title: High-performance thin-layer chromatography as a fast screening tool for phosphorylated peptides.
abstract: This study aimed at developing a rapid chromatographic assay to monitor phosphorylation sites in peptides. For the analysis of nociceptive signal transduction pathways, the detection of phosphorylated proteins/peptides plays a fundamental role. To get further insights in the phosphorylation mechanism of protein kinase C-ε (PKC-ε) and protein kinase A (PKA), potential targets were divided into subsections resulting in peptides that contain only one possible phospho-binding site. The use of high-performance thin-layer chromatography (HPTLC) offers the possibility of a high throughput of samples and the advantage of a quick sample clean-up. A combined strategy of an effect-directed overlay procedure on the TLC plate using specific antibodies (immunostaining, HPTLC-IS) as well as a parallel, direct mass spectrometric methodology by HPTLC-MALDI-TOF-MS was developed. With regard to HPTLC-IS, validation of the data exhibited a lower limit of detection than the traditionally used protein derivatization reagent fluorescamine. Besides the identification of the phosphorylated peptides, a semi-quantitative estimation can be performed with HPTLC-IS.
authors.affiliation: University of Hamburg, Hamburg School of Food Science, Institute of Food Chemistry, Grindelallee 117, D-20146 Hamburg, Germany. Electronic address: lena.morschheuser@chemie.uni-hamburg.de.
authors.forename: Lena
authors.initials: L
authors.lastname: Morschheuser
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytic and Bioprocesses, Am Mühlenberg 13, D-14476 Potsdam/Golm, Germany; Free University of Berlin, Institute for Chemistry and Biochemistry, Takustraße 3, D-14195 Berlin, Germany. Electronic address: sandra.muekusch@izi-bb.fraunhofer.de.
authors.forename: Sandra
authors.initials: S
authors.lastname: Mükusch
authors.affiliation: University of Hamburg, Institute of Organic Chemistry, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany. Electronic address: maria.riedner@chemie.uni-hamburg.de.
authors.forename: Maria
authors.initials: M
authors.lastname: Riedner
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytic and Bioprocesses, Am Mühlenberg 13, D-14476 Potsdam/Golm, Germany. Electronic address: harald.seitz@izi-bb.fraunhofer.de.
authors.forename: Harald
authors.initials: H
authors.lastname: Seitz
authors.affiliation: University of Hamburg, Hamburg School of Food Science, Institute of Food Chemistry, Grindelallee 117, D-20146 Hamburg, Germany. Electronic address: rohn@chemie.uni-hamburg.de.
authors.forename: Sascha
authors.initials: S
authors.lastname: Rohn
mesh_terms: D000595:Amino Acid Sequence; D002855:Chromatography, Thin Layer / Q000379:methods*; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D010766:Phosphorylation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D010455:Peptides
keywords: HPTLC-MALDI-TOF-MS; HPTLC-immunostaining; High-performance thin-layer chromatography; Nociceptive pathway; Phosphopeptide detection