Ultrahigh-Throughput Liquid Chromatography with Tandem Mass Spectrometry Method for Targeted Protein Degradation Compound Screening Using the Orbitrap Astral Mass Spectrometer.

Targeted protein degradation (TPD) is a therapeutic strategy that utilizes small molecules to induce the proximity-driven degradation of disease-causing proteins. Because the efficacy and selectivity of TPD compounds must be validated across thousands of proteins, high-throughput proteomics is essential for the rapid screening and characterization of these novel degraders. Here, we developed a 300 samples per day (SPD) LC-MS/MS method using the Orbitrap Astral mass spectrometer for ultrahigh-throughput TPD compound screening. We identified close to 8000 protein groups from a single cell line with a coefficient of variation (CV) of less than 10%, highlighting the deep proteome coverage and method reproducibility even at 300 SPD. This high degree of precision provides the statistical confidence to detect subtle, yet significant, changes in protein abundance that were previously challenging to quantify in high-throughput workflows. To evaluate the quantitation accuracy of this method, we further mixed the digests from two or three species at different ratios. Our three-proteome mixture results demonstrated highly accurate quantitation for proteins with both small and large fold changes. Moreover, our two-proteome mixture experiment, where 20 to 160 ng of yeast digest was spiked into 200 ng of HeLa digest, showed an R2 of 0.999 for the yeast proteome, underscoring the quantitation accuracy of the method. Utilizing this workflow, we studied dose-dependent protein degradation patterns induced by pomalidomide, iberdomide, and mezigdomide. Our results indicate that mezigdomide may possess enhanced efficacy in T cells by degrading additional proteins such as IKZF2, thereby boosting anticancer immunity. Together, we developed an ultrahigh-throughput LC-MS/MS method with excellent proteome coverage and quantitation accuracy that is highly suitable for chemoproteomics screening of drug libraries.