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 (-SO2H) and
S-sulfonylation (-SO3H). 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 fold-changes, 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
(ENS), such as C-nitroso compounds and electron deficient diazines, offers significant
advantages in simplicity-of-use and target specificity compared to label-free methods.
When feasible, the rich information provided by site-centric quantitative proteomics
should not be tainted by oxidation artifacts from cell lysis. Therefore, chemoselective
probes that function in a native environment with low cytotoxicity, good cell-permeability,
and competitive kinetics are desired in modern redox chemoproteomics approaches. As
our understanding of sulfur chemistry and redox signaling evolves, newly discovered
cysteine OxiPTMs in microorganisms, plants, cells, tissues, and disease models should
innovatively promote mechanistic and therapeutic research.