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.

Nanopore-Based Protein Identification.

The implementation of a reliable, rapid, inexpensive, and simple method for whole-proteome identification would greatly benefit cell biology research and clinical medicine. Proteins are currently identified by cleaving them with proteases, detecting the polypeptide fragments with mass spectrometry, and mapping the latter to sequences in genomic/proteomic databases. Here, we demonstrate that the polypeptide fragments can instead be detected and classified at the single-molecule limit using a nanometer-scale pore formed by the protein aerolysin. Specifically, three different water-soluble proteins treated with the same protease, trypsin, produce different polypeptide fragments defined by the degree by which the latter reduce the nanopore's ionic current. The fragments identified with the aerolysin nanopore are consistent with the predicted fragments that trypsin could produce.

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.

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.