A Protocol on the Use of Titanium Dioxide Chromatography for Phosphoproteomics

  • Martijn W. H. PinkseEmail author
  • Simone Lemeer
  • Albert J. R. Heck
Part of the Methods in Molecular Biology book series (MIMB, volume 753)


Over the past decade phosphoproteomics has become an emerging discipline within proteomics research, focusing on detection of the reversible modification of proteins by phosphorylation of serine, threonine, and tyrosine residues. For successful analysis, phosphopeptide enrichment is often a prerequisite due to their low stoichiometry, heterogeneity, and low abundance. The enrichment of phosphopeptides is often performed manually, which is inherently labor intensive and a major hindrance in large-scale analyses. Automation of the enrichment method would vastly improve reproducibility and thereby facilitate “high-throughput” phosphoproteomics research. Here, we describe the setup of a simple, robust, and automated online TiO2-based nanoscale chromatographic approach to selectively enrich and separate phosphorylated peptides from proteolytic digests of moderate and high complexity.

Key words

Phosphopeptides online enrichment titanium dioxide 2D chromatography nanoscale liquid chromatography 



The authors would like to acknowledge GL Sciences Inc., Tokyo, for the TiO2 Titansphere material. This work was supported by the Netherlands Proteomics Centre (, a program embedded in the Netherlands Genomics Initiative.


  1. 1.
    Mukherji M (2005) Phosphoproteomics in analyzing signaling pathways. Expert Review of Proteomics. 2, 117–128.PubMedCrossRefGoogle Scholar
  2. 2.
    Rogers LD, Foster LJ (2009) Phosphoproteomics–finally fulfilling the promise? Molecular Biosystems. 5, 1122–1129.PubMedCrossRefGoogle Scholar
  3. 3.
    Gafken PR (2009) An overview of the qualitative analysis of phosphoproteins by mass spectrometry. Methods in Molecular Biology. 527, 159–172.PubMedCrossRefGoogle Scholar
  4. 4.
    Thingholm TE, Jensen ON, Larsen MR (2009) Analytical strategies for phosphoproteomics. Proteomics. 9, 1451–1468.PubMedCrossRefGoogle Scholar
  5. 5.
    Reinders J, Sickmann A (2005) State-of-the-art in phosphoproteomics. Proteomics. 5, 4052–4061.PubMedCrossRefGoogle Scholar
  6. 6.
    Lemeer S, Pinkse MW, Mohammed S, et al. (2008) Online automated in vivo zebrafish phosphoproteomics: from large-scale analysis down to a single embryo. Journal of Proteome Research. 7, 1555–1564.PubMedCrossRefGoogle Scholar
  7. 7.
    Mohammed S, Kraiczek K, Pinkse MW, Lemeer S, Benschop JJ, Heck AJ (2008) Chip-based enrichment and nanoLC-MS/MS analysis of phosphopeptides from whole lysates. Journal of Proteome Research. 7, 1565–1571.PubMedCrossRefGoogle Scholar
  8. 8.
    Pinkse MW, Mohammed S, Gouw JW, van Breukelen B, Vos HR, Heck AJ. (2008) Highly robust, automated, and sensitive online TiO2-based phosphoproteomics applied to study endogenous phosphorylation in Drosophila melanogaster. Journal of Proteome Research. 7, 687–697.PubMedCrossRefGoogle Scholar
  9. 9.
    Van Hoof D, Munoz J, Braam SR, et al. (2009) Phosphorylation dynamics during early differentiation of human embryonic stem cells. Cell Stem Cell. 5, 214–226.PubMedCrossRefGoogle Scholar
  10. 10.
    Rappsilber J, Ishihama Y, Mann M (2003) Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Analytical Chemistry. 75, 663–670.PubMedCrossRefGoogle Scholar
  11. 11.
    Schlosser A, Vanselow JT, Kramer A. (2005) Mapping of phosphorylation sites by a multi-protease approach with specific phosphopeptide enrichment and NanoLC-MS/MS analysis. Analytical Chemistry. 77, 5243–5250.PubMedCrossRefGoogle Scholar
  12. 12.
    Meiring HD, van der Heeft E, ten Hove GJ, de Jong APJM (2002) Nanoscale LC-MS(n); technical design and applications to peptide and protein analysis. Journal of Separation Science. 25, 557–568.CrossRefGoogle Scholar
  13. 13.
    Beausoleil SA, Jedrychowski M, Schwartz D, et al. (2004) Large-scale characterization of HeLa cell nuclear phosphoproteins. Proceedings of the National Academy of Sciences of the United States of America. 101, 12130–12135.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Martijn W. H. Pinkse
    • 1
    • 2
    Email author
  • Simone Lemeer
    • 3
    • 2
  • Albert J. R. Heck
    • 4
    • 5
  1. 1.Department of BiotechnologyTechnical University of DelftDelftThe Netherlands
  2. 2.Netherlands Proteomics CentreDelftThe Netherlands
  3. 3.Technische Universitaet MuenchenFreisingGermany
  4. 4.Biomolecular Mass Spectrometry and Proteomics GroupBijvoet Center for Biomolecular ResearchUtrechtThe Netherlands
  5. 5.Utrecht Institute for Pharmaceutical Sciences, Utrecht UniversityUtrechtThe Netherlands

Personalised recommendations