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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

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BEING VIEWED BY OTHERS SINGLE-CELL PROTEOMICS ENABLED BY NEXT-GENERATION SEQUENCING OR MASS SPECTROMETRY Article 02 March 2023 UNCOVERING BIOLOGY BY SINGLE-CELL PROTEOMICS Article Open

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presenting delegates of the 2019 Single-Molecule Protein Sequencing conference (Jerusalem). We thank the PL-Grid and CI-TASK Infrastructure, Poland, for providing their hardware and software

resources. S.S. acknowledges Postdoc Mobility fellowship no. P400PB 180889 from the Swiss National Science Foundation. E.M.M. and E.V.A. acknowledge funding from the NIH (R35 GM122480 and

R01 DK110520 to E.M.M.), Welch Foundation (F1515 to E.M.M. and F-0046 to E.V.A.), Army Research Office grant W911NF-12-1-0390 and Erisyon. E.M.M. and E.V.A. are co-founders and shareholders

of Erisyon. R.T.K. acknowledges funding from NIGMS (R01 GM138931). P.Y. acknowledges funding from an NIH Director’s New Innovator Award (1DP2OD007292), an NIH Transformative Research Award

(1R01EB018659), an NIH Pioneer Award (1DP1GM133052), and the Molecular Robotics Initiative fund at the Wyss Institute for Biologically Inspired Engineering. M.D. acknowledges funding from a

Systems Biology Department Fellowship from Harvard Medical School and a Technology Development Fellowship from Wyss Institute for Biologically Inspired Engineering. C.C. acknowledges the

Peter and Traudl Engelhorn Foundation. C.D. acknowledges the ERC Advanced Grant Looping DNA (no. 883684) and the NWO programs NanoFront and Basyc. E.M.M., E.V.A. and C.J.H. are co-inventors

on patents relevant to this work. S.O. acknowledges the support of the Azrieli fellowship foundation. N.L.K. acknowledges funding from the Paul G. Allen Frontiers Program (11715), the NIH

HuBMAP program (UH3 CA246635) and NIGMS (P41 GM108569). J.P.M. and Z.K. acknowledge internal funding from NIST and are co-inventors on patents relevant to this work. M. Wanunu acknowledges

funding from the NIH (HG009186). K.S. and A.A. acknowledge funding from the NSF (PHY-1430124). C.J., C.D. and R.E. acknowledge funding from NWO-I (SMPS). C.J. acknowledges funding from HFSP

(RGP0026/2019). A.P. acknowledges Bekker fellowship no. PPN/BEK/2018/1/00296 from the Polish National Agency for Academic Exchange. C.M. and S.H. acknowledge funding from the European

Research Council (ERC ‘Enlightened’, GA 616251) and the CEA Transverse Program ‘Instrumentation and Detection’ (PTC-ID VIRIONEMS). Support from the Proteomics French Infrastructure (PROFI)

is also gratefully acknowledged. G.D. acknowledges funding from FNR (C17/BM/11642138). M.M. acknowledges funding from the Adolphe Merkle Foundation, the Michael J. Fox Foundation for

Parkinson’s Research (grant 17924) and the Swiss National Science Foundation (grant no. 200021-169304). A.M. acknowledges funding from the European Union’s Horizon 2020 research and

innovation programme under grant agreement no. 833399-ERC NanoProt-ID and ISF award 3485/19. M.C. acknowledges computational resources from CINECA (NATWE project) and the Swiss National

Super-Computing Centre (CSCS), under projects sm11 and s865. E.C. acknowledges funding from I-Site Lille, Région Hauts-de-France, and the European Union’s Horizon 2020 Marie Skłodowska-Curie

no. 843052. The study was supported by the project ‘International Centre for Cancer Vaccine Science’ that is carried out within the International Agendas Programme of the Foundation for

Polish Science co-financed by the European Union under the European Regional Development Fund. D.G. thanks Genome Canada and Genome British Columbia for financial support for Genomics

Technology Platforms (GTP) funding for operations and technology development (264PRO). We thank V. Globyte for critical reading. AUTHOR INFORMATION Author notes * These authors contributed

equally: Javier Alfaro, Peggy Bohländer, Mingjie Dai, Mike Filius, Cecil J. Howard, Xander F. van Kooten, Shilo Ohayon, Adam Pomorski, Sonja Schmid. * These authors jointly supervised this

work: Javier Alfaro, Amit Meller, Chirlmin Joo. AUTHORS AND AFFILIATIONS * International Centre for Cancer Vaccine Science, University of Gdańsk, Gdańsk, Poland Javier Antonio Alfaro, 

Georges Bedran, David Goodlett & Umesh Kalathiya * Faculty of Applied Sciences, Delft University of Technology, Delft, the Netherlands Peggy Bohländer & Rienk Eelkema * Wyss

Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA Mingjie Dai & Peng Yin * Department of Systems Biology, Harvard Medical School, Boston, MA, USA

Mingjie Dai & Peng Yin * Department of BioNanoScience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands Mike Filius, Adam Pomorski, Cees Dekker, 

Sung Hyun Kim & Chirlmin Joo * Department of Chemistry, University of Texas at Austin, Austin, TX, USA Cecil J. Howard & Eric V. Anslyn * Department of Biomedical Engineering,

Technion–Israel Institute of Technology, Haifa, Israel Xander F. van Kooten, Shilo Ohayon & Amit Meller * NanoDynamicsLab, Laboratory of Biophysics, Wageningen University, Wageningen,

the Netherlands Sonja Schmid * Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL, USA Aleksei Aksimentiev & Kumar Sarthak * Institute of Bioengineering,

School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Chan Cao * Dipartimento di Ingegneria Industriale, Università di Roma Tor Vergata, Rome, Italy

Mauro Chinappi * Univ. Lille, Inserm, CHU Lille, U1192–Protéomique Réponse Inflammatoire Spectrométrie de Masse–PRISM, Lille, France Etienne Coyaud * Department of Infection and Immunity,

Luxembourg Institute of Health, Strassen, Luxembourg Gunnar Dittmar * Department of Life Sciences and Medicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg Gunnar Dittmar *

Department of Physics, Brown University, Providence, RI, USA Nicholas Drachman & Derek Stein * Genome BC Proteomics Centre, University of Victoria, Victoria, British Columbia, Canada

David Goodlett * Université Grenoble Alpes, CEA, LETI, Grenoble, France Sébastien Hentz * Departments of Chemistry and Molecular Biosciences, and the Feinberg School of Medicine,

Northwestern University, Evanston, IL, USA Neil L. Kelleher * Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, USA Ryan T. Kelly * Institute for Bioscience and

Biotechnology Research, National Institute of Standards and Technology, University of Maryland, Rockville, MD, USA Zvi Kelman & John P. Marino * Biomolecular Labeling Laboratory,

Institute for Bioscience and Biotechnology Research, Rockville, MD, USA Zvi Kelman * Chair of Proteomics and Bioanalytics, Technische Universität München, Freising, Germany Bernhard Kuster, 

Patroklos Samaras & Mathias Wilhelm * Bavarian Center for Biomolecular Mass Spectrometry, Freising, Germany Bernhard Kuster * Department of Biochemistry and Molecular Biology, Biofisika

Institute (CSIC, UPV/EHU), Leioa, Spain David Rodriguez-Larrea * Biodesign Institute, School of Molecular Sciences, Department of Physics, Arizona State University, Tempe, AZ, USA Stuart

Lindsay * Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, the Netherlands Giovanni Maglia * Department of Molecular Biosciences, Center for

Systems and Synthetic Biology, University of Texas at Austin, Austin, TX, USA Edward M. Marcotte * Université Grenoble Alpes, CEA, Inserm, BGE U1038, Grenoble, France Christophe Masselon *

Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland Michael Mayer * University of Toronto, Hospital for Sick Children, Toronto, Ontario, Canada Lusia Sepiashvili *

Department of Physics, Northeastern University, Boston, MA, USA Meni Wanunu * Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, USA Meni Wanunu * Russell

Berrie Nanotechnology Institute, Technion–Israel Institute of Technology, Haifa, Israel Amit Meller Authors * Javier Antonio Alfaro View author publications You can also search for this

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CONTRIBUTIONS J.A.A., C.J. and A.M. conceived and initiated, coordinated and supervised the project. The first draft of the manuscript was written by J.A.A., C.J., A.M., P.B., M.F.,

X.F.v.K., S.O., A.P., S.S., C.J.H., M.D., P.S., G.B., M. Wilhelm and L.S. The manuscript was revised and approved by all authors. CORRESPONDING AUTHORS Correspondence to Javier Antonio

Alfaro, Amit Meller or Chirlmin Joo. ETHICS DECLARATIONS 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. ADDITIONAL INFORMATION PEER REVIEW INFORMATION _Nature Methods_ thanks Tae-Young Yoon and the other, anonymous,

reviewer(s) for their contribution to the peer review of this work. Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with

the rest of the editorial team. PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. RIGHTS AND

PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Alfaro, J.A., Bohländer, P., Dai, M. _et al._ The emerging landscape of single-molecule protein sequencing

technologies. _Nat Methods_ 18, 604–617 (2021). https://doi.org/10.1038/s41592-021-01143-1 Download citation * Received: 04 June 2020 * Accepted: 02 April 2021 * Published: 07 June 2021 *

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