Journal Article

Fast and deep phosphoproteome analysis with the Orbitrap Astral mass spectrometer

Peters-Clarke, Trenton M., Arp, Nicholas, Gasch, Audrey P., Fecher, Caroline, Damoc, Eugen, Lancaster, Noah M., Serrano, Lia R., Stewart, Hamish, Pagliarini, David J., Shishkova, Evgenia, Hansen, Juli, Sinitcyn, Pavel, Galmozzi, Andrea, Pashkova, Anna, Fan, Jing, Rojas, Julie, Westphall, Michael S., Smith, Andrew J., Robinson, Margaret Lea, Arrey, Tabiwang N., Coon, Joshua J., Zabrouskov, Vlad, Hock, Christian, Forny, Patrick

2024

Overview

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. Protein phosphorylation plays critical roles in myriad cell processes. In this work, the authors apply new mass spectrometer technology to detect and quantify tens of thousands of protein phosphorylation sites within one hour or less of analysis. This technology has potential to greatly accelerate biological discovery.

• Publisher: Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
• Subject: 13/106 / 631/1647/2067 / 631/1647/296 / 631/45/475 / 639/638/11/296 / 64/60 / 82/16 / 82/58 / 82/81 / Amino acid sequence / Animals / BASIC BIOLOGICAL SCIENCES / Biological activity / Data collection / HeLa Cells / Humanities and Social Sciences / Humans / INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY / Kinases / Mass spectrometers / Mass spectrometry / Mass Spectrometry - methods / Mice / multidisciplinary / Phosphoproteins - analysis / Phosphoproteins - metabolism / Phosphoproteomes / Phosphorylation / Proteins / Proteome - metabolism / Proteomic analysis / Proteomics / Proteomics - methods / Science / Science (multidisciplinary) / Signal transduction / Technology assessment / Uniqueness