P-139 - Developing targeted mass spectrometry approaches for the identification of protein lipoxidation

Abstract

Lipid peroxidation leads to formation of a variety of reactive products, including short-chain aldehydes. These can covalently modify proteins, a process called “lipoxidation”, and are thought to be involved in inflammatory diseases, as well as having potential as biomarkers. Our aim was to develop label-free mass spectrometry (MS) methods for the identification of aldehyde adducts in complex biological samples. Lysozyme and human serum albumin were treated with acrolein and 4-hydroxyhexanal (HHE) and adducts were stabilized by reduction before tryptic digestion. LC-MS/MS of the resulting peptides was used to map the protein-aldehyde modifications. More than 20 different sites and types of modification were observed, and both acrolein- and HHE-specific diagnostic ions identified. These were tested subsequently tested using multiple reaction monitoring (MRM) and precursor ion scanning mass spectrometry approaches. The small diagnostic ions for acrolein were found to offer the best transitions for MRM and allowed targeted identification of acrolein-containing peptides. This method provides a useful tool for analysis of reactive short-chain aldehyde adducts with proteins and is now being applied to more complex samples.