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.

Label-free fluorescence assay for protein kinase based on peptide biomineralized gold nanoclusters as signal sensing probe

“…Domain 1 is CCY which can reduce Au 3 þ into peptide-AuNCs via the phenolic group of tyrosine (Y) under alkaline conditions and capture the clusters via the SH group of cysteine (C) (Cui et al, 2011). Domain 2 is RRRADDSDDDDD the specific peptide substrate for CK2, which can be phosphorylated at serine (S) in the presence of CK2 and ATP (Freeman et al, 2010;Wang et al, 2013b;Zhou et al, 2013). Therefore, this peptide should perform the roles of both peptideAuNCs synthesis and protein phosphorylation.…”

Electrochemical detection of protein kinase activity based on carboxypeptidase Y digestion triggered signal amplification

“…The corresponding linear regressions could be expressed as I ¼-20.32 log c þ120.81 (R ¼0.9932). The detection limit was estimated to be 0.047 unit/mL (S/N ¼ 3), which was lower than those of 10 , 0.5 (Miao et al, 2012;Tan et al, 2013), 0.47 (Xu et al, 2011), 0.2 (Ji et al, 2009), 0.15 (Xu et al, 2009), 0.134 (Zhou et al, 2013), 0.1 , 0.07 and 0.061 unit/mL (Xu et al, 2012).…”

“…Initially, the un-phosphorylated substrate peptide is digested by CPY from the carboxyl, which leads to the release of free amino acids from electrode surface and the decrease of the electrostatic repulsion. Additionally, the CPY can also hydrolyze the amino residues ADD in the phosphorylated substrate peptide, and the further hydrolysis is blocked by the phosphorylation event at serine residue site (Zhou et al, 2013(Zhou et al, , 2014. From these results, we could conclude that the biosensor was successfully fabricated through different modification processes.…”

“…For this aim, some strategies based on the identification of phosphate, which is transferred from ATP to the substrate peptides catalyzed by protein kinase, have been developed, such as fluorescence Zhou et al, 2013), electrochemiluminescence (Chen et al, 2013), quartz crystal microbalance (Xu et al, 2012), microarray-based resonance light scattering assay (Li et al, 2013b), photoluminescence , and electrochemistry (Li et al, 2013a;Wang et al, 2010;Xu et al, 2009;Yang et al, 2011). Among them, electrochemical strategy is proved to be a kind of promising way to achieve labelfree assay of protein kinase activity due to the advantages of easy operation, simple instrument, low cost and high sensitivity.…”

Simple fluorescence-based detection of protein kinase A activity using a molecular beacon probe

“…[1][2][3][4] During phosphorylation, protein kinases catalyze the addition of g-phosphoryl from adenosine-5 0 -triphosphate (ATP) to a free hydroxyl group of serine, threonine, or tyrosine in a peptide or protein substrate. 5,6 It has been demonstrated that aberrant protein kinase activity and abnormal protein phosphorylation are closely associated with various human diseases, including various forms of cancer, diabetes, Alzheimer disease, restenosis, immune deficiencies, endocrinological disorders, and cardiovascular diseases. [7][8][9][10][11][12] It is estimated that 25% of drug development efforts is focused on protein kinase inhibitors.…”