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A Protocol on the Use of Titanium Dioxide Chromatography for Phosphoproteomics

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

Abstract

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 

Notes

Acknowledgments

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

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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

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