结合靶向和发现蛋白质组学的新型混合DIA策略

A Novel Hybrid-DIA Acquisition Strategy Combines Targeted and Discovery Proteomics

Jesper V. Olsen (University of Copenhagen)

In this breakfast seminar, Jesper will present a novel hybrid-DIA acquisition strategy on the Orbitrap Exploris 480 for simultaneous targeted and discovery phosphoproteomics of cell signaling pathways. The hybrid-DIA strategy combines the best of targeted and discovery proteomics through the utilization of the Application Programming Interface (API) to dynamically intercalate data-independent acquisition (DIA) scans with accurate triggering of predefined (phospho)peptide targets. The hybrid-DIA workflow enables standard DIA acquisition while simultaneously analyzing MS1 scans for spiked-in heavy-labeled (phospho)peptides, which then trigger multiplexed (MSX) targeted quantitation scans of heavy-labeled/endogenous peptide pairs. This strategy ensures precise quantitative information of selected phosphopeptide targets in parallel with profiling the global phosphoproteome in just one MS run per sample. We benchmarked hybrid-DIA against SureQuant and conventional DIA by assessing their potential for extracting quantitative information from the full phosphoproteome in parallel with targeting the >130 phosphopeptides on the inclusion list by analyzing HeLa cell phosphoproteomes stimulated with EGF in presence of different kinase inhibitors. The results demonstrate the power of combining targeted and discovery proteomics to maximize the information derived from phosphoproteomics experiments, which can be highly relevant in experimental contexts where samples with a limited amount do not allow for multiple MS runs.