Targeted protein degradation (TPD) is a transformative strategy in drug discovery that utilizes cellular protein degradation processes to selectively eliminate deleterious proteins. Directly designing small molecules that promote induced proximity to E3 ligase to facilitate protein degradation with selective is challenging in practice, Thereby, compound screening with reliable quantitation accuracy is a crucial phase in drug discovery in the TPD space. The need for screening large numbers of compounds with accurate quantitation makes high-throughput mass spectrometry-based workflows an obvious choice for ensuring accurate lead identification. In this study, LFQ-DIA workflows of various gradients with highly accurate quantitation on Orbitrap Astral mass spectrometer were successfully developed to cater the needs of compound screening.

In our pilot experiments, VCaP prostate cancer epithelial cells were treated with different concentrations of ARCC-4, a PROTAC protein degrader for androgen receptor. Cells were lysed and digested with trypsin using an AccelerOme automated sample preparation platform. The ensuing peptides were loaded onto a 15-cm EASY-Spray™ or 5-cm Ionopticks Aurora UHPLC column and separated with nano or capillary flowrate in trap and elute workflow using a Vanquish Neo UHPLC system for a throughput of 60, 100, 180 and 300 samples per day. Eluted peptides were ionized with an Easy-Spray source and transferred into the Orbitrap Astral mass spectrometer for DIA analysis. Acquired data was processed by Spectronaut, DIA-NN or Proteome Discoverer™ software using CHIMERYS™ intelligent search algorithm by MSAID.

The 100 SPD method enabled over 9000 proteins to be reproducibly identified and quantified with a protein group CV of approximately 10%. To explore the possibility of ultra-fast compound screening, we further increased the throughput to 180 and 300 SPD, which led to the identification of over 8500 proteins and close to 8000 protein groups, respectively. To achieve maximized proteome coverage, an essential component for the validation phase, we further extended this workflow to 60 SPD. We successfully identified approximately 10,000 proteins with the 60 SPD method, which signifies a near-complete proteome identification within the cell. In all the throughputs investigated herein, we observed decreased androgen receptor expression upon increasing ARCC-4 concentration while the vast majority of proteome remained unaffected. In addition, the quantitation accuracy for workflows of various throughputs was validated by three proteomes with different ratios. The observed ratio was found to align closely with the theoretical ratio, thereby affirming the reliability of our workflow. Together, our data demonstrate that the Orbitrap Astral mass spectrometer with Vanquish Neo UHPLC system enable high-throughput compound screening and comprehensive on-target/off-target validation in the development of targeted protein degradation.