Abstract
Abstract
Traditionally, studies of post translational modifications (PTMs) by mass analysis have been limited to modifications such as deamidation and oxidation that have a mass shift. Although Asp isomerization is an important PTM, the selective detection of Asp isomers by mass spectrometry was originally thought to be impossible due to the identical mass of the isomers. The recent development of an LC-MS-based method has facilitated rapid and accurate quantitative analysis of Asp isomers in long-lived proteins; however, because the quantification is based on the extracted ion chromatogram acquired by an MS1 scan, this methodology is not always efficient for detecting extremely low-abundance peptides in complex biological samples. In this paper, we evaluated Asp isomer-containing peptides of αA-crystallin present in tryptic digests of human lens samples with different degrees of protein aggregation and different ages using LC coupled with multiple reaction monitoring (MRM). In a single analysis, the LC-MRM method enabled three tryptic peptides containing isomers of Asp58, Asp91/92, and Asp151 to be detected simultaneously. The extent of isomerization and epimerization of these specific Asp sites in αA-crystallin increased with the progress of α-crystallin aggregation. For the analysis of samples known to isomerize at specific Asp residues, MRM gives a more rapid, less laborious, and high-quality separation of Asp isomer-containing peptides relative to the previous MS1-based quantitative method.