• Methods Mol. Biol. · Jan 2021

    Rapid Shotgun Phosphoproteomics Analysis.

    • Mónica Carrera, Benito Cañas, and Daniel Lopez-Ferrer.
    • Department of Food Technology, Institute of Marine Research (IIM), Spanish National Research Council (CSIC), Vigo, Spain. mcarrera@iim.csic.es.
    • Methods Mol. Biol. 2021 Jan 1; 2259: 259-268.

    AbstractIn 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 h.

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