Utilising a degradation mechanism, PROTACs have been reported as label-free probes in chemoproteomics to examine protein functions in whole-proteomes. They assimilate the function of knockdown or knockout of protein target/s and has the potential to degrade ‘undruggable’ target proteins. The synergy of PROTACs with the Orbitrap Astral Mass Spectrometer, known for its capability to perform analysis with faster throughput, deeper coverage and higher sensitivity, accuracy and precision, was utilised in a high-throughput plate-based approach for efficient target deconvolution of bromodomain and extraterminal (BET) targeting PROTACs (MZ1, dBET6).
MZ1 and dBET6 with their appropriate negative controls (cis-MZ1 and dBET6-Me with modified E3 ligase recruiters) were used at 1 µM in human cancerous and noncancerous cell lines, for a duration of 1 h and 24 h. Processed protein pellets were then analysed in a high-throughput 96-well plate-based manner for elucidation of proteins dysregulated at the respective timeline as a result of PROTAC treatment.
This approach successfully identified 3 BET proteins, bromodomain containing 2/3/4 (BRD2, BRD3, BRD4) as the primary targets of PROTAC MZ1 from the entire proteome of 10253 proteins following a short treatment time of 1 h, demonstrating the feasibility of this technique. Moreover, 24 h treatment with MZ1 revealed 10 proteins that were significantly downregulated and 40 upregulated. Abhydrolase domain containing 5 (ABHD5) and zinc finger protein 385A (ZNF385A) were found to be most up- and downregulated, respectively. In addition, the direct interacting myelocytomatosis oncogene (MYC) protein was also identified to be upregulated.
Herein, we present an efficient high-throughput plate-based methodology for target deconvolution using PROTACs probes to identify primary and secondary target proteins perturbed in a time-dependent manner.