Optimized Time-Segmented Acquisition Expands Peptide and Protein Identification in TIMS-TOF Pro Mass Spectrometry

We introduce here a novel approach, termed time-segmented acquisition (Seg), to enhance the identification of peptides and proteins in trapped ion mobility spectrometry (TIMS)-time-of-flight (TOF) mass spectrometry. Our method exploits the positive correlation between ion mobility values and reversed-phase liquid chromatography (LC) retention time to improve ion separation and resolution. By dividing the LC retention time into multiple segments and applying a segment-specific narrower ion mobility range within the TIMS tunnel, we achieved better separation and higher resolution of ion mobility. In comparison to conventional TIMS methods, which typically scan a static ion mobility range (either from 0.6 to 1.6 [Wide] or from 0.85 to 1.3 [Narrow], V × s/cm²), the Seg method demonstrates marked improvements in identification rates. Compared to Wide scanning, the Seg method increases peptide identifications by 17–27% and protein identifications by 6–16% depending on the gradient length and the sample load. The enhancement in peptide identification is even more pronounced when compared to Narrow scanning, with an increase of 34–86%. These findings highlight the potential of the Seg dda-PASEF method in expanding the capabilities of TIMS-TOF mass spectrometry, especially for peptide-focused analyses, such as post-translational modifications and peptidomics.