Enzymatically modified peptide surfaces: towards general electrochemical sensor platform for protein kinase catalyzed phosphorylations.
We hereby present an electrochemical approach for monitoring the three protein kinases sarcoma-related kinase (Src), extracellular signal-regulated kinase 1 (Erk1), and cyclin A-dependent kinase 2 (CDK2/cyclin A). The electrochemical sensor is based on the ability of kinases to transfer a redox-labeled phosphoryl group to surface-bound peptides that are highly specific substrates for the particular protein kinase (EGIYDVP, EPLTPSG, and HHASPRK, respectively). The detection method relies on the use of 5'-γ-ferrocenoyl-ATP (Fc-ATP) as a co-substrate for peptide phosphorylation. The peptides themselves are attached to a Au substrate, which acts as the working electrode. In this process a Fc-phosphoryl group is transferred to the peptide and the presence of the redox active Fc group is detected electrochemically. All peptide films were fully characterized by cyclic voltammetry (CV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS). Particular attention was given to the electron transfer rates, k(ET), in peptide films after Fc-phosphorylation which were found to be on the order of seconds. The slow ET kinetics is presumably a result of the negative charge on the phosphoryl group. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) and X-ray photoelectron spectroscopy (XPS) experiments based on the peptide modified Au surfaces reveal significant ferrocene and phosphate group content introduced using the kinase-catalyzed phosphorylation reaction.
The University of Western Ontario, Chemistry Department, 1151 Richmond Street, London, Ontario, Canada N6A 5B7.
Sanela
S
Martić
Mahmoud
M
Labib
Heinz-Bernhard
HB
Kraatz
D055162:Biocatalysis; D015374:Biosensing Techniques / Q000379:methods; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D058266:Dielectric Spectroscopy; D055664:Electrochemical Techniques / Q000379:methods*; D005296:Ferrous Compounds / Q000737:chemistry; D007700:Kinetics; D000075163:Metallocenes; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013499:Surface Properties; D019061:src-Family Kinases / Q000378:metabolism
D005296:Ferrous Compounds; D000075163:Metallocenes; D010455:Peptides; D011494:Protein Kinases; D019061:src-Family Kinases; D051357:Cyclin-Dependent Kinase 2; D048052:Mitogen-Activated Protein Kinase 3; C004998:ferrocene