title: Fast and easy phosphopeptide fractionation by combinatorial ERLIC-SCX solid-phase extraction for in-depth phosphoproteome analysis.
abstract: Mass spectrometry-based phosphoproteomic analysis is a powerful method for gaining a global, unbiased understanding of cellular signaling. Its accuracy and comprehensiveness stands or falls with the quality and choice of the applied phosphopeptide prefractionation strategy. This protocol covers a powerful but simple and rapid strategy for phosphopeptide prefractionation. The combinatorial use of two distinct chromatographic techniques that address the inverse physicochemical properties of peptides allows for superior fractionation efficiency of multiple phosphorylated peptides. In the first step, multiphosphorylated peptides are separated according to the number of negatively charged phosphosites by electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). A subsequent strong cation exchange (SCX) step separates mostly singly phosphorylated peptides in the ERLIC flow-through according to their positive charge. The presented strategy is inexpensive and adaptable to large and small amounts of starting material, and it allows highly multiplexed sample preparation. Because of its implementation as solid-phase extraction, the entire workflow takes only 2 h to complete.
authors.affiliation: Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, Freiburg, Germany.|Center for Biological Systems Analysis (ZBSA), University of Freiburg, Freiburg, Germany.
authors.forename: Mostafa
authors.initials: M
authors.lastname: Zarei
authors.affiliation: Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, Freiburg, Germany.|Center for Biological Systems Analysis (ZBSA), University of Freiburg, Freiburg, Germany.|Department of Dermatology, Medical Center, University of Freiburg, Freiburg, Germany.|BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany.
authors.forename: Adrian
authors.initials: A
authors.lastname: Sprenger
authors.affiliation: Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, Freiburg, Germany.|Center for Biological Systems Analysis (ZBSA), University of Freiburg, Freiburg, Germany.|Department of Dermatology, Medical Center, University of Freiburg, Freiburg, Germany.
authors.forename: Michal
authors.initials: M
authors.lastname: Rackiewicz
authors.affiliation: Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, Freiburg, Germany.|Center for Biological Systems Analysis (ZBSA), University of Freiburg, Freiburg, Germany.|Department of Dermatology, Medical Center, University of Freiburg, Freiburg, Germany.|BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany.
authors.forename: Joern
authors.initials: J
authors.lastname: Dengjel
mesh_terms: D002852:Chromatography, Ion Exchange / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D052616:Solid Phase Extraction / Q000379:methods*; D055672:Static Electricity; D013499:Surface Properties; D018511:Systems Integration; D013997:Time Factors; D014025:Titanium / Q000737:chemistry
chemical_list: D010748:Phosphopeptides; C009495:titanium dioxide; D014025:Titanium