Fast and deep phosphoproteome analysis with the Orbitrap Astral mass spectrometer
Owing to its roles in cellular signal transduction, protein phosphorylation plays critical roles in myriad cell processes. That said, detecting and quantifying protein phosphorylation has remained a challenge. We describe the use of a novel mass spectrometer (Orbitrap Astral) coupled with data-independent acquisition (DIA) to achieve rapid and deep analysis of human and mouse phosphoproteomes. With this method, we map approximately 30,000 unique human phosphorylation sites within a half-hour of data collection. The technology is benchmarked to other state-of-the-art MS platforms using both synthetic peptide standards and with EGF-stimulated HeLa cells. We apply this approach to generate a phosphoproteome multi-tissue atlas of the mouse. Altogether, we detect 81,120 unique phosphorylation sites within 12 hours of measurement. With this unique dataset, we examine the sequence, structural, and kinase specificity context of protein phosphorylation. Finally, we highlight the discovery potential of this resource with multiple examples of phosphorylation events relevant to mitochondrial and brain biology.
Authors
- Michael S. Westphall (ORCID: https://orcid.org/0000-0002-7694-3801)
- Pavel Sinitcyn (ORCID: https://orcid.org/0000-0002-2653-1702)
- Margaret Lea Robinson (ORCID: https://orcid.org/0000-0002-4019-3770)
- Christian Hock
- Jing Fan (ORCID: https://orcid.org/0000-0002-5326-5358)
- Lia R. Serrano (ORCID: https://orcid.org/0000-0001-5662-5884)
- Julie Rojas (ORCID: https://orcid.org/0000-0001-8714-1532)
- Andrea Galmozzi (ORCID: https://orcid.org/0000-0002-9411-7327)
- David J. Pagliarini (ORCID: https://orcid.org/0000-0002-0001-0087)
- Audrey P. Gasch (ORCID: https://orcid.org/0000-0002-8182-257X)
- Tabiwang N. Arrey (ORCID: https://orcid.org/0009-0001-2888-2055)
- Anna Pashkova
- Evgenia Shishkova (ORCID: https://orcid.org/0000-0001-6097-6097)
- Vlad Zabrouskov (ORCID: https://orcid.org/0000-0003-3567-9407)
- Eugen Damoc (ORCID: https://orcid.org/0000-0002-4422-2866)
- Hamish Stewart (ORCID: https://orcid.org/0009-0000-1510-4353)
- Trenton M. Peters-Clarke (ORCID: https://orcid.org/0000-0002-9153-2525)
- Nicholas L. Arp (ORCID: https://orcid.org/0000-0001-8709-0667)
- Noah M. Lancaster (ORCID: https://orcid.org/0009-0002-3609-7951)
- J N Hansen (ORCID: https://orcid.org/0009-0005-4762-7956)
- Patrick Forny (ORCID: https://orcid.org/0000-0003-1877-2976)
- Joshua J. Coon (ORCID: https://orcid.org/0000-0002-0004-8253)
- Caroline Fecher (ORCID: https://orcid.org/0000-0002-7179-7323)
- Andrew J. H. Smith (ORCID: https://orcid.org/0000-0002-4327-5886)
Institutions
- University of Wisconsin System (US)
- University of Wisconsin–Madison (US)
- Washington University in St. Louis (US)
- Great Lakes Bioenergy Research Center (US)
- Morgridge Institute for Research (US)
- Quantitative BioSciences (US)
- University of Wisconsin Carbone Cancer Center (US)
- Energy Center of Wisconsin (US)
- Thermo Fisher Scientific (United States) (US)
- Thermo Fisher Scientific (Germany) (DE)
Publication Details
- Journal
- Nature Communications
- Published
- 2024-08-15
- DOI
- https://doi.org/10.1038/s41467-024-51274-0
- Citations
- 64
- Primary Topic
- Advanced Proteomics Techniques and Applications
- Type
- article
- Field-Weighted Citation Impact
- 15.41
Funders
- National Science Foundation
- U.S. Department of Energy
- Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung
- National Institutes of Health
- ACS Division of Analytical Chemistry
- Great Lakes Bioenergy Research Center
- National Institute of General Medical Sciences