U.S. Department of Energy

Pacific Northwest National Laboratory

Resin-assisted enrichment of thiols as a general strategy for proteomic profiling of cysteine-based reversible modifications.

TitleResin-assisted enrichment of thiols as a general strategy for proteomic profiling of cysteine-based reversible modifications.
Publication TypeJournal Article
Year of Publication2014
AuthorsGuo J, Gaffrey MJ, Su D, Liu T, Camp DG, Smith RD, Qian W-J
JournalNat Protoc
Abstract

Reversible modifications of cysteine thiols have a key role in redox signaling and regulation. A number of reversible redox modifications, including disulfide formation, S-nitrosylation (SNO) and S-glutathionylation (SSG), have been recognized for their significance in various physiological and pathological processes. Here we describe a procedure for the enrichment of peptides containing reversible cysteine modifications. Starting with tissue or cell lysate samples, all of the unmodified free thiols are blocked using N-ethylmaleimide (NEM). This is followed by the selective reduction of those cysteines bearing the reversible modification(s) of interest. The reduction is achieved by using different reducing reagents that react specifically with each type of cysteine modification (e.g., ascorbate for SNO). This protocol serves as a general approach for enrichment of thiol-containing proteins or peptides derived from reversibly modified proteins. The approach uses a commercially available thiol-affinity resin (thiopropyl Sepharose 6B) to directly capture free thiol-containing proteins through a disulfide exchange reaction, followed by on-resin protein digestion and multiplexed isobaric labeling to facilitate liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based quantitative site-specific analysis of cysteine-based reversible modifications. The overall approach requires a simpler workflow with increased specificity compared with the commonly used biotinylation-based assays. The procedure for selective enrichment and analyses of SNO and the level of total reversible cysteine modifications (or total oxidation) is presented to demonstrate the utility of this general strategy. The entire protocol requires ∼3 d for sample processing with an additional day for LC-MS/MS and data analysis.

DOI10.1038/nprot.2013.161
Alternate JournalNat Protoc
PubMed ID24336471
Grant ListDP2OD006668 / OD / NIH HHS / United States
P41 GM103493 / GM / NIGMS NIH HHS / United States
P41 GM103493 / GM / NIGMS NIH HHS / United States
Area of Research: 
| Pacific Northwest National Laboratory