Global, in situ, site-specific analysis of protein S-sulfenylation - PubMed (original) (raw)

Global, in situ, site-specific analysis of protein S-sulfenylation

Jing Yang et al. Nat Protoc. 2015 Jul.

Abstract

Protein S-sulfenylation is the reversible oxidative modification of cysteine thiol groups to form cysteine S-sulfenic acids. Mapping the specific sites of protein S-sulfenylation onto complex proteomes is crucial to understanding the molecular mechanisms controlling redox signaling and regulation. This protocol describes global, in situ, site-specific analysis of protein S-sulfenylation using sulfenic acid-specific chemical probes and mass spectrometry (MS)-based proteomics. The major steps in this protocol are as follows: (i) optimization of conditions for selective labeling of cysteine S-sulfenic acids in intact cells with the commercially available dimedone-based probe, DYn-2; (ii) tagging the modified cysteines with a functionalized biotin reagent containing a cleavable linker via Cu(I)-catalyzed azide-alkyne cycloaddition reaction; (iii) enrichment of the biotin-tagged tryptic peptides with streptavidin; (iv) liquid chromatography-tandem MS (LC-MS/MS)-based shotgun proteomics; and (v) computational data analysis. We also outline strategies for quantitative analysis of this modification in cells responding to redox perturbations and discuss special issues pertaining to experimental design of thiol redox studies. Our chemoproteomic platform should be broadly applicable to the investigation of other bio-orthogonal chemically engineered post-translational modifications. The entire analysis protocol takes ∼1 week to complete.

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Conflict of interest statement

COMPETING FINANCIAL INTERSESTS

The authors declare no competing financial interests.

Figures

Figure 1

Figure 1

Selective labeling and detection of protein S-sulphenic acid in cells. (a) Cyclohexanedione group of DYn-2 probe selectively reacts with protein sulphenic acid modifications in cells. To the best of our knowledge, this probe has no reactivity toward other functional groups in cells. Moreover, this probe does not change cell viability and redox balance. (b) Alkyne group of DYn-2-labeled proteins can be conjugated to azide-bearing tags (e.g. fluorescent or biotin tag) via click chemistry and detected by immunofluorescence, western blotting (WB), or proteomics.

Figure 2

Figure 2

Two workflows for proteome-wide identification of targeted sites of alkyne tagged probes. (a) Protein capture. Proteome labeled with an alkynyl probe is first conjugated with an azido biotin with cleavable linker via CuAAC chemistry. The biotinylated proteins are then enriched with streptavidin beads and subjected to sequential on-beads tryptic digestion and released by an enzymatic or chemical cleavage mechanism. The resulting peptides are analyzed by mass spectrometry based proteomics for site-localization. (b) Peptide capture, as described in the Procedure. Proteome labeled with an alkynyl probe is first digested into tryptic peptides and then conjugated with an azido biotin with photocleavable linker via CuAAC chemistry. The biotinylated peptides are then enriched with streptavidin beads and released by an enzymatic or chemical cleavage mechanism. Sequential LC-MS analysis and informatics pipeline provide identification of labeled peptides, proteins and their sites of modification.

Figure 3

Figure 3

Identification of Cys238 of NAA15 as the target of S-sulphenylation in RKO cells. (a) Fully annotated HCD MS/MS spectrum with diagnostic fragment ions (zoom window) of a DYn-2-triazo-hexanoic-acid modified peptide from S-sulphenylated NAA15. The highlighted cysteine in the peptide sequence represents the S-sulphenylated site (Cys238) of NAA15. (b) CID MS/MS spectrum of the same peptide.

Figure 4

Figure 4

Identification of Cys152 (upper) and Cys156 (lower) of GAPDH as the targets of S-sulphenylation in RKO cells. Fully annotated HCD MS/MS spectra of the same peptide sequence with different sites of DYn-2-triazo-hexanoic-acid modification are shown. The cysteine in purple color in the peptide sequence represents the S-sulphenylated site.

Figure 5

Figure 5

Quantitative S-sulphenylome analysis. (a) The proteome-wide and site-specific changes in S-sulfenylation in intact RKO cells in response to exogenous H2O2 (500 μM, 5 min), where nine S-sulfenylated cysteines on the FASN protein are shown in red, and all other cysteines by white symbols. The relative change in protein S-sulphenylation between two conditions is determined by the ratio of extracted ion current peak intensities for heavy and light isotope-labeled DYn-2-tagged peptides, using the MS1 filtering function of Skyline software. Ratios are displayed on a log2 scale on the y axis. (b) Extracted ion chromatograms (XIC) for nine S-sulfenylated peptides/sites from FASN are shown, with the profiles for light- and heavy- labeled peptides in red and blue, respectively. Light labels correspond to untreated cells; heavy labels correspond to H2O2-treated cells. The mean calculated experimental ratios from at least four replicates are displayed below the individual chromatogram, respectively. Figure reproduced from ref..

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