UNIT 18.19 Determining In Vivo Phosphorylation Sites Using Mass Spectrometry

  1. Susanne B. Breitkopf1,
  2. John M. Asara1,2

Published Online: 1 APR 2012

DOI: 10.1002/0471142727.mb1819s98

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Breitkopf, S. B. and Asara, J. M. 2012. Determining In Vivo Phosphorylation Sites Using Mass Spectrometry. Current Protocols in Molecular Biology. 98:18.19:18.19.1–18.19.27.

Author Information

  1. 1

    Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, Massachusetts

  2. 2

    Department of Medicine, Harvard Medical School, Boston, Massachusetts

Publication History

  1. Published Online: 1 APR 2012
  2. Published Print: APR 2012


Phosphorylation is the most studied protein post-translational modification (PTM) in biological systems, since it controls cell growth, proliferation, survival, and other processes. High-resolution/high mass accuracy mass spectrometers are used to identify protein phosphorylation sites due to their speed, sensitivity, selectivity, and throughput. The protocols described here focus on two common strategies: (1) identifying phosphorylation sites from individual proteins and small protein complexes, and (2) identifying global phosphorylation sites from whole-cell and tissue extracts. For the first, endogenous or epitope-tagged proteins are typically immunopurified from cell lysates, purified via gel electrophoresis or precipitation, and enzymatically digested into peptides. Samples can be optionally enriched for phosphopeptides using immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO2) and then analyzed by microcapillary liquid chromatography/tandem mass spectrometry (LC-MS/MS). Global phosphorylation site analyses that capture pSer/pThr/pTyr sites from biological sources sites are more resource and time consuming and involve digesting the whole-cell lysate, followed by peptide fractionation by strong cation-exchange chromatography, phosphopeptide enrichment by IMAC or TiO2, and LC-MS/MS. Alternatively, the protein lysate can be fractionated by SDS-PAGE, followed by digestion, phosphopeptide enrichment, and LC-MS/MS. One can also immunoprecipitate only phosphotyrosine peptides using a pTyr antibody followed by LC-MS/MS. Curr. Protoc. Mol. Biol. 98:18.19.1-18.19.27. © 2012 by John Wiley & Sons, Inc.


  • phosphorylation;
  • mass spectrometry;
  • LC-MS/MS;
  • SCX;
  • IMAC;
  • immunoprecipitation;
  • nano-LC;
  • phosphoproteomics