Rapid Shotgun Phosphoproteomics Analysis

10 pages, 4 figuresIn this chapter, we describe a rapid workflow for the shotgun global phosphoproteomics analysis. The strategy is based on the use of accelerated in-solution trypsin digestion under an ultrasonic field by high-intensity focused ultrasound (HIFU) coupled to titanium dioxide (TiO2) selective phosphopeptide enrichment, fractionation by strong cation exchange chromatography (SCX), and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a high-resolution mass spectrometer (LTQ-Orbitrap XL). The strategy was optimized for the global phosphoproteome analysis of Jurkat T-cells. Using this accelerated workflow, HIFU-TiO2-SCX-LC-MS/MS, 15,367 phosphorylation sites from 13,029 different phosphopeptides belonging to 3,163 different phosphoproteins can be efficiently identified in less than 15 hThis work was supported by the EU Marie Curie actions (FP7-PEOPLE-2012-IEF, ref. 332274), by the Ramon Areces Foundation (XVII National grant), by the GAIN-Xunta de Galicia Project (IN607D 2017/01) and by the Spanish AEI/EU-FEDER (PID2019-103845RB-C21) projectPeer reviewe