- Monitoring selectivity in kinase-promoted phosphorylation of densely packed peptide monolayers using label-free electrochemical detection
- Volume | Issue number
- 27 | 17
- Pages (from-to)
- Document type
- Faculty of Science (FNWI)
- Van 't Hoff Institute for Molecular Sciences (HIMS)
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 33P-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.
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- PT: J; TC: 0; UT: WOS:000294373300107
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