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

Fabien Piguet,Juan Pelta,Abdelghani Oukhaled,Philippe Manivet,Manuela Pastoriza-Gallego,Hadjer Ouldali

doi:10.1038/s41467-018-03418-2

Abstract

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.

Highlights

To cite this version: Fabien Piguet, Hadjer Ouldali, Manuela Pastoriza-Gallego, Philippe Manivet, Juan Pelta, et al
The high resolution of aerolysin pore for the highly-sensitive analysis of short peptides is most likely attributed to its geometry, to its highly charged pore wall and to the high affinity between aerolysin and peptides
Nanoporebased single amino acid size-discrimination of several peptide lengths mixed in solution that differ by a single amino acid is subject to four conditions: i) the whole peptide must be entirely fitted en bloc inside the vicinity of the pore, ii) all amino acids must contribute individually to the current blockades, iii) a high size-sensitivity of the pore conductance change and iv) a high affinity between the pore and the peptide

Summary

Introduction

To cite this version: Fabien Piguet, Hadjer Ouldali, Manuela Pastoriza-Gallego, Philippe Manivet, Juan Pelta, et al. A technology allowing the analysis of size and sequence of short peptide molecules of only few molecular copies is still challenging. Classical metabolomics and proteomics techniques, such as mass spectrometry (MS), High Performance Liquid Chromatography (HPLC), or Nuclear Magnetic Resonance (NMR), suffer a lack of reproducibility and sensitivity that prohibit their use for medical diagnostic[2,3]. These techniques are too expensive, time consuming and heavy to handle in the view of developing a portable medical device application[3]. The current challenge is the application of nanopore technology to peptides and proteins identification, fingerprinting and sequencing

Methods

Results

Conclusion