2022
DOI: 10.1021/acsnano.1c11455
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β-Barrel Nanopores with an Acidic–Aromatic Sensing Region Identify Proteinogenic Peptides at Low pH

Abstract: Biological nanopores are emerging as sensitive single-molecule sensors for proteins and peptides. The heterogeneous charge of a polypeptide chain, however, can complicate or prevent the capture and translocation of peptides and unfolded proteins across nanopores. Here, we show that two β-barrel nanopores, aerolysin and cytotoxin K, cannot efficiently detect proteinogenic peptides from a trypsinated protein under a wide range of conditions. However, the introduction of an acidic–aromatic sensing region in the β… Show more

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Cited by 26 publications
(29 citation statements)
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References 52 publications
(87 reference statements)
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“…Future discrimination of individual biomarkers from complex biological samples will require the characterization of additional parameters that could be used for analyte identification. Such parameters could be determined from blockade current fluctuation, including analysis of the standard deviation of the event blockade current , or definition of internal steps. The combination of parameters, including those from alternative conformations such as those observed for FPA-P, could be used to classify individual or combinations of events with clustering algorithms such as fuzzy-c means, hidden Markov models, or density-based methods. The application of machine learning can further exploit such classification. , While these in-depth data analysis approaches are well developed for DNA sequencing, their application to the identification of single analytes is still in development. In the future, these data processing and analysis techniques will facilitate the specific identification of a signature parameter combination for individual biomarkers.…”
Section: Discussionmentioning
confidence: 99%
See 1 more Smart Citation
“…Future discrimination of individual biomarkers from complex biological samples will require the characterization of additional parameters that could be used for analyte identification. Such parameters could be determined from blockade current fluctuation, including analysis of the standard deviation of the event blockade current , or definition of internal steps. The combination of parameters, including those from alternative conformations such as those observed for FPA-P, could be used to classify individual or combinations of events with clustering algorithms such as fuzzy-c means, hidden Markov models, or density-based methods. The application of machine learning can further exploit such classification. , While these in-depth data analysis approaches are well developed for DNA sequencing, their application to the identification of single analytes is still in development. In the future, these data processing and analysis techniques will facilitate the specific identification of a signature parameter combination for individual biomarkers.…”
Section: Discussionmentioning
confidence: 99%
“…Future discrimination of individual biomarkers from complex biological samples will require the characterization of additional parameters that could be used for analyte identification. Such parameters could be determined from blockade current fluctuation, including analysis of the standard deviation of the event blockade current 86,87 or definition of internal steps. 88−92 The combination of parameters, including those from alternative conformations such as those observed for FPA-P, could be used to classify individual or combinations of events with clustering algorithms such as fuzzy-c means, hidden Markov models, or density-based methods.…”
Section: ■ Conclusionmentioning
confidence: 99%
“…Previous works have used the standard deviation of blocking currents in the 2D scatter plots for the characterization of multiple analytes including peptides and proteins [23] . For example, a 2D scatter plot of the event noise ( σIB ${{\sigma }_{{{\rm I}}_{{\rm B}}}}$ ) from the standard deviation of the ionic currents during the event vs. the excluded current ( I ex %) was applied to distinguish and determine the peptide fragments produced by trypsinated lysozyme [23c] . However, to our best knowledge, this work is the first report of 3D data mapping for peptide identification based on the combination of I b / I 0 , log ( t D ) and σ b , where σ b embodies a unique information dimension that different from the current blockage and duration time.…”
Section: Discussionmentioning
confidence: 99%
“…Finally, although we use GdmCl for threading/stretching a protein in the pore, we note that combining this mode with enzyme-driven protein motion is possible, for example, by using an asymmetric buffer in which: 1) protein unfolding is mediated in a denaturant chamber, 2) electroosmotic forces are used to keep the protein taut at the pore, and 3) enzyme-mediated motion on another chamber of the pore is used to move the protein through the pore in a discrete manner. Other improvements such as changing the electrolyte type 59 , pore variant [60][61][62] , and voltage waveform, can be made to further enhance the ability of our method to obtain more unique and unequivocal fingerprint signatures from full-length proteins.…”
Section: Discussionmentioning
confidence: 99%