Current Approaches to Protein Sequencing

Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained by their limited range and inability to analyze unknown proteins. Studying the associated DNA and RNA may help determine protein sequences, but this is an indirect and inefficient approach due to post-translational modifications, alternative splicing, and protein processing, which all influence the protein sequence. Single-molecule protein sequencing is the answer to overcoming many of these challenges, and several groups are making significant advances in the field.

Next-Generation Protein Sequencing Technology with Platinum®
Initially focused on long-read DNA sequencing, Jeff Hawkins, CEO of Quantum-Si, explained that the group shifted toward protein sequencing to address the needs of the field. Their vision was to advance proteomics in the same way that next-generation sequencing transformed the study of DNA. This transition led to the development of Platinum, their flagship protein sequencing platform. Brian Reed, Head of Research at Quantum-Si, explained that Platinum simplifies protein analysis by condensing what would typically require a biophysics lab full of costly equipment into an inexpensive and accessible benchtop instrument.

The key to Platinum’s operation is a combination of amino acid recognizers and aminopeptidases¹. The dye-labeled recognizers bind to the N-terminal amino acid of a peptide chain as the aminopeptidases sequentially degrade the peptide chain. During this process, the device identifies the different amino acids in the peptide by assessing fluorescence intensity, lifetime, and binding kinetics using a semiconductor chip. This approach facilitates real-time sequencing that simplifies the operational steps and reduces the potential for errors, without the need for fluidics. Furthermore, the automated data analysis simplifies this traditionally complex process and allows researchers to identify amino acid sequences without manual intervention.

Hawkins described their Platinum technology as versatile, serving diverse applications across various lab types. This includes in-depth protein analysis in large proteomics cores as well as protein identification in genomics labs without mass spectrometry access. It's also being employed in QA/QC processes by commercial groups, like pharmaceutical and biotech companies, to validate their work. The technology’s versatility and cost-effectiveness, Hawkins emphasized, enable researchers of any experience level to conduct their proteomics in-house. "You can do this yourself and you can get even deeper insights than mass spec,” he stated. “That's the type of transformation we're looking to drive here in the proteomics field."

Quantum-Si recently announced advancements in their technology with the introduction of V2 Sequencing Kits. These kits enhance the performance of the Platinum sequencer, including improvements in the assay process and the addition of a new amino acid recognizer. These enhancements aim to increase the accuracy and efficiency of protein sequencing, allowing for the identification of previously unknown proteins with greater ease. Additional developments to the technology include improvements in the reproducibility of results and a significant reduction in the cost of sequencing each amino acid.

Both Reed and Hawkins believe that protein sequencing will become an integral component of multiomics workflows, and it will fundamentally change the way research is conducted. The ability to directly sequence proteins without reliance on methods like fixed antibody panels opens up new possibilities for research in protein variations, post-translational modifications, and the exploration of proteoforms. “That's really the next frontier of proteomics, and I think Quantum-Si will be at the forefront,” stated Reed.

Fluorosequencing
Founded in 2018, Erisyon grew from a proteomics-focused lab at the University of Texas at Austin, led by mass spectrometrist Edward Marcott. Inspired by frustration with the limitations of mass spectrometry and a desire to develop a proteomics method with the resolution of next-generation sequencing, Marcott and Erisyon’s CTO, Jagannath Swaminathan, conceptualized and developed their proprietary fluorosequencing technology. This strategy combines the precision of fluorescent detection with the specificity of covalent labeling to offer high-accuracy protein sequencing².

"The technology brings a lot of the advantages of next-generation DNA sequencing to proteins," stated Talli Somekh, Co-Founder and CEO of Erisyon. These advantages include single-molecule sensitivity, massive throughput, and absolute quantification with digital counts of proteins in the sample. He emphasized that Erisyon's technology is not intended to compete with established methods like mass spectrometry and affinity assays, but instead, it is used to fill specific gaps for applications that require high sensitivity, unbiased detection, and digital quantification.

Erisyon's protein sequencing technology is based on a degradative strategy, where amino acids are sequentially removed and identified through their fluorescent tags. This labeling, followed by the removal of N-terminal amino acids, generates an incomplete sequence that can be matched against a database to identify the protein. Somekh explained that this process is similar to the popular game show Wheel of Fortune. “You don't need every letter to solve the puzzle, as long as you know the alphabet, you know the dictionary, and you know the category. In this case, since we know the genome and the transcriptome, which determine the proteome, that incomplete sequence is enough to solve the puzzle.”

Somekh highlighted that Erisyon is an applications-oriented group with several collaborators working on specific tools and applications that match the current state of their technology. In particular, fluorosequencing has shown to be valuable in scenarios where other methods fall short in sensitivity, resolution, or fidelity. These key applications include analyzing very low-concentration samples, investigating post-translational modifications like phosphorylation to differentiate healthy from pathological biomarkers, and quantifying specific low-concentration biomarkers in clinical samples such as tumor biopsies.

Several major updates to their fluorosequencing technology were outlined in a recent pre-print³. These developments include the identification of compatible fluorophores, the introduction of extended polyproline linkers to reduce dye-dye interactions, and the expansion of an end-to-end workflow for sample preparation and sequencing. Furthermore, experimental validations demonstrated the platform's capability to sequence peptide mixtures and identify target neoantigens, highlighting its potential for protein and peptide analysis in clinical samples with limited availability.

As for Erisyon’s future, Somekh shared that along with further developments to their technology, their goal is to effectively demonstrate the many applications of fluorosequencing. “That's what we're planning to accomplish in the coming period,” Somekh stated. “Where we're able to demonstrate the tool on specific applications in order to be a lighthouse, to capture people's imagination as to what protein sequencing can be used for.”

Nanopore Sequencing
While no commercially available solution exists for nanopore-based protein sequencing, this is a very promising area of research with industry experts like Oxford Nanopore Technologies (ONT) actively developing solutions. Similar to their DNA sequencing counterparts, nanopore sensors for protein sequencing utilize ionic current flows through nanometer-sized holes in a membrane. The passage of macromolecules through these pores alters the current and generates a signal that can be used to identify individual amino acids.

There are two prevailing strategies for nanopore-based protein sequencing: non-enzymatic and enzymatic methods. The latter is the focus of several labs, like one run by Jeff Nivala, Research Assistant Professor at the University of Washington. Nivala has been developing nanopore sequencing methods since his graduate studies, and his lab’s recent pre-print demonstrates a novel method for long-range, single-molecule protein sequencing using ONT devices⁴. This approach employs a two-step process that uses an electrophoretic force to propel target proteins through a CsgG nanopore, and then a ClpX unfoldase enzymatically pulls the proteins back through the pore. Using these unfoldase motors, proteins are slowly translocated out of nanopores, allowing for the detailed detection of amino acid substitutions and post-translational modifications across long protein sequences. Despite being in the early stages, this technique shows significant promise for protein sequencing.

Giovanni Maglia, Professor of Chemical Biology at the University of Groningen, has made similar breakthroughs using nanopores to identify and fingerprint proteins. The method employed by Maglia's group overcomes the challenges of variable charges of proteins, which prevent their translocation and linearization. An electric field is used to pull charged ions through a CytK nanopore as the unfolded peptides produce distinct current signatures that enable protein identification⁵. This technique was shown to be effective even with proteins that were previously difficult to thread through the nanopore. Maglia plans to commercialize this technology through his startup, Portal Biotech, and make it accessible to labs and medical professionals.

Research groups like the Bayley Lab from the University of Oxford are also making headway toward a nanopore-based protein analysis method. Bayley’s team recently demonstrated the ability to detect structural variants in proteins at the single-molecule level ⁶. Their method involves unfolding proteins into linear chains and using an electro-osmotic flow to pass them through an engineered staphylococcal α-hemolysin (αHL) pore. During this process, changes in electrical current through the nanopores are measured and can be used to detect post-translational modifications. Notably, this technology does not require labels, enzymes, or additional reagents, and can identify modifications like phosphorylation, glutathionylation, and glycosylation in long protein chains. The researchers anticipate that this new method has the potential to be integrated into portable devices for rapid protein analysis in single cells and tissues, opening new possibilities for personalized medicine and diagnostics.

Final Thoughts
The contributions from these innovative groups highlight the significant progress being made in protein sequencing. Through the continual development of their technologies, they are leading the way for advancements in scientific research and diagnostics, offering new capabilities in the sequencing, identification, and detailed quantification of proteins.

References

1. Reed, B. D., Meyer, M. J., Abramzon, V., Ad, O., Ad, O., Adcock, P., Ahmad, F. R., Alppay, G., Ball, J. A., Beach, J., Belhachemi, D., Bellofiore, A., Bellos, M., Beltrán, J. F., Betts, A., Bhuiya, M. W., Blacklock, K., Boer, R., Boisvert, D., Brault, N. D., … Rothberg, J. M. (2022). Real-time dynamic single-molecule protein sequencing on an integrated semiconductor device. Science, 378(6616), 186–192. https://doi.org/10.1126/science.abo7651
2. Swaminathan, J., Boulgakov, A. A., Hernandez, E. T., Bardo, A. M., Bachman, J. L., Marotta, J., Johnson, A. M., Anslyn, E. V., & Marcotte, E. M. (2018). Highly parallel single-molecule identification of proteins in zeptomole-scale mixtures. Nature Biotechnology, 36, 1076–1082. https://doi.org/10.1038/nbt.4278
3. Mapes, J. H., Stover, J., Stout, H. D., Folsom, T. M., Babcock, E., Loudwig, S., Martin, C., Austin, M. J., Tu, F., Howdieshell, C. J., Simpson, Z. B., Blom, T., Weaver, D., Winkler, D., Vander Velden, K., Ossareh, P. M., Beierle, J. M., Somekh, T., Bardo, A. M., Anslyn, E. V., … Swaminathan, J. (2023). Robust and scalable single-molecule protein sequencing with fluorosequencing. bioRxiv, 2023.09.15.558007. https://doi.org/10.1101/2023.09.15.558007
4. Motone, K., Kontogiorgos-Heintz, D., Wee, J., Kurihara, K., Yang, S., Roote, G., Fang, Y., Cardozo, N., & Nivala, J. (2023). Multi-pass, single-molecule nanopore reading of long protein strands with single-amino acid sensitivity. bioRxiv, 2023.10.19.563182. https://doi.org/10.1101/2023.10.19.563182
5. Sauciuc, A., Morozzo Della Rocca, B., Tadema, M. J., Chinappi, M., & Maglia, G. (2023). Translocation of linearized full-length proteins through an engineered nanopore under opposing electrophoretic force. Nature Biotechnology. https://doi.org/10.1038/s41587-023-01954-x
6. Martin-Baniandres, P., Lan, W. H., Board, S., Romero-Ruiz, M., Garcia-Manyes, S., Qing, Y., & Bayley, H. (2023). Enzyme-less nanopore detection of post-translational modifications within long polypeptides. Nature Nanotechnology, 18(11), 1335–1340. https://doi.org/10.1038/s41565-023-01462-8