Dataset Information

Chemical profiling of live cell temporal redox dynamics in a photoautotrophic cyanobacterium

ABSTRACT: We have developed a quantitative chemical probe approach for live cell labeling of proteins that are sensitive to redox modifications. We utilize this in vivo strategy coupled to mass spectrometry-based proteomics to identify 176 proteins undergoing ~5-10 fold dynamic redox change in response to nutrient limitation and subsequent replenishment in the photoautotrophic cyanobacterium, Synechococcus sp. PCC 7002. This method enables the identification of redox changes in as little as 30 seconds after nutrient perturbation, and oscillations in reduction and oxidation for 60 minutes following the perturbation. The redox changes were validated by demonstrating that protein abundances did not change per global proteomic analyses. Peptides identified by MS for global and probe-labeled samples were required to be at least six amino acids in length having a mass spectra generating function score of <=1E-10, which corresponds to an FDR of <1%. Additionally, only peptides unique in identifying a single protein were utilized to estimate protein abundances, and proteins represented by <2 unique peptides were removed. This resulted in the identification of 176 redox probe labeled protein identifications, and 808 protein identifications in the global data.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Synechococcus Sp. (strain Atcc 27264 / Pcc 7002 / Pr-6) (agmenellum Quadruplicatum)

TISSUE(S): Culture Condition:-induced Cell

SUBMITTER: Matthew Monroe

LAB HEAD: Aaron T. Wright

PROVIDER: PXD000329 | Pride | 2015-08-24

REPOSITORIES: Pride

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Publications

Live cell chemical profiling of temporal redox dynamics in a photoautotrophic cyanobacterium.

Sadler Natalie C NC Melnicki Matthew R MR Serres Margrethe H MH Merkley Eric D ED Chrisler William B WB Hill Eric A EA Romine Margaret F MF Kim Sangtae S Zink Erika M EM Datta Suchitra S Smith Richard D RD Beliaev Alexander S AS Konopka Allan A Wright Aaron T AT

ACS chemical biology 20131106 1

Protein reduction-oxidation (redox) modification is an important mechanism that allows microorganisms to sense environmental changes and initiate cellular responses. We have developed a quantitative chemical probe approach for live cell labeling and imaging of proteins that are sensitive to redox modifications. We utilize this in vivo strategy to identify 176 proteins undergoing ∼5-10-fold dynamic redox change in response to nutrient limitation and subsequent replenishment in the photoautotrophi ...[more]

PMID: 24168666

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Project description:Cysteine is unique among all protein-coding amino acids owing to its intrinsically high nucleophilicity. The cysteinyl thiol group can be covalently modified by both a broad range of redox mechanisms and various electrophiles derived from exogenous or endogenous sources. Measuring proteomic cysteines response to redox perturbation or electrophiles is critical for understanding the underlying mechanisms involved. Activity-based protein profiling (ABPP) based on thiol-reactive probes has been the method of choice for such analyses. We therefore adapted this approach and developed a new chemoproteomic platform, termed as QTRP (Quantitative Thiol Reactivity Profiling), that relies on the ability of a commercially available thiol-reactive probe IPM to covalently label, enrich, and quantify the reactive cysteinome in cells and tissues. Here, we provide a detailed and updated workflow of QTRP that includes procedures for (i) labeling of the reactive cysteinome from cell or tissue samples (e.g. control vs treatment) with IPM, (ii) processing the protein samples into tryptic peptides and tagging the probe-modified peptides with isotopically labeled azido-biotin reagents containing a photo-cleavable linker via click chemistry reaction, (iii) capturing biotin-conjugated peptides with streptavidin beads, (iv) identifying and quantifying the photo-released peptides by mass spectrometry (MS)-based shotgun proteomics, (v) interpreting MS data by a streamlined informatic pipeline using a proteomics software, pFind 3, and an automatic post-processing algorithm. We also outline here how to use QTRP for measuring the changes on proteomic cysteines upon redox perturbation and for identifying targeted cysteine sites of thiol-reactive electrophiles. We anticipate that this protocol should find broad applications in both redox/chemical biology and drug discovery. The protocol for sample preparation takes 3 days, whereas MS measurements and data analyses require 75 min and <30 min, respectively, per sample.

Dataset Information

Chemical profiling of live cell temporal redox dynamics in a photoautotrophic cyanobacterium

Publications

Live cell chemical profiling of temporal redox dynamics in a photoautotrophic cyanobacterium.

Similar Datasets

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