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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.

Short Bio Jesper V. Olsen

Jesper Olsen studied analytical chemistry at the University of Southern Denmark in the laboratory of Roman Zubarev. After this, he worked for two years at MDS Proteomics as a staff scientist before joining the laboratory of Matthias Mann as PhD student. He spent 4 years as a post-doctoral fellow at the Max Planck Institute for Biochemistry in Munich. In 2009, Jesper was recruited to head a research group at the newly established Novo Nordisk Foundation Center for Protein Research (CPR) at the University of Copenhagen (UCPH). In 2012, he was promoted to vice director of CPR and in 2014 full professor at UCPH.
Jesper has made seminal contributions to the field of proteomics and high-resolution mass spectrometry and pioneering quantitative phosphoproteomics technology and its application to study cell-signaling networks to answer outstanding questions in biology. His group is developing proteomics technology and their work on offline peptide chromatographic fractionation in combination with fast online LC-MS/MS has enabled a comprehensive analysis of human proteomes. Most recently, his group has worked on the combination of FAIMS and DIA on the Orbitrap Exploris 480 MS for high-throughput analysis of human proteomes and spatial-proteomics revealing phospho-signaling dynamics at subcellular resolution.