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

  • Kasper Engholm-Keller
  • Martin R. LarsenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1355)


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.

Key words

Phosphopeptide Phosphoproteomics Protein phosphorylation TiO2 Capillary HPLC HILIC TiSH Enrichment Fractionation 



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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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Children’s Medical Research InstituteSydneyAustralia
  2. 2.Department of Biochemistry and Molecular BiologyUniversity of Southern DenmarkOdense MDenmark

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