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 (a). 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 (b). 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 (e.g., C. elegans, D. melanogaster, and M. musculus) 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 (c-e). 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 (f-h).
a. Fu L#, Li Z#, Tian C#, Liu K#, He JX, He JY, He F, Xu P, Yang
J. A quantitative thiol reactivity profiling platform to analyze redox and
electrophile reactive cysteine proteomes. Nat Protoc, 2020,
15(9): 2891-2919 #Co-First,
the same below
b. Fu L#, Liu K#, Sun M, Sun R, Tian C, Bentanzos C, Tallman KA, Porter NA, Yang Y, Guo D, Liebler DC*, Yang J*. Systematic and quantitative assessment of hydrogen peroxide reactivity with cysteines across human proteomes. Mol Cell Proteomics, 2017, 16 (10):1815-1828 *Co-Corresponding, the same below
c. Meng J#, Fu L#, Liu K, Tian C, Wu Z, Jung Y, Ferreira R, Carroll KS, Blackwell TK*, Yang J*. Global profiling of distinct cysteine redox forms reveals wide-ranging redox regulation in C. elegans. Nat Commun, 2021 12: 1415
d. Petrova B, Liu K, Tian C, Kitaoka M, Freinkman E, Yang J*, Orr-Weaver TL*. Dynamic redox balance directs the oocyte-to-embryo transition via developmentally controlled reactive cysteine changes. Proc Natl Acad Sci U S A, 2018, 115 (34): E7978-E7986
e. Pei J#, Li X#, Li W#, Gao Q, Zhang Y, Wang X, Fu J, Cui S, Qu J, Zhao X, Hao D, Ju D, Liu N, Carroll KS, Yang J, Zhang EE, Cao J, Chen H*, Liu D*. Diurnal oscillations of endogenous H2O2 sustained by p66Shc regulate circadian clocks. Nat Cell Biol, 2019, 21(12): 1553-1564.
f. Tian C, Sun R, Liu K, Fu L, Liu X, Zhou W, Yang Y, Yang J. Multiplexed thiol reactivity profiling for target discovery of electrophilic natural products. Cell Chem Biol, 2017, 24 (11):1416-1427
g. Wang W#, Yang J#, Zhang J, Liu Y, Tian C, Qu B, Gao C, Xin P, Chen S, Zhang W, Miao P, Li L, Zhang X, Chu J, Zuo J, Li J, Bai Y, Lei X*, Zhou J*. An Arabidopsis secondary metabolite directly targets expression of the bacterial type III secretion system to inhibit bacterial virulence. Cell Host Microbe, 2020, 27(4): 601-613.
h. Sun R#, Shi F#, Liu K, Fu L, Tian C, Yang Y, Tallman KA, Porter NA, Yang J. A chemoproteomic platform to assess bioactivation potential of drugs. Chem Res Toxicol. 2017, 30 (10): 1797-1803
Mapping Cysteine Redox Modifications
Taking advantage of several redox form-specific probes, we have developed a series of redox proteomic methods to directly, site-specifically and quantitatively profile cysteine redox modifications, including sulfenylation (-SOH), sulfinylation (-SO2H), and persulfidation (-SSH, also known as sulfhydration) (a-c, Fig. 1), expanding the inventories of redox-regulated cysteine sites in native biological systems from various species. These datasets not only provided mechanistic support for prioritizing functional redox sites, but also suggested 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 (d-e). In another example, by quantifying sulfiredoxin-dependent changes in the sulfinome, we discovered many new targets of this sulfinic acid reductase, revealing a heretofore unknown layer of thiol-based cellular redox-regulation (b).
a. Shi Y, Fu L, Yang J*, Carroll KS*. Wittig reagents for chemoselective sulfenic acid ligation enables global site stoichiometry analysis and redox-controlled mitochondrial targeting. Nat Chem. Accepted
b. Akter S#, Fu L#, Jung Y, Lo Conte M, Lawson JR, Lowther TW, Sun R, Liu K, Yang J*, Carroll KS*. Chemical proteomics reveals new targets of cysteine sulfinic acid reductase. Nat Chem Biol, 2018, 14 (11): 995-1004 (Featured in a News and Views, Nat Chem Biol, highlighted in Nat Methods and F1000Prime)
c. Fu L#, Liu K#, He J, Tian C, Yu X, Yang J. Direct proteomic mapping of cysteine persulfidation. Antioxid Redox Signal, 2020, 33(15): 1061-1076 (Editors’ choice for open access)
d. Huang J#, Willems P#, Wei B#, Tian C, Ferreira R, Bodra N, Gache S, Wahni K, Liu K, Vertommen D, Gevaert K, Carroll KS, Van Montagu M*, Yang J*, Van Breusegem F*, Messens J*. Mining for protein S-sulfenylation in Arabidopsis uncovers redox-sensitive sites. Proc Natl Acad Sci U S A, 2019, 116 (42): 21256-21261.
e. Huang Y, Li Z, Zhang L, Tang H, Zhang H, Wang C, Chen S, Bu D, Zhang Z, Zhu Z, Yuan P, Li K, Yu X, Kong W, Tang C, Jung Y, Ferreira R, Carroll KS, Du J, Yang J*, Jin H*. Endogenous SO2-dependent Smad3 redox modification controls vascular remodeling. Redox Biol, 2021, 41: 101898
Figure 2. Site-centric quantitative chemoproteomic workﬂow for global proﬁling of unexpected protein PTMs (ABP: Activity-based Probe)
Discovering Unexpected Post-Translational Modifications (PTMs)
We have developed a
streamlined pipeline to discover new
chemotypes in the proteome (Fig. 2). It
combines an experimental setting for isotopically featuring modifications derived
from activity-based probes and informatic tools (existing ones like, TagRecon, pFind-alioth, and a newly developed
one termed pChem)
for blind-searching unexpected mass shifts on detected peptide sequences. For
instance, this pipeline allowed us to identify a previously unknown type of
4-oxo-2-nonenal (an electrophilic metabolite derived from lipid peroxidation)
derived pyrrole-adduct to cysteinyl residues (a). This modification exhibits
crosstalk with many redox sensitive sites and can also be reversed in cells,
providing much-needed mechanistic insights into the cellular signaling and
potential toxicities associated with this important endogenous lipid electrophile.
In addition, we applied this approach to discover a novel type of protein
carbonyl product, N-terminal formyl protein degradants (b). In addition, with our expertise in mass spectrometry, we can manually characterized unforeseeable PTMs on individual proteins (c).
a. Sun R#, Fu L#, Liu K, Tian C, Yang Y, Tallman KA, Porter NA, Liebler DC, Yang J. Chemoproteomics reveals chemical diversity and dynamics of 4-oxo-2-nonenal modifications in cells. Mol Cell Proteomics, 2017, 16 (10):1789-1800 (Editors' Highlight)
b. Tian C#, Liu K#, Sun R, Fu L, Yang J. Chemoproteomics reveals unexpected lysine/arginine-specific cleavage of peptide chains as a potential protein degradation machinery. Anal Chem. 2018, 90 (1): 794-800
c. Ping YQ# , Mao C# , Xiao P# , Zhao RJ# , Jiang Y# , Yang Z, An WT, Shen DD, Yang F, Zhang H, Qu C, Shen Q, Tian C, Li ZJ, Li S, Wang GY, Tao XN, Wen X, Zhong YN, Yang J, Yi F, Yu X, Xu HE*, Zhang Y*, Sun JP*. Structures of the glucocorticoid-bound adhesion receptor GPR97-Go complex. Nature, 2021, 589: 620-626.
1. Redox proteomics
2. Harnessing quantitative chemoproteomics to reveal the target landscape of clinical covalent drugs（81973279）
3. Proteome-wide discovery of the substrates of sulfiredoxin and their roles in regulating oxidative susceptibility of tumor cells（NSFC:31770885）
4. Quantitative chemoproteomic analysis of dynamic and tumor-specific target profile of gambogic acid and its global regulatory network Study（NSFC: 81573395）
5. Oxidative aggregation of APIP and its role in biological regulation (NSFC: 31500666）
6. Oxidation of APIP and its role in redox regulation of apoptosis (BNSF:5162009)
7. Beijing Nova Program (No. Z171100001117014)8. Youth talent support program of Beijing (No. 201700021223ZK16)