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High-Throughput Mass Spectrometry-Based Proteomics with dia-PASEF

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Proteomics in Systems Biology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2456))

Abstract

Ion mobility separation is becoming an integral part in mass spectrometry-based proteomics. Here we describe the use of a trapped ion mobility-quadrupole time-of-flight (TIMS-QTOF) mass spectrometer for high-throughput label-free quantification with data-independent acquisition. The parallel accumulation-serial fragmentation (PASEF) operation mode positions the mass-selecting quadrupole as a function of the TIMS separation, which allows highly efficient data-independent acquisition schemes (dia-PASEF), but also increases complexity in the method design. We provide a step-by-step protocol for instrument setup, method design, data acquisition and ion mobility-aware, library-based data analysis with Spectronaut. We highlight key acquisition parameters and illustrate their optimization for short gradients. Using the EvosepOne liquid chromatography system, we demonstrate expected results for the analysis of a human cancer cell line at a throughput of 60 samples per day, leading to the quantification of about 6000 protein groups with very high reproducibility. Importantly, the protocol can be readily adapted to other gradients and sample types such as modified peptides.

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Acknowledgements

We thank Prof. Dr. Matthias Mann for his generous support of this project. We are grateful to our colleagues in the Department Proteomics and Signal Transduction at the Max Planck Institute of Biochemistry as well as at Bruker Daltonics and Evosep Biosystems for fruitful discussions and valuable feedback. Research in the Department Proteomics and Signal Transduction is supported by the Max Planck Society for the Advancement of Science. F.M. acknowledges support by the Federal Ministry of Education and Research and the Thuringian Ministry for Economic Affairs, Science and a Digital Society through the Joint Federal Government-Länder Tenure-Track Programme and by the Free state of Thuringia and the European Union via the ‘Innovationszentrum für Thüringer Medizintechnik-Lösungen’ (ThIMEDOP; #2018 IZN 002).

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Authors and Affiliations

  1. Department Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany

    Patricia Skowronek & Florian Meier

  2. Functional Proteomics, Jena University Hospital, Jena, Germany

    Florian Meier

Authors
  1. Patricia Skowronek
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  2. Florian Meier
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Correspondence to Florian Meier .

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Editors and Affiliations

  1. Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada

    Jennifer Geddes-McAlister

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Skowronek, P., Meier, F. (2022). High-Throughput Mass Spectrometry-Based Proteomics with dia-PASEF. In: Geddes-McAlister, J. (eds) Proteomics in Systems Biology. Methods in Molecular Biology, vol 2456. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2124-0_2

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  • DOI: https://doi.org/10.1007/978-1-0716-2124-0_2

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2123-3

  • Online ISBN: 978-1-0716-2124-0

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