Dataset Information

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.

SUBMITTER: Yang J

PROVIDER: S-EPMC4608369 | biostudies-other | 2015 Jul

REPOSITORIES: biostudies-other

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

Yang Jing J Gupta Vinayak V Tallman Keri A KA Porter Ned A NA Carroll Kate S KS Liebler Daniel C DC

Nature protocols 20150618 7

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 t ...[more]

PMID: 26086405

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Project description:Due to its compatible oxygen-transporting abilities, hemoglobin (Hb) is a protein of interest in the development of artificial oxygen therapeutics. Despite continuous formulation attempts, extracellular Hb solution often exhibits undesirable reactions when applied in vivo. Therefore, protein engineering is frequently used to examine alternative ways of controlling the unwanted reactions linked to cell-free Hb solutions. In this study, three mutants of human fetal hemoglobin (HbF) are evaluated; single mutants αA12D and αA19D, and a double mutant αA12D/A19D. These variants were obtained by site-directed mutagenesis and recombinant production in E. coli, and carry negative charges on the surface of the α-subunit at the designated mutation sites. Through characterization of the mutant proteins, we found that the substitutions affected the protein in several ways. As expected, the isoelectric points (pIs) were lowered, from 7.1 (wild-type) down to 6.6 (double mutant), which influenced the anion exchange chromatographic procedures by shifting conditions toward higher conductivity for protein elution. The biological and physiological properties of HbF could be improved by these small modifications on the protein surface. The DNA cleavage rate associated with native HbF could be reduced by 55%. In addition, the negatively charged HbF mutant had an extended circulation time when examined in a mouse model using top load Hb additions. At the same time, the mutations did not affect the overall structural integrity of the HbF molecule, as determined by small-angle X-ray scattering. In combination with circular dichroism and thermal stability, modest structural shifts imposed by the mutations could possibly be related to changes in secondary structure or reorganization. Such local deformations were too minor to be determined within the resolution of the structural data; and overall, unchanged oxidation and heme loss kinetics support the conclusion that the mutations did not adversely affect the basic structural properties of Hb. We confirm the value of adding negatively charged residues onto the surface of the protein to improve the global functions of recombinant Hb.

Dataset Information

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

Publications

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

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