High accuracy mass spectrometry in large-scale analysis of protein phosphorylation

Methods Mol Biol. 2009;492:131-42. doi: 10.1007/978-1-59745-493-3_7.


With the appearance of a new generation of high-performance hybrid mass spectrometers, high accuracy (sub-parts-per-million) mass spectrometry is becoming increasingly available to a wider scientific community. Here we discuss the advantages of such mass spectrometric instrumentation in the global analysis of protein phosphorylation. We describe a detailed workflow for fractionation and enrichment of phosphopeptides from digests of whole cell/tissue lysates by strong cation exchange and TiO(2) chromatography and their subsequent measurement on an LTQ-Orbitrap mass spectrometer under several acquisition regimes.

MeSH terms

  • Amino Acid Sequence
  • Chromatography, Ion Exchange
  • Chromatography, Liquid
  • HeLa Cells
  • Humans
  • Mass Spectrometry / methods*
  • Molecular Sequence Data
  • Phosphopeptides / analysis
  • Phosphopeptides / chemistry
  • Phosphopeptides / metabolism
  • Phosphoproteins / analysis*
  • Phosphoproteins / chemistry
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Proteomics
  • Sensitivity and Specificity
  • Titanium / chemistry
  • Trypsin / metabolism


  • Phosphopeptides
  • Phosphoproteins
  • titanium dioxide
  • Titanium
  • Trypsin