Protein phosphorylation analysis based on proton release detection: potential tools for drug discovery.

Phosphorylation is the most important post-translational modification of proteins in eukaryotic cells and it is catalysed by enzymes called kinases. The balance between protein phosphorylation and dephosphorylation is critical for the regulation of physiological processes and its unbalance is the cause of several diseases. Conventional assays used to analyse the kinase activity are limited as they rely heavily on phospho-specific antibodies and radioactive tags. This makes their use impractical for high throughput drug discovery platforms. We have developed two versatile methods to detect the release of protons (H(+)) associated with the protein phosphorylation catalysed by kinases. The first approach is based on the pH-sensitive response of oxide-semiconductor interfaces and the second method detects the pH changes in phosphorylation reaction using a commercial micro-pH electrode. The proposed methods successfully detected phosphorylation of myelin basic protein by PKC-α kinase. These techniques can be readily adopted for multiplexed arrays and high throughput analysis of kinase activity, which will represent an important innovation in biomedical research and drug discovery.