Advertisement

Phosphopeptide Enrichment Using Offline Titanium Dioxide Columns for Phosphoproteomics

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

Abstract

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 

Notes

Acknowledgments

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.

References

  1. 1.
    Salih E (2005) Phosphoproteomics by mass spectrometry and classical protein chemistry approaches. Mass Spectrom Rev 24:828–846PubMedCrossRefGoogle Scholar
  2. 2.
    Yu LR, Issaq HJ, Veenstra TD (2007) Phosphoproteomics for the discovery of kinases as cancer biomarkers and drug targets. Proteomics Clin Appl 1:1042–1057PubMedCrossRefGoogle Scholar
  3. 3.
    Bahl JM, Jensen SS, Larsen MR, Heegaard NH (2008) Characterization of the human cerebrospinal fluid phosphoproteome by titanium dioxide affinity chromatography and mass spectrometry. Anal Chem 80:6308–6316PubMedCrossRefGoogle Scholar
  4. 4.
    Zhou W, Ross MM, Tessitore A et al (2009) An initial characterization of the serum phosphoproteome. J Proteome Res 8:5523–5531PubMedCrossRefGoogle Scholar
  5. 5.
    Carrascal M, Gay M, Ovelleiro D, Casas V, Gelpi E, Abian J (2010) Characterization of the human plasma phosphoproteome using linear ion trap mass spectrometry and multiple search engines. J Proteome Res 9:876–884PubMedCrossRefGoogle Scholar
  6. 6.
    Bodenmiller B, Mueller LN, Mueller M, Domon B, Aebersold R (2007) Reproducible isolation of distinct, overlapping segments of the phosphoproteome. Nat Methods 4:231–237PubMedCrossRefGoogle Scholar
  7. 7.
    Jensen SS, Larsen MR (2007) Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques. Rapid Commun Mass Spectrom 21:3635–3645PubMedCrossRefGoogle Scholar
  8. 8.
    Thingholm TE, Jensen ON, Robinson PJ, Larsen MR (2008) SIMAC (sequential elution from IMAC), a phosphoproteomics strategy for the rapid separation of monophosphorylated from multiply phosphorylated peptides. Mol Cell Proteomics 7:661–671PubMedGoogle Scholar
  9. 9.
    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. J Proteome Res 7:687–697PubMedCrossRefGoogle Scholar
  10. 10.
    Larsen MR, Thingholm TE, Jensen ON, Roepstorff P, Jorgensen TJ (2005) Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics 4:873–886PubMedCrossRefGoogle Scholar
  11. 11.
    Yu LR, Zhu Z, Chan KC, Issaq HJ, Dimitrov DS, Veenstra TD (2007) Improved titanium dioxide enrichment of phosphopeptides from HeLa cells and high confident phosphopeptide identification by cross-validation of MS/MS and MS/MS/MS spectra. J Proteome Res 6:4150–4162PubMedCrossRefGoogle Scholar
  12. 12.
    Pinkse MW, Uitto PM, Hilhorst MJ, Ooms B, Heck AJ (2004) Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. Anal Chem 76:3935–3943PubMedCrossRefGoogle Scholar
  13. 13.
    Kuroda I, Shintani Y, Motokawa M, Abe S, Furuno M (2004) Phosphopeptide-selective column-switching RP-HPLC with a titania precolumn. Anal Sci 20:1313–1319PubMedCrossRefGoogle Scholar
  14. 14.
    Sano A, Nakamura H (2004) Titania as a chemo-affinity support for the column-switching HPLC analysis of phosphopeptides: application to the characterization of phosphorylation sites in proteins by combination with protease digestion and electrospray ionization mass spectrometry. Anal Sci 20:861–864PubMedCrossRefGoogle Scholar
  15. 15.
    Sugiyama N, Masuda T, Shinoda K, Nakamura A, Tomita M, Ishihama Y (2007) Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chromatography for nano-LC-MS/MS in proteomics applications. Mol Cell Proteomics 6:1103–1109PubMedCrossRefGoogle Scholar
  16. 16.
    Kruger M, Kratchmarova I, Blagoev B, Tseng YH, Kahn CR, Mann M (2008) Dissection of the insulin signaling pathway via quantitative phosphoproteomics. Proc Natl Acad Sci USA 105:2451–2456PubMedCrossRefGoogle Scholar

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

Personalised recommendations