research articles signal transduction pathways in vascular endothelial cells, serve as ligands for the scavenger receptor CD36, and facilitate lipid uptake in macrophages and the formation of foam cells.9,32 Most of the implicated phospholipids have electrophilic sub- stituents and could be expected to form covalent protein adducts. However, the identities of proteins covalently modiﬁed by phospholipid electrophiles have not been reported. A major challenge in studying protein damage by lipid electrophiles is the sheer diversity of products generated.33,34 For example, the oxidative decomposition of a phospholipid containing linoleate at the sn-2 position would yield several electrophilic phospholipid products. HNE and 4-oxononenal are formed from the ω-end of linoleate ester,35 but other reactive electrophiles contain the carboxy-end of the linoleate ester. Thus, ketooxododecenoate (KODA), hydroxyoxodode- cenoate (HODA), and other adducts esteriﬁed to phospholipid are expected to be among the electrophile adducts formed with proteins from oxidized linoleate-containing phospholipids (Figure 1). Characterization of the protein targets of the family of electrophiles formed from a phospholipid is also complicated by the relatively low levels of adducts compared to unmodiﬁed proteins in relevant biological systems. Analysis of the protein targets of a family of phospholipid electrophiles requires a means of high afﬁnity capture of the adducted proteins. Recently, we reported the synthesis of the biotinylated phos- phatidylcholine PLPBSO, which is the biotin-sulfoxide analogue of 1-palmitoyl-2-linoleoylglycerylphosphatidyl-choline (PLPC).36 Oxidation of PLPBSO formed KODA, HODA, and other expected linoleate oxidation products and also formed the corresponding electrophile adducts with a model peptide. In that work, we also demonstrated feasibility of streptavidin capture of PLPBSO- adducted human serum albumin from incubation of the protein with oxidized PLPBSO. Here, we describe the use of PLPBSO as a probe to identify major protein targets of oxidized phospholipids in human plasma. This approach identiﬁed apolipoprotein A1 (ApoA1) as the principal target of PLPBSO oxidation products and led us to map adducts from lipid oxidation products on this protein. We report the identiﬁcation of adducts from several phospholipid electrophiles, including carboxy-terminal ad- ducts. We further evaluated the sites of ApoA1 adduction by the prototypical lipid electrophile HNE. The concordance of ApoA1 site-speciﬁc modiﬁcation between exogenous HNE and endogenous phospholipid electrophiles suggests that the iden- tiﬁed targets may be preferred modiﬁcation sites on ApoA1 in oxidative stress in vivo. Experimental Procedures Chemicals and Reagents. PLPBSO was synthesized as de- scribed recently.36 Plasma samples were collected from healthy human donors as part of an IRB-approved study. 2,2′-azobis[2- (2-imidazolin-2-yl)propane]-dihydrochloride (AIPH) was from Wako Chemicals (Osaka, Japan), streptavidin sepharose beads were from GE Healthcare (Piscataway, NJ), and ammonium hydroxide was from Sigma (St. Louis, MO). ApoA1 antibody (rabbit polyclonal antibody to the RLAEYHAKATEH peptide of ApoA1) was from Cayman Chemical (Ann Arbor, MI). Rabbit polyclonal antibody against HNE histidine Michael adducts was from Calbiochem (San Diego, CA). Alexa Fluor 680-conjugates streptavidin and all secondary antibodies were purchased from Molecular Probes (Carlsbad, CA). Protein G agarose was from 4238 Journal of Proteome Research Vol. 7, No. 10, 2008 Szapacs et al. Roche Applied Science (Indianapolis, IN). Mass spectrometry grade trypsin (Trypsin Gold) was from Promega (Madison, WI). Isolation of Plasma. Whole blood from fasting, healthy subjects was collected in a 440 mL ACD blood collection bag (Baxter) containing 2 g of dextrose monohydrate, 1.66 g of sodium citrate dihydrate, 188 mg of anhydrous citric acid, 140 mg of monobasic sodium phosphate monohydrate, and 17.3 mg of adenine. Plasma was subsequently isolated from red blood cells by centrifuging the whole blood bag at 4200 rpm for 10 min at 22 °C. Supplementation of PLPBSO to Plasma and Isolation of HDL. PLPBSO was dissolved in DMSO (50 mM) and was added to plasma at a ﬁnal concentration 100 µ M (<0.3% v). The same volume of DMSO was added to the control plasma. They were placed in 37 °C oil bath and stirred for 1.5 h. Half of the supplemented and native plasma were aliquoted into eppen- dorf tubes and stored at -80 °C until further use. HDL was isolated by ultracentrifugation (1.063 g/mL < d < 1.12 g/mL) from both native and supplemented plasma.37 Oxidation of Native and PLPBSO Supplemented Plasma. Native and PLPBSO supplemented plasma (50 µ L) were oxi- dized with 5 mM AIPH overnight at 37 °C. Lipid electrophile adducts were stabilized by reduction with 30 mM NaCNBH3. A total of 50 µ L of PBS was added to the sample, and 50 µ L of protein-G agarose slurry then was used to remove IgG side chain that nearly comigrates with ApoA1 on SDS-PAGE. The supernatant was collected and ﬁltered through Amicon 10 kDa molecular weight cutoff ﬁlter to remove small molecules residual azo initiator and reducing agent. The samples then were diluted to 300 µ L with 100 mM ammonium bicarbonate, treated with 10 mM DTT at 50 °C for 30 min, and then with 20 mM IAM in the dark at room temperature. Streptavidin Capture of Biotinylated Proteins for Protein Identiﬁcation. Streptavidin sepharose beads (100 µ L slurry) were washed with 3 × 500 µ L of 6 M urea in PBS. The oxidized, reduced, and alkylated native or PLPBSO supplemented plasma samples were added to the washed streptavidin beads and allowed to rotate for 2 h at room temperature. The supernatant was discarded and the beads were washed with 3 × 500 µ L of 6 M urea in PBS with rotation for 15 min at room temperature, then with 3 × 500 µ L of 1 M NaCl with rotation for 15 min at room temperature, and ﬁnally with 3 × 500 µ L of 50 mM ammonium bicarbonate. Elution of biotinylated proteins was done by selective hydrolysis of the PLPBSO acyl ester using 100 µ L of 15% NH4OH38 in 3 M urea overnight at room tempera- ture. After hydrolysis, the supernatant was collected and the beads were rinsed with 2 × 100 µ L of 50 mM ammonium bicarbonate and combined with the eluate, which was placed under house vacuum for 30 min to remove excess NH3. The pH was adjusted to ∼8 with 30% acetic acid. Sample Digestion, Preparation, and LC-MS-MS Analysis of Peptides by Gas-Phase Fractionation. Samples were enzy- matically digested for 18-24 h at 37 °C using 0.5 µ g of trypsin and the solution then was acidiﬁed to pH 3 with formic acid to halt the digestion. Then, the samples were evaporated under vacuum, desalted using Zip-Tips (Millipore C18, Millipore, Billerica, MA), and resuspended in 200 µ L of 0.1% formic acid for LC-MS-MS analysis. LC-MS-MS analyses were performed on a Thermo LTQ linear ion trap instrument (Thermo Electron, San Jose, CA) equipped with a Thermo Surveyor HPLC system, nanospray source, and microautosampler. Peptides were resolved on a 100 µ m × 11 cm fused silica capillary column (Polymicro Tech-