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

Nature Protocols volume 9, pages 64–75 (2014)Cite this article

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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.

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Figure 1: Schematic of the pre-processing and enrichment strategy for different reversible cysteine modifications.
Figure 2: Enrichment of SNO-modified peptides from mouse muscle cells.
Figure 3: Cysteine oxidation in RAW cells

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Acknowledgements

Portions of this work were supported by the US National Institutes of Health (NIH) Director's New Innovator Award Program DP2OD006668 and a US Department of Energy (DOE) Early Career Research Award (to W.-J.Q.), NIH P41 GM103493 (to R.D.S.), and the DOE Office of Biological and Environmental Research Genome Sciences Program under the Pan-omics project. The experimental work was performed in the Environmental Molecular Science Laboratory, a DOE/Biological and Environmental Research (BER) national scientific user facility at the Pacific Northwest National Laboratory (PNNL) in Richland, Washington. PNNL is operated by Battelle for the DOE under contract no. DE-AC05-76RLO-1830.

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Authors and Affiliations

  1. Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA

    Jia Guo, Matthew J Gaffrey, Dian Su, Tao Liu, David G Camp II, Richard D Smith & Wei-Jun Qian

  2. Genentech Inc., South San Francisco, California, USA

    Dian Su

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Contributions

J.G., M.J.G. and D.S. performed the experiments and optimized the protocol; T.L. developed the initial enrichment method; D.G.C. and R.D.S. contributed to development of the measurement capabilities used; W.-J.Q. conceived and supervised the project. J.G., M.J.G. and W.-J.Q. wrote the manuscript.

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Correspondence to Wei-Jun Qian.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Conditions for selective reduction, negative and positive controls for multiple reversible cysteine modifications. (PDF 223 kb)

Supplementary Data

Quantification of total cysteine oxidation levels in RAW cells treated with 0.1 mM and 0.5 mM diamide. (XLSX 220 kb)

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Guo, J., Gaffrey, M., Su, D. et al. Resin-assisted enrichment of thiols as a general strategy for proteomic profiling of cysteine-based reversible modifications. Nat Protoc 9, 64–75 (2014). https://doi.org/10.1038/nprot.2013.161

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