Data-independent acquisition (DIA) approach for comprehensive ubiquitinome profiling in targeted protein degradation

Abstract Targeted protein degradation (TPD) has emerged as a highly promising therapeutic strategy for a wide range of diseases, including cancer and neurodegenerative disorders. The ubiquitin-proteasome system, which is responsible for protein degradation, plays a critical role in this process. Gaining comprehensive insights into the ubiquitylation landscape is essential for the development of selective and efficient targeted protein degradation approaches. Recently, data-independent acquisition (DIA) has gained significant popularity as a robust and unbiased approach for quantitative proteomics. Here, we report a robust workflow that utilizes diGly antibody-based enrichment followed by an optimized Orbitrap-based DIA method for the identification of ubiquitylated peptides. We identify over 40,000 diGly peptides corresponding to more than 7,000 proteins in a single measurement from cells exposed to a proteasome inhibitor, highlighting an exceptional throughput. By applying our optimized workflow, we successfully identify ubiquitylation sites on substrate proteins with various TPD approaches. By also leveraging the cutting-edge Orbitrap Astral technology, we resolve rapid PROTAC-driven ubiquitylation on target proteins, as early as 5 min post-treatment. Therefore, our workflow holds tremendous potential for rapidly establishing mode-of-action for various TPD modalities, including PROTACs and molecular glues.