The Use of Titanium Dioxide Micro-Columns to Selectively Isolate Phosphopeptides from Proteolytic Digests
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 wordsProtein phosphorylation Phosphopeptide enrichment Titanium dioxide chromatogra-phy (TiO2) Glycolic acid Trifluoroacetic acid (TFA) Mass spectrometry
- 9.Pinkse, M. W., Uitto, P. M., Hilhorst, M. J., Ooms, B., and Heck, A. J. (2004) Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. Analytical Chemistry 76, 3 935–43.Google Scholar
- 15.Gobom, J., Nordhoff, E., Mirgorodskaya, E., Ekman, R., and Roepstorff, P. (1999) Sample purification and preparation technique based on nano-scale reversed-phase columns for the sensitive analysis of complex peptide mixtures by matrix-assisted laser desorption/ionization mass spectrometry. Journal of Mass Spectrom-etry 34, 105–16.CrossRefGoogle Scholar
- 16.Liu, S., Zhang, C., Campbell, J. L., Zhang, H., Yeung, K. K., Han, V. K., and Lajoie, G. A. (2005) Formation of phosphopeptide-metal ion complexes in liquid chromatography/elec-trospray mass spectrometry and their influence on phosphopeptide detection. Rapid Communication in Mass Spectrometry 19, 2747–56.CrossRefGoogle Scholar
- 17.Thingholm, T. E., Jensen, O. N., P.J, R., and Larsen, M. R. (2007) SIMAC - A phos-phoproteomic strategy for the rapid separation of mono-phosphorylated from multiply phosphorylated peptides. Published online by MCP on November 27th 2007 (M700362-MCP200).Google Scholar