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Evaluation of FRET X for single-molecule protein fingerprinting

ABSTRACT: Summary Single-molecule protein identification is an unrealized concept with potentially ground-breaking applications in biological research. We propose a method called FRET X (Förster Resonance Energy Transfer via DNA eXchange) fingerprinting, in which the FRET efficiency is read out between exchangeable dyes on protein-bound DNA docking strands and accumulated FRET efficiencies constitute the fingerprint for a protein. To evaluate the feasibility of this approach, we simulated fingerprints for hundreds of proteins using a coarse-grained lattice model and experimentally demonstrated FRET X fingerprinting on model peptides. Measured fingerprints are in agreement with our simulations, corroborating the validity of our modeling approach. In a simulated complex mixture of >300 human proteins of which only cysteines, lysines, and arginines were labeled, a support vector machine was able to identify constituents with 95% accuracy. We anticipate that our FRET X fingerprinting approach will form the basis of an analysis tool for targeted proteomics. Graphical abstract Highlights • We propose a FRET-based single-molecule protein identification method• Peptides are experimentally distinguishable by their fingerprints• Our approach can classify the constituents of complex samples with 95% accuracy Structural biology; Biophysics; Mathematical biosciences; Proteomics

SUBMITTER: de Lannoy C

PROVIDER: S-EPMC8546410 | biostudies-literature |

REPOSITORIES: biostudies-literature

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