Issue 6, 2016

An immunochemical approach to detect oxidized protein tyrosine phosphatases using a selective C-nucleophile tag

Abstract

Protein tyrosine phosphatases are crucial regulators of signal transduction and function as antagonists towards protein tyrosine kinases to control reversible tyrosine phosphorylation, thereby regulating fundamental physiological processes. Growing evidence has supported the notion that reversible oxidative inactivation of the catalytic cysteine residue in protein tyrosine phosphatases serves as an oxidative post-translational modification that regulates its activity to influence downstream signaling by promoting phosphorylation and induction of the signaling cascade. The oxidation of cysteine to the sulfenic acid is often transient and difficult to detect, thus making it problematic in understanding the role that this oxidative post-translational modification plays in redox-biology and pathogenesis. Several methods to detect cysteine oxidation in biological systems have been developed, though targeted approaches to directly detect oxidized phosphatases are still lacking. Herein we describe the development of a novel immunochemical approach to directly profile oxidized phosphatases. This immunochemical approach consists of an antibody designed to recognize the conserved sequence of the PTP active site (VHCDMDSAG) harboring the catalytic cysteine modified with dimedone (CDMD), a nucleophile that chemoselectively reacts with cysteine sulfenic acids to form a stable thioether adduct. Additionally, we provide biochemical and mass spectrometry workflows to be used in conjugation with this newly developed immunochemical approach to assist in the identification and quantification of basal and oxidized phosphatases.

Graphical abstract: An immunochemical approach to detect oxidized protein tyrosine phosphatases using a selective C-nucleophile tag

Supplementary files

Article information

Article type
Paper
Submitted
03 dec 2015
Accepted
08 jan 2016
First published
08 jan 2016

Mol. BioSyst., 2016,12, 1790-1798

Author version available

An immunochemical approach to detect oxidized protein tyrosine phosphatases using a selective C-nucleophile tag

F. J. Garcia and K. S. Carroll, Mol. BioSyst., 2016, 12, 1790 DOI: 10.1039/C5MB00847F

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