Isotope-coded affinity tag (ICAT) approach to redox proteomics: identification and quantitation of oxidant-sensitive cysteine thiols in complex protein mixtures |
| |
Authors: | Sethuraman Mahadevan McComb Mark E Huang Hua Huang Sequin Heibeck Tyler Costello Catherine E Cohen Richard A |
| |
Institution: | Vascular Biology Unit, Cardiovascular Proteomics Center, and Mass Spectrometry Resource, Boston University School of Medicine, Boston, MA 02118, USA. |
| |
Abstract: | 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. |
| |
Keywords: | |
本文献已被 PubMed 等数据库收录! |
|