Phosphate electrical signature helps detect important cellular events

Analysis of post-translational modification of proteins can help us take a closer look at cellular events. Tokyo Tech researchers have developed a new single-molecule detection assay that can do this using electrical measurements. The technique was able to detect peptide phosphorylation with a specificity of 91% and an accuracy of 95%.

Every day, millions of biological processes occur in our body at the cellular level. Studying these processes can help us learn more about how cells work, a field that continues to intrigue researchers. Recently, however, a new player has appeared in this field. A new analytical method, single molecule detection, has gained momentum due to its success in observing specific biologically relevant molecules and the processes associated with them.

Scientists have tested ways to use single-molecule detection assays to study proteins and their post-translational modifications (PTMs). PTMs are enzymatic changes observed after protein synthesis, in which functional groups are added to amino acids in the protein, allowing it to perform a specific function. The study of PTMs can help us understand cell signaling and the origin of various diseases. However, assays designed to do so have to be highly selective and specific for that protein. Given the lack of sensitivity of current techniques, obtaining single-molecule PTM measurements is challenging.

Recently, researchers at the Tokyo Institute of Technology (Tokyo Tech) have found an “electrifying” way to overcome these limitations. In his recent advance, published in the Journal of the American Chemical Society, a team of scientists led by Associate Professor Tomoaki Nishino of Tokyo Tech reported the detection of single-molecule phosphorylation in peptides (short chains of amino acids) and the formation of an orthophosphate bond with the help of electronic signatures. Dr. Nishino explains, “We chose peptide phosphorylation, an archetypal and biologically relevant PTM, for our screening studies. The goal was to develop a tool that could detect even the slightest alteration in the chemical structure of amino acids.”

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To begin with, the team studied the electronic properties of phosphorylated peptides using their inorganic analog, orthophosphoric acid (H3emails4). They prepared a phosphate solution (PO43-) and subjected it to a scanning tunneling microscope (STM)-assisted break junction (BJ) technique. When current was passed between two gold STM electrodes, an orthophosphate group was found to close 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 crossover and its signature that prompted further experiments.

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

The method demonstrated in this study provides an unforeseen perspective into the world of PTMs in proteins. This novel technique will also open new avenues for the use of single-molecule PTM detection in clinical diagnostics and pharmaceutical applications. “There is a strong connection between protein phosphorylation and the pathogenesis of a wide range of diseases. Our method will allow scientists to unravel how phosphorylation regulates cellular events that lead to the origin of a disease and thus help in the treatment development. Dr. Nishino concludes.

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Materials provided by Tokyo Institute of Technology. Note: content can be edited for style and length.

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