Identification of proteins adducted by lipid peroxidation products in plasma and modifications of apolipoprotein A1 with a novel biotinylated phospholipid probe

Abstract

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 linoleoylglycerylphosphatidylcholine probe called PLPBSO ( Tallman et al. Chem. Res. Toxicol. 2007, 20, 227-234 ), 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.

Figure 1

Structure of PLPBSO and application to identification of protein targets of phospholipid electrophiles. See text for discussion.

Figure 2

Base hydrolysis of biotinylated proteins in various concentrations of NH₄OH and different temperatures shown in Alexa Fluor 680-conjugated streptavidin Western blot (left) and visible coomassie-stained gel (right). Disappearance of biotin in the Western shows the degree of hydrolysis of the phospholipid headgroup. The visible gel indicates the stability of the proteins under the hydrolysis conditions.

Figure 3

Immunoblot confirmation of PLPBSO adducts on ApoA1. Oxidation induces a migration shift of ApoA1 in SDS-PAGE analysis and streptavidin labels the PLPBSO-modified ApoA1 protein. Oxidized plasma was immunoblotted with polyclonal ApoA1 antibody followed by Alexa fluor 680 antirabbit (left) or with antistreptavidin (right).

Figure 5

LC-MS−MS of the T*H¹⁶²LAPYSDELR tryptic peptide sequence from ApoA1 modified with KODA (A), with HNE (B) at His 162; of the QKL*H¹³⁵ELQEK tryptic peptide sequence modified with 7-oxoheptanoic acid at His135 (C); and of the LEAL*K¹⁸²ENGGAR tryptic peptide sequence modified with 8-oxooctanoic acid at Lys182 (D).

Figure 4

Isolated HDL treated with HNE and oxidized with AIPH in different concentrations. They are visualized with anti-HNE (left) or colloidal blue (right). Note the migration shift seen in the colloidal blue stained gel in the ApoA1 band between 20−26 kDa is induced by oxidation, but not by HNE.