Relative quantification of oxylipid-modified peptides using a QTrap instrument

摘要

The adduction of proteins and other biomolecules by electrophilic lipid peroxidation products such as 4-hydroxynonenal or acrolein is thought to be an initiating and/or propagating factor in 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 this oxidative damage 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 to their corresponding unmodified peptides. By using an affinity labeling and enrichment technique along with multiple reaction monitoring (MRM) 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. Here we demonstrate the potential of this method to determine the relative abundance of oxidative damage by acrolein occurring in-vivo for several mitochondrial proteins.