A highly sensitive nanomechanical cantilever sensor assay based on an electrical measurement has been developed for detecting activated cyclic adenosine monophosphate (cyclic AMP)-dependent protein kinase (PKA). Employing a peptide derived from the heat-stable protein kinase inhibitor (PKI), a magnetic bead system was first selected as a vehicle to immobilize the PKI-(5-24) peptide for capturing PKA catalytic subunit and the activity assay was applied for indirectly assessing the binding. Synergistic interactions of adenosine triphosphate (ATP) and the peptide inhibitor with the kinase were then investigated by a solution phase capillary electrophoretic assay, and by surface plasmon resonance technology which involved immobilization of the peptide inhibitor. After systemically evaluated by a homogeneous direct binding assay, the ATP-dependent recognition of the catalytic subunit of PKA by PKI-(5-24) was successfully transferred on to the nanomechanical cantilevers at protein concentrations of 6.6 pM-66 nM, exhibiting much higher sensitivity and wider dynamic range than the conventional activity assay. Thus, direct assessment of activated kinases using the cantilever sensor system functionalized with specific peptide inhibitors holds great promise in analytical applications and clinical medicine.
Bibliographical noteFunding Information:
This work was supported by the 21C Frontier Functional Proteomics Research Center funded by the Korean Ministry of Science & Technology, and by the Intelligent Microsystem Center of the 21st Century Frontier R&D Program sponsored by the Korean Ministry of Commerce, Industry and Energy.
- Capillary electrophoresis
- Cyclic adenosine monophosphate-dependent protein kinase
- Fluorescence polarization
- Heat-stable protein kinase inhibitor
- Nanomechanical cantilever
- Peptide inhibitor
- Surface plasmon resonance
ASJC Scopus subject areas
- Analytical Chemistry
- Environmental Chemistry