Systematic and Quantitative Assessment of Hydrogen Peroxide Reactivity With Cysteines Across Human Proteomes

Abstract

Protein cysteinyl residues are the mediators of hydrogen peroxide (HO)-dependent redox signaling. However, site-specific mapping of the selectivity and dynamics of these redox reactions in cells poses a major analytical challenge. Here we describe a chemoproteomic platform to systematically and quantitatively analyze the reactivity of thousands of cysteines toward HO in human cells. We identified >900 HO-sensitive cysteines, which are defined as the HO-dependent redoxome. Although redox sites associated with antioxidative and metabolic functions are consistent, most of the HO-dependent redoxome varies dramatically between different cells. Structural analyses reveal that HO-sensitive cysteines are less conserved than their redox-insensitive counterparts and display distinct sequence motifs, structural features, and potential for crosstalk with lysine modifications. Notably, our chemoproteomic platform also provides an opportunity to predict oxidation-triggered protein conformational changes. The data are freely accessible as a resource at http://redox.ncpsb.org/OXID/.

Proteome-wide analysis of cysteine oxidation reveals metabolic sensitivity to redox stress

“…A recent study using the isoTOP-ABPP platform quantified cysteine reactivities of more than 3000 cysteine residues in a human cell line (with ~9700 cysteine residues quantified across a panel of six lines) 48 . During the preparation of this manuscript, another chemoproteomic approach titled quantitative thiol reactivity profiling (QTRP) was published, which used iodoacetamide-alkyne probes, click chemistry, and subsequent enrichment 49 . QTRP enabled quantification of cysteine reactivity to hydrogen peroxide of up to ~3700 peptides in a single human cell line and up to ~7000 across a panel of four.…”

Isotopically Labeled Clickable Glutathione to Quantify Protein S‐Glutathionylation

“…We also highlighted glutathionylation of muscle‐relevant proteins, providing the opportunity to investigate the functional impact of glutathionylation on specific cysteines in cardiomyocytes. Lastly, in recent years, cysteine reactivity or oxidation profiling has been valuable to predict and annotate hyper‐reactive or functional cysteines . Our quantification strategy developed here could be applicable to profile glutathionylation susceptibility in the future.…”

Proteome‐Wide Analysis of Cysteine S‐Sulfenylation Using a Benzothiazine‐Based Probe

“…Among them, a benzothiazine‐based probe, called BTD, exhibits the highest level of S‐sulfenylome reactivity ( k obs =1700 M −1 sec −1 ), which represents a rate enhancement of more than two orders of magnitude compared to the dimedone‐based probe, DYn‐2 ( k obs =10 M −1 sec −1 ) (Gupta et al., ). BTD demonstrated to be highly compatible with the site‐centric chemoproteomic platform (Fu et al., ; Sun et al., , ; Tian et al., ; Yang et al., ). Therefore, the new BTD probe can be used to globally, site‐specifically map and quantify cysteine S‐sulfenylation in complex proteomes in a much more efficient manner.…”

“…The following protocol describes a well-established, quantitative chemoproteomic platform (Fu et al, 2017;Gupta et al, 2017;Sun et al, 2017a,b;Tian, Liu, Sun, Fu, & Yang, 2018; to enrich and analyze the BTD-labeled S-sulfenylated peptides. First, the BTD-labeled protein samples are digested into tryptic peptides.…”