Robust proteome profiling of cysteine-reactive fragments using label-free chemoproteomics

George S. Biggs, Emma E. Cawood, Aini Vuorinen, William J. McCarthy, Harry Wilders, Ioannis G. Riziotis, Antonie J. Zouwen, Jonathan Pettinger, Luke Nightingale, Peiling Chen, Andrew J. Powell, David House, Simon J. Boulton, J. Mark Skehel, Katrin Rittinger () and Jacob T. Bush ()
Additional contact information
George S. Biggs: Stevenage
Emma E. Cawood: Stevenage
Aini Vuorinen: Stevenage
William J. McCarthy: The Francis Crick Institute
Harry Wilders: Stevenage
Ioannis G. Riziotis: Stevenage
Antonie J. Zouwen: The Francis Crick Institute
Jonathan Pettinger: Stevenage
Luke Nightingale: The Francis Crick Institute
Peiling Chen: GSK
Andrew J. Powell: Stevenage
David House: Stevenage
Simon J. Boulton: The Francis Crick Institute
J. Mark Skehel: The Francis Crick Institute
Katrin Rittinger: The Francis Crick Institute
Jacob T. Bush: Stevenage

Nature Communications, 2025, vol. 16, issue 1, 1-14

Abstract: 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.

Date: 2025
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DOI: 10.1038/s41467-024-55057-5

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