“High-Throughput and Sensitive (Phospho)Proteomics Using FAIMS and directDIA”
Prof. Dr. Jesper Velgaard Olsen (Novo Nordisk Foundation Center for Protein Research, University of Copenhagen)
Abstract: In this webinar, I will present our ongoing work focusing on High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) and Data-Independent Acquisition (DIA) on the Orbitrap Exploris 480 MS for high-throughput analysis of proteomes and phosphoproteomes. Combining DIA with FAIMS using single CVs with short online LC gradients delivered by the Evosep One LC system, we can identify ~2500 peptides per LC gradient minute when analyzing the resulting dataset with a project-specific spectral library in SN13. We compared the new directDIA 2.0 algorithm in the SpectroMine 2.0/SN14 with the SN13 version and found a significant improvement in terms of identifications for short gradients with up to 45% more peptides and 33% more protein groups identified. We also find that directDIA 2.0 in combination with FAIMS and single compensation voltages (CVs), is comparable in HeLa proteome depth achieved with DIA searches using project-specific libraries in SN13. Importantly, internal switching between three CVs ‘on-the-fly’ shows ever greater benefit with directDIA 2.0 for 21min LC gradients resulting in more than 40,000 unique peptides identified per run. Moreover, from single-shot analysis of phosphoproteomes derived from Ti-IMAC enrichment of 200 ug HeLa lysate, we can reproducibly identify and quantify ~15,000 unique phosphopeptides in 21min when looping between four different CVs. This is achieved without the need for spectral libraries when using directDIA 2.0.
Short-Biography: Jesper V. Olsen studied analytical chemistry at the University of Southern Denmark in Odense in the laboratory of Roman Zubarev. After his master studies, he worked two years at MDS Proteomics as staff scientist before joining the laboratory of Matthias Mann as a 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 back to Denmark to head a group at the newly established Novo Nordisk Foundation Center for Protein Research (CPR) at the University of Copenhagen. In 2012, he was promoted to vice director of CPR and in 2014 full professor at the Faculty of Health and Medical Sciences.
Jesper has made a number of seminal contributions to the field of proteomics and high-resolution mass spectrometry, and most importantly to the development of quantitative phosphoproteomics technology and its application to study cell signaling networks and thereby answer outstanding questions in biology. His group is continuously developing the 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 phosphoproteomes.
“Advances in Data-independent Acquisition Strategies: BoxCar-DIA and diaPASEF”
Prof. Dr. Florian Meier (Functional Proteomics - Jena University Hospital)
Abstract: Data-independent acquisition (DIA) methods are increasingly attractive for proteomics analysis of large sample cohorts, which requires a high degree of reproducibility and data completeness. However, current acquisition methods utilize only a small fraction of the available peptide ion current. On the Orbitrap platform, we have recently introduced the BoxCar acquisition method that accumulates ten-fold more low-abundance ions on the MS1 level with a windowed acquisition scheme. Combining BoxCar with DIA holds great promise for the analysis of high dynamic range samples as well as library-free DIA. Another novel acquisition method is diaPASEF, which combines trapped ion mobility spectrometry (TIMS) with fast time-of-flight mass analysis. The method exploits the correlation of ion mass and mobility to sample up to 100% of the peptide precursor ion current and thereby achieves deep proteome coverage and high quantitative accuracy in single runs, even for very low sample amounts.
Short-Biography: Prof. Florian Meier is an independent research group leader at the Jena University Hospital (Germany), where he aims to establish mass spectrometry-based omics in a clinical environment. He is also affiliated with the group of Matthias Mann at the Max-Planck Institute of Biochemistry, where he developed novel data acquisition methods for quantitative mass spectrometry-based proteomics during his post-doc and PhD. He received his PhD in Biochemistry from the Ludwig-Maximilians University Munich in 2018. His research was awarded the MPIB Junior Scientists' Publication Award 2018 and the 2019 Wolfgang Paul Study Award of the German Mass Spectrometry Society.