Mass spectrometry characterization of acrolein protein targets in the liver: Focus on site‐specific analysis

Abstract

Acrolein is the most reactive α, β‐unsaturated aldehyde and this property also makes it extremely cytotoxic. Toxicity is mediated, in part, by carbonylation of amino acids through Michael addition, which potentially alters the structure and function of target proteins. Acrolein can be generated endogenously: It is a product of lipid peroxidation and of metabolism of exogenous substances, such as the chemotherapeutic cyclophosphamide and the herbicide allyl alcohol. Total protein carbonylation is elevated in mouse hepatocytes due to acrolein exposure. However, the specific proteins that are modified by acrolein, and importantly, the amino acid sites changed by the aldehyde are not known. Thus, in this study, we determined the identity of the proteins selectively adducted by acrolein with a focus on characterizing carbonylated amino acid residues. Rat liver microsomes were resuspended in buffer and the non‐denatured mix was treated with acrolein. Microsomal proteins were kept in their native conformations in this experiment in order to determine acrolein accessible sites that may have relevance in vivo. Carbonylated proteins were labeled with biotin hydrazide and digested with trypsin. Then, biotinylated peptides were isolated from the mixture with avidin affinity chromatography and analyzed via LC‐MS/MS. Eighty‐seven different proteins and 125 unique modification sites were discerned using a Mascot score >; 30 and a precursor mass error < 20 ppm. Identified proteins include 9 isotypes of cytochrome P450, which plays an important role in metabolism, and 12 ribosomal proteins that are critical for protein synthesis. Our results validate this peptide‐centric methodology and should facilitate an understanding of the role that protein carbonylation plays in acrolein's toxicity within the liver.