Radiolytic Modification of Sulfur-Containing Amino Acid Residues in

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Anal. Chem. 2005, 77, 2437-2449

Radiolytic Modification of Sulfur-Containing Amino Acid Residues in Model Peptides: Fundamental Studies for Protein Footprinting Guozhong Xu†,‡ and Mark R. Chance*,†,‡,§

Center for Synchrotron Biosciences, Department of Physiology & Biophysics and Biochemistry, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, New York 10461

Protein footprinting based on hydroxyl radical-mediated modification and quantitative mass spectroscopic analysis is a proven technique for examining protein structure, protein-ligand interactions, and structural allostery upon protein complex formation. The reactive and solventaccessible amino acid side chains function as structural probes; however, correct structural analysis depends on the identification and quantification of all the relevant oxidative modifications within the protein sequence. Sulfur-containing amino acids are oxidized readily and the mechanisms of oxidation are particularly complex, although they have been extensively investigated by EPR and other spectroscopic methods. Here we have undertaken a detailed mass spectrometry study (using electrospray ionization mass spectrometry and tandem mass spectrometry) of model peptides containing cysteine (CysSH), cystine (disulfide bonded Cys), and methionine after oxidation using γ-rays or synchrotron X-rays and have compared these results to those expected from oxidation mechanisms proposed in the literature. Radiolysis of cysteine leads to cysteine sulfonic acid (+48 Da mass shift) and cystine as the major products; other minor products including cysteine sulfinic acid (+32 Da mass shift) and serine (-16 Da mass shift) are observed. Radiolysis of cystine results in the oxidative opening of the disulfide bond and generation of cysteine sulfonic acid and sulfinic acid; however, the rate of oxidation is significantly less than that for cysteine. Radiolysis of methionine gives rise primarily to methionine sulfoxide (+16 Da mass shift); this can be further oxidized to methionine sulfone (+32 Da mass shift) or another product with a -32 Da mass shift likely due to aldehyde formation at the γ-carbon. Due to the high reactivity of sulfur-containing amino acids, the extent of oxidation is easily influenced by secondary oxidation events or the presence of redox reagents used in standard proteolytic digestions; when these are accounted for, a reactivity * To whom correspondence should be addressed. Tel.: (718) 430 4136. Fax: (718) 430 8587. E-mail: [email protected]. † Center for Synchrotron Biosciences. ‡ Department of Physiology & Biophysics. § Department of Biochemistry. 10.1021/ac0484629 CCC: $30.25 Published on Web 03/03/2005

© 2005 American Chemical Society

order of cysteine > methionine ∼ tryptophan > cystine is observed. Protein footprinting combining hydroxyl radical-mediated modification and mass spectrometry can sensitively analyze solvent accessibility of side chain residues within the structure of proteins and their macromolecular assemblies.1-10 Experimentally, protein solutions are exposed to conditions that generate hydroxyl radicals, either by radiolysis,5,9 chemical means,6,7 or electrical discharge,8,11 the solvent-accessible and reactive amino acid side chains are oxidized and the modified samples are analyzed by mass spectrometry, typically after proteolysis, and the sites and extents of oxidation are determined. The solvent accessibility of reactive side chains is quantitatively evaluated by examining the oxidation rates of individual peptides, and the sites of oxidation are determined using tandem MS,5,12,13 while the binding interfaces of protein complexes and structural allostery upon binding are mapped through examining the changes in oxidation rates of the target peptides as a function of ligand binding.1-4,10,14,15 The side chains of reactive amino acid residues, including Cys, Met, Phe, Tyr, Trp, His, Leu, Pro, Arg, Lys, Glu, and Asp,13,16,17 as well as Val and Ile (unpublished data) can be used as structural (1) Kiselar, J. G.’ et al. Mol. Cell. Proteomics 2003, 2 (10), 1120-32. (2) Kiselar, J. G.; et al. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (7), 3942-7. (3) Liu, R.; et al. Biochemistry 2003, 42 (43), 12447-54. (4) Guan, J. Q.; Almo, S. C.; Chance, M. R. Acc. Chem. Res. 2004, 37 (4), 2219. (5) Guan, J. Q.; et al. Biochemistry 2002, 41 (18), 5765-75. (6) Sharp, J. S.; Becker, J. M.; Hettich, R. L. Anal. Biochem. 2003, 313 (2), 216-25. (7) Sharp, J. S.; Becker, J. M.; Hettich, R. L. Analysis of protein solvent accessible surfaces by photochemical oxidation and mass spectrometry. Anal. Chem. 2004, 76 (3), 672-83. (8) Wong, J. W.; Maleknia, S. D.; Downard, K. M. Anal. Chem. 2003, 75 (7), 1557-63. (9) Maleknia, S. D.; et al. Anal. Biochem. 2001, 289 (2), 103-15. (10) Gupta, S.; et al. Mol. Cell Proteomics 2004, 3 (10), 950-9. (11) Maleknia, S. D.; Chance, M. R.; Downard, K. M. Rapid Commun. Mass. Spectrom. 1999, 13 (23), 2352-8. (12) Kiselar, J. G.; et al. Int. J. Radiat. Biol. 2002, 78 (2), 101-14. (13) Maleknia, S. D.; Brenowitz, M.; Chance, M. R. Anal. Chem. 1999, 71 (18), 3965-73. (14) Guan, J. Q.; et al. Biochemistry 2003, 42 (41), 11992-2000. (15) Rashidzadeh, H.; et al. Biochemistry 2003, 42 (13), 3655-65. (16) Xu, G., Takamoto, K.; Chance, M. R. Anal. Chem. 2003, 75 (24), 69957007. (17) Xu, G.; Chance, M. R. Anal. Chem. 2004, 76 (5), 1213-21.

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probes in footprinting experiments. Understanding the fundamental chemistry of hydroxyl radical-mediated modifications of amino acid side chains is a prerequisite for successfully analyzing protein oxidations to carry out footprinting research. For aliphatic and aromatic amino acid residues,18-21 oxidation generally leads to modified peptides with mass additions of +16 or +14 Da due to the formation of hydroxyl or carbonyl groups in the amino acid side chains. However, Arg gives rise to a characteristic product with a -43 Da mass change as a result of oxidative elimination of the guanidino group,16 while Asp and Glu yield characteristic products with a -30 Da mass change as a result of oxidative decarboxylation of the side-chain carboxyl groups.17 His produces multiple products with mass changes of -22, -10, +5, and +16 Da as consequence of ring opening and addition.16 Due to the complexity and specific nature of the side-chain oxidation products observed, understanding the various modification products of all side chains is essential to properly analyzing oxidation footprinting data. The sulfur-containing amino acid residues, Cys and Met, along with disulfide have the most reactive side chains of the amino acids.22 They are valuable structural probes both for their reactivity and for their roles in protein structure and function. Cysteine is important for the catalytic activity of many enzymes23 and is the second most conserved residue in the primary structure of homologous proteins after Trp.24 Disulfide bridges are also very important in protein structure, and the classical view that they have been added during evolution for purposes of stability is maturing, as more examples of cleavage of disulfide bonds being important regulatory switches has emerged.25 Methionine, a hydrophobic residue with a sulfur-containing side chain, generally prefers to be buried in protein hydrophobic cores. It shows minimal conservation on the surface of proteins with the conspicuous exception that it is conserved in known ligand binding sites.26 Therefore, conservation of Met on the protein surface can imply a prospective site for a macromolecular interface.26 Cys and Met are both highly reactive with respect to measured rates of reaction with hydroxyl radicals,22 we have found that Met is easily oxidized in many proteins,1-4,12,14 and we have observed Cys oxidation in proteins after radiolysis as well.10 The high reactivity of Cys and Met residues requires a full understanding of their oxidation chemistry and careful control of experimental conditions as well as sample handling before final analysis. Radiolytic oxidation of methionine,27-33 cysteine,27,34-43 and (18) Garrison, W. M. Chem. Rev. 1987, 87, 381-98. (19) Davies, M. J.; Dean, R. T. Radical-mediated protein oxidation: from chemistry to medicine; Oxford University Press: Oxford, U.K., 1997. (20) Hawkins, C. L.; Davies, M. J. Biochim. Biophys. Acta. 2001, 1504 (2-3), 196-219. (21) Stadtman, E. R. Methods Enzymol. 1995, 258, 379-93. (22) Buxton, G. V.; et al. J. Phys. Chem. Ref. Data 1988, 17, 513-886. (23) Claiborne, A.; et al. Adv. Protein Chem. 2001, 58, 215-76. (24) van Vlijmen, H. W.; et al. J. Mol. Biol. 2004, 335 (4), 1083-92. (25) Hogg, P. J. Trends Biochem. Sci. 2003, 28 (4), 210-4. (26) Ma, B.; et al. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (10), 5772-7. (27) Prutz, W. A.; et al. Int. J. Radiat. Biol. 1989, 55 (4), 539-56. (28) Hiller, K.; et al. J. Am. Chem. Soc. 1981, 103 (10), 2734-43. (29) Schoneich, C.; et al. J. Am. Chem. Soc. 2003, 125 (45), 13700-13. (30) Vogt, W. Free Radical Biol. Med. 1995, 18 (1), 93-105. (31) Bobrowski, K.; Holcman, J. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 1987, 52 (1), 139-44. (32) Miller, B. L.; Williams, T. D.; Scho ¨neich, C. J. Am. Chem. Soc. 1996, 118 (45), 11014-25. (33) Lu, C.; Yao, S.; Lin, N. Biochim. Biophys. Acta. 2001, 1525 (1-2), 89-96.

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cystine27,44-50 in peptides and proteins has been the subject of intense study, and the chemistry of sulfur-centered radicals has also been reviewed.51,52 From these studies it is clear that the oxidation of sulfur-containing amino acids is very complicated and leads to multiple products; in addition, the proposed mechanisms of oxidation are not without some controversy. These studies have been carried out primarily by UV spectroscopy and EPR on the sulfur-centered free radicals or intermediates. Footprinting emphasizes the final oxidation products instead of intermediates, and the intense EPR and UV/visible signals detected in intermediate radicals do not necessarily reflect the levels of relevant products. Recently, mass spectrometry has been used to examine the oxidation products of Cys residue oxidized by hydrogen peroxide.53,54 In this study, we have used electrospray ionization mass spectroscopy (ESI-MS) and tandem mass spectrometry (MS/MS) to investigate the radiolytic modification of sulfur-containing residues and disulfides, emphasizing the detection of final products to improve oxidative footprinting approaches. EXPERIMENTAL PROCEDURES Materials. Radiolytic modifications of sulfur-containing residues were investigated using both small peptides for simple and direct observation of modified products and long peptides to further confirm the modifications and identify any characteristic fragmentation pattern of the products by tandem mass spectrometry. The peptides containing methionine sulfoxide (Met(O)) were used to identify any further modification of methionine sulfoxide. The sequences were selected to simplify the peptide oxidation and emphasize the modification of the target residues for study. Peptides Gly-Cys-Gly (GCG), Gly-Met-Gly (GMG), (Gly-Cys)2 ((GC)2, dimer of dipeptide H-Gly-Cys-OH connected by a disulfide bond), Phe-Thr-Leu-Cys-Phe-Arg-NH2 (FTLCFR-NH2), Ala-Asn-ProAsp-Cys-Lys-Thr-Ile-Leu-Lys-Ala-Leu-Gly-Pro-Ala-Ala-Thr (ANPDCKTILKALGPAAT), Gln-Asn-Cys-Pro-Arg-Gly-NH2 (QNCPRGNH2), Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-Ala-CysNH2 disulfide (RSSCFGGRIDRIGAC-NH2 internal disulfide), CysIle-Ile-Arg-Asn-Cys-Pro-Lys-Gly-NH2 disulfide (CIIRNCPKG-NH2 internal disulfide), His-Asp-Met-Asn-Lys-Val-Leu-Asp-Leu (anti(34) Lal, M. Radiat. Phys. Chem. 1994, 43 (6), 595-611. (35) Sevilla, M. D.; Becker, D.; Yan, M. Int. J. Radiat. Biol. 1990, 57 (1), 6581. (36) Hoffman, M. Z.; Hayon, E. J. Phys. Chem. 1973, 77 (8), 990-6. (37) Al-Thannon, A.; Peterson, R. M.; Trumbore, C. N. J. Phys. Chem. 1968, 72 (7), 2395-400. (38) Al-Thannon, A. A.; et al. Int. J. Radiat. Phys. Chem. 1974. 6 (4), 233-48. (39) Dewey, D. L.; Beecher, J. Nature 1965, 206 (991), 1369-70. (40) Markakis, P.; Tappel, A. L. J. Am. Chem. Soc. 1960, 82 (7), 1613-6. (41) Becker, D.; et al. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 1988, 53 (5), 767-86. (42) Wardman, P.; von Sonntag, C. Methods Enzymol. 1995, 251, 31-45. (43) Schoneich, C. Methods Enzymol. 1995, 251, 45-55. (44) Purdie, J. W. Can. J. Chem. 1971, 49, 725-30. (45) Owen, T. C.; Wilbraham, A. C. J. Am. Chem. Soc. 1969, 91 (12), 3365-71. (46) Owen, T. C.; et al. J. Am. Chem. Soc. 1968, 90 (1), 196-200. (47) Purdie, J. W. J. Am. Chem. Soc. 1967, 89 (2), 226-8. (48) Fang, X.; et al. Int. J. Radiat. Biol. 1995, 68 (4), 459-66. (49) Purdie, J. W. Radiat. Res. 1971, 48, 474-83. (50) Bonifacic, M.; Asmus, K. D. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 1984, 46 (1), 35-45. (51) Chatgilialoglu, C.; Asmus, K.-D. Sulfur-Centered Reactive Intermediates in Chemistry and Biology; Plenum Press: New York, 1990. (52) Alfassi, Z. B. S-Centered Radicals; John Wiley & Sons: New York, 1999. (53) Willett, W. S.; Copley, S. D. Chem. Biol. 1996, 3 (10), 851-7. (54) Deutsch, J. C.; Santhosh-Kumar, C. R.; Kolhouse, J. F. J. Chromatogr., A 1999, 862 (2), 161-8.

flammin-2: HDMNKVLDL), Leu-Trp-Met-Arg-Phe-Ala-NH2 (LWMRFA-NH2), Tyr-Gly-Gly-Phe-Met(O)-Arg (YGGFM(O)R, methionine sulfoxide), and Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-LeuMet(O) (GSNKGAIIGLM(O), methionine sulfoxide) were purchased from Bachem California Inc. (Torrance, CA). All peptides had a purity of at least 90% and were used without further purification. B & J Brand high-purity acetonitrile (MeCN) was purchased from Honeywell International Inc. (Muskegon, MI). Solutions of peptides at a concentration of 20-70 µM were prepared in Nanopure water purified by a Millipore Ultrapure Water System. Preparation of Met(O)-Antiflammin-2. In a 0.2-mL Eppendorf tube, 10 µL of 1.0 mM antiflammin-2 (HDMNKVLDL) was mixed with 1.0 µL of 30% H2O2, kept at 4°C for 2 days, and then dried in a Speed-Vac. The oxidized peptide was analyzed by ESIMS and MS/MS, and more than 98% of peptide was converted to Met(O)-antiflammin-2 based on the observed +16 Da mass addition due to the oxidation of Met to methionine sulfoxide. The preoxidized peptide was used to identify the radiolytic oxidation product in antiflamin-2 due to the further oxidation of methionine sulfoxide. Exposure of Peptides to Radiation. Peptide solutions were exposed to either synchrotron X-rays at the X-28C beamline of NSLS at Brookhaven National Laboratory or a cesium-137 γ-ray source (1300 rads/min) at the Albert Einstein College of Medicine. Radiolysis using synchrotron X-rays and low-flux γ-rays generates similar modifications of the biological samples, except on different time scales, i.e., milliseconds for synchrotron X-rays and minutes for low-flux γ-rays. The 10-µL aliquots of 20-70 µM peptide solutions in a 0.2-mL Eppendorf tube were exposed to synchrotron X-rays using a stand and shutter for 10-30 ms at beam currents ranging from 180 to 250 mA in accordance with our established protocols.5,55 For samples exposed to the cesium-137 source, 40 µL of peptide solution in a 1.5-mL Eppendorf tube was exposed for 0.5-30 min. The exposure time was also controlled electronically.16 After irradiation, all samples were stored at -20 °C before analysis. Mass Spectrometric Analysis of Peptides. Most samples were analyzed directly by ESI-MS without chromatographic separation. The peptide concentrations were adjusted to 10 µM with acetonitrile and were infused directly into the ESI-MS at a flow rate of 3 µL/min. Mass spectra were acquired on a Finnigan LCQ quadrupole ion trap mass spectrometer (Finnigan Corp., San Jose, CA) with a mass accuracy of (0.2 amu and unit resolution. The needle voltage was set at 4.5 kV. The instrument was tuned using the known masses of the unmodified peptides. All spectra were recorded in the profile mode as indicated in the results. The sites of amino acid oxidation were identified by the collisioninduced dissociation (CID) MS/MS spectra for selected ions, and the amounts of unmodified and modified peptides are estimated according to the peak intensity of mass spectral signals. RESULTS AND DISCUSSION Radiolytic Oxidation of Cysteine. ANPDCKTILKALGPAAT. This peptide, which lacks other highly reactive residues (e.g., Met, Trp, Tyr, and Phe), was chosen to emphasize the modification of Cys and identify any specific fragmentation pattern of the products. Oxidation of Cys residues may generate sulfenic acid, sulfinic acid, (55) Ralston, C. Y.; et al. Methods Enzymol. 2000, 317, 353-68.

and sulfonic acid through the addition of one, two, or three oxygen atoms at the sulfur atom (corresponding to +16, +32, and +48 Da mass additions). Oxidation can also result in cystine through formation of a disulfide bond. The above Cys-containing peptide was irradiated by synchrotron X-rays for 15 ms, and its positive ESI-MS spectrum is displayed in Figure 1A. The species seen at m/z 1683.7 corresponds to the molecular ion of unmodified peptide, and the species observed at m/z 1705.8 and 1721.7 are the original peptide with sodium and potassium ion adducts. The peak observed at m/z 1731.6 corresponds to the primary oxidation product, which exhibits a mass addition of +48 Da compared to the original peptide. In addition, multiple oxidation products with mass changes of -16, +16, +32, +64, and +80 Da relative to the unmodified peptide are observed at m/z values of 1667.7, 1699.6, 1715.6, 1747.6, and 1763.6, respectively. Tandem mass spectra were acquired to determine the modification sites for each oxidation product using CID. The Biemann nomenclature system was used for naming the peptide fragments.56 Cleavage of the peptide chain in the collision cell can occur at the CR-C, C-N, or N-CR bonds within the peptide, and these cleavage events yield six types of fragments that are labeled an, bn, and cn, when a positive charge is retained on the N-terminal side of the peptide; and xn, yn, and zn, when a positive charge is retained on the C-terminal side, with the n value denoting the size of the fragment in amino acids. The MS/MS spectrum of the ion at m/z 1731.6 (corresponding to the species with +48 Da mass shift), shown in Figure 1B, indicates that the Cys residue within the peptide is specifically oxidized to cysteine sulfonic acid. Unmodified fragment ions corresponding to y8, y9, y10, y11, and y12 are seen at m/z values of 728.3, 841.4, 954.5, 1055.5, and 1183.5, respectively, and no modified ions related to these fragments are present. This indicates that 12 residues on the C-terminal side of Cys do not give rise to the +48 Da mass shift. The fragment ions observed at m/z values of 1334.5 and 1546.5 belong to ions y13 + 48 and y15 + 48, respectively, while no unmodified y13 and y15 ions are observed. The presence of y12 and y13 + 48 and absence of y12 + 48 and y13 indicate the +48 Da modification is located specifically at Cys. The b-ion series fragments are consistent with Cys modification; ions observed at m/z 677.2, 891.3, 1004.3, 1203.5, 1316.5, 1373.5, 1541.3, 1612.5, correspond to b6 + 48, b8 + 48, b9 + 48, b11 + 48, b12 + 48, b13 + 48, b15 + 48, and b16 + 48, respectively. The internal fragment ions PDCKTILKA+48, PDCKTILKAL+48, PDCKTILKALG+48, and PDCKTILKALGPAA+48 are seen at m/z 1018.4, 1131.5, 1188.5, and 1427.6, respectively. These are consistent with the +48 Da oxidation product arising from Cys. The fragment ion at m/z 1649.7 is 82 Da lower than the parent ion. It is a fragment ion characteristic of Cys + 48 oxidation resulting from the loss of a neutral molecular H2SO3 from the side chain of cysteine sulfonic acid.57 The Cys residue is also oxidized to cysteine sulfinic acid. This is supported by the MS/MS spectrum of the product observed at m/z 858.4 corresponding to the doubly charged +32 Da species (the singly charged species is observed at m/z 1715.6) as shown in Figure 1C. The intense peak at m/z 825.3 is due to the loss of 66 Da from the parent ion. The 66 Da mass reduction is consistent (56) Biemann, K, Methods Enzymol. 1990, 886-7. (57) Wang, Y.; et al. J. Am. Soc. Mass Spectrom. 2004, 15 (5), 697-702.

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Figure 1. (A) Positive ESI-MS spectrum of peptide ANPDCKTILKALGPAAT exposed to synchrotron X-rays for 15 ms. (B) MS/MS spectrum of (MH + 48)+ at m/z 1731.6. (C) MS/MS spectrum of (MH2 + 32)2+ at m/z 858.4. (D) MS/MS spectrum of (MH - 16)+ at m/z 1667.7.

with the loss of a neutral H2SO2 from the oxidized cysteine sulfinic acid side chain.57 The peak observed at m/z 398.1 corresponds to the b4 ion, and no b4 + 32 ion is observed. The ions observed at m/z 533.1, 857.5, 875.2, 1116.5, and 1300.4 correspond to b5 + 32, b8 + 32 - H2O, b8 + 32, b10 + 32, and b12 + 32, respectively, while there are no corresponding unmodified b fragments. The presence of b4 and b5 + 32, the loss of H2SO2 fragments, and the absence of b4 + 32 and b5 indicate that the +32 Da derives from cysteine. The conclusion is further supported by an examination of the y-ion series. Ions y4, y5, y6, y7, y8 y9, y10, y11, and y12 are seen at m/z values of 358.9, 416.1, 529.3, 600.2, 723.8, 841.5, 954.5, 1055.5, and 1183.5, respectively, while there are no corresponding +32 Da fragment ions present. Meanwhile, the peaks at m/z 592.4, 659.7, and 765.8 belong to y122+, (y13 + 32) 2+, and (y15 + 32) 2+, respectively. The weak ion observed at m/z 1667.7 corresponds to an oxidation product with -16 Da mass shift that is also derived from Cys. The MS/MS spectrum of m/z 1667.7 is shown in Figure 1D. The unmodified y series ions y8, y9, y10, and y12 are present at m/z 728.4, 841.3, 954.5, and 1183.5, respectively, while there are no corresponding modified fragment ions. The peaks at m/z 1270.5 and 1482.5 correspond to y13 - 16 and y15 - 16, respectively, while 2440 Analytical Chemistry, Vol. 77, No. 8, April 15, 2005

no corresponding unmodified b13 and b15 fragment ions are seen. The presence of y12 and y13 - 16 ions and absence of y12 - 16 and y13 indicate that the -16 Da mass loss takes place at Cys. The result is consistent with the observed modified b series ions at m/z 827.3, 940.5, 1068.3, 1139.6, 1252.5, 1309.5, 1477.5, and 1548.5 corresponding to b8 - 16, b9 - 16, b10 - 16, b11 - 16, b12 - 16, b13 - 16, b15 - 16, and b16 - 16, respectively. The -16 Da product possibly results from conversion of Cys to Ser due to the loss of thiol and formation of hydroxyl group at side chain. The minor oxidation products giving rise to a +16 Da mass shift seen at m/z 1699.6 and +64 Da mass shift seen at m/z 1747.6 were also identified by tandem mass spectrometry (data not shown). MS/MS spectra of the singly charged ion of the +64 Da product (m/z 1747.6) and its corresponding doubly charged ion (at m/z 874.4) clearly indicate that the +16 Da mass addition originates from the oxidation of multiple residues that may include Lys, Thr, Ile, Leu, Pro, and Cys. GCG. The oxidation of the Cys side chain and ionization efficiency of the relevant products are demonstrated more clearly in the simple tripeptide GCG. The peptide was exposed to γ-rays for 6 min, and the positive and negative ESI-MS spectra are presented in Figure 2A and B. All the products were examined

Figure 2. (A) Positive and (B) negative ESI-MS spectra of peptide GCG exposed to γ-rays for 6 min. (C) Positive ESI-MS spectrum of peptide FTLCFR-NH2 exposed to synchrotron X-rays for 15 ms. (D) Positive ESI-MS spectrum of peptide QNCPRG-NH2 exposed to γ-rays for 4 min.

by tandem mass spectrometry (spectra not shown). Presumptive disulfide product was generated, as represented by the ions observed at m/z 469.0 in the positive ESI spectrum (Figure 2A) and at m/z 467.0 in the negative ESI spectrum (Figure 2B). A trace amount of product with -16 Da mass shift is also observed at m/z 220.0 in positive ESI-MS and m/z 218.0 in negative ESIMS. Several oxidation products are observed in the negative ESIMS spectrum but not in the positive spectrum. In the negative ESI-MS spectrum, the strong peak at m/z 282.0 shows a +48 Da mass shift from the original peptide, the major product is likely generated from the oxidation of sulfhydryl in Cys to sulfonic acid. The weak peak at m/z 266.0, with +32 Da mass shift from the unmodified peptide, likely results from the oxidation of the sulfhydryl in Cys to sulfinic acid. The other three weak peaks at m/z 280.0, 298.0, and 314.0, corresponding to +46, +64, and +80 Da mass shifts, respectively, are due to the further oxidation of disulfide, which will be discussed below. The peak m/z 200.0 corresponding to -34 Da mass loss is possibly due to the cleavage of H2S in the instrumental interface. The formation of negatively

charged sulfonic and sulfinic acid groups in these oxidation products decreases the ionization efficiency in positive electrospray; this results in their being observed only in negative ESI. FTLCFR-NH2. The typical oxidation of Cys is also demonstrated in this peptide, which contains multiple highly reactive aromatic residues. This peptide was exposed to synchrotron X-rays for 15 ms, and the positive ESI-MS spectrum is shown in Figure 2C. Oxidation products with mass changes of -16, +16, +32, +48, and +64 Da are found at m/z 769.5, 801.3, 817.3, 833.3, and 849.3, respectively. The shoulder observed at m/z 784.4 to the left of m/z 785.4 likely corresponds to the disulfide dimer. All of the products are identified by tandem mass spectrometry (data not shown). The +48 and +32 Da products likely originate from the formation of sulfonic and sulfinic acid at the Cys side chain, while the +16 Da products result from the oxidation of Phe and Leu residues based on the tandem mass spectrum. QNCPRG-NH2. This is another peptide used to demonstrate the possible sequence dependence of Cys oxidation. The peptide was exposed to γ-rays for 4 min, and the positive ESI-MS spectrum Analytical Chemistry, Vol. 77, No. 8, April 15, 2005

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Scheme 1. Radiolysis of Water by Ionizing Radiation

is shown in Figure 2D. Similar to the above peptide, the +48 Da products assigned to Cys-sulfonic acid is the principal product. Mechanism of Cysteine Oxidation. The radiolysis chemistry of Cys in aqueous solutions has been subjected to extensive study for decades.27,34-40,58,59 A number of intermediate radical species have been identified, and their structure and reactivity have been studied using spin-trap EPR, optical, and conductivity measurements in the presence and absence of oxygen.34,35,38,41,60 A variety of mechanisms have been proposed to account for the various products generated after the formation of the initial radical species as well as the role of oxygen in mediating product formation. Despite this extensive study, the reaction mechanisms for the diverse array of products are not fully understood. Radiolysis of water generates hydroxyl radicals (HO•), solvated electrons (eaq-), and hydronium ions (H3O+);58,61 the latter ions can react with solvated electrons to produce hydrogen radicals (H•) and water.58,61,62 In the presence of oxygen, eaq- and H• can react with O2 leading to superoxide radical anion (O2•-), hydroperoxyl radical (HO2•), and finally hydrogen peroxide (H2O2),61 as shown in Scheme 1. Hydroxyl radicals also self-quench by dimerization to form H2O2.61 Therefore, the oxygen atoms of H2O2 originate from both water and dissolved O2. Scheme 2 displays the likely interactions of these primary radical species with sulfhydryl species and the comprehensive mechanisms of oxidation based on previous studies. Although some of the reaction intermediates may be open to question, the scheme is a reasonable framework for interpreting the results seen here. The initial step in Cys oxidation is the formation of the thiyl radical species (RS•) via hydrogen abstraction from sulfhydryl (RSH) by HO•, H•, and possibly by O2•- and other oxygen reactive species.34,38,63 RS• undergoes two competitive rapid reactions with molecular oxygen (O2) or thiolate (RS-) to generate a thiyl peroxyl radical (RSOO•) or a conjugated disulfide radical anion (RSSR)•- dependent the pH, oxygen, and thiol concentrations.42,43 RS• reacts rapidly (diffusion controlled) yet reversibly with a molecular oxygen (O2) to generate a thiyl peroxyl radical (RSOO•), (58) Armstrong, D. A. Application of pulse radiolysis for the study of short-lived sulfur species, In Sulfur-centered Reactive Intermediates in Chemistry and Biology; Chatgilialoglu, C., Asmus, K.-D., Eds.; Plenum Press: New York, 1990. (59) Owen, T. C.; Brown, M. T. J. Org. Chem. 1969, 34 (4), 1161-2. (60) Asmus, K. D. Methods Enzymol. 1990, 186, 168-80. (61) Draganic, I. G.; Draganic, Z. D. The radiation chemistry of water; Academic Press: New York, 1971. (62) Klapper, M. H.; Faraggi, M. Q. Rev. Biophys. 1979, 12 (4), 465-519. (63) Winterbourn, C. C.; Metodiewa, D. Free Radical Biol. Med. 1999, 27 (34), 322-8.

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Scheme 2. Radiolytic Oxidation of Cysteine Residue

with rate constants of >109 M-1 s-1 for the forward and 105-106 M-1 s-1 for the reverse reactions,64 with an equilibrium constant of 103-104. Based on Scheme 2, RSOO• is seen to be the precursor of sulfenic, sulfinic, sulfonic acid, disulfide, and serine products.34 Sulfenic acid RSOH and sulfinic acid RSO2H are both generated from RSOO•. Thiyl hydroperoxide (RSOOH) is possibly present as an intermediate in the formation of RSOH, RSO2H, and disulfide RSSR. Sulfenic acid (RSOH) is typically unstable and highly reactive;65-67 thus, it is barely observable in these experiments. RSOH can form sulfinic acid (RSO2H) and sulfonic acid (RSO3H) through reaction with H2O2, O2•-, O2, or other oxidizing reagents.47 RSOH can also be converted to disulfides by reacting with another thiol group.38 Sulfinic acid RSO2H may be generated either from RSOO• via hydrogen abstraction from another thiol group (RSH), reaction with hydroperoxyl HO2•, isomerization from thiyl hydroperoxide RSOOH, or further oxidation from sulfenic acid RSOH by H2O2. The sulfonic acid (+48 Da) product presumably dominates in these studies since it requires the participation of no additional radical species, which are present at very low steadystate concentrations, and only requires rearrangements and further reaction with molecular oxygen or hydrogen peroxide H2O2. RSOO• can easily isomerize to the fully sulfur-centered, thermodynamically favored sulfonyl radical RSO2•.35 RSO2• may subsequently react with another oxygen molecule and give rise to sulfonyl peroxyl radical,35 RSO2OO•. The highly reactive intermediate converts ultimately to sulfonic acid RSO3H via interacting with a water molecule.68 Oxygen is necessary for the radiolytic generation of sulfonic acid.47 The further oxidation of sulfenic and sulfinic acids also leads to the sulfonic acid. In the absence of air, sulfinic acid is produced in moderate yield and disulfide in high yield.49 Disulfide is one of the primary products for radiolysis of small cysteine-containing peptides in both aerobic and anaerobic conditions.34,39 There are several avenues to the formation of disulfide. Thiyl radical can react rapidly with thiolate RS- to form disulfide (64) Asmus, K. D. Sulfur-centered reactive intermediates as studied by radiation chemical and complementary techniques. In S-Centered Radicals; Alfassi, Z. B., Ed.; John Wiley & Sons: New York., 1999. (65) Allison, W. S. Acc. Chem. Res. 1976, 9, 293-9. (66) Carballal, S.; et al. Biochemistry 2003, 42 (33), 9906-14. (67) Davis, F. A.; Billmers, R. L. J. Am. Chem. Soc. 1981, 103 (23), 7016-8. (68) Zhang, X.; et al. J. Phy. Chem. 1994, 98, 6541-7.

radical anion (RSSR)•-,58 which reacts with a molecular O2 giving rise to molecular disulfide RSSR and superoxide radical anion O2•-.34 The reactions of RS• with RS- and O2 are two competitive reactions dependent upon pH, thiol, and O2 concentrations.42 Higher pH results in the formation of the disulfide anion by increasing the concentration of thiolate ion; higher thiol concentration and low O2 concentration also favor the formation of disulfide and disfavor formation of oxygen-addition oxidation products. The second approach to generate disulfide is the combination of intermediate RSOOH or RSOH with a parent RSH via loss a H2O2 or water molecule.59 The third route is the combination of two RS• radicals in the absence of oxygen,58 and the probability is greatly reduced in air-saturated solutions because of favored reaction of RS• with O2 in higher concentration around 0.3 mM.49 The yield of disulfide is particularly high under anaerobic conditions due to a chain reaction that can be inhibited by O2. If there are more than one free cysteine- or cystinecontaining peptides in the solution, mixed disulfide R′SSR can also form via thiol-disulfide exchange, which takes place through displacement reaction of disulfide by RS• or RS-.64 The serine product corresponding to a -16 Da mass shift results from the generation of an alanyl radical R•; this carboncentered radical is likely derived from two separate routes. First, it can be generated directly from the original thiol through reaction with a hydrated electron eaq- and loss of SH-34 or reaction with a hydrogen radical H• and loss of H2S.36 Meanwhile, R• can be produced by elimination of neutral SO2 from thiyl peroxyl radical RSOO•. Similar to all other aliphatic hydrocarbon radicals, the alanyl radical R• reacts with O2 to give rise to a peroxyl radical ROO•, which leads to the formation of ROH. The mechanisms leading to the formation of sulfinic and sulfonic acids indicate that both products incorporate oxygen from water or dissolved O2. Our previous radiolysis studies of cysteinecontaining fibronectin peptide (RCDC) in water and 18O-labeled water also indicated the likely prevalence of the cysteine sulfinic and sulfonic acid products and demonstrated that both molecular oxygen and oxygen from water were incorporated in the oxidation products.13 These model peptide data confirm that these products are formed independent of the overall peptide sequence and indicate other possible products, such as serine and disulfide formation. We have also recently demonstrated that, for proteins, solvent-accessible Cys side chains oxidize primarily to the +32 and +48 Da cysteine sulfinic and sulfonic acids.10 Disulfide formation is less likely within proteins during radiolysis, because non-disulfide-bound cysteine residues are generally separated in space. However, disulfides can form during proteolysis and storage, because free cysteine residues are sensitive to oxidation from trace amounts of oxygen or H2O2 generated during sample irradiation, and mixed disulfides can be also formed by thioldisulfide exchange. Thus, care needs to be taken in sample handling after radiolysis exposure. Radiolytic Oxidation of Disulfide Bonds. (GC)2. This is a dimer of dipeptide H-Gly-Cys-OH connected by a disulfide bond. It was selected to allow simple analysis of the reaction products. The oxidation products are not observed in a positive electrospray mass spectrum as in the case of GCG. The negative ESI-MS spectrum of (GC)2 exposed to γ-rays for 8 min is presented at Figure 3A. The observed ion at m/z 353.0 is from the unmodified

peptide. Only trace amounts of possible +16 and +32 Da oxygenaddition products to the disulfide are observed as weak peaks at m/z 368.7 and 385.0. A weak peak seen at m/z 177.1 likely corresponds to the dipeptide GC. The major oxidation products appear at m/z 225.0, 223.0, and 208.9 with mass shifts of +48, +46, and +32 Da relative to GC. Weak ions observed at m/z 257.0 and 241.1 exhibit mass shifts of +80 and +64 Da relative to GC, respectively. The signals of m/z 177 for GC and m/z 208.9 for GC + 32 decrease as a function of irradiation time due to the secondary oxidation by dissolved O2 and other oxidative species such as H2O2 generated during radiolysis. Negative MS/MS of the GC + 48 product at m/z 225.1 was carried out; the CID data are shown in Figure 3B. The fragment ion seen at m/z 81.0 is likely HSO3-, and a complementary ion due to the loss of neutral H2SO3 is present at m/z 143.0. The two fragments at m/z 180.1 and 181.0 are likely due to the decarboxylation of C-terminal carboxyl group; these radiolysis products are well precedented.17 The data indicate the GC + 48 species results from the oxidative cleavage of disulfide bond in (GC)2 and conversion of sulfhydryl to sulfonic acid. Other products were also identified by tandem MS. The negative MS/MS spectrum of GC + 80 Da product at m/z 257.0 is shown in Figure 3C. The ions observed at m/z 81.0 and 175.0 are likely the fragment ion HSO3- and its complementary fragment GC + 80 - H2SO3. The ion observed at m/z 177.0 likely corresponds to GC. These data indicate the presence of the S-SO3H group, and the +80 Da product species is likely GCSSO3H, which is generated by oxidative cleavage of the S-C bond in (GC)2. The MS/MS spectrum of GC + 46 Da at m/z 223.0 also indicates the presence of a sulfonic acid group. It may be similar to the GC + 48 Da product but possibly with a carbon-carbon double bond between the R- and β-carbons in the oxidized Cys residue. The +32 Da product observed at m/z 209.0 is also similar to GC + 48 Da except that the Cys is oxidized to sulfinic acid instead of sulfonic acid, as suggested by MS/MS spectrum (not shown). The time course of oxidation of (GC)2 as a function of time is shown in Figure 3D. The amount of original peptide decreases exponentially with irradiation time. The GC + 32 Da increases up to 2 min and keeps at a relatively constant level of ∼10%, while minor products GC and GC + 80 Da are seen at very low levels. The major products GC + 48 and GC + 46 Da increase proportionally with irradiation time after an initial lag phase. The GC + 46 Da product could be the further oxidation product of GC + 48 Da, as result of hydrogen abstraction from the R-carbon, subsequent loss of another hydrogen from the β-carbon, and then formation of a carbon-carbon double bond. The hydrogen abstraction from the R-carbon is strongly affected by steric hindrance, so the +46 Da product is normally not observed in larger peptides, and is very weak even in the tripeptide GCG as shown in Figure 2B. RSSCFGGRIDRIGAC-NH2 (Internal Disulfide). This long peptide containing an internal disulfide bond was used to confirm the oxidation of cystine and demonstrate the fragmentation pattern of the major product. The peptide was exposed to synchrotron X-rays for 15 ms, and the positive ESI-MS spectrum exhibiting both singly and doubly charged signals is shown in Figure 4A. The peaks observed at m/z 1594.7 and 798.1 are the singly and Analytical Chemistry, Vol. 77, No. 8, April 15, 2005

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Figure 3. (A) Negative ESI-MS spectrum of peptide (GC)2 (dimer of GC connected by disulfide) exposed to γ-rays for 8 min. (B) Negative MS/MS spectrum of (GC + 48)- at m/z 225.0. (C) Negative MS/MS spectrum of (GC + 80)- at m/z 257.0. (D) Decay of unmodified (GC)2 and accumulation of different products shown as fraction of total amount of all species as a function of exposure time.

doubly charged ions corresponding to the unmodified peptide. A series of oxygen-addition oxidation products with multiples of +16 Da mass changes are found as both singly and doubly charged ions at m/z 1610.6, 806.0, 1626.6, 814.0, etc. The characteristic product due to the oxidative deguanidation of Arg is seen at m/z 1551.7 as a singly charged ion and at m/z 776.5 as a doubly charged ion.16 Of particular interest is a product at +98 Da mass shift observed at m/z 1592.6 as a singly charged ion and at m/z 846.9 as a doubly charged ion. Oxidative opening of the disulfide bond to generate two sulfonic acid groups in the two Cys residues would be consistent with this +98 Da mass shift product (+96 Da relative to the disulfide reduced peptide). The MS/MS spectrum of the ion observed at m/z 846.9 is presented in Figure 4B. The fragment ions are labeled based on the reduced form of the peptide. To simplify the labeling, the superscript star symbol * represents +48 Da mass increase from the corresponding unmodified fragment ion, while the double star ** represents a +96 Da mass increase. The ion observed at m/z 805.9 likely results from the loss of neutral H2SO3 (-82 Da) from the parent ion. The peak at m/z 311.1 is the unmodified b3 fragment. The peaks at m/z 482.1, 629.2, 899.3, 1012.3, and 1127.5 correspond to the singly charged modified fragment ions b*4, b*5, b*8, b*9, and b*10, respectively, with +48 Da mass additions compared to the unmodified fragment 2444 Analytical Chemistry, Vol. 77, No. 8, April 15, 2005

ions. The peaks at m/z 642.3, 698.8, 727.3, and 762.9 belong to the doubly charged modified fragment ions b*112+, b*122+, b*132+, and b*142+, respectively, with +48 Da mass additions. There are no unmodified fragment ions observed from b4 to b14. The presence of b3 and b4* and absence of b3* and b4 indicate that one of the +48 Da modifications in located at Cys-4. The presence of the b*142+ fragment indicates the another +48 Da modification is located at the C-terminal Cys residue. The conclusion is confirmed by the y-series fragment ions. The peaks observed at m/z 566.2, 681.1, 950.3, 1007.5, 1064.4, and 1211.5 are modified fragment ions y*5, y*6, y*8, y*9, y*10, and y*11, respectively, that retain the +48 Da mass addition. The peaks at m/z 1362.3 and 1449.6 are y**12 and y**13 ions with +96 Da mass addition. No y*12 and y*13 ions with a +48 Da mass addition are observed. The presence of y*5-y*11 and y**12-y**13 ions and the absence of y*12-y*13 further confirms that +48 Da modifications at each of the two Cys residues. Mechanism of Disulfide Oxidation. Similar to single cysteine residues, the sulfur moiety in the disulfide bond is the principal target of attack resulting in the formation of thiyl radicals. Consequently, the radiolysis products are analogous to those resulting from the radiolysis of single cysteine residues in the presence of oxygen, with cysteine sulfonic acid CysSO3H and regenerated disulfides as the primary radiolytic products.46 The

Figure 4. (A) Positive ESI-MS spectrum of peptide RSSCFGGRIDRIGAC-NH2 (internal disulfide) exposed to synchrotron X-rays for 15 ms. (B) MS/MS spectrum of (MH2 + 98)2+ at m/z 846.9.

Scheme 3. Radiolytic Oxidation of Cystine

proposed mechanisms of disulfide bonds oxidation based on the literatures are displayed in Scheme 3. Hydroxyl radical OH•, hydrated electron eaq-, and hydrogen radical H• are all able to react rapidly with disulfide and generate RS• radicals under anaerobic conditions.49 However, under air-equilibrated solution conditions and micromolar sample concentrations, eaq- and H• react primarily with oxygen (which is present at 0.3 mM concentration49) to give superoxide or hydroperoxy radicals. Solvated electrons (eaq-) attack disulfide RSSR by electron attachment (rate constant ∼1010 M-1 s-1 )69 to give disulfide radical anions (RSSR)•- under anaerobic conditions.44,60 (RSSR)•- can be identified readily by optical or EPR spectroscopy.70 It reacts rapidly

with protons (rate constant ∼1010 M-1 s-1) to give rise to a transient protonated disulfide radical (RSS(H)R)•, which decomposes rapidly into free thiyl radical RS• and thiol components.71 Hydrogen radical H• may also react with RSSR to produce RS• and RSH.40,49 The newly born thiol subsequently reacts with sulfenic acid to produce a new disulfide. (RSSR)•- may also cleave its S-S bond to yield a thiyl radical RS• and a thiolate anion RSthrough a reversible reaction.58 In aerated solutions, these reactions may still occur to some extent since the reaction rates for eaq- with thiol and O2 are similar in magnitude; meanwhile, the (RSSR)•- may react with an O2 to regenerate RSSR and give rise a superoxide radical O2•-. Hydroxyl radical HO• attacks disulfide through two likely pathways. HO• may react with RSSR directly and lead to a thiyl radical RS• and sulfenic acid RSOH.47 HO• may abstract an electron from disulfide bond giving rise to a disulfide radical cation (RSSR)•+,58 which is stabilized by electron delocalization. Heterolysis of (RSSR)•+ produces a thiyl radical RS• and sulfenium cation RS+.45 RS+ may be neutralized by OH- to form sulfenic acid or absorb an O2 to form an sulfonium cation RSO2+, which combines with an OH- to become sulfonic acid RSO3H.46 The thiyl radical RS• and sulfenic acid RSOH produced by the reaction of RSSR with solvated electron eaq- or hydroxyl radical HO• are subject to further oxidation as described in the radiolysis of cysteine yielding sulfinic acid RSO2H, sulfonic acid RSO3H, disulfides, and other products. Radiolytic Oxidation of Methionine. GMG. The oxidation of Met residue can be demonstrated clearly in the simple tripeptide. It was exposed to γ-rays for 12 min, and the positive ESI-MS spectrum is shown in Figure 5A. The peak observed at m/z 264.0 corresponds to the unmodified peptide. The primary product is observed at m/z 280.0 with a +16 Da mass shift from the original peptide. Other minor products are present at m/z 296.0, 250.0, 248.0, 234.0, and 232.0 with mass shifts of +32, -14, -16, -30, and -32 Da, respectively, from the unmodified peptide. The major product at m/z 280.0 is presumed to result from the oxidation of Met to methionine sulfoxide Met(O). Tandem MS data are shown in Figure 5B. The ions observed at m/z 205.0 and 223.0 correspond to the b2 + 16 and y2 + 16 fragment ions, respectively. No b2 and y2 fragment ions are found. The presence of b2 + 16 and y2 + 16 and absence of b2 and y2 indicated the +16 Da modification is located at Met, which is oxidized to Met(O). Methionine sulfoxide can be easily recognized by its lowenergy CID spectrum through the unique elimination of methanesulfenic acid (CH3SOH, 64 Da) from the side chain of Met(O).72,73 This elimination can occur for both the original molecular ion and for sequence fragment ions. The peak at m/z 216.1 is assigned to the loss of 64 Da from the parent ion, and the peak at m/z 181.1 is possibly due to further loss of a H2O and a NH3 from the m/z 216.1 ion. Elimination from the modified b2 (69) Braams, R. Radiat. Res. 1966, 27, 319-29. (70) Gilbert, B. C. Structure and reaction mechanisms in sulfur-radical chemistry revealed by E.S.R. spectroscopy. In Sulfur-Centered Reactive Intermediates in Chemistry and Biology; Chatgilialoglu, C., Asmus, K.-D., Eds.; Plenum Press: New York, 1990. (71) Hoffman, M. Z.; Hayon, E. J. Am. Chem. Soc. 1972, 94 (23), 7950-7. (72) Lagerwerf, F. M.; et al. Rapid Commun. Mass. Spectrom. 1996, 10 (15), 1905-10. (73) Reid, G. E.; et al. J. Proteome Res. 2004, 3 (4), 751-9.

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Figure 5. (A) Positive ESI-MS spectrum of peptide GMG exposed to γ-rays for 12 min. (B) MS/MS spectrum of (MH + 16)+ at m/z 280.0.

and y2 fragment ions is also seen at m/z 141.1 (for b2 + 16 - 64), 159.0 (for y2 + 16 - 64), and 124.0 (for b2 + 16 - 64 - NH3). Other products are also identified by tandem mass spectrometry. The +32 Da product at m/z 296.0 results from the oxidation of Met to methionine sulfone. The -30 Da product at m/z 234.0 and the -14 Da (+16 - 30 Da) product at m/z 250.0 likely result from oxidative decarboxylation of the C-terminal carboxyl group of the unmodified peptide and its +16 Da oxidation product (observed at m/z 280.0), respectively.17 The -32 Da product at m/z 232.0 and -16 Da product at m/z 248.0 also result from the modification of Met and have not been reported previously. The -32 Da product may arise from further oxidation of methionine sulfoxide as a result of loss of a methanesulfinyl group and formation of an aldehyde group at the γ-carbon originally next to sulfur moiety, as shown in Scheme 4. However, this suggestion is just based on the mass change. HDMNKVLDL. This peptide is used to further demonstrate the oxidation of Met and the characteristic fragmentation pattern of the relevant products in tandem mass spectrometry. Antiflammin-2 peptide was irradiated by synchrotron X-rays for 15 ms, and the positive ESI-MS spectrum is presented in Figure 6A. The peak observed at m/z 1084.4 corresponds to the unmodified peptide. Similar to GMG, the major product with +16 Da mass addition is present at m/z 1100.4. Other modification products include m/z 2446 Analytical Chemistry, Vol. 77, No. 8, April 15, 2005

1052.4, 1068.4, 1098.4, 1114.4, and 1116.4 with mass changes of -32, -16, +14, +30, and +32 Da, respectively. The +16 Da oxidation product results from the oxidation of Met residue to methionine sulfoxide Met(O) as indicated by the MS/MS spectrum of m/z 550.7 corresponding to the doubly charged ion of the +16 Da product shown in Figure 6B. The peaks at m/z 247.0, 360.0, and 701.3 belong to unmodified y2, y3, and y6 ions, respectively, and no corresponding +16 Da modified fragment ions are found. The peaks at m/z 848.2 and 963.1 are modified y7 + 16 and y8 + 16, respectively, and no corresponding unmodified y7 and y8 fragments are found. The presence of y6 and y7 + 16 and absence of y6 + 16 and y7 indicate the +16 Da oxidation product is located at the Met residue. This is consistent with the presence of b2, b5 + 16, b6 + 16, (b6 + 16)2+, b7 + 16, (b7 + 16)2+, and (b8 + 16)2+ ions seen at m/z 253.1, 642.1, 741.2, 371.1, 854.2, 427.7, and 485.1, respectively. The oxidation of Met to methionine sulfoxide is further confirmed by the unique fragments resulting from the loss of 64 Da from molecular ion and b and y series ions as indicated by the arrows in the Figure 6B. The Met residue is rarely oxidized to methionine sulfone. The +32 Da products in the peptide result from the oxidation of other residues including Val, Lys, and Leu in addition to the oxidation of Met to sulfoxide. The MS/MS spectrum of doubly charged ion at m/z 558.7 corresponding to the +32 Da product is presented in Figure 6D. One characteristic of the spectrum is that each b + 32 series fragment is always accompanied by a b + 16 Da fragment, indicating multiple positions of oxidation as opposed to multiple oxidations at the same position. For example, the fragments b5 + 32 at m/z 658.1, b6 + 32 at m/z 757.1, (b6 + 32)2+ at m/z 379.3, b7 + 32 at m/z 870.3, (b7 + 32)2+ at m/z 435.7, b8 + 32 at 985.3, and (b8 + 32)2+ at m/z 493.2 are accompanied by corresponding +16 Da fragments including b5 + 16 at m/z 642.2, b6 + 16 at m/z 741.3, (b6 + 16)2+at m/z 371.3, b7 + 16 at m/z 854.1, (b7 + 16)2+ at m/z 427.7, b8 + 16 at 969.5, and (b8 + 16)2+ at m/z 485.2, respectively. Another characteristic of the spectrum is the presence of the unique -64 Da mass loss from the molecular ion at m/z 526.7 and most of the observed b + 32 Da ions as shown as arrows in Figure 6D. The -64 Da mass loss is characteristic of methionine sulfoxide and not observed in the case of methionine sulfone. The presence of b5 + 16 at m/z 642.2 indicates that either Val or either of the two Leu residues were oxidized, while the presence of b5 + 32 with a -64 Da satellite peak indicates a likely +16 Da modification at His or Lys as compared to the less reactive Asp and Asn (unpublished data). The presence of b8 + 16 at m/z 969.5 indicates a +16 Da modification at the C-terminal Leu. The +16 Da modification at Lys, Val, or two Leu residues is also confirmed by the presence of y6 + 16 at m/z 717.3, y6 + 32 at m/z 733.3, y7 + 32 at m/z 864.3, and y8 + 32 at 979.3, with the last two peaks accompanied with the diagnostic -64 Da peaks from methionine sulfoxide. The data clearly indicate the high reactivity of methionine and the significantly lower reactivity of methionine sulfoxide. Initial oxidation to methionine sulfoxide is readily observed, while subsequent oxidations favor residues such as Leu and Val as opposed to further oxidation of the sulfoxide to the sulfone. This can be seen from the comparable intensity of +30 and +32 Da modification products. Aliphatic side chains can be oxidized to either +16 or +14 Da mass shifts due to the formation of alcohol

Scheme 4. Radiolytic Oxidation of Methionine Residue

or carbonyl group, although the +16 Da species is typically more prevalent except in the case of Pro.13 However, +14 Da oxidations are also generated in aliphatic residue oxidation. The comparable intensities of the +32 and +30 Da species, which represent multiple oxidations, confirm that residues including Lys, Val, and Leu contribute to the oxidation in the case of the multiply oxidized species. To specifically explore the reactivity of methionine sulfoxide, the Met residue in the peptide antiflammin-2 was first oxidized to methionine sulfoxide using hydrogen peroxide and then vacuumdried, ESI-MS and MS/MS spectra (data not shown) indicated more than 98% of Met residue was oxidized to methionine sulfoxide with very negligible oxidation of other residues. The resulting Met(O)-antiflammin-2 (concentration of 40 µM) was exposed to γ-rays for 8 min and the positive ESI-MS spectrum is presented in Figure 6C. The +16 Da modification product at m/z 1116.4 shows an signal intensity stronger than that of +14 Da species at m/z 1114.4, as expected from the oxidation of most aliphatic amino acid side chains13 (Xu and Chance, unpublished data). Combining the evidence from tandem mass spectrometry, we can conclude that reactivity of methionine sulfoxide for oxidation to sulfone is less than other reactive residues such as Leu and Val in the peptide. However, methione sulfoxide in the peptide can be further oxidized to generate the -32 Da modification product (relative to unmodified Met) as shown in Figure 6A. The same product (labeled -48) was generated when the Met(O)-antiflammin-2 was irradiated by γ-rays as shown at m/z 1052.5 in Figure 6C. MS/MS spectrum of this product from irradiation of antiflammin-2 is identical to that from irradiation of Met(O)-antiflammin-2 (data not shown). The tandem MS spectrum does not conclusively point to the -32 Da modification at Met due to the fragmentation pattern, however, it does demon-

strate that the -32 Da modification is located at sequence MNK and most likely at Met. To further confirm that the -32 Da modification is located at Met, another peptide GSNKGAIIGLM(O) with oxidized Met (to sulfoxide) in a 40 µM solution was irradiated by γ-rays for 8 min and analyzed by MS and MS/MS (data not shown). A -48 Da product from the Met(O)-peptide (corresponding to -32 Da from Met) with an intensity similar to that of +14 and +16 Da products but about one-forth of that of +32 Da product was observed, and MS/MS spectrum conclude the specific modification at C-terminal Met(O). The -32 Da modification at Met is not always present in an irradiated Met-containing peptide due to the low reactivity of methionine sulfoxide. When another highly reactive amino acid residue such as Trp, Tyr, and Phe is present in the peptide, the -32 Da modification at Met is not observed. Therefore, the -32 Da modification at Met can be found in irradiated peptides such as HDMNKVLDL and GSNKGAIIGLM(O) but not in peptides such as LWMRFA-NH2 and YGGFM(O)R (data not shown). Mechanism of Methionine Oxidation. Radiolytic oxidation of methionine has also been the subject of long investigation, and a number of intermediate radical species have been identified;27-33 however, the explicit mechanism is still unclear. Methionine sulfoxide, with +16 Da mass shift, is the principal oxidation product in most cases. Methionine sulfoxide can be further oxidized to methionine sulfone accompanied by an additional +16 Da mass shift under harsh oxidation conditions. In this study, another product with -32 Da mass shift is also generated from radiolysis of methionine as well as radiolysis of methionine sulfoxide. The product has not been reported before and likely results from the further reaction of the methionine sulfoxide. Analytical Chemistry, Vol. 77, No. 8, April 15, 2005

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Figure 6. (A) Positive ESI-MS of antiflammin-2 HDMNKVLDL exposed to synchrotron X-rays for 15 ms. (B) Positive MS/MS spectrum of (MH2 + 16)2+ at m/z 550.8. (C) Positive ESI-MS spectrum of Met(O)-antiflammin-2 exposed to γ-rays for 8 min. (D) Positive MS/MS spectrum of (MH2 + 32)2+ at m/z 558.8.

Radiolytic oxidation of methionine may involve the addition of HO• radical to the sulfur atom and hydrogen abstraction.28 At low pH (3.0, the HO•-adduct sulfur-centered radical may eliminate a hydroxide anion HO- to give rise to a sulfurcentered radical cation >S•+.27-29,31,74 The process is catalyzed by an electron-rich heteroatom such as O, N, or S on neighboring side chains (Asp, Asn, Glu, Gln, Ser, Thr, Met, etc.) or by adjacent peptide bonds that stabilize the sulfur-center radical cation via formation of an intramolecular three-electron (S∴N, S∴O, S∴S, etc.) bonded cyclic transient.27-29,31,74 The S∴N three-electron bonded radicals may convert intramolecularly into the R-carbon centered radical on the peptide backbone.33 An intermolecular sulfur-sulfur dimeric radical cation >(S∴S)•+< has also been proposed as the reaction intermediate.64 The sulfur-centered radical cation may also deprotonate from the methyl CH3 or methylene CH2 next to the sulfur atom and transform into R-(alkylthio)alkyl radicals in the Met side chain.29 Methionine can (74) Hong, J.; Schoneich, C. Free Radical Biol. Med. 2001, 31 (11), 1432-41.

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be also oxidized by other oxygen-reactive species such as H2O2 via a two-electron mechanism but at much lower rate.32 In proteins, the situation is likely different from that of simple peptides. Intermolecular methionine-methionine interactions are generally unlikely, and the protein structure will also restrict conformation fluctuations and formation of the intermediates mentioned above. According to our previous radiolysis studies, the oxygen atom in the methionine sulfoxide originates from both hydroxyl radical and dissolved O2.13 We propose a comprehensive mechanism as shown in Scheme 4 to rationalize our experimental observations. The addition of hydroxyl radicals to Met at the sulfur atom initially generates a hydroxysulfuranyl radical,28 which likely abstracts an O2 molecule32 and subsequently loses a hydroperoxyl radical HO2•, to give rise to methionine sulfoxide (+16 Da mass shift). We suggest that this radical can rearrange through two different mechanisms, one of which results in the incorporation of oxygen from dissolved O2 while the other results in incorporation of oxygen from water molecules through the HO• produced by radiolysis. The methionine sulfoxide can be further oxidized

to methionine sulfone (+32 Da mass shift) possibly by a similar pathway but at a much lower rate. The novel -32 Da product is likely an aldehyde product resulting from the further oxidization of methionine sulfoxide through hydrogen abstraction by HO• radical from the CH2 next to the sulfur atom to form a carboncentered radical, which can be stabilized by electron delocalization to the sulfoxide group. The carbon-centered radical can react with an O2 molecule to form an aldehyde group with release of a CH3S(O)O• radical. Relative Reactivity of Cys, Disulfide, and Met. We compared the reactivity of Cys, disulfide bond, and Met at high solvent accessibilities to Trp. Two peptide mixtures, one containing 70 µM GCG, 40 µM GMG, and 20 µM GWG and another containing 50 µM (GC)2 and 20 µM GWG, were exposed to γ-rays for different time intervals. The peptides were mixed at different concentrations to obtain comparable mass spectrometric signals, and (GC)2 was analyzed separately to avoid cross reaction with GCG through thiol-disulfide exchange. The samples were analyzed by ESI-MS right after exposure to reduce possible secondary oxidation of Met and Cys after exposure by reactive oxygen-containing species such as H2O2 and O2, which may increase the apparent oxidation rates for Met and Cys (Xu and Chance, unpublished data). With GWG as internal standard, the relative amounts of unmodified GCG, GMG, and (GC)2 are measured as the ratios of their mass spectrometric signals to that of unmodified GWG, and the relative reactivity of GCG, GMG, and (GC)2 compared to GWG is estimated from the change of these signal ratios as a function of exposure time. The signal ratios at different exposure times are normalized to that of control for clear comparison and shown in Figure 7. If a peptide is equally reactive to, more, or less reactive than GWG, then the normalized signal ratio will remain level at 1.0, decrease, or increase from 1.0 as the exposure time increases, respectively. As shown in Figure 7, the relative signal ratio decreases, decreases slightly, and increases for GCG, GMG, and (GC)2, respectively, as exposure time increases. The data indicate a reactivity order of GCG > GMG ∼ GWG > (GC)2 in the presence of minimal secondary oxidations. CONCLUSIONS Radiolytic formation of cysteine sulfonic acid and methionine sulfoxide has been clearly demonstrated in oxidation of cysteine and methionine in peptides and are expected to provide a molecular signature of these residues when they are in solventaccessible environments in proteins in well-controlled footprinting experiments. When irradiated by synchrotron X-rays or γ-rays under aerobic conditions, cysteine residues in peptides are

Figure 7. Change of mass spectral signals of GCG, GMG, and (GC)2 relative to that of GWG as a function of exposure time. Two peptides mixtures, one containing 70 µM GCG, 40 µM GMG, and 20 µM GWG and another containing 50 µM (GC)2 and 20 µM GWG, were exposed to γ-rays for different intervals of times separately. The signal ratios at different exposure times were normalized to that of control for clear comparison.

oxidized to cysteine sulfonic acid (with a +48 Da mass shift) and disulfide as principal products along with other minor products also observed. In protein samples, disulfide bonds are generally not formed during irradiation, but precautions need to be taken to prevent the formation of disulfides and mixed disulfides during enzymatic digestion and sample storage if the protein sample contains multiple cysteine residues. Disulfide bonds are subject to oxidative cleavage by ionizing radiation to generate sulfonic acids as the primary product. Methionine residues are normally oxidized to methionine sulfoxide with +16 Da mass shift as the major product; other minor products are also observed. For fully accessible residues, Cys is more reactive than Trp, and Met is as reactive as Trp, whereas disulfide-bound Cys is less reactive than Trp. ACKNOWLEDGMENT This research is supported by the NIH under grants P41-EB01979, P01-GM-66311, and R21-DK-69952. Manuscript received October 18, 2004. Accepted January 20, 2005. AC0484629

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