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

We introduce a novel approach, termed time-segment 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 achieve better separation and higher resolution of ion mobility, resulting in a subtantial increase in peptide identification. This submission contains two parts of data: 1), timsTOF runs of standard Hela Digest at varying amount (5ng-200ng) and LC gradients (30min, 60 min and 90 min) using three different TIMS scan methods. a) Narrow (with TIMS ion mobility scan range 85-130); b) Seg (with a dynamics ion mobility scan ranges), and c) Wide (with 'std' method from 0.6-1.6 ion mobility). 2). Phoshoproteomics analysis of HeLa cell culture using both methods (Seg and Std ion mobility for TIMS scan). The phosphopeptides were enriched using TiO2 and fractioned by high pH reversed-phased LC (15 fractions from B1-B15, for Seg and Std TIMS method analysis).