title: Monitoring phosphatase reactions of multiple phosphorylated substrates by reversed-phase HPLC.
abstract: In an approach to gain insight into the sequence-dependent dephosphorylation of multiple phosphotyrosyl-containing peptides by the phosphatases SHP-1 and PTP1B, we applied a chromatographic technique for the analysis of the dephosphorylation products. Mono-, bi- and triphosphorylated reference peptides corresponding to positions 1999-2014 in the activation loop of the receptor tyrosine kinase Ros were first analyzed by reversed-phase HPLC and MALDI-TOF/TOF mass spectrometry. Then, the respective products from enzymatic treatment were investigated by HPLC and compared to the standard peptides. The results obtained in this study emphasize the advantage of monitoring phosphatase reactions for mono- and biphosphorylated peptides using the described procedure rather than spectrophotometric and fluorimetric methods that do not allow for a clear identification of the products formed.
authors.affiliation: Institute of Biochemistry and Biophysics, Biological-Pharmaceutical Faculty, Friedrich-Schiller-University, Philosophenweg 12, D-07743 Jena, Germany.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Teichmann
authors.forename: Robert
authors.initials: R
authors.lastname: Winkler
authors.forename: Kornelia
authors.initials: K
authors.lastname: Hampel
authors.forename: Antje
authors.initials: A
authors.lastname: Trümpler
authors.forename: Frank D
authors.initials: FD
authors.lastname: Böhmer
authors.forename: Diana
authors.initials: D
authors.lastname: Imhof
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D010748:Phosphopeptides; D010744:Phosphoric Monoester Hydrolases