Isotope-coded affinity tag (ICAT) approach to redox proteomics: identification and quantitation of oxidant-sensitive cysteine thiols in complex protein mixtures.
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.
Sethuraman
Mahadevan
M
Vascular Biology Unit, Cardiovascular Proteomics Center, and Mass Spectrometry Resource, Boston University School of Medicine, Boston, MA 02118, USA.
McComb
Mark E
ME
Huang
Hua
H
Huang
Sequin
S
Heibeck
Tyler
T
Costello
Catherine E
CE
Cohen
Richard A
RA
D000345:Affinity Labels; D000818:Animals; D002247:Carbon Isotopes; D003545:Cysteine / Q000032:analysis* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis; D009206:Myocardium / Q000737:chemistry; D016877:Oxidants / Q000378:metabolism; D010084:Oxidation-Reduction; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000032:analysis / Q000378:metabolism
D000345:Affinity Labels; D002247:Carbon Isotopes; D008565:Membrane Proteins; D016877:Oxidants; D011506:Proteins; D013438:Sulfhydryl Compounds; D006861:Hydrogen Peroxide; D003545:Cysteine