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.