Site-Specific Protein Adducts of 4-Hydroxy-2(E)-Nonenal in Human THP-1 Monocytic Cells: Protein Carbonylation Is Diminished by Ascorbic Acid

Abstract

The protein targets and sites of modification by 4-hydroxy-2(E)-nonenal (HNE) in human monocytic THP-1 cells after exogenous exposure to HNE were examined using a multipronged proteomic approach involving electrophoretic, immunoblotting, and mass spectrometric methods. Immunoblot analysis using monoclonal anti-HNE antibodies showed several proteins as targets of HNE adduction. Pretreatment of THP-1 cells with ascorbic acid resulted in reduced levels of HNE-protein adducts. Biotinylation of Michael-type HNE adducts using an aldehyde-reactive hydroxylamine-functionalized probe (aldehyde-reactive probe, ARP) and subsequent enrichment facilitated the identification and site-specific assignment of the modifications by LC-MS/MS analysis. Sixteen proteins were unequivocally identified as targets of HNE adduction, and eighteen sites of HNE modification at Cys and His residues were assigned. HNE exposure of THP-1 cells resulted in the modification of proteins involved in cytoskeleton organization and regulation, proteins associated with stress responses, and enzymes of the glycolytic and other metabolic pathways. This study yielded the first evidence of site-specific adduction of HNE to Cys-295 in tubulin alpha-1B chain, Cys-351 and Cys-499 in alpha-actinin-4, Cys-328 in vimentin, Cys-369 in D-3-phosphoglycerate dehydrogenase, and His-246 in aldolase A.