title: Rapid combinatorial ERLIC-SCX solid-phase extraction for in-depth phosphoproteome analysis.
abstract: Protein phosphorylation is an important mechanism of cellular signaling, and many proteins are precisely regulated through the interplay of stimulatory and inhibitory phosphorylation sites. Phosphoproteomics offers great opportunities to unravel this complex interplay, generating a mechanistic understanding of vital cellular processes. However, protein phosphorylation is substoichiometric and, in particular, peptides carrying multiple phosphorylation sites are extremely difficult to detect in a highly complex mixture of abundant nonphosphorylated peptides. Chromatographic methods are employed to reduce sample complexity and thereby significantly increase the number of phosphopeptide identifications. We previously demonstrated that combinatorial strong cation exchange-electrostatic repulsion-hydrophilic interaction chromatography yields a surplus in overall identifications of phosphopeptides compared with single chromatographic approaches. Here we present a simple and rapid strategy implemented as solid-phase extraction not requiring specific instrumentation such as off-line HPLC systems. It is inexpensive, adaptable for high and low amounts of starting material, and saves time by allowing multiplexed sample preparation without any carry-over problem.
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: Christine
authors.initials: C
authors.lastname: Gretzmeier
authors.forename: Joern
authors.initials: J
authors.lastname: Dengjel
mesh_terms: D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis*; D010750:Phosphoproteins / Q000032:analysis*; D059748:Proteolysis; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D052616:Solid Phase Extraction / Q000379:methods*
chemical_list: D010748:Phosphopeptides; D010750:Phosphoproteins