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Mol Cell Proteomics. 2010 Jul;9(7):1400-10. doi: 10.1074/mcp.M900643-MCP200. Epub 2010 Mar 16.

Targeted quantitation of site-specific cysteine oxidation in endogenous proteins using a differential alkylation and multiple reaction monitoring mass spectrometry approach.

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1
double daggerBuck Institute for Age Research, Novato, California 94945, USA.

Abstract

Reactive oxygen species (ROS) are both physiological intermediates in cellular signaling and mediators of oxidative stress. The cysteine-specific redox-sensitivity of proteins can shed light on how ROS are regulated and function, but low sensitivity has limited quantification of the redox state of many fundamental cellular regulators in a cellular context. Here we describe a highly sensitive and reproducible oxidation analysis approach (OxMRM) that combines protein purification, differential alkylation with stable isotopes, and multiple reaction monitoring mass spectrometry that can be applied in a targeted manner to virtually any cysteine or protein. Using this approach, we quantified the site-specific cysteine oxidation status of endogenous p53 for the first time and found that Cys182 at the dimerization interface of the DNA binding domain is particularly susceptible to diamide oxidation intracellularly. OxMRM enables analysis of sulfinic and sulfonic acid oxidation levels, which we validate by assessing the oxidation of the catalytic Cys215 of protein tyrosine phosphatase-1B under numerous oxidant conditions. OxMRM also complements unbiased redox proteomics discovery studies as a verification tool through its high sensitivity, accuracy, precision, and throughput.

PMID:
20233844
PMCID:
PMC2938085
DOI:
10.1074/mcp.M900643-MCP200
[Indexed for MEDLINE]
Free PMC Article
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