Electrical Signature Detects Impending Cellular Events

Using a new analytical technique called single-molecule detection, researchers in Japan have shown that the electrical signature generated by phosphate could be used to detect impending cellular events. The findings open new avenues for the use of single-molecule detection in clinical diagnosis and pharmaceutical applications.

Each day, millions of biological processes occur in our body at a cellular level. Single-molecule detection has gained interest due to its success in observing specific, biologically relevant molecules and the processes associated with them. Scientists have tried to use such assays to study proteins and their post-translational modifications (PTMs), the enzymatic changes observed after protein synthesis, wherein functional groups are added to the amino acids in the protein, enabling it to perform a specific function. The study of PTMs can help us understand cell signaling and the origin of several diseases. However, assays aiming to do so have to be highly selective and specific to that protein. Given the lack of sensitivity of current techniques, obtaining single-molecule PTM measurements is challenging.

Recently, researchers at Tokyo Institute of Technology (Tokyo Tech) have found a way to overcome these limitations. In a recent issue of the Journal of the American Chemical Society, they reported the single-molecule detection of phosphorylation in peptides—short amino acid chains—and the formation of an orthophosphate junction with the help of electronic signatures.

“We chose peptide phosphorylation, an archetypal and biologically relevant PTM, for our detection studies,” says Associate Professor Tomoaki Nishino.  “The aim was to develop a tool that could detect even the slightest alteration in the chemical structure of amino acids.”

The team first studied the electronic properties of phosphorylated peptides using their inorganic analog, orthophosphoric acid (H3PO4). They prepared a phosphate solution (PO43-­) and subjected it to a scanning tunneling microscope (STM)-assisted break-junction (BJ) technique. When the current was passed between two gold STM electrodes, an orthophosphate group was found to bridge the nanogap between the electrodes by forming a stable junction due to the interaction of its negatively charged oxygen atoms with the gold. It was this junction and its signature that drove further experiments.

The single-orthophosphate junction was found to possess a high conductance of 0.4 G0 and distinct electronic properties, the latter of which enabled this procedure to be highly specific and accurately sense the PTM in question (i.e., phosphorylation). To further test their technique, the team carried out in situ single-molecule phosphorylation assays, where they were able to differentiate between phosphorylated and non-phosphorylated peptides with 95% accuracy and 91% specificity.

The method provides an unforeseen perspective into the world of PTMs in proteins. This novel technique will also open new potential for using single-molecule detection of PTMs in clinical diagnosis and pharmaceuticals.

“There is a strong connection between protein phosphorylation and the pathogenesis of a wide range of diseases. Our method will allow scientists to detangle how phosphorylation regulates the cellular events that lead to the origin of a disease and thereby aid in the development of treatments,” says Nishino.