Analysis of protein phosphorylation combining capillary electrophoresis with ATP analog labeling technique. Issue 4 (2nd December 2021)

• Record Type: Journal Article
• Authors: Li, Yue; Liu, Yaoqi; Huang, Xiangyi; Ren, Jicun
• Is Part Of: Electrophoresis. Volume 43:Issue 4(2022)
• Page Start: 548
• Page End: 558
• Publication Date: 2021-12-02
• Subjects: ATP analog -- CE‐LIF -- D–A reaction -- Phosphorylation; Electrophoresis -- Periodicals; Electrophoresis -- Periodicals; 541.372
• DOI: 10.1002/elps.202100251
• Languages: English
• ISSNs: 0173-0835

Abstract

Abstract: Protein phosphorylation is one of the most basic mechanisms for regulating and controlling protein biological activity and function, and it is also a very important posttranslational modification process. Protein phosphorylation participates in and regulates many life activities such as signal transduction, gene expression, cell cycle, and so on. In this paper, we propose a method for the determination of the protein phosphorylation combining capillary electrophoresis (CE) with ATP analog labeling technique. We synthesized two new ATP analogs (ATP‐NB and ATP‐TATD‐NB) functionalized by norbornene. Using Abl kinase as a model, we established a method for the determination of the kinase activity in solution and lysate by CE with laser‐induced fluorescence detection (CE‐LIF). This method was used to evaluate the efficiencies of kinase inhibitors. The IC50 values obtained are basically consistent with the reports. By D–A reaction (inverse electron demand Diels–Alder reaction) to label TZ‐BODIPY fluorescence, we also realized the phosphorylation fluorescence detection of substrate peptide. Then, we used fluorescence confocal microscopy imaging technology to study the phosphorylation of proteins in vivo by the D–A reaction of ATP‐NB and TZ‐BODIPY. Our preliminary results documented that the combination of CE‐LIF with analog ATP‐NB labeling technique is an effective strategy for the determination of the protein phosphorylation and the kinase activity and for screening of kinase inhibitors. The D–A reaction of ATP‐NB and TZ‐BODIPY also laid the foundation for the subsequent in situ study of protein phosphorylation.