Obama Takashi, Kato Rina, Masuda Yutaka, Takahashi Katsuhiko, Aiuchi Toshihiro, Itabe Hiroyuki
Department of Biological Chemistry, School of Pharmaceutical Sciences, Showa University, Shinagawa-ku, Tokyo, Japan.
Proteomics. 2007 Jun;7(13):2132-41. doi: 10.1002/pmic.200700111.
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.
氧化修饰的低密度脂蛋白（oxLDL）是动脉粥样硬化发展过程中的主要因素之一。由于载脂蛋白B-100（apoB-100）的不溶性以及氧化修饰的异质性，oxLDL中apoB-100的修饰结构尚不清楚。我们应用了一种用于对天然和修饰的低密度脂蛋白（LDL）样品中的apoB-100蛋白进行LC-MS/MS分析的膜上样品制备程序，以去除LDL中的脂质成分，随后收集apoB-100的胰蛋白酶消化产物。与常用的胶内消化方案相比，使用聚偏二氟乙烯（PVDF）膜的样品制备程序大大提高了胰蛋白酶肽段的回收率，并在最终分析中提高了序列覆盖率，从而鉴定出铜诱导的oxLDL中的修饰氨基酸残基。检测到一个被主要脂质过氧化产物4-羟基壬烯醛修饰的组氨酸残基，以及氧化组氨酸和色氨酸残基。LC-MS/MS结合膜上样品制备程序是分析apoB-100等高度疏水蛋白质的有用方法。
