title: Quantitative detection of phosphoproteins by combination of two-dimensional difference gel electrophoresis and phosphospecific fluorescent staining.
abstract: Here we combine a standard two-dimensional difference gel electrophoresis (DIGE) protocol with subsequent post-staining of gels with phosphospecific fluorescent Pro-Q Diamond dye. The combination of these two methods for fluorescence detection of proteins allows quantitative detection of phosphoproteins in 2-DE-gels. We established this protocol within a functional proteomics experiment. Mammary epithelial cells (EpH4) were stimulated in culture by epidermal growth factor (EGF), endosomal fractions prepared after subcellular fractionation and phosphorylated proteins successfully detected on endosomes. For instance, Endo A cytokeratin, known as phosphoprotein and differentiation marker inducible by MAPK signaling, was identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). With this protocol, all steps of combined proteome and phosphoproteome profiling experiments are significantly simplified and accelerated, taking full advantage of both methods in terms of specificity, sensitivity and accuracy of quantification.
authors.affiliation: Biocenter, Division of Cell Biology, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: Taras
authors.initials: T
authors.lastname: Stasyk
authors.forename: Sandra
authors.initials: S
authors.lastname: Morandell
authors.forename: Rania
authors.initials: R
authors.lastname: Bakry
authors.forename: Isabel
authors.initials: I
authors.lastname: Feuerstein
authors.forename: Christian W
authors.initials: CW
authors.lastname: Huck
authors.forename: Guenther
authors.initials: G
authors.lastname: Stecher
authors.forename: Guenther K
authors.initials: GK
authors.lastname: Bonn
authors.forename: Lukas A
authors.initials: LA
authors.lastname: Huber
mesh_terms: D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D051379:Mice; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D020543:Proteome / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D010750:Phosphoproteins; D020543:Proteome