Identification of the oxidation states of the active site cysteine in a recombinant protein tyrosine phosphatase by electrospray mass spectrometry using on-line desalting

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 loca...

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Published inRapid communications in mass spectrometry Vol. 12; no. 20; pp. 1457 - 1462
Main Authors DeGnore, Jon P., König, Simone, Barrett, William C., Chock, P. Boon, Fales, Henry M.
Format Journal Article
LanguageEnglish
Published Chichester, UK John Wiley & Sons, Ltd 30.10.1998
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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 μL 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. © 1998 John Wiley & Sons, Ltd.
AbstractList 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.
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 μL 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. © 1998 John Wiley & Sons, Ltd.
Author Fales, Henry M.
König, Simone
DeGnore, Jon P.
Barrett, William C.
Chock, P. Boon
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Snippet 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...
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...
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SubjectTerms Amino Acid Sequence
Binding Sites
Cysteine - chemistry
Humans
Mass Spectrometry
Molecular Sequence Data
Oxidation-Reduction
Protein Tyrosine Phosphatases - chemistry
Recombinant Proteins - analysis
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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
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