title: Identification of total reversible cysteine oxidation in an atherosclerosis model using a modified biotin switch assay.
abstract: Oxidative stress due to the imbalance of reactive oxygen species (ROS) and the resulting reversible cysteine oxidation (CysOX) are involved in the early proatherogenic aspect of atherosclerosis. Given that the corresponding redox signaling pathways are still unclear, a modified biotin switch assay was developed to quantify the reversible CysOX in an atherosclerosis model established by using a monocytic cell line treated with platelet releasate. The accumulation of ROS was observed in the model system and validated in human primary monocytes. Through the application of the modified biotin switch assay, we obtained the first reversible CysOX proteome for this model. A total of 75 peptides, corresponding to 53 proteins, were quantified with oxidative modification. The bioinformatics analysis of these CysOX-containing proteins highlighted biological processes including glycolysis, cytoskeleton arrangement, and redox regulation. Moreover, the reversible oxidation of three glycolysis enzymes was observed using this method, and the regulation influence was verified by an enzyme activity assay. NADPH oxidase (NOX) inhibition treatment, in conjunction with the modified biotin switch method, was used to evaluate the global CysOX status. In conclusion, this versatile modified biotin switch assay provides an approach for the quantification of all reversible CysOX and for the study of redox signaling in atherosclerosis as well as in diseases in other biological systems.
authors.affiliation: §Department of Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1Z1.
authors.forename: Ru
authors.initials: R
authors.lastname: Li
authors.affiliation: §Department of Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1Z1.
authors.forename: Jiqing
authors.initials: J
authors.lastname: Huang
authors.affiliation: §Department of Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1Z1.
authors.forename: Juergen
authors.initials: J
authors.lastname: Kast
mesh_terms: D000595:Amino Acid Sequence; D050197:Atherosclerosis / Q000378:metabolism / Q000473:pathology; D001681:Biological Assay; D001710:Biotin / Q000737:chemistry; D001792:Blood Platelets / Q000737:chemistry; D002457:Cell Extracts / Q000494:pharmacology*; D002460:Cell Line; D002853:Chromatography, Liquid; D003545:Cysteine; D006801:Humans; D053858:Metabolic Networks and Pathways; D008954:Models, Biological; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D009000:Monocytes / Q000737:chemistry / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019255:NADPH Oxidases / Q000737:chemistry; D010084:Oxidation-Reduction; D010455:Peptides / Q000302:isolation & purification*; D061251:Primary Cell Culture; D020543:Proteome / Q000302:isolation & purification*; D017382:Reactive Oxygen Species / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D002457:Cell Extracts; D010455:Peptides; D020543:Proteome; D017382:Reactive Oxygen Species; D001710:Biotin; D019255:NADPH Oxidases; D003545:Cysteine
keywords: atherosclerosis; biotin switch assay; platelet releasate; reversible cysteine oxidation