Protein Sequencing

The large-scale study of the proteins produced by an organism, proteomics, is crucial to understand cellular processes underlining diseases and to identify specific indicators linked to diseases in a biological sample, also known as biomarkers. Perhaps surprisingly, it is still not clear which of the ~20,000 human genes are translated into proteins. Therefore, major efforts are underway to identify these genes, including the Human Proteome Project and the Human Protein Atlas project and ProteomeXchange consortium.

Another problem is that many proteins are modified post-translationally, and such modifications are often present in very few copies. Therefore, bulk technologies such as mass spectrometry are not always capable of capturing the full range of protein modifications.

We are developing a technology based on nanopores to sequence individual proteins. This technology will allow the fast and low-cost analysis of proteins, including low abundance proteins and their post-translational modification

Biological nanopore–based protein sequencing and recognition is challenging due to the folded structure or non-uniform charge of peptides. Over the past few years we have solved many fundamental problems on the analysis of proteins and peptides with nanoopres. In particular we have:

• Developped a FraC nanopore for peptide sequencing

• Showed that we can capture all peptides, despite their charge, using alpha helical and beta barrel nanopores (Nature comm. 2017)

• Showed that for certain conditions the volume (related to the mass) of peptides can be identified from the current blockade (Nature comm. 2019)

• Proteins can be identified from their peptide profile (Nature comm. 2021)

• Post-translational modifications can be identified and quantified (Nano Lett. 2021)

  
  

• A proteasome nanopore to control the transport of proteins and peptides across a nanopore (Nature Chem. 2021)

• Aromatic residues in the nanopore improve the recognition of peptides (ACS Nano 2021)

• Beta barrel nanopores can be engineered to identify all peptides (ACS Nano 2022)

• An electroosmotic flow can be engineered to translocate unstructure polypeptides against electrophoretic forces (Nature Biotech. 2023)

• Isobaric and cyclic peptides can be identified with nanopores (JACS 2023)

BOX 1: DNA SEQUENCING

This approach builds on what is arguably the most successful enterprise of nanopore technology: the sequencing of single DNA molecules. The idea of using a protein nanopore to expedite sequencing at the single-molecule level first appeared in the literature in 1996. Following a decade of investigations of the physical mechanism of the DNA translocation process, in 2005 it was shown that nanopores are capable of single-base recognition in immobilized strands. In 2008 all four DNA base could be distinguished with engineered pores. To date, three nanopores have been described in the literature to discriminate nucleobases (αHL, MspA, and FraC). In 2008 and then in 2010 it was shown that DNA polymerase enzymes are able to ratchet DNA through the nanopore base-by-base.

Building on this work, a commercial device that sequences DNA, the Oxford Nanopore Technologies (ONT) MinION, is being tested in clinics. The device has a low capital cost, is by far the most portable DNA sequencer available, and can produce data in real-time. Because it can perform long reads, it has numerous prospective applications including the ability of improving genome sequence assemblies and resolution of repeat-rich regions. Arrays of thousands of nanopores allow high-throughput analysis. However, owing to the multiple occupancy of nucleobases inside the nanopore used, the raw output in the MinION is generated not by individual bases but by 5-nucleotide stretches known as k-mers.