IDENTIFICATION OF THE HEPATIC PROTEIN TARGETS OF REACTIVE METABOLITESOF ACETAMINOPHEN IN-VIVO IN MICE USING 2-DIMENSIONAL GEL-ELECTROPHORESIS AND MASS-SPECTROMETRY

Liver toxicity following an overdose of acetaminophen is frequently co nsidered a model for drug-induced hepatotoxicity. Extensive studies ov er many years have established that such toxicity is web correlated wi th liver protein arylation by acetaminophen metabolites. Identificatio n of protein targets for covalent modifications is a challenging but n ecessary step in understanding how covalent binding could lead to live r toxicity. Previous approaches suffered from technical limitations, a nd thus over the last 10 years heroic efforts were required to determi ne the identity of only a few target proteins. We present a new mass s pectrometry-based strategy for identification of all target proteins t hat now provides a comprehensive survey of the suite of Liver proteins modified. After administration of radiolabeled acetaminophen to mice, the proteins in the liver tissue lysate were separated by two-dimensi onal polyacrylamide gel electrophoresis. In-gel digestion of the radio labeled gel spots gave a set of tryptic peptides, which were analyzed by matrix-assisted laser desorption ionization mass spectrometry. Inte rrogation of data bases based on experimentally determined molecular a -eights of peptides and product ion tags from postsource decay mass sp ectra was employed for the determination of the identities of modified liver proteins. Using this method, more than 20 new drug-labeled prot eins have been identified.