title: Global, in situ, site-specific analysis of protein S-sulfenylation.
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.
authors.affiliation: 1] Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, USA. [2] Jim Ayers Institute for Precancer Detection and Diagnosis, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Yang
authors.affiliation: Department of Chemistry, The Scripps Research Institute, Jupiter, Florida, USA.
authors.forename: Vinayak
authors.initials: V
authors.lastname: Gupta
authors.affiliation: Department of Chemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: Keri A
authors.initials: KA
authors.lastname: Tallman
authors.affiliation: Department of Chemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: Ned A
authors.initials: NA
authors.lastname: Porter
authors.affiliation: Department of Chemistry, The Scripps Research Institute, Jupiter, Florida, USA.
authors.forename: Kate S
authors.initials: KS
authors.lastname: Carroll
authors.affiliation: 1] Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, USA. [2] Jim Ayers Institute for Precancer Detection and Diagnosis, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Liebler
mesh_terms: D002460:Cell Line; D006801:Humans; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D013434:Sulfenic Acids / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D011506:Proteins; D013434:Sulfenic Acids