Proteogenomics and Differential Ion Mobility Enable the Exploration of the Mutational Landscape in Colon Cancer Cells

The sensitivity and depth of proteomic analyses are limited
by isobaric ions and interferences that preclude the identification
of low abundance peptides. Extensive sample fractionation is often
required to extend proteome coverage when sample amount is not a limitation.
Ion mobility devices provide a viable alternate approach to resolve
confounding ions and improve peak capacity and mass spectrometry (MS)
sensitivity. Here, we report the integration of differential ion mobility
with segmented ion fractionation (SIFT) to enhance the comprehensiveness
of proteomic analyses. The combination of differential ion mobility
and SIFT, where narrow windows of ∼m/z 100 are acquired in turn, is found particularly advantageous
in the analysis of protein digests and typically provided more than
60% gain in identification compared to conventional single-shot LC–MS/MS.
The application of this approach is further demonstrated for the analysis
of tryptic digests from different colorectal cancer cell lines where
the enhanced sensitivity enabled the identification of single amino
acid variants that were correlated with the corresponding transcriptomic
data sets.