title: Investigating redox regulation of protein tyrosine phosphatases using low pH thiol labeling and enrichment strategies coupled to MALDI-TOF mass spectrometry.
abstract: A central feature of the protein tyrosine phosphatase (PTP) catalytic mechanism is an attack of the substrate's phosphate moiety by a thiolate ion in the signature CX5R motif. In addition to being an effective nucleophile in this form, the thiolate ion is also susceptible to reversible redox regulation. This attribute permits temporal inhibition of PTP activities, which affects numerous cellular processes utilizing kinase-mediated signal propagation. Accumulating evidence has revealed diverse mechanisms adopted by PTPs to avoid irreversible thiol oxidation of the active site Cys residue, often involving structurally proximal thiols within the active site region. Therefore, there has been a significant effort made to develop thiol labeling strategies coupled to mass spectrometry to identify and characterize redox sensitive thiols within PTPs as a necessary step in understanding how a particular PTP is regulated by redox signaling. A common drawback to many current methods is the use of neutral pH labeling techniques, requiring special attention with regards to non-specific thiol oxidation during sample preparation. This study describes the use of rapid, low pH thiol labeling methods to overcome this issue. Mercury immobilized metal affinity chromatography (Hg-IMAC) demonstrated high selectivity and specificity while enriching for thiol-containing peptides from the atypical dual specificity phosphatase hYVH1 (also known as DUSP12). This approach revealed several reversibly oxidized thiols within the catalytic domain of hYVH1. Subsequently, use of another low pH labeling reagent, 4,4-dithiopyridine (4-DTP) helped identify novel disulfide linkages providing evidence that hYVH1 utilizes a disulfide exchange mechanism to prevent irreversible oxidation of the catalytic Cys residue in the active site.
authors: Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4, Canada. Electronic address: bonham@uwindsor.ca.
authors: Christopher A
authors: CA
authors: Bonham
authors: Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4, Canada.
authors: Aaron J
authors: AJ
authors: Steevensz
authors: Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4, Canada.
authors: Qiudi
authors: Q
authors: Geng
authors: Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4, Canada. Electronic address: vacratsi@uwindsor.ca.
authors: Panayiotis O
authors: PO
authors: Vacratsis
mesh_terms: D000345:Affinity Labels; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D000345:Affinity Labels; D013438:Sulfhydryl Compounds; D017027:Protein Tyrosine Phosphatases
keywords: DUSP12; Dual specificity phosphatase 12; Immobilized metal affinity chromatography; Mass spectrometry; Organomercurial; Reversible thiol oxidation