Robust proteome profiling of cysteine-reactive fragments using label-free chemoproteomics
Abstract Identifying pharmacological probes for human proteins represents a key opportunity to accelerate the discovery of new therapeutics. High-content screening approaches to expand the ligandable proteome offer the potential to expedite the discovery of novel chemical probes to study protein fun...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-01-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-024-55057-5 |
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| author | George S. Biggs Emma E. Cawood Aini Vuorinen William J. McCarthy Harry Wilders Ioannis G. Riziotis Antonie J. van der Zouwen Jonathan Pettinger Luke Nightingale Peiling Chen Andrew J. Powell David House Simon J. Boulton J. Mark Skehel Katrin Rittinger Jacob T. Bush |
| author_facet | George S. Biggs Emma E. Cawood Aini Vuorinen William J. McCarthy Harry Wilders Ioannis G. Riziotis Antonie J. van der Zouwen Jonathan Pettinger Luke Nightingale Peiling Chen Andrew J. Powell David House Simon J. Boulton J. Mark Skehel Katrin Rittinger Jacob T. Bush |
| author_sort | George S. Biggs |
| collection | DOAJ |
| description | Abstract Identifying pharmacological probes for human proteins represents a key opportunity to accelerate the discovery of new therapeutics. High-content screening approaches to expand the ligandable proteome offer the potential to expedite the discovery of novel chemical probes to study protein function. Screening libraries of reactive fragments by chemoproteomics offers a compelling approach to ligand discovery, however, optimising sample throughput, proteomic depth, and data reproducibility remains a key challenge. We report a versatile, label-free quantification proteomics platform for competitive profiling of cysteine-reactive fragments against the native proteome. This high-throughput platform combines SP4 plate-based sample preparation with rapid chromatographic gradients. Data-independent acquisition performed on a Bruker timsTOF Pro 2 consistently identified ~23,000 cysteine sites per run, with a total of ~32,000 cysteine sites profiled in HEK293T and Jurkat lysate. Crucially, this depth in cysteinome coverage is met with high data completeness, enabling robust identification of liganded proteins. In this study, 80 reactive fragments were screened in two cell lines identifying >400 ligand-protein interactions. Hits were validated through concentration-response experiments and the platform was utilised for hit expansion and live cell experiments. This label-free platform represents a significant step forward in high-throughput proteomics to evaluate ligandability of cysteines across the human proteome. |
| format | Article |
| id | doaj-art-673f3270b83c4fd19cc17022696f96aa |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-673f3270b83c4fd19cc17022696f96aa2025-01-26T12:41:42ZengNature PortfolioNature Communications2041-17232025-01-0116111410.1038/s41467-024-55057-5Robust proteome profiling of cysteine-reactive fragments using label-free chemoproteomicsGeorge S. Biggs0Emma E. Cawood1Aini Vuorinen2William J. McCarthy3Harry Wilders4Ioannis G. Riziotis5Antonie J. van der Zouwen6Jonathan Pettinger7Luke Nightingale8Peiling Chen9Andrew J. Powell10David House11Simon J. Boulton12J. Mark Skehel13Katrin Rittinger14Jacob T. Bush15Crick-GSK Biomedical LinkLabs, GSK, Gunnels Wood Road, StevenageCrick-GSK Biomedical LinkLabs, GSK, Gunnels Wood Road, StevenageCrick-GSK Biomedical LinkLabs, GSK, Gunnels Wood Road, StevenageMolecular Structure of Cell Signalling Laboratory, The Francis Crick InstituteCrick-GSK Biomedical LinkLabs, GSK, Gunnels Wood Road, StevenageCrick-GSK Biomedical LinkLabs, GSK, Gunnels Wood Road, StevenageMolecular Structure of Cell Signalling Laboratory, The Francis Crick InstituteCrick-GSK Biomedical LinkLabs, GSK, Gunnels Wood Road, StevenageSoftware Engineering and AI, The Francis Crick InstituteGSK Chemical Biology, GSKCrick-GSK Biomedical LinkLabs, GSK, Gunnels Wood Road, StevenageCrick-GSK Biomedical LinkLabs, GSK, Gunnels Wood Road, StevenageDSB Repair Metabolism Laboratory, The Francis Crick InstituteProteomics Science Technology Platform, The Francis Crick InstituteMolecular Structure of Cell Signalling Laboratory, The Francis Crick InstituteCrick-GSK Biomedical LinkLabs, GSK, Gunnels Wood Road, StevenageAbstract Identifying pharmacological probes for human proteins represents a key opportunity to accelerate the discovery of new therapeutics. High-content screening approaches to expand the ligandable proteome offer the potential to expedite the discovery of novel chemical probes to study protein function. Screening libraries of reactive fragments by chemoproteomics offers a compelling approach to ligand discovery, however, optimising sample throughput, proteomic depth, and data reproducibility remains a key challenge. We report a versatile, label-free quantification proteomics platform for competitive profiling of cysteine-reactive fragments against the native proteome. This high-throughput platform combines SP4 plate-based sample preparation with rapid chromatographic gradients. Data-independent acquisition performed on a Bruker timsTOF Pro 2 consistently identified ~23,000 cysteine sites per run, with a total of ~32,000 cysteine sites profiled in HEK293T and Jurkat lysate. Crucially, this depth in cysteinome coverage is met with high data completeness, enabling robust identification of liganded proteins. In this study, 80 reactive fragments were screened in two cell lines identifying >400 ligand-protein interactions. Hits were validated through concentration-response experiments and the platform was utilised for hit expansion and live cell experiments. This label-free platform represents a significant step forward in high-throughput proteomics to evaluate ligandability of cysteines across the human proteome.https://doi.org/10.1038/s41467-024-55057-5 |
| spellingShingle | George S. Biggs Emma E. Cawood Aini Vuorinen William J. McCarthy Harry Wilders Ioannis G. Riziotis Antonie J. van der Zouwen Jonathan Pettinger Luke Nightingale Peiling Chen Andrew J. Powell David House Simon J. Boulton J. Mark Skehel Katrin Rittinger Jacob T. Bush Robust proteome profiling of cysteine-reactive fragments using label-free chemoproteomics Nature Communications |
| title | Robust proteome profiling of cysteine-reactive fragments using label-free chemoproteomics |
| title_full | Robust proteome profiling of cysteine-reactive fragments using label-free chemoproteomics |
| title_fullStr | Robust proteome profiling of cysteine-reactive fragments using label-free chemoproteomics |
| title_full_unstemmed | Robust proteome profiling of cysteine-reactive fragments using label-free chemoproteomics |
| title_short | Robust proteome profiling of cysteine-reactive fragments using label-free chemoproteomics |
| title_sort | robust proteome profiling of cysteine reactive fragments using label free chemoproteomics |
| url | https://doi.org/10.1038/s41467-024-55057-5 |
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