What is single-molecule analysis?

When there’s a question about the inner workings of cells, biologists can learn a lot by looking at all the proteins present, also known as the proteome. Broadscale proteomic analyses, like those that compare the proteomes of many different cells, can be incredibly enlightening when it comes to understanding the big picture. They can reveal what biological pathways are active in different cells and point researchers to the molecular basis of a biological function. 

While such analyses are often performed on bulk samples that don’t provide information on individual proteins in isolation, next generation proteomics technologies like the Nautilus Proteome Analysis Platform aim to achieve single-molecule analysis of all proteins in a sample.

Critically, we’re designing a platform capable of single-molecule analysis of intact proteins. Many other technologies only analyze small pieces of proteins called peptides at the single-molecule level. This makes it difficult to determine which peptides came from the same full-length protein and obscures the many possible combinations of modifications present on the full protein. Overall, without the ability to analyze intact proteins at the single-molecule level, it is very difficult to identify the precise proteoforms present.

Powered by platforms like ours, single-molecule protein studies can reveal the exact molecular composition of each protein in a sample, including how individual proteins have been modified. That knowledge could play an important role for functional proteomics studies that attempt to reveal the roles specific proteins play in the body, whether that’s catalyzing chemical reactions or allowing a pathogen to enter our cells. 

In general, single-molecule analyses can reveal mechanistic details that are obscured by bulk measurements, and single-molecule techniques are useful across a wide variety of fields and applications. Here’s a quick snapshot of when single-molecule analysis is most helpful — in proteomics and beyond — and what researchers can learn from it.  

Examples of single-molecule analysis 

Single-molecule measurements give the highest resolution and highest sensitivity possible when quantifying proteins or other molecules present in a sample. A single-molecule analysis can reveal even tiny variations to proteins or other molecules.  

This sort of resolution into the proteome or other types of molecules can be necessary for diagnosing disease, understanding the state of tissues, or finding treatments for disease. For instance, in a recent report in Nature Biotechnology, researchers in Israel developed a single-molecule assay that detects and quantifies protein complexes called nucleosomes that have been modified in various ways. The assay specifically measures these molecules in blood plasma and may enable researchers to use the modified nucleosomes as biomarkers for the early detection of cancer. 

Single-molecule analysis can also enable scientists to look at the mechanics of proteins involved in various pathways or reactions, since individual protein molecules can change over the course of a reaction. Without single-molecule techniques, it would be impossible to make out the precise changes related to the mechanisms of these reactions — lower resolution analyses would instead give a blurry, averaged-over-time picture of the various steps and stages. 

Many such single-molecule techniques are not proteomics techniques, but still provide high resolution insights into protein function. For example, a technique called single-molecule force spectroscopy is increasingly used to study the dynamics of folding and unfolding proteins. In a recent review in Langmuir, a journal of the American Chemical Society, the author shows how this type of single-molecule analysis can be used to investigate various proteins like calcium-binding proteins, which are fundamental to cellular communication. Likewise, researchers in a recent study in Nucleic Acids Research used single-molecule analysis to characterize protein-DNA interactions like those that occur during the repair of damaged DNA. 

Of course, single-molecule measurements aren’t always necessary and could be overkill for certain situations. For example, if there’s a protein biomarker associated with a disease, a broad look may be all that’s needed to show whether the protein abundance is elevated (or reduced) in a sample. In other cases, including early detection of some disease biomarkers, the proteins are in low enough abundance to require single-molecule sensitivity.  

Single-molecule analysis with the Nautilus Platform 

We’re developing the Nautilus Platform to enable more accessible, efficient, and sensitive proteomics. This next-generation proteomics technology is designed to make use of high-throughput, single-molecule analysis that will supercharge discovery for the future. Learn more about our technology and the steps we’re taking to design the ideal proteomic analysis platform here.