Lipid Electrophile Adducts of ApoA1 Table 3. Sites of HNE Adduction on ApoA1 Following Treatment of HDL with HNE or Oxidation of Plasma with AIPHa AIPH (µ M) HNE (µ M) sequence 100 1000 5000 1 10 100 1000 VK*DLATVYVDVLK K12 1/3b DLATVYVDVLK*DSGR K23 1/3 DLEEVK*AK K94 2/3 3/3 AK*VQPYLDDFQK K96 1/3 3/3 K*WQEEMELYR K107 3/3 LH*ELQEK H135 3/3 3/3 AH*VDALR H155 1/3 2/3 3/3 3/3 3/3 3/3 TH*LAPYSDELR H162 2/3 3/3 3/3 2/3 3/3 3/3 3/3 LAEYH*AK H193 3/3 3/3 3/3 3/3 3/3 3/3 3/3 ATEH*LSTLSEK H199 1/3 3/3 3/3 3/3 AK*PALEDLR K208 1/3 3/3 3/3 VSFLSALEEYTK*K K238 1/3 a Mass error (ppm) for measurements of the precursor ions of the observed peptide adducts were analyzed on a Thermo LTQ-Orbitrap instrument and were as follows: K12, 3.2 ppm; K23, 3.8 ppm; K94, 3.9 ppm; K96, 3.8 ppm; K107, 3.7; H135, 4.0 ppm; H155, 3.2 ppm; H162, 2.9 ppm; H193, 3.7; H199, 3.3; K208, 3.8 ppm; K238, 5.9. b Denotes that the adducts were detected in either 1 of 3, 2 of 3, or 3 of 3 independent experiments, respectively. reactive aldehydes, R, -unsaturated aldehydes, epoxy-R, - unsaturated aldehydes, and other products that can form protein adducts that are stabilized with NaBH3CN. Oxidation products in which the electrophile comprises a remnant portion of the linoleate chain at the sn-2 position will covalently link the adducted protein to the biotinylated choline head- group, which enables capture with streptavidin. Thus, capture of biotinylated proteins following oxidation enables capture and analysis of the protein targets of PLPBSO-derived phospholipid electrophiles. We used NaBH3CN to stabilize adducts formed with PLPBSO oxidation products in order to detect both HODA and KODA adducts. Although NaBH4 traps Michael adducts more ef- ﬁciently, it also reduces KODA ketones to hydroxyls. On the other hand, NaBH3CN reduces imines and reduces carbonyls very slowly. The latter reaction was sufﬁciently slow in our studies to potentially allow redistribution of adducts between Michael and imine forms. Model studies (not shown) with an HNE-adducted peptide indicated that reduction with NaBH4 for 4 h at room temperature yielded only the Michael adduct, whereas reduction with NaBH3CN under the same conditions yielded the reduced Michael adduct and nonreduced aldehyde and imine adducts. Because our product analyses were es- sentially qualitative in nature, this effect probably did not signiﬁcantly bias our results. However, this chemistry would pose challenges for quantitative analyses of KODA adducts. Although digestion and LC-MS-MS analysis of the captured proteins will enable their identiﬁcation, the adduct peptides would most likely go undetected because the phospholipid would suppress ionization or induce fragmentations in the phospholipid moiety that would compete with peptide frag- mentation, thus, precluding identiﬁcation by database search of MS-MS spectra. We developed a mild ammonia hydrolysis procedure that cleaves the phospholipid fatty acyl ester bonds with minimal concomitant hydrolysis of the adducted protein (Figure 1). Selective ammonia hydrolysis enabled release of the biotiny- lated proteins from the streptavidin beads and left protein-lipid electrophile adducts containing a carboxy-terminal group research articles (Figure 2). Stringent wash of the streptavidin beads containing captured proteins with 6 M urea and 1 M NaCl reduced nonspeciﬁc binding to the streptavidin. Following a tryptic digest of the released proteins, both adducted and unadducted peptides were detected by LC-MS-MS analysis. Identiﬁcation of Proteins Adducted by PLPBSO and Map- ping of Adducts. Oxidation of plasma supplemented with PLPBSO was initiated using the water soluble free radical initiator AIPH. The water soluble free radical azo initiator, AIPH, was used to initiate a true free radical chain reaction. The use of the azo initiator allows the rate of oxidation to be readily controlled by adjusting the concentration of AIPH and the temperature of the reaction. Full-scan LC-MS chromatograms of the tryptic digests indicate that the streptavidin beads captured a signiﬁcant amount of biotinylated protein in the PLPBSO-supplemented, oxidized plasma, as indicated by the strong ion current in the LC-MS of the corresponding tryptic digests (Figure S1A in Supporting Information). In contrast, very little protein was captured on streptavidin from the nonsupple- mented, oxidized plasma samples (Figure S1B in Supporting Information). To identify proteins adducted by PLPBSO, these tryptic digests then were subjected to LC-MS-MS analysis using a gas-phase fractionation procedure and the data then were searched against the IPI Human protein database. The experiment was repeated four times and 21 proteins were conﬁdently identiﬁed (2+ peptide identiﬁcations at 95% con- ﬁdence). The proteins represented by the highest sequence coverage, reﬂecting those present in the PLPBSO-labeled protein pool in greatest abundance, are listed in Table 1 (A complete listing of identiﬁed proteins is provided in Table S2 in Supporting Information). ApoA1 was the most abundantly represented PLPBSO target. Conﬁrmation of ApoA1 Adduction with Oxidized Biotiny- lated Lipid. To conﬁrm ApoA1 adduction using immunoblot- ting techniques, PLPBSO supplemented plasma or native plasma both were oxidized with AIPH. Immunoblotting was done with either a polyclonal antibody raised against ApoA1 peptides or Alexa Fluor 680-conjugated streptavidin. The anti- ApoA1 immunoblot indicated that, after oxidation in both the PLPBSO supplemented plasma and native plasma, the ApoA1 band is shifted to higher apparent molecular weight (Figure 3). The Alexa Fluor 680-conjugated streptavidin immunoblot shows biotin labeling of the higher molecular weight band, which is due to the covalent adduction of phospholipid to the protein. Identiﬁcation of Carboxylated Electrophile Adducts in ApoA1. To identify the adducts with carboxy-terminal electro- philes in ApoA1, PLPBSO-supplemented plasma was oxidized with AIPH followed by NaCNBH3 reduction to stabilize the adducts and PLPBSO-adducted proteins were captured with streptavidin beads. After stringent washes to remove nonspe- ciﬁcally bound protein, the beads were treated with 15% ammonium hydroxide to selectively hydrolyze the phospholipid esters. The eluted proteins (enriched in carboxyl-terminal lipid adducts) were resolved by SDS-PAGE and the ApoA1 band was excised for in-gel trypsin digestion followed by LC-MS-MS analysis. Analysis of the MS-MS data with the P-Mod algo- rithm42 searched ApoA1 tryptic peptide sequences against the MS-MS data to identify peptides that correspond to ApoA1 peptides, including adducts. This approach does not require the user to specify anticipated adduct masses or sequence speciﬁcities (e.g., His modiﬁcation), but instead performs an unbiased search for spectra that match search sequences with Journal of Proteome Research Vol. 7, No. 10, 2008 4241