Abstract
Photoaffinity probes are routinely utilized to identify proteins that interact with small molecules. However, despite this common usage, resolving the specific sites of these interactions remains a challenge. Here we developed a chemoproteomic workflow to determine precise protein binding sites of photoaffinity probes in cells. Deconvolution of features unique to probe-modified peptides, such as their tendency to produce chimeric spectra, facilitated the development of predictive models to confidently determine labeled sites. This yielded an expansive map of small-molecule binding sites on endogenous proteins and enabled the integration with multiplexed quantitation, increasing the throughput and dimensionality of experiments. Finally, using structural information, we characterized diverse binding sites across the proteome, providing direct evidence of their tractability to small molecules. Together, our findings reveal new knowledge for the analysis of photoaffinity probes and provide a robust method for high-resolution mapping of reversible small-molecule interactions en masse in native systems.
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Data availability
The Uniprot Homo sapiens proteome database (downloaded July 2020; 74,782 sequences) was used for proteomic searches. MS datasets have been deposited on ProteomeXchange as follows: Benchmark SoL (PXD044869) and whole protein (PXD044870). TMT pilot SoL (PXD044886) and whole protein (PXD044887). TMT dose nonenantiomers SoL (PXD044881) and whole protein (PXD044882). TMT dose enantiomers SoL (PXD044883) and whole protein (PXD044884). Molecular modeling.pdb files have been uploaded to the Zenodo repository and can be accessed through https://doi.org/10.5281/zenodo.8326534. Source data are provided with this paper.
Code availability
Scripts developed in this work are available at https://github.com/jmwozniak/DizcoProcessing and have been uploaded to Zenodo85.
Change history
11 January 2024
A Correction to this paper has been published: https://doi.org/10.1038/s41589-024-01546-z
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Acknowledgements
This work was supported by the National Institute of Allergic and Infectious Diseases NIAID/R01 AI156268 (C.G.P.), 1U19AII71443-01 (C.G.P. and S.F.) and T32AI007244-39 (J.M.W.) as well as National Institutes of Health grant R01GM069832 (S.F.).
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Contributions
C.G.P. and J.M.W. conceived the project. J.M.W. and W.L. developed chemoproteomic methods and performed chemoproteomic experiments. J.M.W. developed the chemoproteomic analytical workflow with input from A.D. W.L. and L.-Y.C. performed gel-based and CETSA validation experiments. A.J. synthesized compounds. S.F. and P.G. performed molecular docking analyses. All authors contributed to data analysis and interpretation. C.G.P. and J.M.W. wrote the paper with input from all authors.
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C.G.P. is a cofounder and scientific advisor to Belharra Therapeutics, a biotechnology company interested in using chemical proteomic methods to develop small-molecule therapeutics. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Comparison of MultiPSM and ptmRS probe localization.
(a) Overlap of MultiPSM and ptmRS locations for all unique peptides for each benchmark probe. (b) Example spectra where ptmRS calls a single probe modified location (L9) but there is more evidence for other locations. Ions are colored according to whether they are consistent with probe N-terminal to L9 (red), C-terminal to L9 (blue) or localized to L9 and other locations (black). Unmatched fragment ions are shown in light gray. (c) Quantification of fragment ions shown in panel b.
Extended Data Fig. 2 Development of a predictive model for spectra containing photo-affinity probe labeled peptides.
(a) Predictive abilities of spectral features for identifying probe-labeled peptides (all probes merged in plots). (b) Confusion matrix of final predictive model trained on and applied to all probes. (c) Split confusion matrix of predictive models trained on individual probes and applied to all other individual probes.
Extended Data Fig. 3 Integration of Dizco model with MSFragger.
(a) ROC curves for predicting probe labeled peptides generated from MSFragger output (using a custom delta score = hyperscore - nextscore and retention time difference from unlabeled peptide of same length). (b) Overlap of unique probe-labeled peptides from Sequest and MSFragger searches.
Extended Data Fig. 4 Extended validation of stereoselective probe-target interactions.
(a) GSTM3 concentration plot and probe label site proximal to the active site (PDB: 3GTU). (b) Immunoblot analysis and quantification of GSTM3 stereoselective probe binding. (c) Immunoblot analysis of competitive blockade of probe (S)-9-NAMPT interaction using a cognate competitor molecule (S)-9c in cells. (d) Immunoblot analysis of competitive blockade of probe (R)-9-NAMPT interaction using a cognate competitor molecule (R)-9c in cells. Each immunoblot displayed is representative of two independent experiments. (PD = pulldown).
Extended Data Fig. 5 Proteins possessing multiple binding sites with varying EC50 values.
For all structures, residues labeled by probes are colored red or light red for probe 3 and blue or light blue for probe 8. The remainder of each detected peptide is colored black. Active/other indicated sites are colored green and co-resolved ligands are colored yellow. CYP51A1 probe 3 concentration plot (a), peptide plots (b) and label sites (c; PDB: 6UEZ). SoL-2a/b refers to two unique peptides that support the same high EC50 binding site (SoL-2b is absent from presented PDB structure, but proximity to SoL-2a was determined from Alpha Fold structure). NENF probe 3 concentration plot (d), peptide plots (e) and label sites (f; AF-Q9UMX5-F1-model_v2). SoL-1a/b refers to two unique peptides that support the same low EC50 binding site. SLC25A15 probe 8 concentration plot (g), peptide plots (h) and label sites (i; AF-Q9Y619-F1-model_v2). All calculated EC50 values are approximations.
Extended Data Fig. 6 Extended orthogonal validation of probe-target interactions.
(a) Cellular thermal shift assay (CETSA) temperature gradient and quantification of probe 8-ACAT2 interaction. (b) CETSA dose analysis of probe 8-ACAT2 interaction. (c) Probe 8-ACAT2 concentration plot from proteomics experiment. (d) CETSA temperature gradient and quantification of probe 3-EPHX1 interaction. (e) CETSA dose analysis of probe 3-EPHX1 interaction. (f) Probe 3-EPHX1 concentration plot from proteomics experiment. (g) CETSA temperature gradient and quantification of probe 6-PMPCA interaction. (h) CETSA dose analysis of probe 6-PMPCA interaction. Each immunoblot displayed is representative of two independent experiments.
Extended Data Fig. 7 Extended orthogonal validation of functional sites.
(a) MTHFD2 probe 6 label sites overlapping with LY345899-binding site (PDB: 5TC4). (b) Immunoblot analysis of competitive blockade of probe 6-MTHFD2 interaction using LY345899 in cells. (c) ACAT2 probe 8 label sites overlapping with CoA-binding site (PDB: 1WL4). (d) Immunoblot analysis of competitive blockade of probe 8-ACAT2 interaction using CoA. (e) Immunoblot analysis of competitive blockade of probe 3-ABHD12 interaction using DO264 (see Supplementary Figure 12a for corresponding ABHD12 structure and probe 3 peptide plot) in cells. Each immunoblot displayed is representative of two independent experiments. (PD = pulldown).
Extended Data Fig. 8 Extended orthogonal validation of sites of unknown function.
(a) Depiction of probe label sites overlapping with sites of unknown function for probe 6-PMPCA (AF-Q10713-F1-model_v2) interaction. Depiction of probe label sites overlapping with sites of unknown function and immunoblot analysis and quantification of probe 6-ACAD9 (AF-Q9H845-F1-model_v2) (b-c), and probe 3-PCYO1XL (AF-Q8NBM8-F1-model_v2) (d-e) interactions in cells. Immunoblot analysis and quantification of probe 3-GDI2 (f) and probe 6-CDK1 (g) interactions (see Fig. 6i,k for corresponding structures and peptide plots) in cells. Each immunoblot displayed is representative of two independent experiments. (PD = pulldown).
Supplementary information
Supplementary Information
Supplementary Figs. 1–15, notes, chemical synthesis and characterization.
Supplementary Data 1
Whole-protein and site-of-labeling proteomics data in HEK293T cells (Supplementary Tables 1–16). Table titles and legends are within the file.
Source data
Source Data Fig. 5
Uncropped blots of western blot images.
Source Data Fig. 5
Quantification of western blot images.
Source Data Fig. 6
Uncropped blots of western blot images.
Source Data Extended Data Fig. 4
Uncropped blots of western blot images.
Source Data Extended Data Fig. 4
Quantification of western blot images.
Source Data Extended Data Fig. 6
Uncropped blots of western blot images.
Source Data Extended Data Fig. 6
Quantification of western blot images.
Source Data Extended Data Fig. 7
Uncropped blots of western blot images.
Source Data Extended Data Fig. 8
Uncropped blots of western blot images.
Source Data Extended Data Fig. 8
Quantification of western blot images.
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Wozniak, J.M., Li, W., Governa, P. et al. Enhanced mapping of small-molecule binding sites in cells. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-023-01514-z
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DOI: https://doi.org/10.1038/s41589-023-01514-z
