title: Isotope-coded affinity tag approach to identify and quantify oxidant-sensitive protein thiols.
abstract: 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.
authors.affiliation: Vascular Biology Unit, Boston University School of Medicine, MA 02118, USA.
authors.forename: Mahadevan
authors.initials: M
authors.lastname: Sethuraman
authors.forename: Mark E
authors.initials: ME
authors.lastname: McComb
authors.forename: Tyler
authors.initials: T
authors.lastname: Heibeck
authors.forename: Catherine E
authors.initials: CE
authors.lastname: Costello
authors.forename: Richard A
authors.initials: RA
authors.lastname: Cohen
mesh_terms: D000345:Affinity Labels / Q000032:analysis* / Q000138:chemical synthesis / Q000378:metabolism; D000818:Animals; D003402:Creatine Kinase / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007460:Iodoacetamide / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry; D018482:Muscle, Skeletal / Q000201:enzymology; D016877:Oxidants / Q000494:pharmacology*; D020543:Proteome / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000737:chemistry*
chemical_list: D000345:Affinity Labels; D016877:Oxidants; D020543:Proteome; D013438:Sulfhydryl Compounds; D006861:Hydrogen Peroxide; D003402:Creatine Kinase; D003545:Cysteine; D007460:Iodoacetamide