Quantitative phosphoproteomic analysis of the tumor necrosis factor pathway

J Proteome Res. 2006 Jan;5(1):127-34. doi: 10.1021/pr050270m.


Protein phosphorylation has become a focus of many proteomic studies due to the central role that it plays in biology. We combine peptide-based gel-free isoelectric focusing and immobilized metal affinity chromatography to enhance the detection of phosphorylation events within complex protein samples using LC-MS. This method is then used to carry out a quantitative phosphoproteomic analysis of the tumor necrosis factor (TNF) pathway using HeLa cells metabolically labeled with 15N-containing amino acids, where 145 phosphorylation sites were found to be up-regulated upon the activation of the TNF pathway.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Chromatography, Affinity
  • Chromatography, Liquid
  • HeLa Cells
  • Humans
  • Isoelectric Focusing
  • Isotope Labeling
  • Mass Spectrometry
  • Nitrogen Isotopes / analysis
  • Nitrogen Isotopes / metabolism
  • Peptide Fragments / analysis
  • Phosphopeptides / analysis
  • Phosphoproteins / analysis*
  • Phosphorylation
  • Proteome / analysis*
  • Proteomics / methods*
  • Tumor Necrosis Factor-alpha / metabolism*


  • Nitrogen Isotopes
  • Peptide Fragments
  • Phosphopeptides
  • Phosphoproteins
  • Proteome
  • Tumor Necrosis Factor-alpha