title: Sequential phosphorylation analysis using dye-tethered peptides and microfluidic isoelectric focusing electrophoresis.
abstract: We report a simple method for analyzing sequential phosphorylation by protein kinases using fluorescent peptide substrates and microfluidic isoelectric focusing (μIEF) electrophoresis. When a dye-labeled peptide substrate was sequentially phosphorylated by two consecutive protein kinases (mitogen-activated protein kinase (MAPK) and glycogen synthase kinase 3 (GSK3)), its differently phosphorylated forms were easily separated and visualized by fluorescent focusing zones in the μIEF channel based on a change in the isoelectric point (pI) by phosphorylation. As a result, ratiometric and quantitative analysis of the fluorescent focusing regions shifted by phosphorylation enabled the analysis of phosphorylation efficiency and the relevant inhibition of protein kinases (MAPK and GSK3) with high simplicity and selectivity. Furthermore, the GSK3 activity in the cell lysates was elucidated by μIEF electrophoresis in combination with immunoprecipitation. Our results suggest that this method has great potential for analyzing the sequential phosphorylation of multiple protein kinases that are implicated in cellular signaling pathways.
authors.affiliation: Department of Life Science, Hanyang University, Seoul 133-791, Republic of Korea; Research Institute for Natural Sciences, Hanyang University, Seoul 133-791, Republic of Korea.
authors.forename: Hoseok
authors.initials: H
authors.lastname: Choi
authors.affiliation: Department of Mechanical Convergence Engineering, Hanyang University, Seoul 133-791, Republic of Korea.
authors.forename: Nakchul
authors.initials: N
authors.lastname: Choi
authors.affiliation: Department of Life Science, Hanyang University, Seoul 133-791, Republic of Korea; Research Institute for Natural Sciences, Hanyang University, Seoul 133-791, Republic of Korea.
authors.forename: Butaek
authors.initials: B
authors.lastname: Lim
authors.affiliation: Department of Life Science, Hanyang University, Seoul 133-791, Republic of Korea; Research Institute for Natural Sciences, Hanyang University, Seoul 133-791, Republic of Korea.
authors.forename: Tae-Wuk
authors.initials: TW
authors.lastname: Kim
authors.affiliation: Department of Mechanical Convergence Engineering, Hanyang University, Seoul 133-791, Republic of Korea; Institute of Nano Science and Technology, Hanyang University, Seoul 133-791, Republic of Korea. Electronic address: simonsong@hanyang.ac.kr.
authors.forename: Simon
authors.initials: S
authors.lastname: Song
authors.affiliation: Department of Life Science, Hanyang University, Seoul 133-791, Republic of Korea; Research Institute for Natural Sciences, Hanyang University, Seoul 133-791, Republic of Korea; Institute of Nano Science and Technology, Hanyang University, Seoul 133-791, Republic of Korea. Electronic address: ypilkim@hanyang.ac.kr.
authors.forename: Young-Pil
authors.initials: YP
authors.lastname: Kim
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation*; D004396:Coloring Agents; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D007525:Isoelectric Focusing / Q000295:instrumentation*; D056656:Lab-On-A-Chip Devices; D061986:MCF-7 Cells; D010455:Peptides; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D015398:Signal Transduction
chemical_list: D004396:Coloring Agents; D010455:Peptides; D047428:Protein Kinase Inhibitors; D048049:Extracellular Signal-Regulated MAP Kinases; D038362:Glycogen Synthase Kinase 3
keywords: Immunoprecipitation; Inhibition assay; Isoelectric focusing; Microfluidic; Protein kinase; Sequential phosphorylation