Deep Ubiquitination Site Profiling by Data-Independent Acquisition Mass Spectrometry Reveals Usp7 Targets on a Proteome-Wide Scale

Abstract

Mass spectrometry-based ubiquitinomics has enormous potential to provide new insights into ubiquitin-mediated cellular processes. However, its routine application is limited by laborious sample preparation, low quantification accuracy, lack of data completeness, and considerable throughput limitations. We report a highly optimized method for in-depth ubiquitin site profiling, which combines efficient protein extraction and data-independent acquisition mass spectrometry (DIA-MS). Instead of conventional spectral library building by data-dependent acquisition (DDA) MS, we use library-free DIA to generate a far more comprehensive library, comprising 111,707 distinct ubiquitination sites. With this, we quantify up to 70,000 ubiquitinated peptides in single DIA-MS runs with high precision and throughput. Using a specific inhibitor, we apply our method to identify targets of the deubiquitinase Usp7. Instead of just monitoring a single treatment time, we performed time-course experiments without proteasome inhibitors and quantify more than 40,000 ubiquitinated peptides and 10,000 proteins in each sample. This allows us for the first time to connect early ubiquitination events to later changes in protein abundance on a global scale. Besides confirming known substrates, we uncover a set of new likely Usp7 targets, demonstrating the power of our workflow to resolve ubiquitination and protein degradation dynamics with an unprecedented analytical depth.