Variable-Velocity Traveling-Wave Ion Mobility Separation Enhancing Peak Capacity for Data-Independent Acquisition Proteomics.

High mass accuracy, data-dependent acquisition is the current standard method in mass spectrometry-based peptide annotation and quantification. In high complexity samples, limited instrument scan speeds often result in under-sampling. In contrast, all-ion data-independent acquisition methods bypass precursor selection, alternating high and low collision energies to analyze product and precursor ions across wide mass ranges. Despite capturing data for all events, peptide annotation is limited by inadequate alignment algorithms or overlapping ions. Ion mobility separation can add an orthogonal analytical dimension, reducing ion interference to improve reproducibility, peak capacity, and peptide identifications to rival modern hybrid quadrupole orbitrap systems. Despite the advantages of ion mobility separation in complex proteomics analyses, there has been no quantitative measure of ion mobility resolution in a complex proteomic sample. Here, we present TWIMExtract, a data extraction tool to export defined slices of liquid chromatography/ion mobility/mass spectrometry (LC-IM-MS) data, providing a route to quantify ion mobility resolution from a commercial traveling-wave ion mobility time-of-flight mass spectrometer. Using standard traveling-wave ion mobility parameters (600 m/s, 40 V), 90% of the annotated peptides occupied just 23% of the ion mobility drift space, yet inclusion of ion mobility nearly doubled the overall peak capacity. Relative to fixed velocity traveling-wave ion mobility settings, ramping the traveling-wave velocity increased drift space occupancy, amplifying resolution by 16%, peak capacity by nearly 50%, and peptide/protein identifications by 40%. Overall, variable-velocity traveling-wave ion mobility-mass spectrometry significantly enhances proteomics analysis in all-ion fragmentation acquisition.