등전점 미세유체칩을 이용한 순차적 인산화 분석 연구

Abstract

Protein kinase cascades are ubiquitous signaling effectors, leading to sequential phosphorylation events to mediate a myriad of physiological events in cells. Herein, a simple method for analyzing sequential phosphorylation by protein kinases is demonstrated using fluorescent peptide substrates and microfluidic isoelectric focusing (μIEF) electrophoresis. The μIEF was implemented to make it possible to generate a rapid pH gradient by adding ampholytes to the running solution under the applied electrical field and to allow for easy separation with substrate peptides. This resulted in reliable analysis of phosphorylation degree and efficiency with respect to single or consecutive kinase reaction. In addition, inhibitor effect of mitogen-activated protein kinases (MAPK) or glycogen synthase kinase 3 (GSK3) was effectively analyzed in a ratiometric and quantitative manner. Furthermore, protein kinase activity in cell lysates was detected by the fluorescent μIEF electrophoresis in combination with immunoprecipitation. These results suggest that the developed method has great potential for analyzing the sequential phosphorylation of multiple protein kinases that are implicated in cellular signaling pathways.