title: Monitoring selectivity in kinase-promoted phosphorylation of densely packed peptide monolayers using label-free electrochemical detection.
abstract: This paper describes remarkably high sensitivities in the label-free detection of kinase-promoted phosphorylation for 14 different peptide substrates on electrode-immobilized monolayers (gold or nitride) using serine/threonine kinases PKA, PKC, and CaMK2. Peptide substrates were preselected using (33)P-labeling in a microarray of 1024 substrates. The three most active peptides (A1-A3, C1-C3, and M1-M3) were investigated using electrochemical impedance spectroscopy (EIS) and ion-sensitive field effect transistors (ISFETs). Some of the peptide substrates, for example, the PKC-specific substrate PPRRSSIRNAH (C1), showed a remarkably high sensitivity in the EIS-based sensor measurements. Our studies revealed that this high sensitivity is primarily due to the monolayer's packing density. Nanoscopic studies demonstrated a distinct disordering of the C1-monolayer upon phosphorylation, while phosphatase-promoted dephosphorylation regenerated the highly ordered peptide monolayer. As a matter of fact, the initial surface packing of the peptide monolayer mainly determined the level of sensitivity, whereas electrostatic repulsion of the redox-active species was found to be much less important.
authors.affiliation: Institute of Chemistry and The Nanoscience and Nanotechnology Center, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
authors.forename: Elza
authors.initials: E
authors.lastname: Snir
authors.forename: Jos
authors.initials: J
authors.lastname: Joore
authors.forename: Peter
authors.initials: P
authors.lastname: Timmerman
authors.forename: Shlomo
authors.initials: S
authors.lastname: Yitzchaik
mesh_terms: D055664:Electrochemical Techniques; D004566:Electrodes; D006046:Gold / Q000737:chemistry; D009570:Nitriles / Q000737:chemistry; D010316:Particle Size; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry / Q000378:metabolism*; D013499:Surface Properties
chemical_list: D009570:Nitriles; D010455:Peptides; D006046:Gold; D010770:Phosphotransferases