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.

Machine Learning Assisted Simultaneous Structural Profiling of Differently Charged Proteins in a Mycobacterium smegmatis Porin A (MspA) Electroosmotic Trap

“…4 Besides, neither method can extract characteristics of a single protein from the ensemble. Single-molecule fluorescent protein fingerprinting could address these limitations, 5 but it also faces other problems such as dark reads, which are common in most single-molecule fluorescence methods. 2 Further, none of these strategies possess a resolution to directly resolve conformational change of a protein.…”

Single-Molecule Peptide Identification Using Fluorescence Blinking Fingerprints

“…Unlike other single-molecule fluorescence approaches, , blinkognition does not rely on chemical degradation or proteolysis steps; thus, it is faster, simpler, and less prone to artifacts. Although other single-molecule fluorescence fingerprinting approaches exist, , blinkognition has the advantage of providing information about the presence of PTMs on amino acids that have not been labeled. Compared to nanopore sequencing, blinkognition does not require denaturation of the peptide, can be applied to both negatively and positively charged peptides (e.g., C1–C4 and E1–E3 , respectively), and could be massively parallelized to produce millions of single-molecule reads, similarly to next-generation DNA sequencing.…”

“…Single-molecule fluorescence-based techniques are massively parallelizable, but some of these methods rely on multiple cycles of chemical or enzymatic degradation, , making data acquisition long and prone to errors. Other fluorescence-based methods can detect accurately the position of selected amino acids, , but it is unclear how sensitive these methods are to the nature of PTMs present in other amino acids in the peptide. Here, we provide proof of a fundamentally new approach to identifying single-peptide molecules.…”

Engineering Biological Nanopore Approaches toward Protein Sequencing

“…On a last note, combining the nanopore-based approach with other methods of protein analysis is likely to prove beneficial. Several recent protein fingerprinting methods, based on the readout of a subset of residue types, have begun to adopt such an integrative approach toward biomolecule sequencing. ,,,, For example, the combination of MS and a nanopore ion source was developed for sequencing single proteins . In this concept, the nanopore electrospray was used to guide a protein into a linear configuration by delivering individual AA ions directly into a mass spectrometer sequentially; thus, the ions could be efficiently detected using their mass-to-charge ratios .…”