Development of a peptide inhibitor-based cantilever sensor assay for cyclic adenosine monophosphate-dependent protein kinase.
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.
Kwon
Hyuk-Sung
HS
Life Sciences Division, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul, Republic of Korea.
Han
Ki-Cheol
KC
Hwang
Kyo Seon
KS
Lee
Jeong Hoon
JH
Kim
Tae Song
TS
Yoon
Dae Sung
DS
Yang
Eun Gyeong
EG
D000255:Adenosine Triphosphate / Q000737:chemistry; D015374:Biosensing Techniques; D020134:Catalytic Domain; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D000242:Cyclic AMP / Q000737:chemistry*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004563:Electrochemistry; D019075:Electrophoresis, Capillary / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006358:Hot Temperature; D008280:Magnetics; D053758:Nanoparticles / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D020349:Surface Plasmon Resonance
D004791:Enzyme Inhibitors; D010455:Peptides; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases