Lipid Electrophile Adducts of ApoA1 fatty acyl chain. These detected mass shifts corresponded to Schiff base adducts of hexanal, heptanal, octanal, and nonanal (Table S1 in Supporting Information). Detection of these species was less consistent from experiment-to-experiment than was detection of the carboxylated adducts described above. This may reﬂect the fact that the streptavidin capture and wash protocol would select against ω-terminal adducts, unless ApoA1 molecules modiﬁed by these species also were covalently labeled with the phospholipid. Although adducts were found throughout the protein, the adduction of H162 is of interest because this residue is within a sequence known to interact with Lecithin-cholesterol acyl- transferase (LCAT), an enzyme that contributes to HDL particle maturation.44 Analysis of HNE Adducts in ApoA1 Following Oxidation of HDL Particles with AIPH or Treatment with HNE. Our analyses of the streptavidin-captured ApoA1 (see above) identi- ﬁed relatively few adducts corresponding to modiﬁcation by noncarboxylated electrophiles originating from the ω-terminus of the fatty acid (e.g., HNE). To determine if HNE adducts are formed on ApoA1 upon oxidation of native HDL associated lipids, HDL particles were isolated from plasma and then oxidized overnight with different concentrations of AIPH or treated with HNE. Adducts from both treatments were stabi- lized by reduction with NaBH4. Anti-HNE immunoblotting revealed immunoreactivity of the ApoA1 band (∼20-26 kDa) from HDL particles oxidized with 5 mM AIPH or treated with either 100 µ M or 1 mM HNE (Figure 4A). The visible gel in Figure 4B shows that a molecular weight shift and band broadening for ApoA1 (similar to that observed in Figure 2, above) was induced by as little as 100 µ M AIPH, but not by HNE treatment. This suggests that the ApoA1 migration is affected perhaps by multiple modiﬁcations of the ApoA1 protein that affect protein charge, shape and/or SDS binding, but that HNE adduction alone does not produce these effects. HNE adduction sites on ApoA1 were mapped by LC-MS-MS analysis of in-gel tryptic digests of the ApoA1 band from AIPH- oxidized and HNE-treated samples described above. MS-MS spectra were acquired by data-dependent scanning mode, which automatically samples intact peptide ion signals for MS-MS.45 The MS-MS spectra were searched against a human protein sequence database with the Sequest algorithm with allowance for variable modiﬁcations on histidine and lysine residues for borohydride-reduced HNE Schiff base adducts (+140 amu) or Michael adducts (+158 amu). As a complemen- tary approach, the sequences of ApoA1 tryptic peptides were searched against the MS-MS data with the P-Mod algorithm to enable detection of spectra corresponding to modiﬁed peptides. P-Mod hits corresponding to the expected +140 and +158 mass shifts on histidine or lysine residues were recorded. All putative spectra of adducts were subjected to manual inspec- tion to verify the quality of sequence matches. Ten sites of adduction were identiﬁed, including K12, K23, K94, K96, K107, H135, H155, H162, H193, H199, K208, and K238 (Table 3). All of these modiﬁcations were identiﬁed as borohydride-reduced HNE Michael adducts. Lysine adducts were found on missed tryptic cleavage sites. Accurate mass measurements for the adduct precursor ions with a Thermo LTQ-Orbitrap instrument con- ﬁrmed calculated values to within 3-6 ppm (Table 3). In replicate experiments in which HDL was treated with either HNE or oxidized with AIPH, histidines in the sequences TH162LAPYSDELR and LAEYH193AK were repeatedly found to be modiﬁed even with exposure to as little as 1 µ M HNE (Table research articles 3). His162, which lies within the LCAT-interacting domain of ApoA1,44 was a target for modiﬁcation by both HNE and the carboxyated electrophile KODA, representative MS-MS spectra for which are both shown in Figure 5. Discussion Although lipid electrophiles generated during oxidative stress are widely thought to play critical roles in injury and adaptation to stress, relatively little is known about the protein targets of these species. Target identiﬁcation is complicated by the great diversity of potential targets, the low abundance of modiﬁca- tions, and the difﬁculty of selectively capturing and analyzing the modiﬁed proteins. We have approached this problem by employing afﬁnity-tagged lipid substrates that form oxidation products representative of the same lipid class, but that also enable high afﬁnity capture of the lipid electrophile adducts. The PLPBSO probe generates oxidation products similar to the corresponding phosphatidylcholine analogue and also forms adducts to proteins.36 Use of PLPBSO enabled detection of ApoA1 as a major protein target of phospholipid electrophiles in plasma. Subsequent analysis of ApoA1 from oxidized plasma identiﬁed novel lipid electrophile adducts derived from phos- pholipid oxidation products. Moreover, adduct mapping studies revealed adduction in a functionally critical region of the protein. ApoA1 is the core protein component of HDL particle, which mediates reverse cholesterol transport from tissues to the liver and also exerts antiatherogenic effects.44,46,47 ApoA1 and plasma HDL cholesterol also are inversely correlated with coronary artery disease.48 Recent work indicates that HDL function is impaired in cardiovascular disease and systemic inﬂammatory disorders.47 Functional impairment of HDL has been attributed to oxidative modiﬁcations of ApoA1 by my- eloperoxidase 49-54 or reactive products of lipid oxidation.55,56 Thus, our results indicating that ApoA1 is a major target of reactive phospholipid electrophiles led us to focus attention on the types and site-speciﬁcity of ApoA1 adduction. The biotin tag on the choline headgroup in PLPBSO allows afﬁnity capture of protein adducts that contain the phospho- lipid headgroup. These adducts are derived from electrophiles esteriﬁed to the phospholipid and form carboxylated adducts upon ammonia hydrolysis. Nevertheless, not all adducts identi- ﬁed from streptativin-captured protein are necessarily derived from PLPBSO. Some of the identiﬁed adducts may originate from endogenous ApoA1 lipid esters and are formed on ApoA1 that is also adducted with PLPBSO. The formation of carboxy- lated lipid electrophile adducts has been studied to only a limited extent. Williams and colleagues characterized KODA and HODA adducts formed by decomposition of linoleate hydroperoxide in the presence of puriﬁed cytochrome c.17 We also observed adducts with 7-oxoheptanoic acid and 8-oxooc- tanoic acid on ApoA1 lysine residues. Although the formation of these imine adducts is not unexpected, this is the ﬁrst report of the direct detection of these adducts, to our knowledge. The covalent binding of aldehyde-containing phospholipids has been shown previously to generate antigenic components of oxidized low density lipoprotein (LDL).57,58 These changes, in turn, promote recognition of oxidized LDL by C-reactive protein59 and functional alterations in monocytes exposed to oxidized LDL.60 A notable characteristic of ApoA1 subjected to free radical oxidation is the alteration of its migration characteristics in SDS-PAGE (Figure 3). This may reﬂect the formation of Journal of Proteome Research Vol. 7, No. 10, 2008 4243