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

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 E to improve the signal intensities of fragment ions by providing improved ion beam sampling efficiency, which we termed therefore signal-enhanced MS E (SEMS E ). The theoretical expectation that SEMS E 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 i ) in comparison to five established TWIMS-DDA and TWIMS-MS E 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 E provides 6.8- to 11.5-fold larger peak areas than established MS E techniques. While this clearly shows the advantages of SEMS E , 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 E data sets were integrated using Skyline.