Broadscale proteomics on the Nautilus Platform

When talking about the revolutionary proteomics work we hope to enable with the Nautilus Proteome Analysis Platform, we think about two general research modalities: 

• Broadscale proteomics 
• Targeted proteoform studies 

Broadscale proteomics is an approach that provides researchers with a snapshot of protein identities and abundances across the entire proteome. Targeted proteoform studies, on the other hand, zoom in on a particular protein or set of proteins and quantify their many possible modifications and variations (their proteoforms). Both modalities are designed to generate novel and impactful biological insights and both are necessary to ensure the proteomics revolution bears fruit. 

In this blog post, we cover the technical aspects of broadscale proteomics on the Nautilus Proteome Analysis Platform. We also describe the overarching problems in proteomics expected to be solved by the platform and discuss a few ways broadscale proteomics may be applied in future research efforts. In a companion blog post, we’ll dive into targeted proteoform studies.

Broadscale proteomics on the Nautilus Platform

We aim to achieve broadscale proteomics using  “Protein Identification by Short Epitope Mapping” or PrISM. We cover this method in detail in our recent preprint, but, in brief, it is designed to enable our platform to identify and quantify more than 95% of the proteome in a comprehensive, sensitive, reproducible, and rapid way. It is expected that this methodology will cover the wide dynamic range of the proteome with an accessible, integrated workflow from sample to insight. 

Using PrISM , our platform is designed to repeatedly interrogate billions of intact protein molecules with a diverse set of novel multi-affinity probes. Single, intact proteins from a sample are first attached to individual landing pads on a 10 billion landing pad array. Then, the platform flows multi-affinity probes over the array in many independent cycles. Each multi-affinity probe is designed to bind short amino acid sequences present in many proteins. High-resolution fluorescent imaging techniques determine which probes bind at which landing pads on the array in each cycle. Although an individial cycle cannot determine protein identity since the short sequences are present in many proteins, binding patterns unique to each protein emerge through iterative cycles of interrogation and are recognized with machine learning. This enables detection of >95% of the proteome with roughly 300 probes. Because proteins are identified at the single-molecule level, protein quantification is performed by counting how many times a protein is identified. 

The 10 billion landing pads on our nano-fabricated arrays should enable us to measure proteins varying in abundance over 9 orders of magnitude of dynamic range with more than 95% proteome coverage across species and sample types.  

Aiming to solve major issues in broadscale proteomics

Older proteomics technologies like mass spectrometry are routinely used for broadscale analysis but rarely cover the full dynamic range of the proteome. Thus, signals from high abundance proteins often drown out those from low abundance proteins and likely mask essential biological insights. These technologies also infer protein identity from pools of protein fragments (peptides) instead of full-length proteins. This makes it difficult to generate reproducible, quantitative results. Other protein analysis techniques rely upon the creation of highly specific affinity reagents for each and every protein in the proteome. This is very difficult to scale to the 20,000+ proteins in the human proteome and makes it challenging to do cross-species comparisons in translational studies. 

On our platform, multi-affinity probes, single-molecule, intact protein analysis, and robust machine learning algorithms are designed to make it easier to capture the breadth and depth of the proteome. 

Applications of broadscale proteomics

To understand how such analyses could be used, it’s informative to look at previous proteomics studies. We hope to enable scientists to vastly expand on similar work in the future, and recent publications represent just the tip of iceberg in terms of what researchers can reveal with comprehensive broadscale proteomics. 

For example, researchers working with Nautilus Scientific Advisory Board member Professor Ruedi Aebersold used a multiomics approach to discover biological differences across HeLa cell lines. These rapidly growing cells are commonly used in many studies of human cell biology, but acquire important physiological differences as they are propagated across labs . Broadscale proteomics revealed that differences in protein levels across HeLa cell lines lead to vast differences in cellular morphology, growth rates, response to micro RNA transfection, and susceptibility to Salmonella infection. Here, broadscale proteomics revealed differences in a common research tool that could have vast impacts on how that tool is used in the future. These analyses may lead to more reproducible results across many future research efforts using HeLa cells. 

While this study focused on the basic biology of an important research tool, broadscale proteomics has many, many applications in applied biology including: 

• Biomarker discovery 
• Drug development 

• Toxicology 

In addition, our recent white papers dive into applications of proteomics in basic research, precision medicine, and novel cancer treatments. The potential applications of accessible broadscale proteomics are nothing short of inspiring and we’re only scratching the surface. 

An exciting future for broadscale proteomics

Broadscale proteomics studies reveal a wealth of data that can lead to many further experiments and insights. These can truly change the way researchers think about and even conduct experiments. We are excitedly working to make such studies far more accessible, robust, and comprehensive with our novel platform. We cannot wait for the treasure trove of insights we hope to enable.