Detection of aberrant protein phosphorylation in cancer using direct gold-protein affinity interactions

- A new methodology for protein phosphorylation detection is reported.
- Phosphorylation modifications were found to alter protein's inherent ability to interact with bare gold surfaces.
- It is the first report employing proteins-gold affinity for pinpointing the presence of phosphorylation within the protein sequence.
- The method does not require phospho-specific antibodies or any other phospho-recognizing tag molecule.
- The method specifically circumvents the current paradigm in most electrochemical biosensors of prior immobilization of kinase target peptide onto the sensor surface.

Protein phosphorylation is one of the most prominent post-translational mechanisms for protein regulation, which is frequently impaired in cancer. Through the covalent addition of phosphate groups to certain amino-acids, the interactions of former residues with nearby amino-acids are drastically altered, resulting in major changes of protein conformation that impacts its biological function. Herein, we report that these conformational changes can also disturb the protein's ability to interact with and adsorb onto bare gold surfaces. We exploited this feature to develop a simple electrochemical method for detecting the aberrant phosphorylation of EGFR protein in several lung cancer cell lines. This method, which required as low as 10 ng/µL (i.e., 50 ng) of purified EGFR protein, also enabled monitoring cell sensitivity to tyrosine kinase inhibitors (TKI) ― a common drug used for restoring the function of aberrantly phosphorylated proteins in lung cancer. The reported strategy based on direct gold-protein affinity interactions avoids the conventional paradigm of requiring a phospho-specific antibody for detection and could be a potential alternative of widely used mass spectrometry.

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