Single-Molecule Peptide Fingerprinting

Ginkel, Jetty van; Filius, Mike; Szczepaniak, Malwina; Tulinksi, Pawel; Meyer, Anne S.; Joo, Chirlmin

doi:10.1101/226712

Cited by 6 publications

(5 citation statements)

References 41 publications

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“…This percentage is expected to be reduced significantly if an enzyme, such as ClpX, is used to enable controlled protein translocation through the nanopore as previously shown by Nivala et al 33 ClpX is a robust enzyme that can process modified substrates and can significantly reduce the translocation time of polypeptides. 34 Finally, event traces with more than one peak may be attributed to multiple readings of the label due to thermal motion of the peptide. Again, using a processing enzyme to control the translocation speed of the peptide through the pore would reduce the occurrence of these events.…”

Section: Resultsmentioning

confidence: 99%

Resolving Chemical Modifications to a Single Amino Acid within a Peptide Using a Biological Nanopore

et al. 2019

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While DNA sequencing is now amply available, fast, and inexpensive, protein sequencing remains a tremendous challenge. Nanopores may allow for developing a protein sequencer with single-molecule capabilities. As identification of 20 different amino acids currently presents an unsurmountable challenge, fingerprinting schemes are pursued, in which only a subset of amino acids is labeled and detected. This requires modification of amino acids with chemical structures that generate a distinct nanopore ionic current signal. Here, we use a model peptide and the fragaceatoxin C nanopore to characterize six potential tags for a fingerprinting approach using nanopores. We find that labeled and unlabeled proteins can be clearly distinguished and that sensitive detection is obtained for labels with a spectrum of different physicochemical properties such as mass (427–1275 Da), geometry, charge, and hydrophobicity. Additionally, information about the position of the label along the peptide chain can be obtained from individual current-blockade event features. The results represent an important advance toward the development of a single-molecule protein-fingerprinting device with nanopores.

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Section: Resultsmentioning

confidence: 99%

Resolving Chemical Modifications to a Single Amino Acid within a Peptide Using a Biological Nanopore

et al. 2019

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“…After the incubation time, we washed the mixture trice by centrifugation at 2,000 g for 30 s and replacement of the supernatant with fresh buffer. The last replacement of the supernatant was done with imaging buffer (0.8% dextrose, 1 mg/mL glucose oxidase, 170 mg/mL catalase, and 1 mM Trolox [(±) − 6 − hydroxy − 2, 5, 7, 8 − tetramethylchromane − 2 − carboxylic acid; 238813; Merck]) (45) to reduce the bleaching of the fluorophores during imaging.…”

Section: Methodsmentioning

confidence: 99%

Direct visualization of superselective colloid-surface binding mediated by multivalent interactions

Linne

Visco

Angioletti‐Uberti

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Reliably distinguishing between cells based on minute differences in receptor density is crucial for cell–cell or virus–cell recognition, the initiation of signal transduction, and selective targeting in directed drug delivery. Such sharp differentiation between different surfaces based on their receptor density can only be achieved by multivalent interactions. Several theoretical and experimental works have contributed to our understanding of this “superselectivity.” However, a versatile, controlled experimental model system that allows quantitative measurements on the ligand–receptor level is still missing. Here, we present a multivalent model system based on colloidal particles equipped with surface-mobile DNA linkers that can superselectively target a surface functionalized with the complementary mobile DNA-linkers. Using a combined approach of light microscopy and Foerster resonance energy transfer (FRET), we can directly observe the binding and recruitment of the ligand–receptor pairs in the contact area. We find a nonlinear transition in colloid-surface binding probability with increasing ligand or receptor concentration. In addition, we observe an increased sensitivity with weaker ligand–receptor interactions, and we confirm that the timescale of binding reversibility of individual linkers has a strong influence on superselectivity. These unprecedented insights on the ligand–receptor level provide dynamic information into the multivalent interaction between two fluidic membranes mediated by both mobile receptors and ligands and will enable future work on the role of spatial–temporal ligand–receptor dynamics on colloid-surface binding.

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“…A proof of concept was experimentally demonstrated by the same group this year 36 . Using a donor-labelled ClpP (the proteolytic chamber that binds ClpX), the authors sequentially read out FRET signals from acceptor-labelled substrates.…”

Section: Protein Fingerprinting Using Fluorescencementioning

confidence: 98%

Paving the way to single-molecule protein sequencing

2018

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Proteins are major building blocks of life. The protein content of a cell and an organism provides key information for the understanding of biological processes and disease. Despite the importance of protein analysis, only a handful of techniques are available to determine protein sequences, and these methods face limitations, for example, requiring a sizable amount of sample. Single-molecule techniques would revolutionize proteomics research, providing ultimate sensitivity for the detection of low-abundance proteins and the realization of single-cell proteomics. In recent years, novel single-molecule protein sequencing schemes that use fluorescence, tunnelling currents and nanopores have been proposed. Here, we present a review of these approaches, together with the first experimental efforts towards their realization. We discuss their advantages and drawbacks, and present our perspective on the development of single-molecule protein sequencing techniques.

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Single-Molecule Peptide Fingerprinting

Abstract: peer-reviewed)

Cited by 6 publications

References 41 publications

Resolving Chemical Modifications to a Single Amino Acid within a Peptide Using a Biological Nanopore

Resolving Chemical Modifications to a Single Amino Acid within a Peptide Using a Biological Nanopore

Direct visualization of superselective colloid-surface binding mediated by multivalent interactions

Paving the way to single-molecule protein sequencing

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