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 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. This resulted in a substantial increase in peptide identification. In comparison to conventional TIMS methods, which typically scan a static ion mobility range (either from 0.6 to 1.6 or from 0.85 to 1.3), the Seg method significantly enhances the identification of peptides, proteins and average sequence coverage per protein. These findings highlight the potential of the Seg method in expanding the capabilities of TIMS-TOF mass spectrometry, especially for peptide-focused analysis such as post-translational modifications and peptidomics.