title: Comparison of ERLIC-TiO2, HILIC-TiO2, and SCX-TiO2 for global phosphoproteomics approaches.
abstract: Reversible phosphorylations play a critical role in most biological pathways. Hence, in signaling studies great effort has been put into identification of a maximum number of phosphosites per experiment. Mass spectrometry (MS)-based phosphoproteomics approaches have been proven to be an ideal analytical method for mapping of phosphosites. However, because of sample complexity, fractionation of phosphopeptides prior to MS analysis is a crucial step. In the current study, we compare the chromatographic strategies electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), hydrophilic interaction liquid chromatography (HILIC), and strong cation exchange chromatography (SCX) for their fractionation behavior of phosphopeptides. In addition, we investigate the use of repetitive TiO(2)-based enrichment steps for a maximum identification of phosphopeptides. On the basis of our results, SCX yields the highest number of identified phosphopeptides, whereas ERLIC is optimal for the identification of multiphosphorylated peptides. Consecutive incubations of fractions and flow-through by TiO(2) beads enrich qualitatively different sets of phosphopeptides, increasing the number of identified phosphopeptides per analysis.
authors.affiliation: Freiburg Institute for Advanced Studies (FRIAS), School of Life Sciences-LifeNet, University of Freiburg, Albertstr. 19, 79104 Freiburg, Germany.
authors.forename: Mostafa
authors.initials: M
authors.lastname: Zarei
authors.forename: Adrian
authors.initials: A
authors.lastname: Sprenger
authors.forename: Fabian
authors.initials: F
authors.lastname: Metzger
authors.forename: Christine
authors.initials: C
authors.lastname: Gretzmeier
authors.forename: Joern
authors.initials: J
authors.lastname: Dengjel
mesh_terms: D002853:Chromatography, Liquid; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D010750:Phosphoproteins / Q000737:chemistry*; D040901:Proteomics; D014025:Titanium / Q000737:chemistry*
chemical_list: D010750:Phosphoproteins; C009495:titanium dioxide; D014025:Titanium