Activity-Based Sensing for Site-Specific Proteomic Analysis of Cysteine Oxidation

Abstract

Oxidative post-translational modifications (OxiPTMs) of cysteine residues are the molecular foundation of thiol-based redox regulation that modulates physiological events such as cell proliferation, differentiation, and migration and, when dysregulated, can lead to biomolecule damage and cell death. Common OxiPTMs of cysteine thiols (─SH) include reversible modifications such as S-sulfenylation (─SOH), S-glutathionylation (─SSG), disulfide formation (─SSR), S-nitrosylation (─SNO), and S-sulfhydration (─SSH) as well as more biologically stable modifications like S-sulfinylation (─SO 2 H) and S-sulfonylation (─SO 3 H). In the past decade, our laboratory has developed first-in-class chemistry-based tools and proteomic methods to advance the field of thiol-based redox biology and oxidative stress. In this Account, we take the reader through the historical aspects of probe development and application in our laboratory, highlighting key advances in our understanding of sulfur chemistry, in the test tube and in living systems.Offering superior resolution, throughput, accuracy, and reproducibility, mass spectrometry (MS)based proteomics coupled to chemoselective "activity-based" small-molecule probes is the most rigorous technique for global mapping of cysteine OxiPTMs. Herein, we describe the evolution of this field from indirect detection to state-of-the-art site-centric quantitative chemoproteomic approaches that enable mapping of physiological and pathological changes in cysteine oxidation. These methods enable protein and site-level identification, mechanistic studies, mapping foldchanges, and modification stoichiometry. In particular, this Account focuses on activity-based methods for profiling S-sulfenylation, S-sulfinylation, and S-sulfhydration with an eye toward new reactions and methodologies developed in our group as well as their applications that have shed new light on fundamental processes of redox biology. Among several classes of sulfenic acid probes, dimedone-based C-nucleophiles possess superior chemical selectivity and compatibility with tandem MS. Cell-permeable dimedone derivatives with a bioconjugation handle are capable of detecting of S-sulfenylation in living cells. In-depth screening of a C-nucleophile library has yielded several entities with significantly enhanced reactivity over dimedone while maintaining selectivity, and reversible linear C-nucleophiles that enable controlled target release. C-Nucleophiles have also been implemented in tag-switch methods to detect S-sulfhydration. Most recently, activity-based detection of protein S-sulfinylation with electrophilic nitrogen species *

Wittig reagents for chemoselective sulfenic acid ligation enables global site stoichiometry analysis and redox-controlled mitochondrial targeting

“…Herein, we described Wittig reagents as next-generation probes for protein sulfenic acids, featuring three key properties: First, the probes display high nucleophilicity under physiological conditions, offering fast reaction kinetics while precluding cross-reactivity in biological systems 16 To date, site-specific chemoproteomic profiling of protein sulfenic acids in intact cells has been predicated almost exclusively on DYn-2, a 1,3-cyclohexadione-based probe, which is quite selective, but hampered by slow reaction kinetics ( Supplementary Fig. 1c).…”

Type III intermediate filaments as targets and effectors of electrophiles and oxidants

“…Protein oxidation can occur upon exposure to diverse oxidant species and induce multiple structural alterations. Several excellent recent reviews have addressed the oxidative modifications of proteins [ 41 , 42 ], and of cysteine residues in particular [ 43 , 44 ], and provided detailed methodological information for their assessment. In addition, oxidative stress can increase the production of electrophilic species eliciting additional PTMs.…”

Immunological Techniques to Assess Protein Thiol Redox State: Opportunities, Challenges and Solutions

“…The second-order bimolecular reaction between an alkylating agent and SOH is, however, significantly slower than the thiol labelling reaction [149], suggesting it could be mitigated by titrating the molar mass used and reaction time. If methylsulfonyl benzothiazole is used to label reduced thiols without reacting with SOH species, then the ability of the resultant SO 2 species to distort downstream analysis by reacting with RSNO should be considered [153]. As the development of tunable cysteine reactive phosphate tags attests [91], untuned alkylating agents can fail to label many proteins despite their molar excess.…”

“…However, the ability of TCO/Tz functionalized reagents to permeate the cell is unclear and should be tested before live cell labelling is undertaken. The development of SH/Sselective tools coupled to reagents to directly probe each modification type without reduction by forming a diagnostic product allied to carefully buffer preparation should make it possible to advance redox proteomic and immunological analysis [153]; especially for contexts when only destructive analysis is currently possible (e.g., human skeletal muscle). At present, many reaction based technologies (e.g., a mutant glutathione synthase able to incorporate azide functionalized alanine instead of glycine into GSH to study RSSG [163]) would need to be modified (e.g., synthesis of TCO alanine) to be compatible with IEDDA Click PEGylation.…”