Stoichiometric Thiol Redox Proteomics for Quantifying Cellular Responses to Perturbations

Abstract

Post-translational modifications regulate the structure and function of proteins that can result in changes to the activity of different pathways. These include modifications altering the redox state of thiol groups on protein cysteine residues, which are sensitive to oxidative environments. While mass spectrometry has advanced the identification of protein thiol modifications and expanded our knowledge of redox-sensitive pathways, the quantitative aspect of this technique is critical for the field of redox proteomics. In this review, we describe how mass spectrometry-based redox proteomics has enabled researchers to accurately quantify the stoichiometry of reversible oxidative modifications on specific cysteine residues of proteins. We will describe advancements in the methodology that allow for the absolute quantitation of thiol modifications, as well as recent reports that have implemented this approach. We will also highlight the significance and application of such measurements and why they are informative for the field of redox biology.

SP3-Enabled Rapid and High Coverage Chemoproteomic Identification of Cell-State–Dependent Redox-Sensitive Cysteines

“…As multi-cysteine-containing peptides are ubiquitous in redox motifs (e.g. CXXC), this limitation is J o u r n a l P r e -p r o o f particularly problematic for measures of thiol-oxidation state (35,36,37). Furthermore, the relative performance of these tools for chemoproteomic applications remains unexplored.…”

A Deep Redox Proteome Profiling Workflow and Its Application to Skeletal Muscle of a Duchene Muscular Dystrophy Model

“…Furthermore, the multiple replicates of genotype-specific total thiol channels, which are representative of all free and reversibly oxidized thiols present in the sample, made quantification of thiol oxidation at the stoichiometric level possible. Stoichiometry-based analysis offers an advantage towards understanding changes in thiol oxidation [32]. There are also several caveats to consider for the current approach.…”

RedoxiFluor: A microplate assay to quantify protein thiol redox state in percentages and moles

“…Recent technical developments enable percentage cysteine residue redox state to be quanti ed on a proteome wide scale (i.e., array and context) 9,35 . RedoxiFluor measures all target speci c redox modi able thiols; which, for many targets is a breakthrough achievement (i.e., proteomics has yet to detect 94 and 50% of the thiols in IRAK1 and PP2A, respectively).…”