Monitoring selectivity in kinase-promoted phosphorylation of densely packed peptide monolayers using label-free electrochemical detection

Elza Snir, Jos Joore, Peter Timmerman*, Shlomo Yitzchaik

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

33 Scopus citations

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 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.

Original languageAmerican English
Pages (from-to)11212-11221
Number of pages10
JournalLangmuir
Volume27
Issue number17
DOIs
StatePublished - 6 Sep 2011

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