Protein Fingerprinting using the Aerolysin Nanopore

Abstract

Recent work has highlighted the performance of the aerolysin nanopore (Ael) as a sensor for poly(ethyleneglycol) (1), DNA (2) and peptides (3). Here we show that Ael nanopore enables discrimination of proteins based on the differentiation of pepetide fragments generated by specific enzymes without limitation on the size, the conformation and the charge of the protein of interest. Alternatively, we explore the detection of peptide fragments created by physical degradation (light, sound). Statistical analysis of the current blockades induced by the different protein fragments, and the observation of populations specific to each type of fragment (e.g.; preferential current blockade levels depending on the type of fragment) allow generating a fragmentation profile characteristic of the initial protein, in a form roughly analogous to the signal from a mass spectrometer. This signature could then allow the identification of the initial protein by comparison with a database generated using identified proteins. (1) Baaken, G., Halimeh, I., Bacri, L., Pelta, J., Oukhaled, A., & Behrends, J. C. (2015). High-resolution size-discrimination of single nonionic synthetic polymers with a highly charged biological nanopore. ACS Nano, 9(6), 6443-6449. (2) Cao, C., Ying, Y. L., Hu, Z. L., Liao, D. F., Tian, H., & Long, Y. T. (2016). Discrimination of oligonucleotides of different lengths with a wild-type aerolysin nanopore. Nature Nanotechnology, 11(8), 713. (3) Piguet, F., Ouldali, H., Pastoriza-Gallego, M., Manivet, P., Pelta, J., & Oukhaled, A. (2018). Identification of single amino acid differences in uniformly charged homopolymeric peptides with aerolysin nanopore. Nature Communications, 9(1), 966.