Identiﬁcation of Proteins Adducted by Lipid Peroxidation Products in Plasma and Modiﬁcations of Apolipoprotein A1 with a Novel Biotinylated Phospholipid Probe Matthew E. Szapacs,†,§ Hye-Young H. Kim,‡,§ Ned A. Porter,‡ and Daniel C. Liebler*,† Departments of Biochemistry and Chemistry, Vanderbilt University, Nashville, Tennessee 37232-0146 Received February 14, 2008 Reactive electrophiles generated by lipid peroxidation are thought to contribute to cardiovascular disease and other oxidative stress-related pathologies by covalently modifying proteins and affecting critical protein functions. The difﬁculty of capturing and analyzing the relatively small fraction of modiﬁed proteins complicates identiﬁcation of the protein targets of lipid electrophiles. We recently synthesized a biotin-modiﬁed linoleoylglycerylphosphatidycholine probe called PLPBSO (Tallman et al. Chem. Res. Toxicol. 2007, 20, 227-234000), which forms typical linoleate oxidation products and covalent adducts with model peptides and proteins. Supplementation of human plasma with PLPBSO followed by free radical oxidation resulted in covalent adduction of PLPBSO to plasma proteins, which were isolated with streptavidin and identiﬁed by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Among the most highly modiﬁed proteins was apolipoprotein A1 (ApoA1), which is the core component of high density lipoprotein (HDL). ApoA1 phospholipid adduct sites were mapped by LC-MS-MS of tryptic peptides following mild base hydrolysis to release esteriﬁed phospholipid adducts. Several carboxylated adducts formed from phospholipid-esteriﬁed 9,12-dioxo-10(E)-dodecenoic acid (KODA), 9-hydroxy, 12-oxo-10(E)-dodecenoic acid (HODA), 7-oxoheptanoic acid, 8-oxooctanoic acid, and 9-oxononanoic acid were identiﬁed. Free radical oxidations of isolated HDL also generated adducts with 4-hydroxynonenal (HNE) and other noncarboxylated electrophiles, but these were only sporadically identiﬁed in the PLPBSO-adducted ApoA1, suggesting a low stoichiometry of modiﬁcation in the phospholipid-adducted protein. Both phospholipid electrophiles and HNE adducted His162, which resides in an ApoA1 domain involved in the activation of Lecithin-cholesterol acyltransferase and maturation of the HDL particle. ApoA1 lipid electrophile adducts may affect protein functions and provide useful biomarkers for oxidative stress. Keywords: Lipid electrophile • protein adduct • afﬁnity probe • apolipoprotein A1 Introduction The formation of oxidants is a hallmark of chemical toxicity, inﬂammation, and other types of environmental stresses.1,2 Oxidative stress and oxidants also are involved in human diseases that account for signiﬁcant morbidity and mortality, including cancer, atherosclerosis, and neurodegenerative diseases.3-7 Although oxidative stress derives fundamentally from the excessive ﬂux of reduced oxygen species, such as superoxide and hydrogen peroxide, secondary products of lipid oxidation may play critical roles in oxidant-associated molec- ular pathologies. Lipid electrophiles such as malondialdehyde, hydroxyalkenals, oxoalkenals, epoxyalkenals, and γ-ketoalde- hydes readily react with proteins, and the formation of covalent adducts is hypothesized to contribute to oxidant-related dis- * Address correspondence to: Daniel C. Liebler, Room 1213 MRBIII, 465 21st Avenue South, Nashville, TN 37232-6350. Fax, 615-343-8372; e-mail, daniel.liebler@vanderbilt.edu. § These authors contributed equally to this work. † Department of Biochemistry, Vanderbilt University. ‡ Department of Chemistry, Vanderbilt University. 10.1021/pr8001222 CCC: $40.75  2008 American Chemical Society eases and toxicities.8,9 Because they reﬂect the occurrence of oxidative stress, protein adducts from lipid electrophiles also may serve as biomarkers for oxidant-mediated pathologies.10,11 Most studies of protein modiﬁcation by lipid electrophiles have examined a few prototypical lipid oxidation products, such as 4-hydroxynonenal (HNE), 4-oxononenal, and γ-ketoalde- hydes (levuglandins) using mass spectrometry (MS). This published work has identiﬁed relevant protein adduction chemistries with amino acid side chain nucleophiles12-17 and in some cases mapped adducts to speciﬁc sequences in modiﬁed proteins.17-25 Collectively, the adduct mapping data indicate signiﬁcant site-speciﬁcity in modiﬁcation, which is further reﬂected by a recent analysis of the kinetics of compet- ing alkylation reactions of HNE with human serum albumin.26 Polyunsaturated fatty acids in living systems are largely present as phospholipid esters and recent studies have focused on novel biological properties of phospholipids bearing ep- oxyisoprostane and epoxycyclopentenone substituents27-29 or a reactive R, -unsaturated carbonyl oxidation product at the sn-2 position.30,31 These products stimulate inﬂammatory Journal of Proteome Research 2008, 7, 4237–4246 4237 Published on Web 09/09/2008