Single-molecule peptide fingerprinting

Ginkel, Jetty van; Filius, Mike; Szczepaniak, Malwina; Tuliński, Paweł; Meyer, Anne S.; Joo, Chirlmin

doi:10.1073/pnas.1707207115

Cited by 74 publications

(79 citation statements)

References 41 publications

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“…It is a small step from there to cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) (70), which uses oligonucleotide-labeled antibodies to integrate protein and transcriptome measurements together for an efficient readout. Separate from methods that leverage DNA/RNA sequencing, two strategies are scrutinized for fluorescent protein "fingerprinting" that use a few specific fluorescently labeled residues along with fluorescence microscopy to identify peptide fragments from proteins after they are subjected to consecutive rounds of degradation (71)(72)(73). The sparse fluorescent sequence acquired this way is assigned to a specific protein by alignment to a reference database, which is similar to the workflow used in BU-MS.…”

Section: The First Tentative Steps Beyond Msmentioning

confidence: 99%

“…An elaboration on this same theme, single-molecule fluorescence resonance energy transfer (FRET)-based "peptide fingerprinting" instead harnesses the AAA+ protease, ClpXP, to scan peptides ( Fig. 4B) (72,73). The ClpXP protein complex is an enzymatic motor that first unfolds the protein using adenosine triphosphate hydrolysis and then degrades it, forcing it to translocate progressively through a central orifice in the molecule.…”

Section: Fluorescent "Protein Fingerprinting"mentioning

confidence: 99%

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Beyond mass spectrometry, the next step in proteomics

2020

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Proteins can be the root cause of a disease, and they can be used to cure it. The need to identify these critical actors was recognized early (1951) by Sanger; the first biopolymer sequenced was a peptide, insulin. With the advent of scalable, single-molecule DNA sequencing, genomics and transcriptomics have since propelled medicine through improved sensitivity and lower costs, but proteomics has lagged behind. Currently, proteomics relies mainly on mass spectrometry (MS), but instead of truly sequencing, it classifies a protein and typically requires about a billion copies of a protein to do it. Here, we offer a survey that illuminates a few alternatives with the brightest prospects for identifying whole proteins and displacing MS for sequencing them. These alternatives all boast sensitivity superior to MS and promise to be scalable and seem to be adaptable to bioinformatics tools for calling the sequence of amino acids that constitute a protein.

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Section: The First Tentative Steps Beyond Msmentioning

confidence: 99%

Section: Fluorescent "Protein Fingerprinting"mentioning

confidence: 99%

Beyond mass spectrometry, the next step in proteomics

2020

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“…To enhance surface passivation against non-specific protein adsorption, we injected 5% Tween-20 solution in buffer B into the channel of the flowcell, incubated for 10 min and washed out with 600 µl of buffer A. 23…”

Section: Characterization Of Printed Protein Features the Width And mentioning

confidence: 99%

Oriented Soft DNA Curtains for Single Molecule Imaging

Kopūstas

Ivanovaite

Rakickas

et al. 2020

Preprint

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Over the past twenty years, single-molecule methods have become extremely important for biophysical studies. These methods, in combination with new nanotechnological platforms, can significantly facilitate experimental design and enable faster data acquisition. A nanotechnological platform, which utilizes flow-stretch of immobilized DNA molecules, called DNA Curtains, is one of the best examples of such combinations. Here, we employed new strategies to fabricate a flowstretch assay of stably immobilized and oriented DNA molecules using protein template-directed assembly. In our assay a protein template patterned on a glass coverslip served for directional assembly of biotinylated DNA molecules. In these arrays, DNA molecules were oriented to one another and maintained extended either by single-or both-ends immobilization to the protein templates. For oriented both-end DNA immobilization we employed heterologous DNA labeling and protein template coverage with the anti-digoxigenin antibody. In contrast to the single-end, both-ends immobilization does not require constant buffer flow for keeping DNAs in an extended configuration, allowing us to study protein-DNA interactions at more controllable reaction conditions. Additionally, we increased immobilization stability of the biotinylated DNA molecules using protein templates fabricated from traptavidin. Finally, we demonstrated that double-tethered Soft DNA Curtains can be used in nucleic acid-interacting protein (e.g. CRISPR-Cas9) binding assay that monitors binding location and position of individual fluorescently labeled proteins on DNA.

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“…Some exciting ideas have been recently reported and the reader can find a comprehensive review on approaches to single-molecule protein sequencing in Restrepo-Perez et al [4]. The methods reported in the literature are based on single molecule techniques as nanopores [6][7][8][9][10][11][12][13], fluorescence [14] and tunneling currents across nanogaps [15,16]. Every method has its own advantages and drawbacks.…”

Section: Introductionmentioning

confidence: 99%

“…Only very recently, two reports demonstrated, by means of numerical simulations, that optimized solid-state [11] and biological nanopore [17] are able to distinguish among high numbers of amino-acids. Other recent works proposed to take advantage of comprehensive protein sequence databases which enable the identification of a protein by labelling and detecting a limited number of amino-acids [14,18]. By knowing this limited subset of the whole sequence is then possible to select the corresponding protein within the available database.…”

Section: Introductionmentioning

confidence: 99%

Adaptive nanopores: A bioinspired label-free approach for protein sequencing and identification

et al. 2020

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Single molecule protein sequencing would tremendously impact in proteomics and human biology and it would promote the development of novel diagnostic and therapeutic approaches. However, its technological realization can only be envisioned, and huge challenges need to be overcome. Major difficulties are inherent to the structure of proteins, which are composed by several different amino-acids. Despite long standing efforts, only few complex techniques, such as Edman degradation, liquid chromatography and mass spectroscopy, make protein sequencing possible. Unfortunately, these techniques present significant limitations in terms of amount of sample required and dynamic range of measurement. It is known that proteins can distinguish closely similar molecules. Moreover, several proteins can work as biological nanopores in order to perform single molecule detection and sequencing. Unfortunately, while DNA sequencing by means of nanopores is demonstrated, very few examples of nanopores able to perform reliable protein-sequencing have been reported so far. Here, we investigate, by means of molecular dynamics simulations, how a re-engineered protein, acting as biological nanopore, can be used to recognize the sequence of a translocating peptide by sensing the “shape” of individual amino-acids. In our simulations we demonstrate that it is possible to discriminate with high fidelity, 9 different amino-acids in a short peptide translocating through the engineered construct. The method, here shown for fluorescence-based sequencing, does not require any labelling of the peptidic analyte. These results can pave the way for a new and highly sensitive method of sequencing.

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Single-molecule peptide fingerprinting

Cited by 74 publications

References 41 publications

Beyond mass spectrometry, the next step in proteomics

Beyond mass spectrometry, the next step in proteomics

Oriented Soft DNA Curtains for Single Molecule Imaging

Adaptive nanopores: A bioinspired label-free approach for protein sequencing and identification

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