Journal
MOLECULAR & CELLULAR PROTEOMICS
Volume 3, Issue 3, Pages 273-278Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/mcp.T300011-MCP200
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Funding
- NCRR NIH HHS [P41 RR10888] Funding Source: Medline
- NHLBI NIH HHS [T32 HL07224, T32 HL007969] Funding Source: Medline
- PHS HHS [268200248178C] Funding Source: Medline
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An approach is described for identifying and quantifying oxidant- sensitive protein thiols using a cysteine- specific, acid- cleavable isotope- coded affinity tag ( ICAT) reagent ( Applied Biosystems, Foster City, CA). The approach is based on the fact that only free cysteine thiols are susceptible to labeling by the iodoacetamide- based ICAT reagent, and that mass spectrometry can be used to quantitate the relative labeling of free thiols. To validate our approach, creatine kinase with four cysteine residues, one of which is oxidant- sensitive, was chosen as an experimental model. ICAT- labeled peptides derived from creatine kinase were used to evaluate the relative abundance of the free thiols in samples subjected ( or not) to treatment with hydrogen peroxide. As predicted, hydrogen peroxide decreased the relative abundance of the unmodified oxidant- sensitive thiol residue of cysteine- 283 in creatine kinase, providing proof of principle that an ICAT- based quantitative mass spectrometry approach can be used to identify and quantify oxidation of cysteine thiols. This approach opens an avenue for proteomics studies of the redox state of protein thiols.
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