Identification of the Covalent Binding Sites of the Cycllopenttenone Prostaglandin 15-Deoxy-DELTA12,14-Prostaglandin J_(2) (15d-PGJ_(2)) in Ubiquitin Carboxy-Terminal Hydrolase L1 (UCH-L1)

摘要

Multiple MS platforms, using both intact protein as well as bottom-up analyses of trypsin or chymotrypsin digests, were used to detect 15d-PGJ_(2) adducts of UCH-L1. The adducts were formed by in vitro reaction of 15d-PGJ_(2) with recombinant UCH-L1 in PBS buffer, pH 7.4 . Both ESI-TOF-MS and MALDI-TOF-MS of the intact protein showed as many as five 15d-PGJ_(2) moieties bound to UCH-L1. The sites of adduction were determined by bottom-up MS and MS/MS analyses. In-gel digests of the samples allowed detection of 15d-PGJ_(2) bound to a peptide containing C220. No other cysteines modified with 15d-PGJ_(2) were detected, although C90 and C152 were detected as unmodified by 15d-PGJ_(2) (i.e., they were detected as their carbamidomethylated form after reaction with iodoacetamide). In-solution digestion using trypsin or chymotrypsin allowed detection of all six cysteines. Only C132 was found to not be modified by 15d-PGJ_(2). The methods presented here will be used to develop an assay to detect 15d-PGJ_(2) adducts of UCH-L1 formed in vivo.