylated without reducing any Cys34 residues which had been oxidized to cysteic aci Fifty-seven fractions were collected after HPLC anal- ysis of a tryptic digest of native HSA and each fraction was analyzed by ESI MS and LSIMS. A mass spectral map of the HSA protein rapidly confirmed 72% of the sequence. A peptide in fraction 54 (retention time = 61. min) gave an (M + H)' ion signal at mass 2491.3 corre- sponding to the amino acid sequence 21-41, containing “ Al m/z values listed correspond to the mass of the monaisotopic (°C) molecular in.TABLE It FINCH AL Cys34, which was carboxymethylated. The LSIMS mass spectrum of this peptide is shown in Fig. 1a. HPLC analysis of the tryptic digest of H,O,-treated HSA showed noticeable differences after R, 45 min, in- cluding the appearance of new peaks as illustrated in Fig. 2. MS analysis of the fractions revealed that oxidation of } Cys34 occurred as well as oxidation of Met residues. In | the case of oxidized HSA, peptides for both the unmodified and oxidized Cys34 residue were detected for sequence 21-41, The mass spectrum for the HPLC fraction con- taining the oxidized peptide is shown in Fig. 1b; the (M + H)" ion, m/z 2481.3, matches the calculated mass for the peptide containing Cys34, which has been converted ls to eysteic acid (RSO}H). For oxidized HSA, only weak Zi/> > 2 & 3 | ion signals were detected for tryptic peptides containing | unmodified Met residues, with no ion signal for the se~ ‘quence 546-557 containing unmodified Met548. However, strong ion signals were observed for new peptides at lower R. (Fig. 2b), which gave (M + H)" ion signals correspond- ing to a mass shift of +16 over the peptides containing unmodified Met residues, indicating conversion of each Met to Met sulfoxide. ‘A summary of the reversed-phase HPLC and MS data for each peptide containing an oxidation site is shown in ‘Table I for both native and oxidized HSA cleaved with trypsin. The extent of oxidation for the peptides was es- timated on a scale of 1 to 5 (1—lowest, 5—highest), based on the relative (M + H)* ion intensities. In the case of Cys34, the ion signal of the oxidized peptide was approx- imately half the intensity of the peptide containing the unmodified residue (rating 1). In the case of Met548, the oxidized peptide gave a strong ion signal intensity, while the corresponding unmodified peptide was not detected; therefore a rating of 5 was assigned, denoting complete oxidation of this residue. Although ion-suppression effects are known to exist in LSIMS for mixtures of peptides (14), these results showed very distinct differences in the ion intensities, which suggests that the extent of oxidation for each residue varies depending upon location in the protein sequence. Relative peptide ion intensities were reproducible from two different HPLC runs where the same amount of digested protein was injected onto the column. Tandem MS was carried out to identify the oxidized peptides; however, only limited sequence information was obtained. This result was not unexpected since the masses of all but one of the oxidized peptides were in the range ‘of 2200-3000, above the upper mass limit where current tandem MS methods normally yield complete sequence information (15, 16). Therefore, manual Edman degra- dation followed by LSIMS analysis was carried out on HPLC fractions containing the oxidized peptides to con- firm their identity. Results from these experiments are summarized in Table II. The first three N-terminal amino acid residues were confirmed for each peptide containing an oxidized residue, except sequence 287-313, in which AA lost R ne BI v 1662 969 rd Badman cycle 99.1 2208.8 roa. ue 22919 21978 (mie) 996 -99.1 147 1 2nd Redman cycle i 2388.8 2296.9 Peptides Containing an Oxidized Residue Das 128 am ony /MS Analysi Ist Bem MH (rj) 25646 2904.2 idman Degeadati Manual Me ria) 2067.4 586 29012 292.4 2481.3 Sequence ine sulfoxide, C*—eysteie acid SHCIARVENDE M*PADLPSLAA DFVESK RM*PCAEDYLS EK & 445-466 ‘Teyptie peptideOXIDATION OF HUMAN § the first two were confirmed. In two cases, peptide se- quences 445-466 and 546-557, loss of the Met sulfoxide residue (Am = —147) was observed from the N-terminus of the peptide. DISCUSSION For HSA exposed to 1.5% HO» for 4 h, five sites of oxidation have been identified using reversed-phase HPLC and MS: Cys34, Met123, Met298, Met446, and Met548. It is possible that Met87 and Met329 are also oxidized; however, only small ion signals were observed for peptides 82-93 and 324-337 in the digest of native albumin and no corresponding oxidized peptides were ob- served in the digest of oxidized albumin. While oxidation of the sulfhydryl group on the test peptide TPTPTQFLC was complete within 30 min, only partial oxidation of Cys34 in HSA to cysteic acid (RSO,H) occurred for a 4-h exposure time. This result supports the theory that the Cys34 residue is located within a cleft of the protein which may be hydrophobic in nature, limiting its reactivity (17). The theory of a free sulfhydryl group in albumin located in a cleft is also supported by the work of Takabayashi and co-workers (18), who discovered that oxidation of the free cysteine in bovine albumin by DTNB enhanced binding of fatty acids, and that binding of fatty acids also enhanced oxidation of the sulfhydryl. Further- more, based on crystallography data of HSA recorded at the 2.8-A level, Carter and He (19) have recently reported that Cys34 is partially protected from the solvent due to its location in a turn between two of the helical subdo- mains. In contrast to the limited oxidation of Cys34, HPLC and MS data indicate extensive oxidation of the Met res- idues. The Met548 residue was completely oxidized, which suggests that the C-terminus is highly exposed. Variations in the extent of oxidation of the other Met residues could be related to the three-dimensional structure of the pro- tein. The data also suggest that Met residues play an im- portant role in the antioxidant properties of HSA, par ticularly under conditions of high oxidant stress. In fact, the methionine residues in HSA may play an even more important role than Cys34 in neutralizing HO, and ox- ygen radical intermediates. In conclusion, the combination of HPLC and mass spectrometry has been demonstrated to be effective in locating oxidation sites in HSA after exposure to HsO.. ‘This approach should prove useful for examining oxida- tive modifications of HSA in the cornea or proteins in the lens under conditions of high oxidative stress. The oxidation of lens proteins has been shown to accompany the development of senile nuclear cataract (20, 21). More 599) $ERUM ALBUMIN generally, the application of mass spectrometry to identify protein alterations in tissues exposed to high oxidant stress could be useful for damage assessment as well as for identifying proteins which can act as sacrificial an- tioxidants. Finally, protein oxidation is often implicated in the aging process and the methods described here could be of importance in understanding structural modifica- tions involved in aging. ACKNOWLEDGMENTS We thank K. Thornburg for his technical assistance and Dr. S. Ro: senaweig for the gift of the synthetic test peptide, This work was sup- ported hy DIR 8804502 from the National Science Foundation and E (05757, EY-04939, EY.08259 from the National Institutes of Health J.W F. was supported by a postdoctoral training grant, HLD7260, from the National Institutes of Health REFERENCES 1. Halliwell, B. (1988) Biochem. Pharmacol. 87, 569-871 2. Bishop, C."T., Mieza, Z., Crapo, J. D. and Freeman B. A. (1985) In Vitro Cell Dev. Biol 21, 229° 236. 8. Stocks, J, Gutteridge, M. C., Sharp, RJ, and Dormandy, T. L. (1974) Clin, Sei, 47, 225-233, 4. Wasil, B., Halliwell, B,, Hutchison, D.C. S., and Baum, H. (1987) Biochem. J. 243, 219-223 5. Radi, R., Bush, K, M,, Cosgrove, T. P., and Freeman, B.A. (1991) Arch, Biochem. Biophys. 286, 117-125. 6. Zhu, Land Crouch, R. K. (1992) Cornea 11, 561-572, 7. Kreranoski. J. Z., and Houlsby, RD. (1988) J. Arm. Optom. Assoc. 59, 198-197. 8. Davies, K. J. A. (1986) J. Free Radicals Biol. Med. 2, 156-173. 9. Davies, K. J. A. 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