Oral Presentation 31st Annual Lorne Proteomics Symposium 2026

Systematic Proteome-Wide Target Interrogation of PROTAC activity by High-Throughput DIA-MS on the Orbitrap Astral Platform (133360)

Chuhui Huang 1 , Yijia (Jessica) Ji 2 , Thanh Nhan (Nathan) Nguyen 3 , Amanda De Paoli 2 , Joshua Morrow 2 , Darren J Creek 2 , Manuela Jorg 4 , Ghizal Siddiqui 2
  1. Medicinal Chemistry, Monash Institute of Pharmaceutical Science, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia
  2. Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville Campus, Parkville, Victoria, Australia
  3. Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia
  4. Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia

Proteomics provides a comprehensive and unbiased means to profile the proteome, enabling simultaneous quantification of thousands of proteins across experimental conditions. An efficient degradation-based proteomics workflow was established using data-independent acquisition-mass spectrometry (DIA-MS) and the Orbitrap Astral platform to investigate the mode of actions and on target specificity of bromodomain and extra-terminal protein-targeting PROTACs, MZ1 and dBET6. Human cancerous and noncancerous cell lines (HeLa, MDA-MB-231 and HEK293) were treated with 1 µM of the PROTACs and their respective negative control controls (cis-MZ1 and dBET6Me) for 1 h and 24 h. Following treatment, cell pellets were harvested, proteins were processed and analysed in a 96-well plate format to identify proteins dysregulated at each timepoint.

This approach successfully identified the three BET family members, bromodomain containing proteins 2/3/4 (BRD2, BRD3, BRD4) as the primary targets of MZ1 and dBET6 after 1 h treatment. Extended 24 h treatment with MZ1 and dBET6 further revealed 51, 57 and 115 secondary proteins significantly perturbed in HEK293, HeLa and MDA-MB-231 cells, respectively.

This approach was further applied to proteolysis targeting chimeras (CLIPTACs), which form PROTACs within the cellular system via click chemistry, and to the degradation tag (dTAG) system, which utilises a single-point mutant of (F36V) as the TAG domain with corresponding Degraders that selectively recruit FKBP12F36V over wild-type FKBP1.

Overall, this work demonstrates the feasibility of identifying direct PROTAC targets following short incubations, as well as elucidating downstream proteomic alterations upon prolonged treatment, surpassing the resolution achievable by traditional immunoblotting techniques.