title: Graphene oxide-peptide nanocomplex as a versatile fluorescence probe of protein kinase activity based on phosphorylation protection against carboxypeptidase digestion.
abstract: The research on complicated kinomics and kinase-target drug discovery requires the development of simple, cost-effective, and multiplex kinase assays. Herein, we propose a novel and versatile biosensing platform for the detection of protein kinase activity based on graphene oxide (GO)-peptide nanocomplex and phosphorylation-induced suppression of carboxypeptidase Y (CPY) cleavage. Kinase-catalyzed phosphorylation protects the fluorophore-labeled peptide probe against CPY digestion and induces the formation of a GO/peptide nanocomplex resulting in fluorescence quenching, while the nonphosphopeptide is degraded by CPY to release free fluorophore as well as restore fluorescence. This GO-based nanosensor has been successfully applied to sensitively detect two model kinases, casein kinase (CKII) and cAMP-dependent protein kinase (PKA) with low detection limits of 0.0833 mU/μL and 0.134 mU/μL, respectively. The feasibility of this GO-based sensor was further demonstrated by the assessment of kinase inhibition by staurosporine and H-89, in vitro kinase assay in cell lysates, and simultaneous detection of CKII and PKA activity. Moreover, the GO-based fluorescence anisotropy (FA) kinase assay has been also developed using GO as a FA signal amplifier. The proposed sensor is homogeneous, facile, universal, label-free, and applicable for multiplexed kinase assay, presenting a promising method for kinase-related biochemical fundamental research and inhibitor screening.
authors.affiliation: State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, China.
authors.forename: Jiang
authors.initials: J
authors.lastname: Zhou
authors.forename: Xiahong
authors.initials: X
authors.lastname: Xu
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.forename: Xin
authors.initials: X
authors.lastname: Liu
authors.forename: Zhou
authors.initials: Z
authors.lastname: Nie
authors.forename: Meng
authors.initials: M
authors.lastname: Qing
authors.forename: Lihua
authors.initials: L
authors.lastname: Nie
authors.forename: Shouzhuo
authors.initials: S
authors.lastname: Yao
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D002268:Carboxypeptidases / Q000032:analysis / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006108:Graphite / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D061986:MCF-7 Cells; D053758:Nanoparticles / Q000737:chemistry* / Q000378:metabolism; D010087:Oxides / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology
chemical_list: D005456:Fluorescent Dyes; D010087:Oxides; D006108:Graphite; D017868:Cyclic AMP-Dependent Protein Kinases; D002268:Carboxypeptidases