Abstract
With an eye toward expanding chemistries used for covalent ligand discovery, we elaborated an umpolung strategy that exploits the ‘polarity reversal’ of sulfur when cysteine is oxidized to sulfenic acid, a widespread post-translational modification, for selective bioconjugation with C-nucleophiles. Here we present a global map of a human sulfenome that is susceptible to covalent modification by members of a nucleophilic fragment library. More than 500 liganded sulfenic acids were identified on proteins across diverse functional classes, and, of these, more than 80% were not targeted by electrophilic fragment analogs. We further show that members of our nucleophilic fragment library can impair functional protein–protein interactions involved in nuclear oncoprotein transport and DNA damage repair. Our findings reveal a vast expanse of ligandable sulfenic acids in the human proteome and highlight the utility of nucleophilic small molecules in the fragment-based covalent ligand discovery pipeline, presaging further opportunities using non-traditional chemistries for targeting proteins.
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Data availability
All data associated with this study are available in the published article and its Supplementary Information. All raw proteomics data are uploaded to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifiers PXD029761 and PXD039908. Source data are provided with this paper.
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Acknowledgements
This work was supported by grants from the National Key R&D Program of China (2022YFA1303000) to L.F., the Natural Science Foundation of China (21922702 and 81973279) to J.Y. and the US National Institutes of Health (R01 GM102187 and R01 CA174864) to K.S.C.
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Authors and Affiliations
Contributions
L.F. designed and performed the chemoproteomic and functional experiments, analyzed data and drafted the manuscript. Y.J. designed, synthesized and characterized the nucleophile fragment library. C.T. helped with the functional experiments. R.B.F. synthesized alkyne reporter fragments. F.C.H. supervised the work. J.Y. and K.S.C. conceived the project, supervised the work, analyzed data and the wrote the manuscript, with input from all authors.
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Extended data
Extended Data Fig. 1 Global analysis of nucleophilic fragment-SOH interactions.
(a) Rank plot showing R10:1 (5 mM vs 0.5 mM, black) and R1:1 (5 mM vs 5 mM, blue) values of BTD-labeled SOH sites across the MDA-MB-231 proteome. MDA-MB-231 cell lysates were labeled with BTD at concentration as indicated, and digested by trypsin. The resulting BTD-modified peptides were conjugated to light and heavy Az-UV-biotin, respectively, via click chemistry. The light and heavy labeled samples then were mixed equally in amount and subjected to streptavidin-based enrichment. After several washing steps, the modified peptides were selectively eluted from beads under 365 nm wavelength of UV light for LC-MS/MS-based proteomic analysis. (b) Line series plot showing the comparison of RH/L values obtained from common sites in the 10:1 and 1:1 dataset. (c) Representative XICs showing changes in BTD-labeled peptides from proteins as indicated. The profiles for light- and heavy-labeled peptide are shown in red and blue, respectively. The average RH/L values calculated from biological duplicates are displayed below each XIC.
Extended Data Fig. 2 Sequence motif analysis of sulfenated cysteines with different intrinsic reactivities.
Comparison of calculated sequence motif for hyper-reactive (upper), moderate-reactive (middle) and unreactive (lower) SOH sites. Images were generated with pLogo and scaled to the height of the largest column within the sequence visualization. The red horizontal lines on the pLogo plots denote P = 0.05 thresholds.
Extended Data Fig. 3 Comparison of proteomic cysteines quantified by two independent methods.
Upper panel: Chemical structures of 1 (left) and 2 (right); Lower panel: Venn diagrams showing the overlap of cysteines quantified by QTRP in this study and those by isoTOP ABPP from Backus, et al., Nature, 2016.
Extended Data Fig. 4 Chemical structures of nucleophilic fragments used in this study.
The observed binding constant (kobs) and reaction rate constant (M-1S-1) were listed below each fragment.
Extended Data Fig. 5 Quality control of the BTD-based SulfenQ analyses.
(a) Distribution of coefficient of variation values for SulfenQ analysis of each fragment. (b) Accumulating number of sulfenylated sites profiled by BTD across all datasets. (c) Frequency of quantification of all SOHs across all SulfenQ analyses performed with nucleophilic fragments.
Extended Data Fig. 6 Interactions between cyano-acetamide-based fragments and the MDA-MB-231 sulfenylome.
(a) Scatter plots showing correlation of sulfenylome reactivity of each cyano-acetamide-based fragment with the corresponding structural features, including number of free NH (Left), molecular weight (MW, middle), and CLogP value (Right). (b) Scatter plots showing correlation of sulfenylome reactivity of each cyano-acetamide-based fragment with the corresponding kinetic parameters, including the observed binding constant (kobs, Left) and reaction rate constant (M-1S-1, Right). (c) Representative XICs showing 5a-induced changes in BTD-labeled peptides bearing protein cysteines as indicated. The profiles for light- and heavy-labeled peptide are shown in red and blue, respectively. The average RH/L values calculated from biological duplicates are displayed below each XIC.
Extended Data Fig. 7 Comparison of sulfenylome reactivity of nucleophilic fragments with the same structural scaffold for each.
Sulfenylome reactivity values of nucleophilic fragments were calculated as the percentage of all quantified SOH sites with RH/L values ≥4 for each fragment.
Extended Data Fig. 8 Validation of liganded proteins.
(a) MDA-MB-231 cell lysates were pre-treated non-clickable BTD (BnBTD, 5 mM, 1 h), followed by treatment of 10a or 10b at the concentration of 5 mM for additional 1 h, clicked with azido biotin and analyzed by western blotting. (b) Recombinant, purified wild-type GSTO1, ACAT1 and GAPDH proteins were pretreated with H2O2 (100 μM, 10 min), followed by treatment with 500 μM 10b probe for 1 h. Protein samples were ‘clicked’ with azide-biotin and analyzed by western blotting. (c) Recombinant, purified wild-type GSTO1, ACAT1 and GAPDH proteins were treated with 5b as indicated concentrations followed by treatment with 10b probe, ‘clicked’ with azide-biotin and analyzed by western blotting. (d) Recombinant, purified wild-type and the corresponding cysteine-to-serine mutants as GSTO1, ACAT1 and GAPDH proteins were treated with 10b at the indicated concentration, clicked and analyzed by western blotting. (e,f) Validation of liganded proteins by 5b in cellulo. MDA-MB-231 cells recombinantly expressing wild-type and the corresponding cysteine-to-serine mutants as Flag epitope-tagged proteins (HDGF and PRXL2A) were treated with 5b as indicated concentrations followed by treatment with 10b probe. Protein samples were harvested, ‘clicked’ with azide-biotin, immunoprecipitated with streptavidin, eluted and separated with SDS-PAGE. Western blotting was performed using antibodies as indicated. Representative data from at least two independent experiments are shown. (g) HEK293T cells recombinantly expressing wild-type and the corresponding cysteine-to-serine mutant as Flag epitope-tagged BCCIP were treated with 8b as indicated concentrations followed by treatment with BTD probe (5 mM, 1 h). Protein samples were harvested, ‘clicked’ with azide-biotin, immunoprecipitated with streptavidin, eluted and separated with SDS-PAGE. Western blotting was performed using antibodies as indicated. Each experiment was repeated three times with similar results.
Extended Data Fig. 9 Functional analysis of interactions between nucleophilic fragments and their liganded enzymes.
(a) Gel blots showing the purity of each recombinant proteins as indicated. (b) Bar charts showing that the redox-dependent changes in enzymatic activities can be perturbed by the indicated nucleophilic fragments. Data are mean ± s.d. (representative data from biological triplicates are shown, two-sided Student’s t-test, P < 0.05 was considered significant). Each experiment was repeated three times with similar results. (c) 3D protein structures of enzymes (PDB#:1EEM for GSTO1; PDB#:2F2S for ACAT1; PDB#:1U8F for GAPDH) were visualized with Pymol 2.1.1. Peptide containing liganded sites are highlighted in blue. Enzyme substrates or ligand are highlighted in red. (d) Representative XICs showing changes in BTD-labeled peptides from proteins as indicated. The profiles for light- and heavy-labeled peptide are shown in red and blue, respectively. The average RH/L values measured from biological duplicates are displayed below each XIC.
Supplementary information
Supplementary Information
Supplementary Information
Supplementary Data 1
Quantitative chemoproteomic analysis of sulfenome reactivity in the human proteome. The protein lysates from A549 cells were labeled with 500 µM and 5 mM BTD, respectively, and subjected to tryptic digestion. The resulting BTD-modified peptides were further conjugated to light and heavy azido-biotin reagents with a photocleavable linker (Az-UV-biotin), respectively, via copper-catalyzed alkyne-azide cycloaddition reaction (CuAAC). The light and heavy labeled samples then were mixed equally in amount and subjected to streptavidin-based enrichment. After several washing steps, the modified peptides were selectively eluted from beads under 365-nm wavelength of UV light and subjected to LC–MS/MS-based proteomic analysis. Hyperreactive cysteines would be expected to label to completion at low probe concentrations, and less reactive cysteines should show concentration-dependent increases in BTD labeling.
Supplementary Data 2
Comparing nucleophilic fragments with electrophilic fragments showed distinct target profiles of electrophilc and nucleophilc fragments with the same scaffold.
Supplementary Data 3
Global profiling of sulfenic acid ligandability in the human proteome. The protein lysates from MDA-MB-231 cells were first labeled with 0 mM or 2 mM fragment, then labeled with 5 mM BTD and finally subjected to tryptic digestion. The resulting BTD-modified peptides were further conjugated to light and heavy azido-biotin reagents with a photocleavable linker (Az-UV-biotin), respectively, via copper-catalyzed alkyne-azide cycloaddition reaction. The light and heavy labeled samples were then mixed equally in amount and subjected to streptavidin-based enrichment. After several washing steps, the modified peptides were selectively eluted from beads under 365-nm wavelength of UV light and subjected to LC–MS/MS-based proteomic analysis. For each cysteine detected, we calculated a control/fragment ratio (RH/L). Fragment-liganded sulfenic acid would enable less BTD-derived chemoselective conjugation, thereby rendering relatively high RH/L values.
Supplementary Data 4
Redox-dependent ligandability of a nucleophilic fragment. MDA-MB-231 cells were pre-treated with or without H2O2 (0.5 mM, 15 min), followed by treatment of vehicle (DMSO) or 5b (2 mM, 1 h) and subsequent probe labeling with BTD (5 mM, 1 h). The probe-labeled proteomes were harvested, clicked with light and heavy azido-UV-biotin reagents and mixed equally. The click reaction mixtures were then subjected to streptavidin enrichment, followed photorelease and LC–MS/MS analysis.
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Fu, L., Jung, Y., Tian, C. et al. Nucleophilic covalent ligand discovery for the cysteine redoxome. Nat Chem Biol 19, 1309–1319 (2023). https://doi.org/10.1038/s41589-023-01330-5
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DOI: https://doi.org/10.1038/s41589-023-01330-5
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