Limits...
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2664612&req=5

fig4: 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.

Mentions: Our analyses of the streptavidin-captured ApoA1 (see above) identified relatively few adducts corresponding to modification by noncarboxylated electrophiles originating from the ω-terminus of the fatty acid (e.g., HNE). To determine if HNE adducts are formed on ApoA1 upon oxidation of native HDL associated lipids, HDL particles were isolated from plasma and then oxidized overnight with different concentrations of AIPH or treated with HNE. Adducts from both treatments were stabilized by reduction with NaBH4. Anti-HNE immunoblotting revealed immunoreactivity of the ApoA1 band (∼20−26 kDa) from HDL particles oxidized with 5 mM AIPH or treated with either 100 μM or 1 mM HNE (Figure 4A). The visible gel in Figure 4B shows that a molecular weight shift and band broadening for ApoA1 (similar to that observed in Figure 2, above) was induced by as little as 100 μM AIPH, but not by HNE treatment. This suggests that the ApoA1 migration is affected perhaps by multiple modifications of the ApoA1 protein that affect protein charge, shape and/or SDS binding, but that HNE adduction alone does not produce these effects.


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)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: 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.
Mentions: Our analyses of the streptavidin-captured ApoA1 (see above) identified relatively few adducts corresponding to modification by noncarboxylated electrophiles originating from the ω-terminus of the fatty acid (e.g., HNE). To determine if HNE adducts are formed on ApoA1 upon oxidation of native HDL associated lipids, HDL particles were isolated from plasma and then oxidized overnight with different concentrations of AIPH or treated with HNE. Adducts from both treatments were stabilized by reduction with NaBH4. Anti-HNE immunoblotting revealed immunoreactivity of the ApoA1 band (∼20−26 kDa) from HDL particles oxidized with 5 mM AIPH or treated with either 100 μM or 1 mM HNE (Figure 4A). The visible gel in Figure 4B shows that a molecular weight shift and band broadening for ApoA1 (similar to that observed in Figure 2, above) was induced by as little as 100 μM AIPH, but not by HNE treatment. This suggests that the ApoA1 migration is affected perhaps by multiple modifications of the ApoA1 protein that affect protein charge, shape and/or SDS binding, but that HNE adduction alone does not produce these effects.

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