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

Kasper Engholm-Keller, Martin R Larsen

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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.

Original languageEnglish
Title of host publicationPhospho-Proteomics : Methods and Protocols
EditorsLouise von Stechow
VolumePart III
PublisherSpringer
Publication date2016
Edition2.
Pages161-177
Chapter11
ISBN (Print)978-1-4939-3048-7
ISBN (Electronic)978-1-4939-3049-4
DOIs
Publication statusPublished - 2016
SeriesMethods in Molecular Biology
Volume1335
ISSN1064-3745

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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 L. V. Stechow (Ed.), Phospho-Proteomics : Methods and Protocols (2. ed., Vol. Part III, pp. 161-177). Springer. Methods in Molecular Biology, Vol.. 1335 https://doi.org/10.1007/978-1-4939-3049-4_11