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

Szapacs ME, Kim HY, Porter NA, Liebler DC - J. Proteome Res. (2008)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232-0146, USA.

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.

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Structure of PLPBSO and application to identification of protein targets of phospholipid electrophiles. See text for discussion.
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fig1: Structure of PLPBSO and application to identification of protein targets of phospholipid electrophiles. See text for discussion.

Mentions: 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), hydroxyoxododecenoate (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).


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

Szapacs ME, Kim HY, Porter NA, Liebler DC - J. Proteome Res. (2008)

Structure of PLPBSO and application to identification of protein targets of phospholipid electrophiles. See text for discussion.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2664612&req=5

fig1: Structure of PLPBSO and application to identification of protein targets of phospholipid electrophiles. See text for discussion.
Mentions: 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), hydroxyoxododecenoate (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).

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232-0146, USA.

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.

Show MeSH
Related in: MedlinePlus