Evaluating Peptide Fragment Ion Detection Using Traveling Wave Ion Mobility Spectrometry with Signal-Enhanced MS (SEMS).

The inherent poor sampling of fragment ions in time-of-flight mass analyzers was recently improved for data-dependent acquisition (DDA) by considering their drift times in traveling wave ion mobility spectrometry (TWIMS). Here, we extend this TWIMS-DDA approach to the data-independent acquisition (DIA) mode MS to improve the signal intensities of fragment ions by providing improved ion beam sampling efficiency, which we termed therefore signal-enhanced MS (SEMS). The theoretical expectation that SEMS improves the number of identified peptides, the number of quantifiable peptides, and the lower limit of quantitation in wideband DIA was evaluated on an electrospray ionisation-ion mobility spectrometry-quadrupole-time-of-flight-MS (ESI-IMS-Q-TOF-MS) (Synapt G2-S) in comparison to five established TWIMS-DDA and TWIMS-MS methods with respect to the number of peptide identifications, the spectral quality of supporting peptide spectra matches, and (most importantly) fragment ion signal sensitivity. A comparison of the fragment signals clearly indicated that SEMS provides 6.8- to 11.5-fold larger peak areas than established MS techniques. While this clearly shows the advantages of SEMS, the inherent limitations of the current software tools do not allow using all benefits in routine analyses. As the simultaneous fragmentation of co-eluting peptides limited peptide identification, DDA and MS data sets were integrated using Skyline.