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Phospho-Proteomics pp 161–177Cite as

Improving the Phosphoproteome Coverage for Limited Sample Amounts Using TiO2-SIMAC-HILIC (TiSH) Phosphopeptide Enrichment and Fractionation

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1355))

Abstract

Obtaining high phosphoproteome coverage requires specific enrichment of phosphorylated peptides from the often extremely complex peptide mixtures generated by proteolytic digestion of biological samples, as well as extensive chromatographic fractionation prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Due to the sample loss resulting from fractionation, this procedure is mainly performed when large quantities of sample are available. To make large-scale phosphoproteomics applicable to smaller amounts of protein we have recently combined highly specific TiO2-based phosphopeptide enrichment with sequential elution from immobilized metal affinity chromatography (SIMAC) for fractionation of mono- and multi-phosphorylated peptides prior to capillary scale hydrophilic interaction liquid chromatography (HILIC) based fractionation of monophosphorylated peptides. In the following protocol we describe the procedure step by step to allow for comprehensive coverage of the phosphoproteome utilizing only a few hundred micrograms of protein.

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Acknowledgements

This work was supported by the Lundbeck Foundation (M.R.L., Junior Group Leader Fellowship, K.E.-K., postdoctoral fellowship), The Danish Council for Independent Research and the European Union FP7 Marie Curie Actions—COFUND programme (K.E.-K., MOBILEX postdoc fellowship, grant ID DFF–1325-00154) and the Sehested Hansen Foundation (M.R.L., K.E.-K.).

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Authors and Affiliations

  1. Children’s Medical Research Institute, Westmead, Sydney, NSW, Australia

    Kasper Engholm-Keller

  2. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230, Odense M, Denmark

    Kasper Engholm-Keller & Martin R. Larsen

Authors
  1. Kasper Engholm-Keller
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  2. Martin R. Larsen
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Corresponding author

Correspondence to Martin R. Larsen .

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Editors and Affiliations

  1. University of Copenhagen, Center for Protein Research, Copenhagen Nm, Denmark

    Louise von Stechow

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Engholm-Keller, K., Larsen, M.R. (2016). Improving the Phosphoproteome Coverage for Limited Sample Amounts Using TiO2-SIMAC-HILIC (TiSH) Phosphopeptide Enrichment and Fractionation. In: von Stechow, L. (eds) Phospho-Proteomics. Methods in Molecular Biology, vol 1355. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-3049-4_11

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  • DOI: https://doi.org/10.1007/978-1-4939-3049-4_11

  • Publisher Name: Springer, New York, NY

  • Print ISBN: 978-1-4939-3048-7

  • Online ISBN: 978-1-4939-3049-4

  • eBook Packages: Springer Protocols

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