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Highly selective enrichment of phosphorylated peptides using titanium dioxide

Nature Protocols volume 1, pages 1929–1935 (2006)Cite this article

Abstract

The characterization of phosphorylated proteins is a challenging analytical task since many of the proteins targeted for phosphorylation are low in abundance and phosphorylation is typically substoichiometric. Highly efficient enrichment procedures are therefore required. Here we describe a protocol for selective phosphopeptide enrichment using titanium dioxide (TiO2) chromatography. The selectivity toward phosphopeptides is obtained by loading the sample in a 2,5-dihydroxybenzoic acid (DHB) or phthalic acid solution containing acetonitrile and trifluoroacetic acid (TFA) onto a TiO2 micro-column. Although phosphopeptide enrichment can be achieved by using TFA and acetonitrile alone, the selectivity is dramatically enhanced by adding DHB or phthalic acid since these compounds, in conjunction with the low pH caused by TFA, prevent binding of nonphosphorylated peptides to TiO2. Using an alkaline solution (pH ≥ 10.5) both monophosphorylated and multiphosphorylated peptides are eluted from the TiO2 beads. This highly efficient method for purification of phosphopeptides is well suited for the characterization of phosphoproteins from both in vitro and in vivo studies in combination with mass spectrometry (MS). It is a very easy and fast method. The entire protocol requires less than 15 min per sample if the buffers have been prepared in advance (not including lyophilization).

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Figure 1: Packing TiO2 micro-columns.
Figure 2
Figure 3: MALDI Q-TOF MS spectra of 1 pmol peptide mixture before and after TiO2 purification.
Figure 4: Zoom of m/z 1,600–1,800 from Figure 3.
Figure 5: MALDI Q-TOF mass spectrum of the TiO2 enriched phosphopeptides from 50 fmol peptide mixture.

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Acknowledgements

This work was supported by the Danish Natural Science Research Council (grant 21-03-0167 to M.R.L.) and the Danish Strategic Research Council (to O.N.J.).

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230, Odense, Denmark

    Tine E Thingholm, Thomas J D Jørgensen, Ole N Jensen & Martin R Larsen

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Correspondence to Martin R Larsen.

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Thingholm, T., Jørgensen, T., Jensen, O. et al. Highly selective enrichment of phosphorylated peptides using titanium dioxide. Nat Protoc 1, 1929–1935 (2006). https://doi.org/10.1038/nprot.2006.185

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