Deep ubiquitination site profiling by single-shot data-independent acquisition mass spectrometry

We report a highly optimized proteomics method for in-depth ubiquitination profiling, which combines efficient protein extraction and data-independent acquisition mass spectrometry (DIA-MS). Employing DIA for both spectral library generation and single-shot sample analysis, we quantify up to 70,000 ubiquitinated peptides per MS run with high precision, data completeness and throughput. Our approach resolves the dynamics of ubiquitination and protein degradation with an unprecedented analytical depth.