Chung Woon-Gye, Maier Claudia S
Oregon State University, Department of Chemistry, Corvallis, Oregon, USA.
Curr Protoc Toxicol. 2008;Chapter 17:Unit 17.9. doi: 10.1002/0471140856.tx1709s35.
The modification of proteins by reactive products of lipid peroxidation is associated with a large number of diseases and biological aging; thus, methods that enable the characterization of oxylipid-protein and/or peptide conjugates are highly in demand. This unit outlines a chemical labeling approach to identifying and characterizing proteins modified by lipid peroxidation products. It also outlines two approaches for mass spectrometry-based identification and detailed characterization of oxylipid conjugates. The first combines chemical labeling of oxylipid-protein conjugates using an aldehyde-specific biotinylation reagent, electrophoretic separation, and mass spectrometry-based identification of the biotinylated proteins. In the second approach, protein extracts are treated with the aldehyde-specific reagent, proteolyzed using trypsin, and the biotinylated peptides are enriched using immobilized monomeric avidin. The enriched peptide fractions are submitted to tandem mass spectrometry for determining the peptide sequence information, site of the modification, and chemical nature of the oxylipid.
脂质过氧化反应产物对蛋白质的修饰与大量疾病及生物衰老相关；因此，能够对氧化脂质-蛋白质和/或肽缀合物进行表征的方法需求极大。本单元概述了一种化学标记方法，用于鉴定和表征被脂质过氧化产物修饰的蛋白质。它还概述了两种基于质谱的方法，用于氧化脂质缀合物的鉴定和详细表征。第一种方法是使用醛特异性生物素化试剂对氧化脂质-蛋白质缀合物进行化学标记、电泳分离，并基于质谱鉴定生物素化的蛋白质。在第二种方法中，蛋白质提取物用醛特异性试剂处理，用胰蛋白酶进行蛋白水解，然后使用固定化的单体抗生物素蛋白富集生物素化的肽段。将富集的肽段组分进行串联质谱分析，以确定肽序列信息、修饰位点以及氧化脂质的化学性质
