On possible trypsin-induced biases in peptides analysis with aerolysin nanopore.

Nanopore-based single-molecule analysis technique is a promising approach in the field of proteomics. In this Technical Brief, the interaction between the biological nanopore of Aerolysin (AeL) and peptides is investigated, focusing on potential biases depending on the AeL activation protocol. Our results reveal that residual trypsin, which may be unintentionally introduced in analyte solution when using a classical AeL activation protocol, can induce a significant formation of shorter peptides by enzymatic degradation of longer ones, which may lead to unwanted effects and/or misinterpretations. AeL free-trypsin activation protocol eliminates this bias and appears more appropriate for peptide/proteins analysis, specifically in the perspective of nanopore-based molecular fingerprinting or of low-abundance species characterization.

Pore-forming toxins as tools for polymer analytics: From sizing to sequencing.

We report here on the nanopore resistive pulse sensing (Np-RPS) method, involving pore-forming toxins as tools for polymer analytics at single molecule level. Np-RPS is an electrical method for the label-free detection of single molecules. A molecule interacting with the pore causes a change of the electrical resistance of the pore, called a resistive pulse, associated with a measurable transient current blockade. The features of the blockades, in particular their depth and duration, contain information on the molecular properties of the analyte. We first revisit the history of Np-RPS, then we discuss the effect of the configuration of the molecule/nanopore interaction on the molecular information that can be extracted from the signal, illustrated in two different regimes that either favor molecular sequencing or molecular sizing. Specifically, we focus on the sizing regime and on the use of two different pore-forming toxins, staphylococcal α-hemolysin (αHL) and aerolysin (AeL) nanopores, for the characterization of water-soluble polymers (poly-(ethylene glycol), (PEG)), homopeptides, and heteropeptides. We discuss how nanopore sizing of polymers could be envisioned as a new approach for peptide/protein sequencing.

Electrical recognition of the twenty proteinogenic amino acids using an aerolysin nanopore.

Efforts to sequence single protein molecules in nanopores1-5 have been hampered by the lack of techniques with sufficient sensitivity to discern the subtle molecular differences among all twenty amino acids. Here we report ionic current detection of all twenty proteinogenic amino acids in an aerolysin nanopore with the help of a short polycationic carrier. Application of molecular dynamics simulations revealed that the aerolysin nanopore has a built-in single-molecule trap that fully confines a polycationic carrier-bound amino acid inside the sensing region of the aerolysin. This structural feature means that each amino acid spends sufficient time in the pore for sensitive measurement of the excluded volume of the amino acid. We show that distinct current blockades in wild-type aerolysin can be used to identify 13 of the 20 natural amino acids. Furthermore, we show that chemical modifications, instrumentation advances and nanopore engineering offer a route toward identification of the remaining seven amino acids. These findings may pave the way to nanopore protein sequencing.

Aerolysin, a Powerful Protein Sensor for Fundamental Studies and Development of Upcoming Applications.

The nanopore electrical approach is a breakthrough in single molecular level detection of particles as small as ions, and complex as biomolecules. This technique can be used for molecule analysis and characterization as well as for the understanding of confined medium dynamics in chemical or biological reactions. Altogether, the information obtained from these kinds of experiments will allow us to address challenges in a variety of biological fields. The sensing, design, and manufacture of nanopores is crucial to realize these objectives. For some time now, aerolysin, a pore forming toxin, and its mutants have shown high potential in real time analytical chemistry, size discrimination of neutral polymers, oligosaccharides, oligonucleotides and peptides at monomeric resolution, sequence identification, chemical modification on DNA, potential biomarkers detection, and protein folding analysis. This review focuses on the results obtained with aerolysin nanopores on the fields of chemistry, biology, physics, and biotechnology. We discuss and compare as well the results obtained with other protein channel sensors.

Identification of single amino acid differences in uniformly charged homopolymeric peptides with aerolysin nanopore.

There are still unmet needs in finding new technologies for biomedical diagnostic and industrial applications. A technology allowing the analysis of size and sequence of short peptide molecules of only few molecular copies is still challenging. The fast, low-cost and label-free single-molecule nanopore technology could be an alternative for addressing these critical issues. Here, we demonstrate that the wild-type aerolysin nanopore enables the size-discrimination of several short uniformly charged homopeptides, mixed in solution, with a single amino acid resolution. Our system is very sensitive, allowing detecting and characterizing a few dozens of peptide impurities in a high purity commercial peptide sample, while conventional analysis techniques fail to do so.

Probing driving forces in aerolysin and α-hemolysin biological nanopores: electrophoresis versus electroosmosis.

The transport of macromolecules through nanopores is involved in many biological functions and is today at the basis of promising technological applications. Nevertheless the interpretation of the dynamics of the macromolecule/nanopore interaction is still misunderstood and under debate. At the nanoscale, inside biomimetic channels under an external applied voltage, electrophoresis, which is the electric force acting on electrically charged molecules, and electroosmotic flow (EOF), which is the fluid transport associated with ions, contribute to the direction and magnitude of the molecular transport. In order to decipher the contribution of the electrophoresis and electroosmotic flow, we explored the interaction of small, rigid, neutral molecules (cyclodextrins) and flexible, non-ionic polymers (poly(ethylene glycol), PEG) that can coordinate cations under appropriate experimental conditions, with two biological nanopores: aerolysin (AeL) and α-hemolysin (aHL). We performed experiments using two electrolytes with different ionic hydration (KCl and LiCl). Regardless of the nature of the nanopore and of the electrolyte, cyclodextrins behaved as neutral analytes. The dominant driving force was attributed to EOF, acting in the direction of the anion flow and stronger in LiCl than in KCl. The same qualitative behaviour was observed for PEGs in LiCl. In contrast, in KCl, PEGs behaved as positively charged polyelectrolytes through both AeL and aHL. Our results are in agreement with theoretical predictions about the injection of polymers inside a confined geometry (ESI). We believe our results to be of significant importance for better control of the dynamics of analytes of different nature through biological nanopores.

High Temperature Extends the Range of Size Discrimination of Nonionic Polymers by a Biological Nanopore.

We explore the effect of temperature on the interaction of polydisperse mixtures of nonionic poly(ethylene glycol) (PEG) polymers of different average molar masses with the biological nanopore α-hemolysin. In contrast with what has been previously observed with various nanopores and analytes, we find that, for PEGs larger than a threshold molar mass (2000 g/mol, PEG 2000), increasing temperature increases the duration of the PEG/nanopore interaction. In the case of PEG 3400 the duration increases by up to a factor of 100 when the temperature increases from 5 °C to 45 °C. Importantly, we find that increasing temperature extends the polymer size range of application of nanopore-based single-molecule mass spectrometry (Np-SMMS)-type size discrimination. Indeed, in the case of PEG 3400, discrimination of individual molecular species of different monomer number is impossible at room temperature but is achieved when the temperature is raised to 45 °C. We interpret our observations as the consequence of a decrease of PEG solubility and a collapse of PEG molecules with higher temperatures. In addition to expanding the range of application of Np-SMMS to larger nonionic polymers, our findings highlight the crucial role of the polymer solubility for the nanopore detection.