Chemoproteomics is a powerful tool in drug discovery owing to its ability to elucidate phenotypic mechanisms, characterise protein ligandability and identify the binding of novel chemical matter. The rapid development of chemoproteomic techniques and cutting-edge technologies can reshape the field of drug discovery, particularly in covalent drug development.
Substantial efforts dedicated to mapping amino acid side-chain reactivity in complex biological systems, particularly cysteine residues, has improved our understanding of protein function, redox and signaling pathways and potential therapeutic targets. Although previously considered to increase chances of drug toxicity, intentional and intelligent design strategies have seen the re-emergence of covalent drugs as a popular tactic to address challenging drug targets.
We have recently redesigned the streamlined cysteine activity-based proteome profiling (SLC-ABPP) platform which utilises tandem-mass tag (TMT) multiplexing and enrichment via a desthiobiotin iodoacetamide probe. The next-generation plate-based proteomic platform has significantly improved throughput, miniaturisation and quantitative accuracy. We were able to simultaneously reduce the starting material input 5-fold and increased cysteine quantification 3-fold compared to the previously published method. However, as only 1 in 4 of all cysteines in the proteome have been previously profiled, there remains a desire to further increase cysteine coverage.
The integration of the latest Thermo Fisher mass spectrometer, the Orbitrap Astral, into this platform has further enhanced throughput and sensitivity in routine multiplexed proteome profiling. This is achieved due to the Orbitrap Astral’s new high-resolution accurate mass analyser which boasts ultra-fast scan speeds of up to 200 Hz, resolving power of 80k and narrow isolation windows as low as 0.4 Th.
Since developing our multiplexed cysteine profiling platform, our lab can routinely access ~30k reactive cysteines on the Orbitrap Astral in a single experiment, improving the coverage of the cysteinome by a further 50%. This ensures deeper datasets and extensive data completeness, essential for advancing effective drug development strategies.
Together, TMT multiplexing and the Orbitrap Astral facilitate the efficient discovery of novel protein-ligand interactions across the expansive human proteome and their further development from hits to lead-like compounds.