Believe the Hype: Nanopore Proteomics Is Moving Forward

GEN BiotechnologyVol. 1, No. 1 Views & NewsFree AccessBelieve the Hype: Nanopore Proteomics Is Moving ForwardYujia Qing and Hagan BayleyYujia QingDepartment of Chemistry, University of Oxford, Oxford, United Kingdom.Search for more papers by this author and Hagan Bayley*Address correspondence to: Hagan Bayley, Department of Chemistry, University of Oxford, Oxford, UK, E-mail Address: hagan.bayley@chem.ox.ac.ukDepartment of Chemistry, University of Oxford, Oxford, United Kingdom.Search for more papers by this authorPublished Online:16 Feb 2022https://doi.org/10.1089/genbio.2022.29008.qbaAboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail A Dutch team led by Cees Dekker reports a significant step in applying nanopore characterization to peptides.Following the success of DNA and RNA sequencing with nanopore technology over the past decade—developed commercially by Oxford Nanopore Technologies—research on polypeptide “sequencing” with nanopores is in vogue. While sequencing itself may be a distant and perhaps needless goal, the identification of modifications to polypeptides and the pinpointing of their locations within polypeptides chains is a pressing need. This is because the 20,000 or so protein-encoding genes found in humans spawn several million different proteoforms, which perform critical roles in cells that cannot be predicted from the genome or even the transcriptome.There are around 400 distinct covalent post-translational modifications (PTM) of polypeptide chains, common examples being phosphorylation and glycosylation, which occur in myriad combinations and permutations. For example, the p53 tumor suppressor protein contains 24 phosphorylation sites. Most PTMs are reversible. In addition, the irreversible production of protein variants also occurs through phenomena including proteolytic maturation, RNA editing, RNA splicing and somatic mutation and hypermutation.In an article published in late 2021 in Science, Cees Dekker's team at the Delft University of Technology (TU Delft) in the Netherlands adapted the nanopore nucleic acid sequencing platform for peptide discrimination (Fig. 1A).1 Previously, DNA or RNA sequencing has been enabled by motor proteins that can ratchet polynucleotides base-by-base through a nanopore detector. To characterize peptides in the same way, Dekker's team conjugated a peptide of interest with an oligonucleotide and used a helicase capable of half-nucleotide (∼0.33 nm) stepping, which is close to the distance between amino acids in an extended polypeptide chain.FIG. 1. Nanopore characterization of peptides and proteins.(A) A system reported by Cees Dekker's team is composed of a helicase (green), which ratchets an oligonucleotide-peptide conjugate through an MspA pore (gray). Re-reading of the peptide was enabled at a high helicase concentration.(B) In an idealized nanopore-based system for protein characterization, an intact protein (pink) would be unfolded during translocation through the nanopore, during which, for example, post-translational modifications could be read.The researchers next moved three negatively charged synthetic peptides that differed by a single amino acid (Asp, Gly, Trp) at a selected position through an MspA pore. After a read, at high concentrations of helicase, a second bound copy of the enzyme allowed the DNA to retract into the pore and be translocated again. In this way, hundreds of re-reads were possible, generating consensus signals that allowed the three “mutants” to be distinguished with high confidence. A Chinese team established a similar system at around the same time with a phi29 DNA polymerase as the motor protein,2 but this enzyme does not allow multiple reads of the same peptide.Despite the appeal of re-reading, the approach has limitations. It is confined to short reads within peptides; about 12 steps were demonstrated in the Science article. The peptides were not typical, comprising entirely negatively charged Asp and Glu residues, except for the “mutated” residue, so that they could be pulled into the nanopore and stretched in an applied potential. We should also note that the amino acid sequences of the polypeptides were not read, rather the signals from the three variants were distinguished. Further, the single-site alterations were manifested in signal changes that occurred over 8 steps and each read took more than 10 seconds.This approach might serve as a variant of peptide fingerprinting, providing more information than the simple recording of dwell times and blockade amplitudes of peptides released in a digest.3 However, it is obvious that the approach cannot be used to sequence or map variants of protein subunits of say 100 or 1,000 residues. To achieve this, proteins must be translocated in their entirety and variations mapped as the extended polymers pass through a nanopore (Fig. 1B).Early efforts in this area showed that proteins could be unfolded with denaturants before translocation or sequentially unfolded during translocation by pulling with a force as low as 10 pN either with a covalently attached leader DNA4 or by using a proteasome-associated translocase.5 Some remarkable nanopore engineering in this area6 lately suggests that significant improvements in polypeptide translocation are imminent. There has been a recent flurry of articles on the identification of amino acids and small peptides.7 But the findings have hardly been surprising given early results in stochastic sensing in which small molecules,8 including enantiomers,9 were distinguished. Indeed, simple patterns of phosphorylation have already been distinguished in a translocating polypeptide chain, albeit in an especially favorable circumstance.10Required are signal processing techniques that account for the effects of flanking residues in the vicinity of a nanopore reading head. One flanking residue on each side of an amino-acid residue generates 400 environments (202) and two on each side 160,000: potentially a formidable problem but mitigated by a ratcheting process and knowledge gained from nucleic acid sequencing. If these issues concerning polypeptide translocation and amino-acid identification can be solved, we should over the coming decade make strong headway in producing inventories of proteins in cells, documenting their variants including PTMs, and performing these tasks at the single-cell level.Sensing with nanopores was conceived more than two decades ago as a means to enumerate a wide variety of substances at the single-molecule level.8 The present ability to identify small molecules and ions, including reactive substances, and to sequence nucleic acids, when complemented with the characterization of polypeptides, oligosaccharides and additional biomolecules, will ultimately yield an inexpensive portable diagnostic device capable of impressing even Bones McCoy (Star Trek).Author Disclosure StatementH.B. is the founder of Oxford Nanopore Technologies.References1. Brinkerhoff H, Kang ASW, Liu J, et al. Multiple rereads of single proteins at single–amino acid resolution using nanopores. Science. 2021;374:1509–1513. DOI: 10.1126/science.abl4381. Crossref, Medline, Google Scholar2. Yan S, Zhang J, Wang Y, et al. Single molecule ratcheting motion of peptides in a Mycobacterium smegmatis porin A (MspA) nanopore. Nano Letters. 2021;21:6703–6710. DOI: 10.1021/acs.nanolett.1c02371. Crossref, Medline, Google Scholar3. Lucas FLR, Versloot RCA, Yakovlieva L, et al. Protein identification by nanopore peptide profiling. Nat Commun. 2021;12:1–9. DOI: 10.1038/s41467-021-26046-9. Crossref, Medline, Google Scholar4. Rodriguez-Larrea D, Bayley H. Multistep protein unfolding during nanopore translocation. Nat Nanotechnol. 2013;8:288–295. DOI: 10.1038/nnano.2013.22. Crossref, Medline, Google Scholar5. Nivala J, Marks DB, Akeson M. Unfoldase-mediated protein translocation through an α-hemolysin nanopore. Nat Biotechnol. 2013;31:247–250. DOI: 10.1038/nbt.2503. Crossref, Medline, Google Scholar6. Zhang S, Huang G, Versloot RCA, et al. Bottom-up fabrication of a proteasome–nanopore that unravels and processes single proteins. Nat Chem. 2021;13:1192–1199. DOI: 10.1038/s41557-021-00824-w. Crossref, Medline, Google Scholar7. Ouldali H, Sarthak K, Ensslen T, et al. Electrical recognition of the twenty proteinogenic amino acids using an aerolysin nanopore. Nat Biotechnol. 2019;38:176-181. DOI: 10.1038/s41587-019-0345-2. Crossref, Medline, Google Scholar8. Bayley H, Cremer PS. Stochastic sensors inspired by biology. Nature. 2001;413:226–230. DOI: 10.1038/35093038. Crossref, Medline, Google Scholar9. Kang XF, Cheley S, Guan X, Bayley H. Stochastic detection of enantiomers. J Am Chem Soc. 2006;128:10684–10685. DOI: 10.1021/ja063485l. Crossref, Medline, Google Scholar10. Rosen CB, Rodriguez-Larrea D, Bayley H. Single-molecule site-specific detection of protein phosphorylation with a nanopore. Nat Biotechnol. 2014;32:179–181. DOI: 10.1038/nbt.2799. Crossref, Medline, Google ScholarFiguresReferencesRelatedDetails Volume 1Issue 1Feb 2022 InformationCopyright 2022, Mary Ann Liebert, Inc., publishersTo cite this article:Yujia Qing and Hagan Bayley.GEN Biotechnology.Feb 2022.28-29.http://doi.org/10.1089/genbio.2022.29008.qbaPublished in Volume: 1 Issue 1: February 16, 2022PDF download