Mass spectrometry-based quantification of myocardial protein adducts with acrolein in an in vivo model of oxidative stress.

Abstract

Acrolein (ACR) exposure leads to the formation of protein-ACR adducts. Protein modification by ACR has been associated with various chronic diseases including cardiovascular and neurodegenerative diseases. Here, we report an analytical strategy that enables the quantification of Michael-type protein adducts of ACR in mitochondrial proteome samples using liquid chromatography in combination with tandem mass spectrometry and selected ion monitoring (LC-MS/MS SRM) analysis. Our approach combines site-specific identification and relative quantification at the peptide level of protein-ACR adducts in relation to the unmodified protein thiol pool. Treatment of 3-month-old rats with CCl(4) , an established in vivo model of acute oxidative stress, resulted in significant increases in the ratios of distinct ACR-adducted peptides to the corresponding unmodified thiol-peptides obtained from proteins that were isolated from cardiac mitochondria. The mitochondrial proteins that were found adducted by ACR were malate dehydrogenase, NADH dehydrogenase [ubiquinone] flavoprotein 1, cytochrome c oxidase subunit VIb isoform 1, ATP synthase d chain, and ADP/ATP translocase 1. The findings indicate that protein modification by ACR has potential value as an index of mitochondrial oxidative stress.