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-barrel dramatically increased the dwell time and the discrimination of peptides in the nanopore at acidic pH. Surprisingly, despite the fact that the two β-barrel nanopores have a similar diameter and an acidic–aromatic construction, their capture mechanisms differ. The electro-osmotic flow played a dominant role for aerolysin, while the electrophoretic force dominated for cytotoxin K. Nonetheless, both β-barrel nanopores allowed the detection of mixtures of trypsinated peptides, with aerolysin nanopores showing a better resolution for larger peptides and cytotoxin K showing a better resolution for shorter peptides. Therefore, this work provides a generic strategy for modifying nanopores for peptide detection that will be most likely be applicable to other nanopore-forming toxins.

Nanopore Discrimination of Coagulation Biomarker Derivatives and Characterization of a Post-Translational Modification

“…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.…”

“…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.…”

3D Blockage Mapping for Identifying Familial Point Mutations in Single Amyloid‐β Peptides with a Nanopore

“…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 ($${{\sigma }_{{{\rm I}}_{{\rm B}}}}$$ ) 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.…”

Unidirectional Single-File Transport of Full-Length Proteins Through a Nanopore

“…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.…”