Phosphotyrosine proteomic study of interferon alpha signaling pathway using a combination of immunoprecipitation and immobilized metal affinity chromatography

Mol Cell Proteomics. 2005 Jun;4(6):721-30. doi: 10.1074/mcp.M400077-MCP200. Epub 2005 Jan 19.


Tyrosine phosphorylation is a type of post-translational modification that plays a crucial role in signal transduction. Thus, the study of this modification at the proteomic level has great biological significance. However, because of the low abundance of tyrosine-phosphorylated proteins in total cell lysate, it is difficult to evaluate the dynamics of tyrosine phosphorylation at a global level. In this work, proteins carrying phosphotyrosine (pTyr) were first purified from whole cell lysate by immunoprecipitation using anti-pTyr monoclonal antibodies. After tryptic digestion, phosphopeptides were further enriched by IMAC and analyzed by LC-MS. Quantitative changes of tyrosine phosphorylation at the global level were evaluated using isotopic labeling (introduced at the methyl esterification step prior to IMAC). Using this double enrichment approach, we characterized interferon alpha (IFNalpha)-induced pTyr proteomic changes in Jurkat cells. We observed induced phosphorylation on several well documented as well as novel tyrosine phosphorylation sites on proteins involved in IFNalpha signal transduction, such as Tyk2, JAK1, and IFNAR subunits. A specific site on alpha-tubulin (Tyr-271) was observed to be phosphorylated upon treatment as well. Furthermore, our results suggest that LOC257106, a CDC42 GAP-like protein, is potentially involved in this pathway.

MeSH terms

  • Antineoplastic Agents / pharmacology*
  • Chromatography, Affinity*
  • Chromatography, Liquid
  • Humans
  • Immunoprecipitation
  • Interferon-alpha / pharmacology*
  • Janus Kinase 1
  • Jurkat Cells
  • Metals
  • Phosphorylation / drug effects
  • Phosphotyrosine / metabolism*
  • Protein Processing, Post-Translational
  • Protein-Tyrosine Kinases / metabolism
  • Proteomics*
  • Receptors, Interferon / classification
  • Receptors, Interferon / metabolism*
  • Signal Transduction*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • TYK2 Kinase
  • Tyrosine / metabolism


  • Antineoplastic Agents
  • Interferon-alpha
  • Metals
  • Receptors, Interferon
  • Phosphotyrosine
  • Tyrosine
  • Protein-Tyrosine Kinases
  • JAK1 protein, human
  • Janus Kinase 1
  • TYK2 Kinase
  • TYK2 protein, human