Targeted proteoform studies 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 known as 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 targeted proteoform studies on the Nautilus Proteome Analysis Platform. We also describe the overarching problems expected to be solved by the platform and discuss a few ways targeted proteoform studies may be applied in future research efforts. In a companion blog post, we cover broadscale proteomics.

Targeted proteoform studies on the Nautilus Proteome Analysis Platform

When performing targeted proteoform studies, researchers generally want to quantify all the proteoforms or a specific subset of proteoforms of their protein of interest. Today people can look at a single or small handful of modifications in bulk with affinity reagents or they can map full proteoforms with top down mass spectrometry. We aim to leverage off the shelf affinity reagents to analyze proteoforms at more depth than possible today. The Nautilus Proteome Analysis Platform’s single-molecule resolution is designed to enable researchers to determine the quantities of specific proteforms with unparalleled precision.

Targeted proteoform studies are expected to follow a similar workflow to broadscale proteomics on the Nautilus platform. First, a researcher would isolate proteins (or a subset of proteins) from their sample of interest and functionalize the proteins for automated deposition on our 10-billion landing pad arrays. However, instead of flowing multi-affinity probes over the array as in broadscale proteomics, the instrument would flow protein and proteoform-specific probes over the array to identify landing pads containing their proteins of interest and the precise proteoforms present.

Solving challenges in targeted proteoform studies

One of the major difficulties in targeted proteoform studies on other platforms lies in distinguishing individual proteins modified in multiple ways from multiple proteins modified in separate ways. This is because other proteomics platforms often make bulk measurements or break proteins into peptides losing data on individual, intact proteins. In contrast, the Nautilus platform is designed to enable researchers to probe single, intact proteins. Thus, researchers can determine the extent of modification to individual proteins and get a more precise understanding of individual proteoform quantities in a sample.

A second major difficulty on other platforms involves the creation of probes targeting distinct modifications on specific proteins. Often it can be difficult to design probes that are specific enough to differentiate one modified protein from another. On our platform, hyper-specific affinity reagents are not necessary for all modifications. Once a protein has been identified on a landing pad using our broadscale approach, researchers can use reagents that identify a class of modifications, such as pan-phosporylation antibodies. If these localize to landing pads known to contain paritcular proteins, researchers can be confident that those proteins are modified.

Applications of targeted proteoform studies

Different proteoforms derived from a single gene can function in very different ways. For example, many cellular signaling pathways are activated by phosphorylation. Abberant activation of such pathways can lead to increased cell growth and give rise to cancer. In another example, tau protein hyperphosphorylation is known to be associated with the progression of Alzheimer’s disease. Looking to more basic biology, in both heath and disease, modifications to histones scaffolding DNA can change how genes are expressed. The collection of proteoforms in a cell can thereby have huge impacts on gene expression and cellular function. Measuring proteoforms at high resolution can thus lead to the identification of novel biomarkers as well as new avenues in drug development.

Indeed, researchers in the Kelleher lab at Northwestern University recently used top-down proteomics (a type of mass spectrometry-based proteomics) to identify proteoforms present in five different tissues. Many of the proteoforms detected appear to relate to the functions of these specific tissues. This kind of work is very challenging now and often requires customized workflows and instrument set-ups but could become far more accessible with new technologies like the Nautilus platform. Similar targeted proteoform studies are expected to lead to a much better understanding of the ways proteins dictate cellular function. This kind of knowledge is essential for the development of novel drugs that target dysfunctional biological pathways specific to certain tissues or organs.

An exciting future for targeted proteoform studies

We hope to make targeted proteoform studies accessible to researchers in many different fields through the Nautilus Proteome Analysis Platform. These studies should complement broadscale proteomics to provide researchers with a clear picture of the many different proteoforms in a biological sample. We expect such understanding to enable scientists to find new biomarkers for disease, develop novel drugs, create a new paradigm in precision medicine, augment biological functions for biotechnological purposes, and find new ways to approach existential crises facing humanity.

Check out some of our other blog posts for more on the fascinating applications of proteomics.