IDENTIFICATION OF THE OXIDATION-STATES OF THE ACTIVE-SITE CYSTEINE INA RECOMBINANT PROTEIN-TYROSINE-PHOSPHATASE BY ELECTROSPRAY MASS-SPECTROMETRY USING ONLINE DESALTING

The oxidation state of the cysteine residue at the active site of huma n protein tyrosine phosphatase (PTP-1B) greatly affects its enzymatic activity. We wished to examine peroxide-treated preparations for modif ications of this enzyme with electrospray mass spectrometry in order t o determine the locations and oxidation states of the cysteines or oth er residues involved in the process. Since these reaction products con tained large amounts of salts and buffers, they required desalting pri or to analysis. Existing on- and off-line methods presented certain di fficulties in handling and sample usage. Based on recent experience wi th direct syringe admission of sample, we developed a procedure as a s imple, inexpensive alternative to full highperformance liquid chromato graphy systems that provides on-line desalting using only a few mu L o f sample. The method was applied to the analysis of oxidized PTP-1B pr eparations where conversion of cysteine 215 to both sulfinic and sulfo nic acid residues was demonstrated. (C) 1998 John Wiley & Sons, Ltd.