research articles carboxylated adducts on histidine and lysine residues, which would generate a variety of ApoA1 forms with modiﬁed charge characteristics and possibly altered SDS binding. In contrast, treatment with HNE does not produce this effect, even when treatment produced comparable levels of HNE adduction. Our analyses of ApoA1 adducts from PLPBSO-supplemented, oxidized plasma that was captured with streptavidin identiﬁed several carboxylated adducts, but relatively few adducts cor- responding to electrophiles derived from the ω-terminus of the fatty acid (e.g., HNE). On the other hand, ApoA1 was modiﬁed by HNE formed during oxidation of isolated HDL with AIPH and by exogenously added HNE (Table 3). In both cases, the distribution of HNE adducts was similar. This conﬁrms that ApoA1 can undergo modiﬁcation both by diffusible HNE-like electrophiles and phospholipid-bound electrophiles. The some- what sporadic detection of HNE and other noncarboxylated adducts in PLPBSO-supplemented plasma following strepta- vidin capture probably reﬂects loss of the nonphospoholipid adducted ApoA1 proteins during the wash steps to remove nonbiotinylated proteins. This also implies that, under the conditions of our experiments, most ApoA1 molecules bear a total of one or two adducts and that these are distributed between all the observed sites. Had the protein been more heavily modiﬁed, one would have expected to consistently observe HNE adducts and other noncarboxylated adducts as well as carboxylated adducts in the streptavidin-captured protein. We identiﬁed adducts in two target sequences that have been identiﬁed previously in studies of oxidized ApoA1. Lys195 was modiﬁed by 9-oxononanoic acid and the nearby His193 was adducted by HNE. These sites are immediately adjacent to Tyr192, which was identiﬁed by the Hazen and Heinecke groups as sites of nitration and chlorination in ApoA1.49,53,54,61 The latter reaction is catalyzed by myeloperoxidase, which binds to this sequence in ApoA1 in HDL.44 We also mapped KODA and HNE adducts to His162, which resides within a sequence recently shown to be critical for interaction with LCAT.44 Tyr166 undergoes chlorination and nitration in athero- mas in vivo and this modiﬁcation blocks the activation of LCAT by ApoA1, which is an obligatory step in HDL particle matura- tion.44 His162 is also a solvent-exposed residue in the LCAT- activating loop that contains Tyr16644 and this raises the possibility that His162 adduction may have functional conse- quences similar to those reported for Tyr166 modiﬁcations. Our studies demonstrate that His 162 and His193 are among the most easily detected adducts at low HNE exposure con- centrations in HDL (Table 3). It might be tempting to conclude that this indicates high kinetic reactivity of these residues with electrophiles. However, detection in LC-MS-MS analyses depends not only on relative levels of the adducts, but also on ionization efﬁciency of the adducted peptides. In a recent study of the kinetics of human albumin adduction at different sites by HNE, we noted that the rate constant for formation of the most readily detected adduct (His67) was over 10-fold lower than that for the most reactive site (His242), which was less readily detected in data-dependent LC-MS-MS analyses.26 Thus, we are planning additional studies of the kinetics of ApoA1 modiﬁcation in plasma and HDL to characterize the hierarchy of reactivity with lipid electrophiles. Recent studies have highlighted the utility and potential clinical signiﬁcance of oxidatively modiﬁed ApoA1.44,49,61 Our results suggest that covalent adducts with lipid electrophiles can mechanistically link lipid peroxidation, oxidative stress and 4244 Journal of Proteome Research Vol. 7, No. 10, 2008 Szapacs et al. disease mechanisms in a similar manner. In addition, lipid electrophile adducts of ApoA1 merit evaluation as potential biomarkers of oxidative stress in vivo. Acknowledgment. This work was supported by National Institutes of Health Grants ES013125 and ES000267. Supporting Information Available: Tables S1 and S2 and Figures S1 and S2. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Ames, B. N. Dietary carcinogens and anticarcinogens. Oxygen radicals and degenerative diseases. Science 1983, 221, 1256–1264. (2) Halliwell, B. Free radicals, antioxidants, and human disease: curiosity, cause, or consequence? [see comments]. Lancet 1994, 344 (8924), 721–724. (3) Berliner, J. A.; Watson, A. D. A role for oxidized phospholipids in atherosclerosis. N. Engl. J. Med. 2005, 353 (1), 9–11. (4) Butterﬁeld, D. A. Amyloid beta-peptide (1-42)-induced oxidative stress and neurotoxicity: implications for neurodegeneration in Alzheimer’s disease brain. A review. Free Radical Res. 2002, 36 (12), 1307–1313. (5) Perry, G.; Nunomura, A.; Hirai, K.; Zhu, X.; Perez, M.; Avila, J.; Castellani, R. J.; Atwood, C. S.; Aliev, G.; Sayre, L. M.; Takeda, A.; Smith, M. A. Is oxidative damage the fundamental pathogenic mechanism of Alzheimer’s and other neurodegenerative diseases. Free Radical Biol. Med. 2002, 33 (11), 1475–1479. (6) Beckman, K. B.; Ames, B. N. The free radical theory of aging matures. Physiol. Rev. 1998, 78 (2), 547–581. (7) Ames, B. N. DNA damage from micronutrient deﬁciencies is likely to be a major cause of cancer. Mutat. Res. 2001, 475 (1-2), 7–20. (8) Marnett, L. J.; Riggins, J. N.; West, J. D. Endogenous generation of reactive oxidants and electrophiles and their reactions with DNA and protein. J. Clin. Invest. 2003, 111 (5), 583–593. (9) Berliner, J. A.; Zimman, A. Future of toxicology-lipidomics, an important emerging area for toxicologists: focus on lipid oxidation products. Chem. Res. Toxicol. 2007, 20 (6), 849–853. (10) Uchida, K. Future of toxicology-lipid peroxidation in the future: from biomarker to etiology. Chem. Res. Toxicol. 2007, 20 (1), 3–5. (11) Kadiiska, M. B.; Gladen, B. C.; Baird, D. D.; Germolec, D.; Graham, L. B.; Parker, C. E.; Nyska, A.; Wachsman, J. T.; Ames, B. N.; Basu, S.; Brot, N.; Fitzgerald, G. A.; Floyd, R. A.; George, M.; Heinecke, J. W.; Hatch, G. E.; Hensley, K.; Lawson, J. A.; Marnett, L. J.; Morrow, J. D.; Murray, D. M.; Plastaras, J.; Roberts, L. J.; Rokach, J.; Shigenaga, M. K.; Sohal, R. S.; Sun, J.; Tice, R. R.; Van Thiel, D. H.; Wellner, D.; Walter, P. B.; Tomer, K. B.; Mason, R. P.; Barrett, J. C. Biomarkers of Oxidative Stress Study II: Are oxidation products of lipids, proteins, and DNA markers of CCl(4) poisoning. Free Radical Biol. Med. 2005, 38 (6), 698–710. (12) Doorn, J. A.; Petersen, D. R. Covalent modiﬁcation of amino acid nucleophiles by the lipid peroxidation products 4-hydroxy-2- nonenal and 4-oxo-2-nonenal. Chem. Res. Toxicol. 2002, 15 (11), 1445–1450. (13) Brame, C. J.; Salomon, R. G.; Morrow, J. D.; Roberts, L. J. Identiﬁcation of extremely reactive gamma-ketoaldehydes (isole- vuglandins) as products of the isoprostane pathway and charac- terization of their lysyl protein adducts. J. Biol. Chem. 1999, 274 (19), 13139–13146. (14) Brame, C. J.; Boutaud, O.; Davies, S. S.; Yang, T.; Oates, J. A.; Roden, D.; Roberts, L. J. Modiﬁcation of proteins by isoketal-containing oxidized phospholipids. J. Biol. Chem. 2004, 279 (14), 13447–13451. (15) Lin, D.; Lee, H. G.; Liu, Q.; Perry, G.; Smith, M. A.; Sayre, L. M. 4-Oxo-2-nonenal is both more neurotoxic and more protein reactive than 4-hydroxy-2-nonenal. Chem. Res. Toxicol. 2005, 18 (8), 1219–1231. (16) Zhu, X.; Sayre, L. M. Long-lived 4-oxo-2-enal-derived apparent lysine michael adducts are actually the isomeric 4-ketoamides. Chem. Res. Toxicol. 2007, 20 (2), 165–170. (17) Williams, M. V.; Wishnok, J. S.; Tannenbaum, S. R. Covalent adducts arising from the decomposition products of lipid hydro- peroxides in the presence of cytochrome c. Chem. Res. Toxicol. 2007, 20 (5), 767–775. (18) Liu, Z.; Minkler, P. E.; Sayre, L. M. Mass spectroscopic character- ization of protein modiﬁcation by 4-hydroxy-2-(E)-nonenal and 4-oxo-2-(E)-nonenal. Chem. Res. Toxicol. 2003, 16 (7), 901–911. (19) Bolgar, M. S.; Yang, C. Y.; Gaskell, S. J. First direct evidence for lipid/protein conjugation in oxidized human low density lipopro- tein. J. Biol. Chem. 1996, 271 (45), 27999–28001.