Unlocking the Proteome's Secrets: The Advancement of Single-Molecule Protein Sequencing Techniques

Even though characterizing the proteome, the complete set of proteins made by a cell or organism, can yield important insights into health and disease, it is still a challenging task. Proteins, in contrast to nucleic acids, are constructed from a wider variety of building blocks, including 20 different amino acids. Additionally, proteins cannot be amplified (e.g. via PCR), meaning that protein analysis methods must work with the limited amount of material available. Mass spectrometry, a method that profiles protein mixtures based on their mass and charge, is currently used in the vast majority of proteomic analyses. However, while this method can quantify thousands of proteins at once, sometimes it fails to correctly identify the molecules and misses proteins that are present in low abundance.

Many, if not all, of the proteins in a sample can now be sequenced thanks to advances in single-molecule technologies. Edward Marcotte of the University of Texas at Austin developed a method called fluorosequencing. Individual amino acids are fluorescently labeled and then sheared off one at a time from the end of a surface-coupled protein, while the resulting fluorescent signal is captured by a camera in a step-by-step chemical process known as fluorosequencing.

The use of fluorescently labeled 'binder' proteins to identify particular peptide sequences at the proteins' termini is another method. Meanwhile, other scientists are working on methods to profile polypeptides based on the changes they induce in an electric current as they pass through nanopores, mimicking the sequencing of DNA using these miniature channels.

One such method, employing protein nanopores, was demonstrated by biophysicist Cees Dekker and his colleagues at Delft University of Technology in the Netherlands. In order to facilitate high-throughput analyses of many individual protein molecules at once, the lab of biomedical engineer Amit Meller at the Technion - Israel Institute of Technology in Haifa is looking into solid-state nanopore devices made from silicon-based materials. While proof-of-concept for single-molecule protein sequencing is still ongoing, commercialization is on the horizon.

Earlier this year, Quantum-Si announced that it would begin shipping its first generation of instruments, and in November 2022, a conference on protein sequencing was held in Delft, with a panel discussion devoted to new companies in the field. Marcotte, a pioneer in the field of protein sequencing who co-founded Erisyon in Austin, Texas, is certain that the only question is not whether or not the technology will be successful, but rather how soon it will be available to the general public.

Topics: Emerging Technologies