Advancing Biomedicine With Single-Molecule Proteomics

Unlocking more and more information from our proteome could deepen our understanding of disease, improve disease detection and influence drug selection to provide more personalized patient care.

Single-molecule proteomics approaches measure each protein in the proteome, one molecule at a time, enabling low-abundance proteins to be detected and analyzed, as well as providing a broad overview of the proteins present. It can also bring new insights to the various proteoforms – post-translationally modified variants of a protein – that are present in a sample.

To find out more about the applications of single-molecule proteomics in biomedicine, and to hear about an emerging next-generation approach, we spoke to Dr. Parag Mallick, chief scientist and co-founder of Nautilus, a biotechnology company dedicated to democratizing access to and unlocking the complexity of the human proteome.

Katie Brighton (KB): Firstly, can you tell me a bit about what single-molecule proteomics is and how this approach might help advance biomedicine?

Parag Mallick (PM): Proteomics is the large-scale study of proteins. By understanding the protein composition of a sample of cells or blood (i.e., what proteins are present and how much of each), it may be possible to understand a disease’s origins, how to detect it as early as possible and how to most effectively treat it. Within any given sample, there are tens of thousands of different types of proteins that span a huge range of abundance, from a handful of molecules to hundreds of millions of molecules. Typical proteomics methods perform “bulk measurement”, in which one measurement is made of all the molecules of a given type of protein or protein fragment.

In single-molecule methods, each individual protein molecule is measured on its own – one at a time. That measurement determines the identity of each individual protein molecule in a sample. To assess how much of each type of protein is present, one simply counts up the identifications. A single-molecule approach enables unprecedented sensitivity and quantitative accuracy. This is particularly crucial for those low-abundance proteins which are present in only a handful of copies per sample. 

Additionally, if such an approach is employed on full-length proteins, rather than protein fragments, it becomes possible to measure the molecular diversity of proteoforms – the myriad of forms of a given type of protein that have been altered with diverse post-translational modifications, such as phosphorylations and methylations. 

Drug target discovery, toxicology studies, drug targeting and disease detection are key areas of application in biomedicine. Proteins are the main drivers of cellular functions, and thus are the most common targets for drugs. Novel insights into their abundance, location and interaction could help physicians evaluate the individual proteomes of their patients and personalize how and when drugs are administered. Additional advances can be made on a broader scale to enhance the accuracy of diagnostics and therapeutics, potentially addressing diseases for entire populations before or at their early onset. If the abnormal presence of a protein can be precisely identified in diseased tissue, said molecule could be further studied as a potential driver of toxicity to guide new treatments. Highly sensitive technologies will enable researchers to locate and differentiate rare protein targets, examine how lead compounds affect cells and more.

KB: Protein Identification by Short epitope Mapping (PrISM) is a next-generation approach to proteomics. Can you explain how it works?

PM: PrISM is the framework that underpins our single-molecule proteomics platform. To enable the analysis of a broad set of proteins or targeted search for a proteoform, we designed PrISM to repeatedly interrogate billions of individual, intact protein molecules with a diverse array of novel affinity protein-binding reagents.

In PrISM, proteins from the sample are first attached to special nanoparticle scaffolds that have been designed so that each scaffold can only hold exactly one protein molecule. Next, these scaffold-protein conjugates are deposited onto a hyper-dense, nanofabricated chip. This chip is capable of holding billions of intact protein molecules.

Across hundreds of cycles, fluorescently labelled antibodies are introduced to the chip where they bind the immobilized proteins. Using a sophisticated imaging system, we detect when probes have bound to a particular protein molecule. Each antibody targets short epitopes of 3-4 amino acids. We have designed these probes to stochastically bind to many different protein molecules with high affinity and low specificity, eventually generating the unique binding patterns of each molecule to be decoded by our machine learning algorithms for protein identity. The protein molecules are then counted to achieve quantification.

Enabled by our PrISM approach, the Nautilus Proteome Analysis Platform is inclusive of the system, consumables and software our team has designed to comprehensively, rapidly and reproducibly measure proteins in any given sample.

KB: What new insights have been gained by using PrISM?

PM: Our PrISM approach can be applied to broadscale proteomics or targeted proteoform studies across the life sciences. With simulations showing that PrISM can identify more than 95% of the proteomes of a wide range of organisms – far beyond the capabilities of existing technologies – the potential for improvements to healthcare, agriculture and basic science research is immense.

In broadscale proteomics, researchers can use PrISM to determine which and how many proteins are present in a sample. Our specially designed, proprietary and multi-affinity probes are applied parallel to each protein molecule on the chip in iterative cycles, building a detailed picture of the existing proteins.

To confirm whether a specific variant of a protein is present, a targeted probe is introduced rather than a mixture of multi-affinity probes. This process determines which coordinates on the chip contain their proteoform of interest.

Our team is currently working with biopharmaceutical and academic partners to target key disease areas including cancer, neurodegeneration and cardiovascular disorders to move society closer to revolutionary drugs for these conditions.

KB: Nautilus recently attended the Human Proteome Organization (HUPO) conference in Cancun and the US HUPO event in Chicago, what were your main takeaways from the events?

PM: The research shared through talks and posters at the global and US HUPO conferences are vital to spurring advancement across the entire field. This year, we had the opportunity to present our single-molecule approach to proteomics, and we saw attendees come away with incredible enthusiasm for these new technologies that offer improved access to the proteome.

Single-molecule approaches are becoming increasingly attractive to researchers, from those who study the mechanisms behind diseases such as cancer, to others who focus on translational applications for drug development. At HUPO Cancun, next-generation methods that are currently being leveraged for disease research demonstrated the possibilities of measuring proteoforms and their effect on key biological processes that may shift an individual’s state of health to one of sickness.

These themes were encouraged by our introduction of the Nautilus First Access Challenge, a competition for early access to single-molecule proteomic insights from our PrISM technology. At the US HUPO conference, we selected three research proposals as winners of the First Access Challenge; researchers from the University of Southern California, the Buck Institute for Research on Aging, and Brigham Young University will be awarded early access to proteomic data through Nautilus’ single-molecule protein analysis platform to address critical research areas including gliomas, acute kidney injuries and pulmonary fibrosis, respectively.

KB: How do you expect the single-molecule proteomics landscape to evolve in the next half of 2023 and beyond?

PM: We are currently in the midst of a broad proteomics revolution for breakthrough technologies and research that will continue to build momentum this year. Industry scientists are now looking to the rapidly advancing single-molecule approach due to its long-sought comprehensive proteome coverage, sensitivity and ease of use. I anticipate that the development of such technologies will become significantly more focused with further study and validation, not only in the lab but in clinical applications as well.

Access to these technologies will broaden with additional advancement and growth within the industry, in many ways mirroring the rise of genomics in recent decades. For example, our team continues to hone the Nautilus platform and pilot single-molecule applications with both longtime collaborators like Genentech and new parties including the Translational Genomics Research Institute, with whom we initiated a collaboration in January.

KB: How will your new partnership with the Translational Genomics Research Institute (TGen) help advance cancer research?

PM: We’ve partnered with TGen to apply single-molecule proteomics to the study of a rare and often fatal childhood cancer called diffuse intrinsic pontine glioma (DIPG). A high-grade malignant brain tumor, DIPG is characterized by genetic mutations that affect how histone proteins in the brainstem are programmed. Investigating the proteins and post-translational modifications that cause these epigenetic changes are the chief focus of our collaboration, which provides TGen with advance access to data generated on our platform that is not obtainable with existing protein analysis techniques. Together we will achieve a better understanding of the proteoform variation that may enable novel ways to diagnose and treat this deadly disease, and additionally apply our approach to other cancers of critical need.

This program marks Nautilus’ fifth early collaboration with leading research teams in oncology, neurodegenerative disease and cardiac disorders. Through these partnerships and the ongoing advancement of our platform, our goal is to provide proteomic insight for research at any stage, from early phases to clinical studies.

KB: With the opening of a new office in San Diego, how does Nautilus plan to expand its team and platform this year?

PM: We are thrilled to establish roots in San Diego, another top biotech and life sciences community well-suited for our scientific commitment and culture. Our talented team is poised for growth over the year as we move closer to launching our platform, both of which our new office and laboratory space in San Diego will support. The expansion comes at an opportune time for our organization. This year, we plan to broaden access to our proteome analysis technology ahead of commercial launch in 2024. It is an exciting time for Nautilus and the proteomics industry as a whole – we urge everyone seeking to measure the proteome at the single-molecule level and at scale to stay tuned.

Dr. Parag Mallick was speaking to Katie Brighton, Scientific Copywriter for Technology Networks.