title: Analysis of modified apolipoprotein B-100 structures formed in oxidized low-density lipoprotein using LC-MS/MS.
abstract: Oxidatively modified low-density lipoprotein (oxLDL) is one of the major factors involved in the development of atherosclerosis. Because of the insolubility of apolipoprotein B-100 (apoB-100) and the heterogeneous nature of oxidative modification, modified structures of apoB-100 in oxLDL are poorly understood. We applied an on-Membrane sample preparation procedure for LC-MS/MS analysis of apoB-100 proteins in native and modified low-density lipoprotein (LDL) samples to eliminate lipid components in the LDLs followed by collection of tryptic digests of apoB-100. Compared with a commonly used in-gel digestion protocol, the sample preparation procedure using PVDF membrane greatly increased the recovery of tryptic peptides and resulted in improved sequence coverage in the final analysis, which lead to the identification of modified amino acid residues in copper-induced oxLDL. A histidine residue modified by 4-hydroxynonenal, a major lipid peroxidation product, as well as oxidized histidine and tryptophan residues were detected. LC-MS/MS in combination with the on-Membrane sample preparation procedure is a useful method to analyze highly hydrophobic proteins such as apoB-100.
authors.affiliation: Department of Biological Chemistry, School of Pharmaceutical Sciences, Showa University, Shinagawa-ku, Tokyo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Obama
authors.forename: Rina
authors.initials: R
authors.lastname: Kato
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Masuda
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Takahashi
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Aiuchi
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Itabe
mesh_terms: D000171:Acrolein / Q000737:chemistry; D000447:Aldehydes / Q000737:chemistry; D000595:Amino Acid Sequence; D053299:Apolipoprotein B-100 / Q000032:analysis / Q000737:chemistry*; D002725:Chloroform / Q000737:chemistry; D002853:Chromatography, Liquid; D019327:Copper Sulfate / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007737:Kynurenine / Q000032:analysis; D008077:Lipoproteins, LDL / Q000032:analysis / Q000737:chemistry*; D008239:Lysine / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D011145:Polyvinyls / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry; D005465:Fluorocarbon Polymers
chemical_list: D000171:Acrolein; D000447:Aldehydes; D053299:Apolipoprotein B-100; D002725:Chloroform; D019327:Copper Sulfate; D003545:Cysteine; D006639:Histidine; D007737:Kynurenine; D008077:Lipoproteins, LDL; D008239:Lysine; D010446:Peptide Fragments; D011145:Polyvinyls; D014357:Trypsin; D014364:Tryptophan; C113380:N(epsilon)-(3-formyl-3,4-dihydropiperidino)lysine; C482330:N(epsilon)-(3-methylpyridinium)lysine; C099252:oxidized low density lipoprotein; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C027576:4-hydroxy-2-nonenal