Unlocking the Future of Proteomics with Nanopore Protein Sequencing
Fedor Lipskerov
Founder Synaptiflora | PhD Student in Molecular Biology at Technion | Science Communicator
Hey everyone! As many of you know, I'm deeply involved in studying proteins and their functions. Usually, we use Mass Spectrometry (MS) - the gold standard in large-scale protein characterization. While powerful, MS has its limitations and today we are going to discuss the next big thing in single-cell proteomics-Nanopore Protein Sequencing.
The Challenge with Current Techniques
In a single mammalian cell, protein copy numbers can range from 1 to 10^8. The lack of a PCR-like amplification strategy for proteins makes these challenges even more pressing. Mass spectrometry, while accessible, can fall short on sensitivity and coverage.?
Nanopore Protein Sequencing
The rapidly advancing field of nanopore protein sequencing technology holds tremendous promise, especially in the realm of single-cell proteomics. It offers:
A Glimpse into Recent Research?
In my view, one of the pioneers in the field of protein nanopore science is Giovanni Maglia . Today, I'd like to highlight some of his team papers that are significantly advancing us toward the reality of protein sequencing.
20S proteasome nanopore
As someone doing PhD on the proteasome, I was fascinated by the research that incorporates a 20S proteasome into an artificial nanopore. This clever assembly acts as a multi-component molecular machine, that controls the unfolding and linear movement of proteins through the nanopore. This nanopore leverages proteasome functions by unfolding selected substrate proteins and allowing for the analysis of fragmented peptides and intact polypeptides.
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CytK nanopore
Traditionally, the non-uniform charge of proteins has posed challenges for their translocation and identification through nanopores. However, using an electroosmotic flow (EOF) in the CytK nanopore can enable the unidirectional movement of unstructured polypeptides, even against a strong electrophoretic force.
This method does not require any denaturants or electrophoretic tags, thus allowing the transport of unmodified, native proteins. Molecular dynamics simulations reveal that the electroosmotic-dominated force has a comparable strength to the electric force on single-stranded DNA, making it a promising mechanism for protein identification and sequencing.
What sets this work apart is its potential to extend the utility of EOFs not just for proteins but also for other polymers that may be weakly or non-uniformly charged. This breakthrough lays the foundation for a more universal application of nanopore technology in the study of diverse biomolecules, setting the stage for possible advances in protein sequencing and beyond.?
A Sea Change in Accessibility ??
The technology's accessibility is expected to expand significantly, thanks to simpler instrumentation, lower costs, and reduced expertise required. This promises to democratize the world of proteomics.
The Future is Bright
Nanopore protein sequencing offers unique advantages such as higher sensitivity, wider throughput, and novel data modalities compared to MS. I am incredibly optimistic about the unique potential of this technology, and I definitely want to use it someday!
Feel free to share your thoughts and insights on this fascinating topic. Let's keep the conversation going! ????????????
Papers:
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#Proteomics #NanoporeSequencing #MassSpectrometry #SingleCell #MolecularBiology #Innovation #Technology