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Identification of multiple target sites for a glutathione conjugate on glutathione-S-transferase by matrix-assisted laser desorption/ionization mass spectrometry

✍ Scribed by Jespersen, S.; Ploemen, J. H. T. M.; van Bladeren, P. J.; Niessen, W. M. A.; Tjaden, U. R.; van der Greef, J.

Publisher: John Wiley and Sons
Year: 1996
Tongue: English
Weight: 576 KB
Volume: 31
Category: Article
ISSN: 1076-5174
DOI: 10.1002/(sici)1096-9888(199601)31:1<101::aid-jms265>3.0.co;2-j

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✦ Synopsis

A mass spectrometric method providing qualitative site-specific information regarding covalent modification of proteins is described. The method involves comparison of unmodified and modified proteins by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) peptide mapping in combination with site-specific mutagenesis of possible target amino acids. The approach is demonstrated through the mapping of glutathione-& transferases (GSH transferases) before and after inhibition with the glutathione conjugate 2-(9glutathionyl)-3,5,6-trichloro-1,4-benzoquinone (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, and Tyr,,, and the sulphydryl group of CyslI4, are target sites for GSTCBQ. Although only one GSTCBQ molecule per active site was detected, it appears to be distributed among all three target sites. In addition, MALDI MS peptide mapping covered 81% of the cDNA deduced amino acid sequence for GSH transferase and site-directed mutagenesis corresponding to a single amino acid substitution were verified.

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