title: Biotin Switch Assays for Quantitation of Reversible Cysteine Oxidation.
abstract: Thiol groups in protein cysteine residues can be subjected to different oxidative modifications by reactive oxygen/nitrogen species. Reversible cysteine oxidation, including S-nitrosylation, S-sulfenylation, S-glutathionylation, and disulfide formation, modulate multiple biological functions, such as enzyme catalysis, antioxidant, and other signaling pathways. However, the biological relevance of reversible cysteine oxidation is typically underestimated, in part due to the low abundance and high reactivity of some of these modifications, and the lack of methods to enrich and quantify them. To facilitate future research efforts, this chapter describes detailed procedures to target the different modifications using mass spectrometry-based biotin switch assays. By switching the modification of interest to a biotin moiety, these assays leverage the high affinity between biotin and avidin to enrich the modification. The use of stable isotope labeling and a range of selective reducing agents facilitate the quantitation of individual as well as total reversible cysteine oxidation. The biotin switch assay has been widely applied to the quantitative analysis of S-nitrosylation in different disease models and is now also emerging as a valuable research tool for other oxidative cysteine modifications, highlighting its relevance as a versatile, robust strategy for carrying out in-depth studies in redox proteomics.
authors.affiliation: The Biomedical Research Centre, University of British Columbia, Vancouver, BC, Canada; University of British Columbia, Vancouver, BC, Canada.
authors.forename: R
authors.initials: R
authors.lastname: Li
authors.affiliation: The Biomedical Research Centre, University of British Columbia, Vancouver, BC, Canada; University of British Columbia, Vancouver, BC, Canada; Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada. Electronic address: kast@brc.ubc.ca.
authors.forename: J
authors.initials: J
authors.lastname: Kast
mesh_terms: D001681:Biological Assay / Q000379:methods*; D001710:Biotin / Q000032:analysis*; D003545:Cysteine / Q000737:chemistry*; D010084:Oxidation-Reduction
chemical_list: D001710:Biotin; D003545:Cysteine
keywords: Biotin switch assay; Disulfide formation; Reversible cysteine oxidation; S-glutathionylation; S-nitrosylation; S-sulfenylation