IDENTIFICATION OF MULTIPLE-TARGET SITES FOR A GLUTATHIONE CONJUGATE ON GLUTATHIONE-S-TRANSFERASE BY MATRIX-ASSISTED LASER DESORPTION IONIZATION MASS-SPECTROMETRY/

A mass spectrometric method providing qualitative site-specific inform ation regarding covalent modification of proteins is described. The me thod involves comparison of unmodified and modified proteins by matrix -assisted laser desorption/ionization mass spectrometry (MALDI MS) pep tide mapping in combination with site-specific mutagenesis of possible target amino acids, The approach is demonstrated through the mapping of glutathione-S-transferases (GSH transferases) before and after inhi bition with the glutathione conjugate 2-(S-glutathionyl)-3,5,6-trichlo ro-1,4-benzo quinone (GSTCBQ). The results demonstrate the utility of site-specific mutagenesis in combination with MALDI MS peptide mapping , Evidence is presented that three residues in or near the active site , including the hydroxyl groups of Tyr(6) and Tyr(115) and the sulphyd ryl group of Cys(114), are target sites for GSTCBQ. Although only one GSTCBQ molecule per active site was detected, it appears to be distrib uted among all three target sites, In addition, MALDI MS peptide mappi ng covered 81% of the cDNA deduced amino acid sequence for GSH transfe rase and site-directed mutagenesis corresponding to a single amino aci d substitution were verified.