Review

. 2021 Jun;18(6):604-617.

doi: 10.1038/s41592-021-01143-1. Epub 2021 Jun 7.

The emerging landscape of single-molecule protein sequencing technologies

Javier Antonio Alfaro^#¹, Peggy Bohländer^#², Mingjie Dai^#^{3

4}, Mike Filius^#⁵, Cecil J Howard^#⁶, Xander F van Kooten^#⁷, Shilo Ohayon^#⁷, Adam Pomorski^#⁵, Sonja Schmid^#⁸, Aleksei Aksimentiev⁹, Eric V Anslyn⁶, Georges Bedran¹⁰, Chan Cao¹¹, Mauro Chinappi¹², Etienne Coyaud¹³, Cees Dekker⁵, Gunnar Dittmar^{14

15}, Nicholas Drachman¹⁶, Rienk Eelkema², David Goodlett^{10

17}, Sébastien Hentz¹⁸, Umesh Kalathiya¹⁰, Neil L Kelleher¹⁹, Ryan T Kelly²⁰, Zvi Kelman^{21

22}, Sung Hyun Kim⁵, Bernhard Kuster^{23

24}, David Rodriguez-Larrea²⁵, Stuart Lindsay²⁶, Giovanni Maglia²⁷, Edward M Marcotte²⁸, John P Marino²¹, Christophe Masselon²⁹, Michael Mayer³⁰, Patroklos Samaras²³, Kumar Sarthak⁹, Lusia Sepiashvili³¹, Derek Stein¹⁶, Meni Wanunu^{32

33}, Mathias Wilhelm²³, Peng Yin^{3

4}, Amit Meller^#^{34

35}, Chirlmin Joo^#³⁶

Affiliations

¹ International Centre for Cancer Vaccine Science, University of Gdańsk, Gdańsk, Poland. javier.alfaro@proteogenomics.ca.
² Faculty of Applied Sciences, Delft University of Technology, Delft, the Netherlands.
³ Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA.
⁴ Department of Systems Biology, Harvard Medical School, Boston, MA, USA.
⁵ Department of BioNanoScience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands.
⁶ Department of Chemistry, University of Texas at Austin, Austin, TX, USA.
⁷ Department of Biomedical Engineering, Technion-Israel Institute of Technology, Haifa, Israel.
⁸ NanoDynamicsLab, Laboratory of Biophysics, Wageningen University, Wageningen, the Netherlands.
⁹ Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
¹⁰ International Centre for Cancer Vaccine Science, University of Gdańsk, Gdańsk, Poland.
¹¹ Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.
¹² Dipartimento di Ingegneria Industriale, Università di Roma Tor Vergata, Rome, Italy.
¹³ Univ. Lille, Inserm, CHU Lille, U1192-Protéomique Réponse Inflammatoire Spectrométrie de Masse-PRISM, Lille, France.
¹⁴ Department of Infection and Immunity, Luxembourg Institute of Health, Strassen, Luxembourg.
¹⁵ Department of Life Sciences and Medicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg.
¹⁶ Department of Physics, Brown University, Providence, RI, USA.
¹⁷ Genome BC Proteomics Centre, University of Victoria, Victoria, British Columbia, Canada.
¹⁸ Université Grenoble Alpes, CEA, LETI, Grenoble, France.
¹⁹ Departments of Chemistry and Molecular Biosciences, and the Feinberg School of Medicine, Northwestern University, Evanston, IL, USA.
²⁰ Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, USA.
²¹ Institute for Bioscience and Biotechnology Research, National Institute of Standards and Technology, University of Maryland, Rockville, MD, USA.
²² Biomolecular Labeling Laboratory, Institute for Bioscience and Biotechnology Research, Rockville, MD, USA.
²³ Chair of Proteomics and Bioanalytics, Technische Universität München, Freising, Germany.
²⁴ Bavarian Center for Biomolecular Mass Spectrometry, Freising, Germany.
²⁵ Department of Biochemistry and Molecular Biology, Biofisika Institute (CSIC, UPV/EHU), Leioa, Spain.
²⁶ Biodesign Institute, School of Molecular Sciences, Department of Physics, Arizona State University, Tempe, AZ, USA.
²⁷ Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, the Netherlands.
²⁸ Department of Molecular Biosciences, Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX, USA.
²⁹ Université Grenoble Alpes, CEA, Inserm, BGE U1038, Grenoble, France.
³⁰ Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland.
³¹ University of Toronto, Hospital for Sick Children, Toronto, Ontario, Canada.
³² Department of Physics, Northeastern University, Boston, MA, USA.
³³ Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, USA.
³⁴ Department of Biomedical Engineering, Technion-Israel Institute of Technology, Haifa, Israel. ameller@technion.ac.il.
³⁵ Russell Berrie Nanotechnology Institute, Technion-Israel Institute of Technology, Haifa, Israel. ameller@technion.ac.il.
³⁶ Department of BioNanoScience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands. c.joo@tudelft.nl.

^# Contributed equally.

PMID: 34099939
PMCID: PMC8223677
DOI: 10.1038/s41592-021-01143-1

Review

The emerging landscape of single-molecule protein sequencing technologies

Javier Antonio Alfaro et al. Nat Methods. 2021 Jun.

. 2021 Jun;18(6):604-617.

doi: 10.1038/s41592-021-01143-1. Epub 2021 Jun 7.

Authors

Affiliations

¹ International Centre for Cancer Vaccine Science, University of Gdańsk, Gdańsk, Poland. javier.alfaro@proteogenomics.ca.
² Faculty of Applied Sciences, Delft University of Technology, Delft, the Netherlands.
³ Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA.
⁴ Department of Systems Biology, Harvard Medical School, Boston, MA, USA.
⁵ Department of BioNanoScience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands.
⁶ Department of Chemistry, University of Texas at Austin, Austin, TX, USA.
⁷ Department of Biomedical Engineering, Technion-Israel Institute of Technology, Haifa, Israel.
⁸ NanoDynamicsLab, Laboratory of Biophysics, Wageningen University, Wageningen, the Netherlands.
⁹ Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
¹⁰ International Centre for Cancer Vaccine Science, University of Gdańsk, Gdańsk, Poland.
¹¹ Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.
¹² Dipartimento di Ingegneria Industriale, Università di Roma Tor Vergata, Rome, Italy.
¹³ Univ. Lille, Inserm, CHU Lille, U1192-Protéomique Réponse Inflammatoire Spectrométrie de Masse-PRISM, Lille, France.
¹⁴ Department of Infection and Immunity, Luxembourg Institute of Health, Strassen, Luxembourg.
¹⁵ Department of Life Sciences and Medicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg.
¹⁶ Department of Physics, Brown University, Providence, RI, USA.
¹⁷ Genome BC Proteomics Centre, University of Victoria, Victoria, British Columbia, Canada.
¹⁸ Université Grenoble Alpes, CEA, LETI, Grenoble, France.
¹⁹ Departments of Chemistry and Molecular Biosciences, and the Feinberg School of Medicine, Northwestern University, Evanston, IL, USA.
²⁰ Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, USA.
²¹ Institute for Bioscience and Biotechnology Research, National Institute of Standards and Technology, University of Maryland, Rockville, MD, USA.
²² Biomolecular Labeling Laboratory, Institute for Bioscience and Biotechnology Research, Rockville, MD, USA.
²³ Chair of Proteomics and Bioanalytics, Technische Universität München, Freising, Germany.
²⁴ Bavarian Center for Biomolecular Mass Spectrometry, Freising, Germany.
²⁵ Department of Biochemistry and Molecular Biology, Biofisika Institute (CSIC, UPV/EHU), Leioa, Spain.
²⁶ Biodesign Institute, School of Molecular Sciences, Department of Physics, Arizona State University, Tempe, AZ, USA.
²⁷ Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, the Netherlands.
²⁸ Department of Molecular Biosciences, Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX, USA.
²⁹ Université Grenoble Alpes, CEA, Inserm, BGE U1038, Grenoble, France.
³⁰ Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland.
³¹ University of Toronto, Hospital for Sick Children, Toronto, Ontario, Canada.
³² Department of Physics, Northeastern University, Boston, MA, USA.
³³ Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, USA.
³⁴ Department of Biomedical Engineering, Technion-Israel Institute of Technology, Haifa, Israel. ameller@technion.ac.il.
³⁵ Russell Berrie Nanotechnology Institute, Technion-Israel Institute of Technology, Haifa, Israel. ameller@technion.ac.il.
³⁶ Department of BioNanoScience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands. c.joo@tudelft.nl.

^# Contributed equally.

PMID: 34099939
PMCID: PMC8223677
DOI: 10.1038/s41592-021-01143-1

Abstract

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.

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Conflict of interest statement

Competing interests

S.H. and C.M. are co-inventors on the patent application EP14158255. E.M.M. and E.V.A. are co-inventors on patent 9625469. D.S. is sponsored by Oxford Nanopore for his work on nanotip MS. E.M.M. and E.V.A. are co-founders and shareholders of Erisyon. B.K. and M. Wilhelm are founders and shareholders of OmicScouts and MSAID. They have no operational role in either company. M.D. and P.Y. are co-inventors on US patent 10006917. P.Y. is an inventor on US patent 10697974 and provisional patent and patent applications on various aspects of DNA nanotechnology–based protein sequencing methods described in this article. P.Y. is a co-founder, director and consultant of Ultivue Inc. and Spear Bio Inc. All remaining authors declare no competing interests. Some authors may be bound by confidentiality agreements that prevent them from disclosing their competing interests in this work; the corresponding authors are not aware of such cases.

Figures

**Fig. 1 |. The emerging landscape of single-molecule protein sequencing and fingerprinting technologies.**
The new technologies address a range of analytes, methods of protein identification and target niches. Various techniques, particularly those involving complex readout signals, are suitable for characterizing short peptide sequences, while others are primed to characterize full-length proteins or larger complexes. The method of protein identification may fingerprint certain classes of amino acids (AA fingerprint) or reveal each amino acid down to its physiochemical class or better (AA sequencing). Technologies might characterize proteins by their mass or the mass of their fragments (mass spectrum). Other methods aim to characterize the properties of folded proteins (structure fingerprint). PTM, post-translational modification; PPI, protein–protein interaction; NEMS-MS, nanoelectromechanical systems MS.

**Fig. 2 |. The renaissance of classic techniques.**
a,b, High-throughput fluorosequencing by Edman degradation featuring amino acid-specific chemical modification of peptides with fluorophores (a) and N-terminal amino acid recognition using a plurality of probes (b). c, Neutral-particle MS is a promising technique to characterize proteoforms. Currently, the technology can be used to characterize large megadalton-scale complexes using silicon-based nanosensors. Graphene nanosensors and further developments may push the technology toward smaller and smaller proteins and potentially lead to increased sequence coverage in global proteomics. ESI, electrospray ionization. d, Nanopore electrospray is a marriage of nanopores, classical electrospray and single-particle detection techniques to sequence single proteins by measuring amino acids one at a time. Panel a adapted with permission from ref., Springer Nature.

**Fig. 3 |. DNA-facilitated protein sequencing.**
a, Schematic of specific amino acid labeling on a denatured protein with DNA strands. Each DNA strand contains a barcode for the specific amino acid and (optionally) a UMI. b–e, Various readout strategies of DNA-labeled samples for protein identification. b, Protein kinetic fingerprinting using qPAINT. c, Protein linear barcoding using molecular-resolution DNA-PAINT. d, DNA proximity recording. e, Protein structural fingerprinting using FRET-X.

**Fig. 4 |. Three strategies of nanopore-based protein sequencing and sensing.**
In all cases, a voltage bias is applied across an insulating membrane (left panels) and the analytes translocate through the nanopore from top to bottom (red arrows). a, Reading unlabeled proteins or peptides using a biological nanopore. b, Identification of whole proteins or peptides by fingerprinting with deep learning algorithms. Residue-specific fluorescent labels (for example, at lysine, cysteine and methionine) can be used to fingerprint proteins and peptides alongside electrical current sensing. c, Identification of folded proteins using lipid tethering. Other possible tethers include DNA carriers, DNA origami anchors and plasmonic trapping.

None — **Sequence coverage in global proteomics studies.**
MS-based global proteomics studies identify and quantify proteins with variable sequence coverage. The single best run from the 47 publications present in ProteomicsDB shows how sample-specific protein sequence coverage improves with sample preparation methods. Sequence coverage generally decreases with sample complexity and increases with time (cost) dedicated to studying the sample.

See this image and copyright information in PMC

References

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1. Smith LM et al. Proteoform: a single term describing protein complexity. Nat. Methods 10, 186–187 (2013). - PMC - PubMed
1. Seattle Times Business Staff. Seattle biotech startup Nautilus to get $350 million, stock listing in blank-check deal. The Seattle Times https://www.seattletimes.com/business/seattle-biotech-startup-nautilus-t... (8 February 2021).
1. Reuters Staff. Protein sequencing firm Quantum-Si to go public via $1.46 billion SPAC merger. Reuters https://www.reuters.com/article/us-quantum-si-m-a-highcape-capital-idUSK... (18 February 2021).
1. Cohen L & Walt DR Single-molecule arrays for protein and nucleic acid analysis. Annu. Rev. Anal. Chem 10, 345–363 (2017). - PubMed

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The emerging landscape of single-molecule protein sequencing technologies

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The emerging landscape of single-molecule protein sequencing technologies

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Conflict of interest statement

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