title: Electrochemical detection of protein kinase activity based on carboxypeptidase Y digestion triggered signal amplification.
abstract: An effective assay method for monitoring protein kinase activity and screening inhibitors is greatly beneficial to kinase-related drug discovery, early diagnosis of diseases, and therapeutic effect evaluation. Herein, we develop a simple electrochemical method for detecting the activity of casein kinase II (CK2) based on phosphorylation against carboxypeptidase Y (CPY) digestion triggered signal amplification, where CK2 catalyzed phosphorylation event protects the substrate peptide from the digestion of CPY, maintains the repulsive force of the substrate peptide towards the redox probe, and results in a weak electrochemical signal. Whereas, without phosphorylation, the substrate peptide is digested by CPY and a strong electrochemical signal is obtained. The detection feasibility is demonstrated for the assay of CK2 activity with low detection limit of 0.047unit/mL. Moreover, the biosensor was used for the analysis of kinase inhibition. Based on the electrochemical signal dependent inhibitor concentration, the IC50 value of ellagic acid was estimated to be 39.77nM. The proposed method is also successfully applied to analyze CK2 activity in cell lysates, proving the applicability in complex biological samples.
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China.
authors.forename: Huanshun
authors.initials: H
authors.lastname: Yin
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China.
authors.forename: Xinxu
authors.initials: X
authors.lastname: Wang
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China.
authors.forename: Yunlong
authors.initials: Y
authors.lastname: Guo
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China. Electronic address: zhyl@sdau.edu.cn.
authors.forename: Yunlei
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China. Electronic address: ashy@sdau.edu.cn.
authors.forename: Shiyun
authors.initials: S
authors.lastname: Ai
mesh_terms: D000595:Amino Acid Sequence; D015374:Biosensing Techniques / Q000379:methods; D047390:Casein Kinase II / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D043402:Cathepsin A / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods; D055664:Electrochemical Techniques / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D056945:Hep G2 Cells; D006801:Humans; D057230:Limit of Detection; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology
chemical_list: D010455:Peptides; D047428:Protein Kinase Inhibitors; D047390:Casein Kinase II; D043402:Cathepsin A
keywords: Carboxypeptidase Y; Electrochemical biosensor; Phosphorylation; Protein kinase