The importance of single-molecule proteomics

You may have seen or heard the term “single-molecule” thrown around recent scientific papers and talks. While it can certainly be a buzzword, true single-molecule technologies have the impressive ability to interrogate their target analytes individually and independently. They provide information on each and every analyte rather than averaging information across a sample. Thus, their output data is provided at the greatest possible resolution. Single-molecule analyses reveal information that would be imperceptible through bulk measurement techniques. 

Below we describe why single-molecule analysis is essential to get a complete picture of the proteome. In addition, we share how we’re designing the Nautilus Proteome Analysis Platform to provide single-molecule protein analysis at an unprecedented scale. We expect the Nautilus Platform to give researchers novel insights into the proteome, the mechanisms underlying disease, and the fundamental functions of life itself.  

Single molecule analyses theoretically provide the greatest level of sensitivity and can precisely map protein modifications for truly comprehensive proteomic analyses

Traditionally, proteomics technologies analyze proteins in bulk. That is, they assess proteins and their properties using technologies that measure signals coming from multiple proteins at once. These signals cannot be deconvoluted back to individual proteins. For example, in mass spectrometry, this comes in the form of identifying peptides derived from many proteins in the sample. However, it’s not usually possible to determine if the identified peptides came from the same original intact protein.  

These bulk measurement technologies are useful in that they can identify abundant proteins, but single-molecule analyses can go further: 
  

• Single-molecule analyses provide the greatest level of sensitivity possible to detect the rarest proteins in a sample 

Bulk measurement technologies are rarely sensitive enough to confidently measure proteins present in small amounts. For example, to identify proteins with mass spectrometry, researchers may need to detect a certain number of peptides that bioinformatics tools can confidently associate with one protein species and no or few others. They must therefore infer the identity of intact proteins from an assortment of peptides. With similar sequences across a variety of proteins and a lack of full protein sequence coverage, such inferences can be difficult to make and are sometimes inaccurate, especially for low abundance proteins.  

Single-molecule analysis technologies are, by definition, able to detect the lowest amount of protein possible – a single protein molecule. Such technologies can theoretically detect all proteins in a sample regardless of their levels. Researchers just need to be able to make enough single-protein measurements to identify all the protein molecules across the dynamic range of proteome with confidence (more on this later).  

• Single-molecule analyses may be able to detect all modifications found on individual proteins  

Proteins can be modified in many different ways to produce millions of proteoforms from thousands of genetically encoded protein species, and one protein can also be modified in multiple ways. Technologies that detect such modifications in bulk samples usually cannot distinguish single proteins with multiple modifications from multiple proteins with single modifications. For example, mass spectrometers usually cannot distinguish which proteins each modified peptide came from. Thus, it will be unclear if the original, intact protein had multiple modifications or multiple protein molecules had some subset of the modifications. There are efforts to improve “top-down” proteomics methods that analyze intact proteins and retain such information, but these are not widely used today.  

Technologies that assess modifications on intact proteins at the single-molecule level can theoretically identify all the specific modifications present on a single protein. If these technologies can interrogate single proteins in multiple ways either simultaneously or sequentially, they should be able to show researchers all the different ways single proteins are modified.   

The Nautilus Proteome Analysis Platform is designed to achieve comprehensive single-molecule analysis

There are a variety of next-generation single-molecule proteome analysis methods. These all aim to achieve highly sensitive and comprehensive proteomic analysis as outlined above, but can have a few issues:  

• The protein inference problem – Many technologies, like single-molecule sequencing technologies, still analyze peptides and not intact proteins. Like mass spectrometry, these technologies must infer full protein sequences from peptides and it will not always be clear which peptides were from which original protein molecules. 
    

• Limited dynamic range – Many single-molecule proteomics platforms cannot cover the full dynamic range of the proteome. They cannot isolate enough peptides or intact proteins to reveal quantities of both high and low abundance proteins. For technologies that analyze peptides, this is a particular problem because they must isolate and analyze many peptides to infer the identity of a single protein.
    
• Destructive analysis – Many single molecule proteomics platforms destroy proteins and peptides as they analyze them. Thus, these proteins and peptides cannot be re-analyzed to gain more information about them or to increase confidence in their identification.  

We’ve designed the Nautilus Proteome Analysis Platform to achieve true single-molecule analysis while avoiding the problems described above. On the Nautilus Platform, we aim to isolate individual, intact protein molecules and repeatedly interrogate them with fluorescent probes non-destructively. Such probes are designed to determine protein identity through our PrISM framework. In addition, probes designed to detect modifications can potentially identify all the ways single proteins are modified. Thus, with the Nautilus Proteome Analysis Platform we aim to enable researchers to interrogate even the rarest proteins and determine how they’ve been modified.   

Importantly, we’re designing our platform with arrays that can accommodate up to 10 billion single-molecule protein landing pads that can be imaged independently. With super-Poisson loading, these arrays are designed to be highly occupied by billions of proteins during every sample run. This should enable researchers to use broadscale proteomics to identify proteins across the majority of the dynamic range of the proteome for most sample types.   

With this technology, we aim to provide researchers with unprecedented access to the proteome. We hope this will enable a revolution in biological research with applications across basic research, neuroscience, oncology, drug development, biomarker discovery, and much more!