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Research Article

Single-molecule peptide fingerprinting

Jetty van Ginkel, Mike Filius, Malwina Szczepaniak, Pawel Tulinski, Anne S. Meyer, and View ORCID ProfileChirlmin Joo
  1. aKavli Institute of Nanoscience, Delft University of Technology, 2629HZ Delft, The Netherlands;
  2. bDepartment of Bionanoscience, Delft University of Technology, 2629HZ Delft, The Netherlands

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PNAS March 27, 2018 115 (13) 3338-3343; first published March 12, 2018; https://doi.org/10.1073/pnas.1707207115
Jetty van Ginkel
aKavli Institute of Nanoscience, Delft University of Technology, 2629HZ Delft, The Netherlands;
bDepartment of Bionanoscience, Delft University of Technology, 2629HZ Delft, The Netherlands
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Mike Filius
aKavli Institute of Nanoscience, Delft University of Technology, 2629HZ Delft, The Netherlands;
bDepartment of Bionanoscience, Delft University of Technology, 2629HZ Delft, The Netherlands
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Malwina Szczepaniak
aKavli Institute of Nanoscience, Delft University of Technology, 2629HZ Delft, The Netherlands;
bDepartment of Bionanoscience, Delft University of Technology, 2629HZ Delft, The Netherlands
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Pawel Tulinski
aKavli Institute of Nanoscience, Delft University of Technology, 2629HZ Delft, The Netherlands;
bDepartment of Bionanoscience, Delft University of Technology, 2629HZ Delft, The Netherlands
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Anne S. Meyer
aKavli Institute of Nanoscience, Delft University of Technology, 2629HZ Delft, The Netherlands;
bDepartment of Bionanoscience, Delft University of Technology, 2629HZ Delft, The Netherlands
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  • For correspondence: anne@annemeyerlab.org c.joo@tudelft.nl
Chirlmin Joo
aKavli Institute of Nanoscience, Delft University of Technology, 2629HZ Delft, The Netherlands;
bDepartment of Bionanoscience, Delft University of Technology, 2629HZ Delft, The Netherlands
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  • ORCID record for Chirlmin Joo
  • For correspondence: anne@annemeyerlab.org c.joo@tudelft.nl
  1. Edited by Alan R. Fersht, Gonville and Caius College, Cambridge, United Kingdom, and approved February 21, 2018 (received for review May 1, 2017)

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Significance

Protein sequencing remains a challenge for small samples. A sensitive sequencing technology will create the opportunity for single-cell proteomics and real-time screening for on-site medical diagnostics. To resolve protein identity, we previously developed a computational algorithm that analyzes the ordered sequence of only two types of amino acids within a protein species. Through modification of a biological nanomachine, here, we developed single-molecule fluorescence technology to linearize protein molecules and to read signals from labeled amino acids in an ordered manner. This proof of concept of single-molecule fingerprinting will open the door to single-molecule protein sequencing and pave the road toward the development of a new, fast, and reliable diagnostic tool.

Abstract

Proteomic analyses provide essential information on molecular pathways of cellular systems and the state of a living organism. Mass spectrometry is currently the first choice for proteomic analysis. However, the requirement for a large amount of sample renders a small-scale proteomics study challenging. Here, we demonstrate a proof of concept of single-molecule FRET-based protein fingerprinting. We harnessed the AAA+ protease ClpXP to scan peptides. By using donor fluorophore-labeled ClpP, we sequentially read out FRET signals from acceptor-labeled amino acids of peptides. The repurposed ClpXP exhibits unidirectional processing with high processivity and has the potential to detect low-abundance proteins. Our technique is a promising approach for sequencing protein substrates using a small amount of sample.

  • single-molecule FRET
  • ClpXP
  • protein analysis
  • peptides
  • single-molecule protein sequencing

Footnotes

  • ↵1To whom correspondence may be addressed. Email: anne{at}annemeyerlab.org or c.joo{at}tudelft.nl.
  • Author contributions: J.v.G., A.S.M., and C.J. designed research; J.v.G., M.F., M.S., and P.T. performed research; J.v.G. and M.F. analyzed data; and J.v.G., M.S., A.S.M., and C.J. wrote the paper.

  • Conflict of interest statement: J.v.G., C.J., and A.S.M. hold a patent (“Single molecule protein sequencing”; WO2014014347).

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1707207115/-/DCSupplemental.

Published under the PNAS license.

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Single-molecule peptide fingerprinting
Jetty van Ginkel, Mike Filius, Malwina Szczepaniak, Pawel Tulinski, Anne S. Meyer, Chirlmin Joo
Proceedings of the National Academy of Sciences Mar 2018, 115 (13) 3338-3343; DOI: 10.1073/pnas.1707207115

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Single-molecule peptide fingerprinting
Jetty van Ginkel, Mike Filius, Malwina Szczepaniak, Pawel Tulinski, Anne S. Meyer, Chirlmin Joo
Proceedings of the National Academy of Sciences Mar 2018, 115 (13) 3338-3343; DOI: 10.1073/pnas.1707207115
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Proceedings of the National Academy of Sciences: 115 (13)
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