SpectroDive™ 11: Straightforward Validation of Your Protein Targets
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Precise Absolute Quantification of Biomarkers Using SpectroDive
Streamlined Integration of Targeted Proteomics for the Validation of Disease Models and the Detection of Proteins Secreted from Rare Primary Cells
Véronique Laforte (Biognosys)
During her talk, Veronique Laforte will show the different improvements that were added to SpectroDive 11. The newly added support for acquisitions using ion mobility (prm-PASEF from Bruker Daltonics, FAIMS-PRM from Thermo Fisher Scientific), as well as the addition of absolute quantification using calibration curves, leads to increased sensitivity and precision of sample absolute quantities. She will focus particularly on how to use SpectroDive to measure assay performance including limit of detection and lower limit of quantification, which measure assay sensitivity and precision.
Therese Dau (Leibniz Institute on Aging)
The introduction of data-independent acquisition (DIA) has improved protein quantitation considerably. Yet, targeted approaches remain important due to their increased sensitivity and more accurate quantitation for challenging samples or questions. Here, we introduce two examples of using parallel reaction monitoring (PRM) and Biognosys’ SpectroDive™ software to solved questions that could not have been answered with shotgun proteomics alone.
In the first project, we analyzed mutants from microcephalin 1 (MCPH1) that have been reported to lead to human primary microcephaly. A deletion in the N-terminal part of MCPH1 reduces brain size and increases ovary tumours in mice. Given its low abundance in the mouse brain, MCPH1 is challenging to detect by shotgun proteomics. Instead, we confirmed the deletion of the N-terminal using PRM.
The second project looked at the impact of the niche on muscle stem cells on aging. We found that the secreted protein SMOC2 accumulated in skeletal muscles of aged mice, contributing to impaired muscle regeneration. Fibro-adipogenic precursor cells as a potential source of SMOC2 was confirmed using a combination of targeted analysis and DIA.
These examples demonstrate the streamlined integration of targeted proteomics analysis for the validation of animal models of disease and protein-level confirmation of single-cell transcriptomic data from primary cells.
Short Bio Véronique Laforte:
With a background in biochemistry and electrical engineering, Veronique obtained a PhD at McGill University in Montreal, Canada, in biomedical engineering and neuroscience, where she focused on microtechnology, ELISA-based multiplexed assay development and validation of potential biomarkers for clinical studies. Veronique then joined the Biognosys team as lead software developer for SpectroDive where she applies her knowledge of assay development to improve the targeted proteomics analyses.
Short Bio Therese Dau:
Therese Dau is currently the head of the proteomics core facility from the Leibniz Institute on Aging, Fritz-Lipmann Institute in Jena, Germany. The facility offers its expertise on mass spectrometry to researchers with age-related projects. While she has worked in the field of protein biochemistry since her PhD (received in June 2014), she started working with mass spectrometry during her PostDoc with Juri Rappsilber in July 2016.
Comparison of DIA and SureQuant/PQ500 Acquisition Methods for Plasma and Serum Samples
Teresa Barth (Ludwig Maximilian University of Munich)
Measuring the protein content of blood samples promises insights into the molecular basics of clinical disease patterns. However, due to the high dynamic range of protein abundance in blood plasma, it is difficult to achieve protein analysis to a great depth. Here, we compare different MS acquisition methods on a Thermo Exploris 480 mass spectrometer to increase protein identifications in plasma and serum samples.
We compared data-independent acquisition (DIA) and SureQuant targeted acquisition using Biognosys’ PQ500™ Reference Peptides kit as internal standard peptides and Biognosys’ SpectroDive™ and Spectronaut® software for data analysis. We found a significant increase in protein identifications when using the SureQuant/PQ500 workflow, despite this acquisition method being limited to the number of peptides in the PQ500 Reference Peptides kit.
Short Bio Teresa Barth:
Teresa Barth studied Biology in Würzburg from 2003-2008 and did he master’s thesis and PhD thesis (chromatin research with mass spectrometry) in the lab of Prof. Axel Imhof in Munich finishing in 2014. From 2016-2018, she worked with Hexal AG/Sandoz on the subject of physicochemical characterization of biosimilars, followed by two years at Helmholtz Zentrum München working in the Core Facility PROT (Research Unit Protein Science). Since 2020, Teresa back with Axel Imhof in the Protein analysis unit ZfP (Zentrallabor für Proteinanalytik) working on Clinical Mass Spectrometry.