Journal
JOURNAL OF PROTEOME RESEARCH
Volume 3, Issue 6, Pages 1228-1233Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr049887e
Keywords
oxidation; cysteine; thiol; iodoacetamide; isotope coded affinity tag (ICAT); proteomics; post-translational modification
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Funding
- NCRR NIH HHS [P41 RR 10888-06] Funding Source: Medline
- NHLBI NIH HHS [R01 HL 31607, T32 HL 007969, T32 HL 07224, N01 HV 28178] Funding Source: Medline
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An approach is described for the simultaneous identification and quantitation of oxidant-sensitive cysteine thiols in a complex protein mixture using a thiol-specific, acid-cleavable isotope-coded affinity tag (ICAT) reagent (Applied Biosystems, USA). The approach is based on the fact that only free cysteine thiols are susceptible to labeling by the iodoacetamide-based ICAT, and that mass spectrometry can be used to quantitate the relative labeling of free thiols. Applying this approach, we have identified cysteine thiols of proteins in a rabbit heart membrane fraction that are sensitive to a high concentration of hydrogen peroxide. Previously known and some novel proteins with oxidant-sensitive cysteines were identified. Of the many protein thiols labeled by the ICAT, only relatively few were oxidized more than 50% despite the high concentration of oxidant used, indicating that oxidant-sensitive thiols are relatively rare, and denoting their specificity and potential functional relevance.
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