One-Hour Phosphoproteome Analysis with the Orbitrap Astral Mass Spectrometer.

As a key mechanism for cellular signal transduction, protein phosphorylation plays critical roles in myriad cellular processes. This modification, however, is highly dynamic and occurs at sub-stoichiometric levels. Mass spectrometry is an essential tool for studying this modification on a global scale; however, the technology's impact suffers from four main limitations: need for site localization, dynamic range, reproducibility, and throughput. Here we describe the use of a novel mass spectrometer (Orbitrap Astral) coupled with data-independent acquisition (DIA) to achieve detection of close to 40,000 unique phosphorylation sites within one hour of analysis. We applied this approach to generate a phosphoproteome atlas of the mouse. Altogether, we detected 81,120 unique phosphorylation sites within 12 hours of measurement. With this unique dataset, we examine the sequence and structural context of protein phosphorylation. Finally, we highlight the discovery potential of this resource with multiple examples of novel phosphorylation events relevant to mitochondrial and brain biology.