Every day, tens of millions of biological processes occur in our body at a cellular level. Studying these processes might help us learn more about how cells function, a field that has continued to intrigue researchers. Recently, nonetheless, there was a recent player on this field. A recent analytical method-;single-molecule detection-;has gained momentum on account of its success in observing specific, biologically relevant molecules and the processes related to them.

Scientists have tried ways to make use of single-molecule detection assays to check proteins and their post-translational modifications (PTMs). PTMs are enzymatic changes observed after protein synthesis, wherein functional groups are added to the amino acids within the protein, enabling it to perform a selected function. The study of PTMs might help us understand cell signaling and the origin of several diseases. Nevertheless, assays aiming to achieve this need to be highly selective and specific to that protein. Given the dearth of sensitivity of current techniques, obtaining single-molecule PTM measurements is difficult.

Recently, researchers at Tokyo Institute of Technology (Tokyo Tech) have found an “electrifying” method to overcome these limitations. Of their recent breakthrough, published within the Journal of the American Chemical Society, a team of scientists led by Associate Professor Tomoaki Nishino from Tokyo Tech reported the single-molecule detection of phosphorylation in peptides-;short amino acid chains-;and the formation of an orthophosphate junction with the assistance of electronic signatures. Dr. Nishino explains, “We selected peptide phosphorylation, an archetypal and biologically relevant PTM, for our detection studies. The aim was to develop a tool that would detect even the slightest alteration within the chemical structure of amino acids.”

To begin with, the team 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 present was passed between two gold STM electrodes, an orthophosphate group was found to bridge the nanogap between the electrodes by forming a stable junction on account of the interaction of its negatively charged oxygen atoms with the gold. It was this junction and its signature that drove further experiments.

The one-orthophosphate junction was found to own 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 query (i.e., phosphorylation). To further test their technique, the team carried out in situ single-molecule phosphorylation assays, where they were capable of differentiate between phosphorylated and non-phosphorylated peptides with 95% accuracy and 91% specificity.

The tactic demonstrated on this study provides an unexpected perspective into the world of PTMs in proteins. This novel technique can even open up recent avenues for using single-molecule detection of PTMs in clinical diagnosis and pharmaceutical applications. “There may be a powerful connection between protein phosphorylation and the pathogenesis of a big selection of diseases. Our method will allow scientists to detangle how phosphorylation regulates the cellular events that result in the origin of a disease and thereby aid in the event of treatments,” concludes Dr. Nishino.

Molecular level detection for large-scale disease eradication!

Source:

Tokyo Institute of Technology

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