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

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 (MOA) and on target specificity of bromodomain and extra-terminal (BET) 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 from the total proteomes of 9,641, 8,157 and 8,988 quantified proteins in HEK293, HeLa and MDA-MB-231 cells, respectively, after short (1 h) treatment. Extended treatment (24 h) with MZ1 and dBET6 further revealed 51, 57 and 115 secondary proteins significantly perturbed (|log2 fold change (log2FC)| ≥ 1 and adjusted p (adj.p) ≤ 0.05) in HEK293, HeLa and MDA-MB-231 cells, respectively. Overall, this work demonstrates the feasibility of the developed approach for 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.