The Use of Titanium Dioxide Micro-Columns to Selectively Isolate Phosphopeptides from Proteolytic Digests

  • Tine E. Thingholm
  • Martin R. Larsen
Part of the Methods in Molecular Biology™ book series (MIMB, volume 527)


Titanium dioxide has very high affinity for phosphopeptides and it has become an efficient alternative to already existing methods for phosphopeptide enrichment from complex samples. Peptide loading in a highly acidic environment in the presence of 2,5-dihydroxybenzoic acid (DHB), phthalic acid, or glycolic acid has been shown to improve selectivity significantly by reducing unspecific binding from nonphosphorylated peptides. The enriched phosphopeptides bound to the titanium dioxide are subsequently eluted from the micro-column using an alkaline buffer. Titanium dioxide chromatography is extremely tolerant towards most buffers used in biological experiments. It is highly robust and as such it has become one of the methods of choice in large-scale phospho-proteomics. Here we describe the protocol for phosphopeptide enrichment using titanium dioxide chromatography followed by desalting and concentration of the sample by reversed phase chromatography prior to MS analysis.

Key words

Protein phosphorylation Phosphopeptide enrichment Titanium dioxide chromatogra-phy (TiO2Glycolic acid Trifluoroacetic acid (TFA) Mass spectrometry 


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Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Tine E. Thingholm
    • 1
  • Martin R. Larsen
    • 1
  1. 1.Department of Biochemistry and Molecular BiologyUniversity of Southern DenmarkOdenseDenmark

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