Abstract
Alterations in protein phosphorylation, a posttranslational modification (PTM) that regulates many processes in living cells, is a fundamental mechanism of many diseases, including cancer. Phosphoproteomics, with the combined use of affinity chromatography and electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, is shedding light into phosphorylation signaling pathways at the proteome level and helps to solve difficulties related to sample complexity and phosphopeptide enrichment. One of the most frequent and efficient methods used to enrich samples for the phosphorylated components is titanium dioxide chromatography. Titanium dioxide has a high affinity for phosphopeptides and can also be selective in specific experimental conditions. Here, we describe a protocol for the use of a MALDI plate covered with titanium dioxide nanostructured film, a device developed for a rapid and efficient study of phosphorylated peptides.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
de Graauw, M., Hensbergen, P., van de Water, B. (2006) Phospho-proteomic analysis of cellular signaling. Electrophoresis 27, 2676–2686.
Macek, B., Mann, M., Olsen, J.V. (2009) Global and site-specific quantitative phosphoproteomics: principles and applications. Annu Rev Pharmacol Toxicol 49, 199–221.
Obenauer, J.C., Cantley, L.C., Yaffe, M.B. (2003) Scansite 2.0: Proteome-wide prediction of cell signaling interactions using short sequence motifs. Nucleic Acids Res 31, 3635–3641.
Pinkse, M.W., Uitto, P.M., Hilhorst, M.J., et al. (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, 3935–3943.
Beausoleil, S.A., Jedrychowski, M., Schwartz, D., et al. (2004) Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc Natl Acad Sci U S A 101, 12130–12135.
Thingholm, T.E., Jorgensen, T.J., Jensen, O.N., et al. (2006) Highly selective enrichment of phosphorylated peptides using titanium dioxide. Nat Protoc 1, 1929–1935.
Thingholm, T.E., Jensen, O.N. (2009) Enrichment and characterization of phosphopeptides by immobilized metal affinity chromatography (IMAC) and mass spectrometry. Methods Mol Biol 527, 47–56, xi.
Larsen, M.R., Thingholm, T.E., Jensen, O.N., et al. (2005) Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics 4, 873–886.
Jensen, S.S., Larsen, M.R. (2007) Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques. Rapid Commun Mass Spectrom 21, 3635–3645.
Chen, C.T., Chen, Y.C. (2005) Fe3O4/TiO2 core/shell nanoparticles as affinity probes for the analysis of phosphopeptides using TiO2 surface-assisted laser desorption/ionization mass spectrometry. Anal Chem 77, 5912–5919.
Liang, S.S., Makamba, H., Huang, S.Y., et al. (2006) Nano-titanium dioxide composites for the enrichment of phosphopeptides. J Chromatogr A 1116, 38–45.
Di Fonzo, F.B., Russo, A., Baserga, V., et al. (2006) Synthesis and characterization of nanostructured tungsten and tungsten oxide films. Catalysis Today 116, 69–73.
Bailini, A., Di Fonzo, F., Fusi, M., et al. (2007) Pulsed laser deposition of tungsten and tungsten oxide thin films with tailored structure at the nano- and mesoscale. Applied Surface Science 253, 8130–8135.
Torta, F., Fusi, M., Casari, C.S., et al. (2009) Titanium dioxide coated MALDI plate for on target analysis of phosphopeptides. J Proteome Res 8, 1932–1942.
Fusi, M., Russo, V., Casari, C.S., et al. (2009) Titanium oxide nanostructured films by reactive pulsed laser deposition. Applied Surface Science 255, 5334–5337.
Di Fonzo, F., Casari, C.S., Russo, V., et al. (2009) Hierarchically organized nanostructured TiO2 for photocatalysis applications. Nanotechnology 20, 1–7.
Laugesen, S., Roepstorff, P. (2003) Combination of two matrices results in improved performance of MALDI MS for peptide mass mapping and protein analysis. J Am Soc Mass Spectrom 14, 992–1002.
Imami, K., Sugiyama, N., Kyono, Y., et al. (2008) Automated phosphoproteome analysis for cultured cancer cells by two-dimensional nanoLC-MS using a calcined titania/C18 biphasic column. Anal Sci 24, 161–166.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Torta, F., Fusi, M., Casari, C.S., Bassi, A.L., Bachi, A. (2011). Nanostructured TiO2 Thin Films for Phosphoproteomics Studies with MALDI Mass Spectrometry. In: Toms, S., Weil, R. (eds) Nanoproteomics. Methods in Molecular Biology, vol 790. Humana Press. https://doi.org/10.1007/978-1-61779-319-6_13
Download citation
DOI: https://doi.org/10.1007/978-1-61779-319-6_13
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-318-9
Online ISBN: 978-1-61779-319-6
eBook Packages: Springer Protocols