What are proteoforms?

You may have heard that there are roughly 20,000 genes in the human genome. One might assume that, since genes encode proteins, there can be no more than 20,000 different proteins in any given cell. However, this is far from the case. In the process of transcribing the information in a gene and later translating into a protein, numerous biological processes can result in modifications to the final protein:  

• The transcription process may begin at different places in the gene 
• Segments of the gene may be edited out 
• Transcription may end prematurely resulting in truncation of the protein 
• Once this information is used to create a protein, the protein itself can be modified with a variety of attachments such as small molecules, sugars, and even other proteins  

All this rearranging and modification explodes the number of possible “proteoforms” that could theoretically be produced in a human cell from 20,000 to millions (Aebersold et al 2018). 

The modifications that make up proteoforms can drive biologically interesting changes to protein function. Indeed, the precise mix of proteoforms in a cell can have great impacts on cell processes, organ function, and total body system function. Thus, knowing more about the proteoforms present in healthy and diseased cells may give scientists great insights into how cells and tissues operate. Unfortunately, most proteomics technologies used today cannot distinguish proteoforms, can only see a small fraction of them, or do not have the resolution to capture multiple modifications and their precise composition within a sample.​
 

• Low sensitivity may limit the proteoforms that can be observed:
  There are many different ways proteins can be modified to create a wide array of proteoforms. However, for any given cell it’s possible that only a few proteoforms will be present in high enough abundance to detect with current methods. There may be low abundance proteoforms that have functional impacts on the cell, but they will be hard to see – tyrosine phosphorylation is a good example, it is known to be extremely important in some proteins but overall levels of tyrosine phosphorylation are very low in most samples.
   

• Limitations due to protein digestion:
  On standard proteomics platforms, full length proteins are often digested into small peptides to facilitate analysis. These peptides are identified, and analysis software makes assumptions when piecing together full proteins from the identified peptides. This makes it difficult to map which individual proteins have modifications and instead provides a bulk report of modifications in aggregate. Additionally, not all the peptides that make up a full protein will be observed. Some peptides will be left out of the analysis and those peptides could have modifications that are missed.
   
• Bulk measurements:​
  ​When affinity reagents are used to detect specific types of proteoforms, they are typically used to analyze bulk samples of many proteins at once and individual proteins cannot be resolved. Thus it can be difficult to detect whether individual proteins are modified in multiple ways and to what extent they are modified. For example, it may be difficult to determine if a sample contains three sets of a single protein species modified in three separate ways or one set with three modifications on each protein molecule. 

The benefits of detecting proteoforms with single-molecule precision

There are many unknowns when it comes to the world of proteoforms. Scientists have made theoretical predictions about the number of proteoforms that could possibly exist, but it is not at all clear what fraction of these proteoforms are actually made, how they might be distributed across cells, and what functional consequences they have.  

Nonetheless, we do know that some protein modifications are highly consequential. For example, the addition of methyl groups to proteins that scaffold DNA can turn off genes that would otherwise suppress cancer development and similar modifications altering gene expression are associated with various cancer outcomes (Nebbioso et al 2018). With the ability to identify specific proteoforms at the single-molecule level in cells and tissues, scientists can more confidently associate proteoforms with various states of health and disease. Such research may lead to better biomarkers that more accurately indicate when a person has a particular disease or the identification of specific proteoforms that may make better targets for novel drugs. 

Enhancing proteoform detection with the Nautilus platform

 At Nautilus, we’re developing a proteomics platform that is designed to make it easier to identify proteoforms with increased precision. 

• The platform analyzes single protein molecules in isolation:
  This gives the platform high sensitivity and thus more potential to detect relatively rare, but possibly important, proteoforms. Single-molecule detection also makes it possible to determine the extent to which individual proteins are modified and overcomes the problems of bulk measurement.
   
• The platform analyzes full-length proteins:
  The platform does not identify proteins from peptide data, so there are no assumptions about how peptides might map to a full-length protein. With full-length analysis, it is theoretically possible to identify modifications across the entire length of a protein and map the precise composition of individual proteoforms within a sample.​

By getting a more in-depth view of the proteoforms that exist across samples, we’ll take the first step toward understanding how these proteoforms impact health and disease. Once scientists observe differences in proteoform abundance across samples, they can investigate whether those differences are functionally significant. The Nautilus Proteome Analysis Platform aims to bring us a long way toward accomplishing these goals.