Rapid Shotgun Phosphoproteomics Analysis

Methods Mol Biol. 2021:2259:259-268. doi: 10.1007/978-1-0716-1178-4_17.

Abstract

In 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.

Keywords: High-intensity focused ultrasound (HIFU); Jurkat T-cells; Mass spectrometry (MS); Phosphoproteomics; Proteomics; Strong cation exchange chromatography (SCX); Titanium dioxide (TiO2).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chemical Fractionation / methods
  • Chromatography, Ion Exchange / economics
  • Chromatography, Ion Exchange / methods
  • Humans
  • Jurkat Cells
  • Phosphopeptides / analysis*
  • Phosphopeptides / isolation & purification
  • Phosphoproteins / analysis*
  • Phosphoproteins / isolation & purification
  • Phosphorylation
  • Proteome / analysis
  • Proteome / isolation & purification
  • Proteomics / economics
  • Proteomics / methods*
  • Tandem Mass Spectrometry / economics
  • Tandem Mass Spectrometry / methods
  • Time Factors
  • Titanium / chemistry
  • Workflow

Substances

  • Phosphopeptides
  • Phosphoproteins
  • Proteome
  • titanium dioxide
  • Titanium