Journal
JOURNAL OF PROTEOME RESEARCH
Volume 14, Issue 5, Pages 2026-2035Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.jproteome.5b00133
Keywords
reversible cysteine oxidation; biotin switch assay; platelet releasate; atherosclerosis
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Funding
- Canadian Institutes of Health Research
- Natural Sciences and Engineering Research Council of Canada
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Oxidative stress due to the imbalance of reactive oxygen species (ROS) and the resulting reversible cysteine oxidation (Cys(OX)) 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 Cys(OX) 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 Cys(OX) proteome for this model. A total of 75 peptides, corresponding to 53 proteins; were quantified with oxidative modification. The bioinfomatics analysis of these Cys(OX)-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 Cys(OX) status. In conclusion, this Versatile modified biotin switch assay provides an approach for the quantification of all reversible Cys(OX) and for the study of redox Signaling in atherosclerosis as well as in diseases in other biological systems.
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