Quantifying the Reactive Cysteine Proteome
We have developed a chemoproteomic platform, termed as QTRP (Quantitative Thiol Reactivity Profiling, Fig. 1), to systematically and quantitatively analyze the reactivity of thousands of proteomic cysteines toward redox perturbation in various species (Nat Protoc 2020). We initially benchmarked QTRP to identify hundreds of previously unknown redox-sensitive cysteines in human cell lines, which substantially expands the scope of the observable redoxome and suggests a change in functional paradigm from a small set of conserved switches to a much larger, adaptable and cell type specific system (Mol Cell Proteomics 2017). This analysis also provides a basis for greatly expanded exploration of the complex networks controlled by redox sensing, transduction and cellular adaptive responses. Furthermore, the QTRP platform has been applied to multicellular animals, such as C. elegans (Nat Commun 2021), D. melanogaster (Proc Natl Acad Sci U S A 2018), and M. musculus (Nat Cell Biol 2019) for identifying proteome-wide alterations in reactive cysteines upon oxidative stress or genetically regulated redox perturbation, providing several valuable resources for uncovering the mechanisms of redox-modulated control in these model organisms. More generally, QTRP can also provide a potential framework for target profiling of thiol-reactive chemicals, such as naturally occurring electrophiles, reactive drug metabolites, covalent inhibitors, and so on (Cell Host Microbe 2020; Cell Chem Biol 2017; Chem Res Toxicol 2017).
Mapping Cysteine Redox Modifications
Taking advantage of redox
form-specific probes, we have developed a series of redox proteomic methods to
directly, site-specifically map and quantify cysteine redox
modifications, including sulfenylation (-SOH, Nat Chem 2021), sulfinylation (-SO2H, Nat Chem Biol 2018),
and persulfidation (-SSH, also known as sulfhydration,
Antioxid Redox Signal 2020), expanding the
inventories of redox-regulated cysteine sites in native biological systems from
various species. Of note, we recently provided a robust approximation of %SOH in cysteines on a proteome-wide scale in cells (Fig. 2)(Nat Chem 2021). These data sets not only provide mechanistic support for prioritizing
functional redox sites, but also offer novel redox mechanisms. For example,
quantitative sulfenome analyses allowed us to uncover the biochemical and/or
pathophysiological functions of sulfenylation events on MAPK4C181
and SMAD3C64 in plant and mouse, respectively (Proc
Natl Acad Sci U S A 2019; Redox Biol 2021). In another
example, by quantifying the sulfinome, we
discovered many new targets of
sulfiredoxin, the sulfinic acid reductase, revealing a
heretofore unknown layer of thiol-based cellular redox-regulation (Nat Chem Biol 2018).
Figure 2. Proteome-wide analysis of cysteine sulfenic acid site stoichiometry (%SOH).
Discovering New Post-Translational Modifications (PTMs)
We have developed a
streamlined pipeline to discover new
chemotypes in the proteome (Fig. 3). It
combines an experimental setting for isotopically featuring modifications derived
from activity-based probes and a newly developed
informatic tool called pChem (BioRxiv 2021)
for blind-searching unexpected mass shifts on detected peptide sequences. This pipeline allowed us to identify several previously unknown PTMs, including a pyrrole-adduct
on cysteine by 4-oxo-2-nonenal (an electrophilic metabolite derived from lipid peroxidation) (Mol Cell Proteomics 2017), N-termini formylation derived from oxidative cleavage of proteins
(Anal Chem 2018), and
the formaldehyde-driven transformation of protein lysine adduction by endogenous dicarbonyls (Chem Sci 2021). The discovery of these modifications provides much-needed mechanistic insights into the cellular signaling and potential toxicities associated with endogenous electrophiles. In addition, with our expertise in mass spectrometry, we manually characterized unforeseeable PTMs on individual proteins (Nature 2021).