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.

Identification of ion series using ion mobility mass spectrometry: the example of alkyl-benzothiophene and alkyl-dibenzothiophene ions in diesel fuels.

Ion mobility-mass spectrometry (IMMS) has been presented as a promising method for analysis of highly complex mixtures. This coupling adds an additional postionization separation dimension to MS. The IM separation of ions is obtained in the millisecond time scale and can be particularly helpful when chromatographic separation is not possible. For obtaining relevant information about the samples, data processing is usually the bottleneck because of the high amount of data generated with IMMS. In the current work, we present a new workflow using specific comparison software dedicated to IMMS data, which allows one to compare m/z-drift time plots to highlight differences between samples. Two diesel fuels have been compared, i.e., the feed and the product of hydrodesulfurization (HDS) process, and this approach allowed us to clearly highlight the variation of intensity of several ions distributed along the plots of both samples. Accurate mass measurements and post IM collision induced dissociation experiments allowed us to identify two series of polycyclic aromatic sulfur-containing heterocycle (PASH) compounds among the matrix ions.