title: Improving the Phosphoproteome Coverage for Limited Sample Amounts Using TiO2-SIMAC-HILIC (TiSH) Phosphopeptide Enrichment and Fractionation.
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.
authors.affiliation: Children's Medical Research Institute, Westmead, Sydney, NSW, Australia.|Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230, Odense M, Denmark.
authors.forename: Kasper
authors.initials: K
authors.lastname: Engholm-Keller
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230, Odense M, Denmark. mrl@bmb.sdu.dk.
authors.forename: Martin R
authors.initials: MR
authors.lastname: Larsen
mesh_terms: D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D006801:Humans; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014025:Titanium / Q000737:chemistry*; D057188:Workflow
chemical_list: D010750:Phosphoproteins; C009495:titanium dioxide; D014025:Titanium
keywords: Capillary HPLC; Enrichment; Fractionation; HILIC; Phosphopeptide; Phosphoproteomics; Protein phosphorylation; TiO2; TiSH