Recent advances in covalent and non-covalent drug discovery with TMT quantitative chemoproteomics

 

Chemoproteomics has become a major approach to discover new protein-ligand interactions for biomedical research and drug discovery. The success of multiple covalent drugs against proteins once deemed “undruggable” has fueled growing interest in electrophiles that covalently bind amino acids such as cysteine. However, current technology can routinely access only a small fraction (<25%) of the human cysteinome due to limited sensitivity. Large compound screening campaigns often opt for fewer replicates due to cost and time constraints, which in turn increase the need for robust quantification accuracy. In addition, screenings against therapeutic targets are frequently plagued by missing values due to the stochastic nature inherent in discovery proteomics. Here, we introduce a platform which integrates tandem mass tag-based sample multiplexing, an intelligent targeted proteomics strategy—GoDig, and a high-depth activity-based cysteine profiling to overcome the challenges and revamp covalent drug discovery. The platform enables high-throughput, targeted interrogation of cysteine engagement with significantly reduced missing values. After identifying confident ligands to proteins of interest, selectivity evaluation can be assessed across ~30K reactive cysteines in a single assay using the platform. We further benchmarked TMT quantification using MS3 technology on the Thermo Scientific™ Orbitrap™ Tribrid™ MS system and found it offers better precision, improved accuracy, and a higher success rate in detecting significant changes compared to using either TMT- or DIA-based quantification on the Thermo Scientific™ Orbitrap™ Astral™ MS.”