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.

蛋白质组学分析的灵敏度与深度受限于同重离子（isobaric ions）与各类干扰，此类干扰会妨碍低丰度肽段的鉴定。当样本总量不受限制时，通常需借助大规模样本分级分离以拓展蛋白质组覆盖度。离子迁移谱设备可作为一种可行的替代方案，用于分离干扰离子并提升峰容量与质谱（mass spectrometry, MS）灵敏度。本研究将差分离子迁移技术与分段离子分级分离（segmented ion fractionation, SIFT）相结合，以提升蛋白质组学分析的全面性。研究发现，差分离子迁移与SIFT的联用方案可依次采集约100质荷比（m/z）的窄窗口，在蛋白质酶解物分析中优势显著；相较于传统单针液相色谱-串联质谱（LC–MS/MS），其肽段鉴定数量可提升60%以上。本研究进一步将该方案应用于不同结直肠癌细胞系的胰蛋白酶酶解物分析，凭借提升的灵敏度，成功鉴定出与对应转录组数据集相关的单氨基酸变异体。