Resin-assisted enrichment of thiols as a general strategy for proteomic profiling of cysteine-based reversible modifications - PubMed (original) (raw)

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

Jia Guo et al. Nat Protoc. 2014 Jan.

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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Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1

Figure 1

Schematic of the pre-processing and enrichment strategy for different reversible cysteine modifications. (a) Sample pre-processing strategies for different types of reversible cysteine modifications. The free thiols are initially blocked by alkylation and the different types of modified cysteines are selectively reduced to free thiols by using individual sets of reagents. Note that the term “total oxidation” is not exact as S-acylation is not a form of oxidation; however, the level of S-acylation is negligible compared to other forms of redox modifications. Also note that the breaking of S-acylation thioester bonds by DTT may not be effective since the reaction between DTT and thioesters is a much slower reaction compared to hydroxylamine. (b) Enrichment method for quantitative analysis of reversible cysteine modifications. After preprocessing, the free thiols derived from modified cysteines are captured by Thiopropyl Sepharose resin. Enriched proteins can be directly eluted for gel electrophoresis. On-resin digestion and on-resin isobaric labeling are performed for quantitative LC MS/MS analysis.

Figure 1

Figure 1

Schematic of the pre-processing and enrichment strategy for different reversible cysteine modifications. (a) Sample pre-processing strategies for different types of reversible cysteine modifications. The free thiols are initially blocked by alkylation and the different types of modified cysteines are selectively reduced to free thiols by using individual sets of reagents. Note that the term “total oxidation” is not exact as S-acylation is not a form of oxidation; however, the level of S-acylation is negligible compared to other forms of redox modifications. Also note that the breaking of S-acylation thioester bonds by DTT may not be effective since the reaction between DTT and thioesters is a much slower reaction compared to hydroxylamine. (b) Enrichment method for quantitative analysis of reversible cysteine modifications. After preprocessing, the free thiols derived from modified cysteines are captured by Thiopropyl Sepharose resin. Enriched proteins can be directly eluted for gel electrophoresis. On-resin digestion and on-resin isobaric labeling are performed for quantitative LC MS/MS analysis.

Figure 2

Figure 2

Enrichment of SNO-modified peptides from mouse muscle cells. (a) Experimental design and workflow with 4-plex iTRAQ labeling (114–117 m/z) for LC MS/MS analysis. (b) An MS/MS spectrum of the Cys-peptide #STEECLSYFGVSETTGLTPDQVK# from SERCA1 (UniProt:AT2A1_Mouse). The symbol # stands for the iTRAQ label on N-terminus and lysine. (c) The zoom-in spectrum of the reporter-ion region showing the increases of SNO levels on the identified peptide in response to GSNO treatment.

Figure 2

Figure 2

Enrichment of SNO-modified peptides from mouse muscle cells. (a) Experimental design and workflow with 4-plex iTRAQ labeling (114–117 m/z) for LC MS/MS analysis. (b) An MS/MS spectrum of the Cys-peptide #STEECLSYFGVSETTGLTPDQVK# from SERCA1 (UniProt:AT2A1_Mouse). The symbol # stands for the iTRAQ label on N-terminus and lysine. (c) The zoom-in spectrum of the reporter-ion region showing the increases of SNO levels on the identified peptide in response to GSNO treatment.

Figure 3

Figure 3

(a) SDS-gel image of enriched SNO-modified proteins from RAW 264.7 cells treated with CysNO for 15 min. UV exposure was performed in a 60 mm petri dish, 6 cm from a 15-watt UV lamp (λmax 365 nm) for 15 min. (b) SDS-gel image of enriched oxidized cysteine-containing peptides (the first 3 lanes) treated with diamide (0 mM, 0.1 mM and 0.5 mM) for 30 min and the total cysteine-containing peptides (lane 4) from RAW cells. (c) Stoichiometry of oxidation as measured by LC-MS/MS of the iTRAQ-labeled samples. Plotted here is the average stoichiometry across all identified peptides under different conditions. The stoichiometry of oxidation of each peptide was calculated as a percentage by dividing the reporter ion intensity for each channel (oxidized thiol) against the intensity of total thiol channel (enriched without NEM blocking).

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