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Related Experiment Video

Updated: Jul 3, 2025

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope
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Full-length single-molecule protein fingerprinting.

Mike Filius1, Raman van Wee1, Carlos de Lannoy1,2

  • 1Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands.

Nature Nanotechnology
|February 13, 2024
PubMed
Summary
This summary is machine-generated.

We developed a single-molecule protein fingerprinting method using fluorescence resonance energy transfer (FRET) to precisely map amino acids and modifications on individual proteins. This technique enhances proteoform analysis and biomarker discovery.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biophysics

Background:

  • Proteins are crucial cellular components, and their diverse forms (proteoforms) are biologically significant.
  • Current analytical methods struggle to distinguish proteoforms, particularly regarding post-translational modifications.
  • Mass spectrometry often yields ambiguous data on modification sites and co-existing modifications.

Purpose of the Study:

  • To introduce a novel single-molecule fluorescence resonance energy transfer (FRET)-based method for protein fingerprinting.
  • To map the precise location of amino acids and post-translational modifications on individual, full-length proteins.
  • To overcome limitations in current proteoform analysis and enable sensitive identification.

Main Methods:

  • Utilized single-molecule FRET via DNA exchange to probe individual amino acids.
  • Achieved subnanometer resolution for fingerprinting both intrinsically disordered and folded globular proteins.
  • Applied machine learning for accurate quantification of protein isoforms in mixtures.

Main Results:

  • Demonstrated successful fingerprinting of alpha-synuclein, including mapping O-GlcNAc modifications and quantifying isoforms.
  • Successfully fingerprinted globular proteins: Bcl-2-like protein 1, procalcitonin, and S100A9.
  • Achieved high-resolution mapping of molecular features on single protein molecules.

Conclusions:

  • Single-molecule FRET protein fingerprinting offers unprecedented sensitivity for proteoform identification.
  • This technique can analyze diverse protein types, including disordered and globular structures.
  • Potential applications include advanced proteomics research and biomarker-based diagnostics.