Drift time-specific collision energies enable deep-coverage data-independent acquisition proteomics

A data-independent acquisition (DIA) mass spectrometry approach, ultradefinition (UD)MS E , offers high reproducibility and improved proteome coverage over alternative DIA and data-dependent acquisition workflows. We present a data-independent acquisition mass spectrometry method, ultradefinition (UD) MS E . This approach utilizes ion mobility drift time-specific collision-energy profiles to enhance precursor fragmentation efficiency over current MS E and high-definition (HD) MS E data-independent acquisition techniques. UDMS E provided high reproducibility and substantially improved proteome coverage of the HeLa cell proteome compared to previous implementations of MS E , and it also outperformed a state-of-the-art data-dependent acquisition workflow. Additionally, we report a software tool, ISOQuant, for processing label-free quantitative UDMS E data.