title: Identification of the oxidation states of the active site cysteine in a recombinant protein tyrosine phosphatase by electrospray mass spectrometry using on-line desalting.
abstract: The oxidation state of the cysteine residue at the active site of human protein tyrosine phosphatase (PTP-1B) greatly affects its enzymatic activity. We wished to examine peroxide-treated preparations for modifications of this enzyme with electrospray mass spectrometry in order to determine the locations and oxidation states of the cysteines or other residues involved in the process. Since these reaction products contained large amounts of salts and buffers, they required desalting prior to analysis. Existing on- and off-line methods presented certain difficulties in handling and sample usage. Based on recent experience with direct syringe admission of sample, we developed a procedure as a simple, inexpensive alternative to full high-performance liquid chromatography systems that provides on-line desalting using only a few microL of sample. The method was applied to the analysis of oxidized PTP-1B preparations where conversion of cysteine 215 to both sulfinic and sulfonic acid residues was demonstrated.
authors.affiliation: Laboratory of Biophysical Chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: J P
authors.initials: JP
authors.lastname: DeGnore
authors.forename: S
authors.initials: S
authors.lastname: König
authors.forename: W C
authors.initials: WC
authors.lastname: Barrett
authors.forename: P B
authors.initials: PB
authors.lastname: Chock
authors.forename: H M
authors.initials: HM
authors.lastname: Fales
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry*; D011994:Recombinant Proteins / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D011994:Recombinant Proteins; D017027:Protein Tyrosine Phosphatases; D003545:Cysteine