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

We present a data-independent acquisition mass spectrometry method, ultradefinition (UD) MSE. This approach utilizes ion mobility drift time-specific collision-energy profiles to enhance precursor fragmentation efficiency over current MSE and high-definition (HD) MSE data-independent acquisition techniques. UDMSE provided high reproducibility and substantially improved proteome coverage of the HeLa cell proteome compared to previous implementations of MSE, 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 UDMSE data.