Phosphopeptide Enrichment Using Offline Titanium Dioxide Columns for Phosphoproteomics

  • Li-Rong Yu
  • Timothy Veenstra
Part of the Methods in Molecular Biology book series (MIMB, volume 1002)


Identification of phosphoproteins or phosphopeptides as cancer biomarkers is an emerging field in phosphoproteomics. Owing to the low stoichiometric nature of protein phosphorylation, phosphoproteins or phosphopeptides must be enriched prior to downstream mass spectrometry analysis. Titanium dioxide (TiO2) has been prevalently used to enrich phosphopeptides from complex proteome samples due to its high affinity for phosphopeptides, and the method is straightforward. In this protocol, an offline phosphopeptide enrichment procedure using TiO2 columns is described. Peptides from a proteome lysate are loaded onto a TiO2 column in an acidic environment, followed by column washing with aqueous, organic, and ammonium glutamate (NH4Glu) buffers at acidic conditions. Phosphopeptides are eluted using an ammonia solution at high pH. Use of NH4Glu significantly reduces nonspecific bindings while a high recovery rate (84 %) of phosphopeptides is retained. The method is optimized for large-scale phosphoproteomic analysis and phosphoprotein biomarker discovery starting from sub-milligram or milligrams of proteome samples.

Key words

Phosphoproteomics Phosphopeptide enrichment Titanium dioxide Biomarker Proteomics Ammonium glutamate Mass spectrometry MS/MS Liquid chromatography Cancer 



This study was supported in part with funds from the National Center for Toxicological Research, US Food and Drug Administration (NCTR/FDA) and in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-CO-12400. The content of this publication does not necessarily reflect the views or policies of the US Food and Drug Administration, the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the United States Government.


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Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Li-Rong Yu
    • 1
  • Timothy Veenstra
    • 2
  1. 1.National Center for Toxicological Research, FDAJeffersonUSA
  2. 2.Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc., Frederick National Laboratory for Cancer ResearchFrederickUSA

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