Mass Spectrometry-based Quantification of Acrolein-modified Cys-containing Peptides in an In Vivo Model of Oxidative Stress

摘要

The adduction of proteins and other biomolecules by electrophilic lipid peroxidation products, such as 4-hydroxynonenal or acrolein, is thought to be linked to the pathophysiology of several diseases, such as atherosclerosis, diabetes, Alzheimer's, Parkinson's and other age-related disorders. Determining the extent or relative amounts of these oxidative modifications could provide valuable insights into the molecular mechanisms of these disorders. Relative quantitation of oxylipid-modified proteins in biological samples is a challenging problem because of the complexity and extreme dynamic range that characterize these samples. Here we demonstrate a method for relative quantification of oxylipid-modified peptides compared to their corresponding unmodified peptides. By using an affinity labeling and enrichment technique along with selected reaction monitoring (SRM) mode on a hybrid linear ion trap mass spectrometer we are able to detect modified peptides of very low natural abundance along with the more abundant native peptide with high selectivity. We also demonstrate a method for relative quantification of acrolein-modified Cys-containing peptides to their corresponding unmodified peptides in rats treated with CCl_(4), an established in vivo model of oxidative stress.