Resin-assisted enrichment of thiols as a general strategy for proteomic profiling of cysteine-based reversible modifications
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Genentech Inc., South San Francisco, CA (United States)
Reversible modifications on cysteine thiols play a significant role in redox signaling and regulation. A number of reversible redox modifications, including disulfide formation, S-nitrosylation, and S-glutathionylation, have been recognized for their significance in various physiological and pathological processes. Here we describe in detail a resin-assisted thiol-affinity enrichment protocol for both biochemical and proteomics applications. This protocol serves as a general approach for specific isolation of thiol-containing proteins or peptides derived from reversible redox-modified proteins. This approach utilizes thiol-affinity resins to directly capture thiol-containing proteins or peptides through a disulfide exchange reaction followed by on-resin protein digestion and on-resin multiplexed isobaric labeling to facilitate LC-MS/MS based quantitative site-specific analysis of redox modifications. The overall approach requires a much simpler workflow with increased specificity compared to the commonly used biotin switch technique. By coupling different selective reduction strategies, the resin-assisted approach provides the researcher with a useful tool capable of enriching different types of reversible modifications on protein thiols. Procedures for selective enrichment and analyses of S-nitrosylation and total reversible cysteine oxidation are presented to demonstrate the utility of this general strategy.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 1115800
- Report Number(s):
- PNNL-SA-96060; 42901; KP1501021
- Journal Information:
- Nature Protocols, 9(1):64-75, Vol. 9, Issue 1; ISSN 1754-2189
- Country of Publication:
- United States
- Language:
- English
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