WO2025003492A1

WO2025003492A1 - Uses of aerolysin nanopores

Info

Publication number: WO2025003492A1
Application number: PCT/EP2024/068392
Authority: WO
Inventors: Chan CAO; Matteo DAL PERARO; Hilal Lashuel
Original assignee: Ecole Polytechnique Federale De Lausanne (Epfl)
Priority date: 2023-06-30
Filing date: 2024-06-28
Publication date: 2025-01-02

Abstract

The invention relates to use of an aerolysin nanopore for the discrimination of different protein isoforms bearing different post-translational modifications (PTMs) and to methods for detecting adverse medical conditions associated with the presence of peptides, polypeptides or proteins bearing particular PTMs.

Description

Uses of aerolysin nanopores Technical Field [0001] The invention relates to use of an aerolysin nanopore for the discrimination of different protein isoforms bearing different post- translational modifications (PTMs) and to methods for detecting adverse medical conditions associated with the presence of peptides, polypeptides or proteins bearing particular PTMs. Background Art [0002] Using biological nanopores to sequence biopolymers, particularly nucleic acids, was proposed many years ago. Recent advances in enzyme-based control of DNA translocation and in DNA nucleotide resolution using mutated biological pores have satisfied the needs for a functional DNA sequencing biological device. [0003] Nanopore sensing is an approach that relies on the exploitation of individual binding or interaction events between to-be-analysed molecules and pore-forming macromolecules. Nanopore sensors can be created by placing nanometric-scaled pore peptide structures in an insulating membrane and measuring voltage-driven ionic transport through the pore in the presence of peptides, polypeptides or proteins. The identity of a peptide, polypeptide or protein can be ascertained through its peculiar electric signature, particularly the duration and extent of current block and the variance of current levels. Two of the essential components of nanopore sensing are (1) the control of analyte movement through the pore and (2) the discrimination of nucleotides as the analyte is moved through the pore. [0004] Pore-forming proteins are produced by a variety of organisms and are often involved in defence or attack mechanisms. One notable feature is that they are produced as soluble proteins that subsequently oligomerize and convert into a transmembrane pore in the target membrane. The most extensively characterized pore-forming proteins are the bacterial pore-forming toxins (PFTs), which, depending on the secondary structure elements that cross the bilayer, have been classified as α- or β-PFTs. [0005] Aerolysin, produced by Aeromonas species, is the founding member of a large superfamily that spans all of the kingdoms of life. Although it was the first β-PFT for which the X-ray structure of the soluble form was solved, the structure of the pore has remained elusive for long time. Aerolysin forms a heptameric beta-barrel in biological membranes. It is secreted as a monomer that binds to the outer membrane of susceptible cells. Upon binding, the monomers oligomerize to form a water-filled transmembrane channel that facilitates uncontrolled permeation of water, ions, and small organic molecules. Rapid discharge of vital molecules, such as ATP, dissipation of the membrane potential and ionic gradients, and irreversible osmotic swelling leading to rupture or lysis of the cell wall, frequently causing death of the host cell. This pore-forming property has been identified as a major mechanism by which protein toxins cause damage to cells. [0006] Aerolysin is the most promising biological nanopore for identifying the subtle differences between detected molecules, because of its unique structure profile. Cao C. et al. (Nat Nanotechnol. 2016 Apr 25. doi: 10.1038/nnano.2016.66) demonstrated the ability of aerolysin nanopore to resolve at high resolution individual short oligonucleotides that are 2 to 10 bases long without any extra chemicals or modifications, useful for single-molecule analysis of oligonucleotides. [0007] Additionally, Cao C. et al. (Nature Communications volume 9, Article number: 2823, 2018) described nanopore experimental results and molecular simulations based on an aerolysin structural model to map the sensing spots for ssDNA translocation. Computational and experimental results revealed two critical sensing spots (R220, K238) generating two constriction points along the pore lumen. Taking advantage of the sensing spots, all four nucleobases, cytosine methylation and oxidation of guanine can be clearly identified in a mixture sample. [0008] Beyond sequencing of biological polymers, there is a need for methods that make it possible to identify protein post-translational modifications. Protein post-translational modifications (PTMs) play crucial roles in biology and have emerged as reliable biomarkers for several diseases. However, only a handful of techniques are available to accurately measure their levels, capture their complexity at a single molecule level and characterize their multifaceted roles in health and disease. [0009] The standard methods currently available for the detection of PTMs are mass spectrometry (MS) and enzyme linked antibody-based assays. Recently great progress has been made towards developing and improving MS methods and immunoassays to map, detect and quantify modified proteins in biological samples. For instance, proximity extension assays and single molecule array have shown a high sensitivity for detecting PTMs. However, these techniques have fundamental drawbacks in terms of detection of the simultaneous occurrence of multiple PTMs, thus precluding correct profiling of PTMs and their functional cross-talk, as well as bulkiness, expensiveness and inherent complexity of use of MS systems. Therefore, alternative, user-friendly methods to accurately identify protein PTMs are highly desirable and essential, as increasing evidence suggests the PTM code is a combinatorial code that involves complex interplay and cross-talk among PTMs. [0010] The use of nanopores for the detection of post-translational modifications of peptides, polypeptides and proteins has been addressed in few prior art publications. For example, WO2015/040423A1 discloses that transmembrane nanopores can be used to determine the presence, number or position(s) of one or more post-translational modifications in a peptide, polypeptide or protein. This detection method is however limited to the determination of the presence, number or positions of PTM(s) of known type. This document does not teach determination of the type of PTM. [0011] Restrepo-Pérez et al., Nano Lett.2019, 19, 7957−7964 discloses the use of aerolysin for discriminating between phosphorylation and O- glycosylation in model peptides, wherein only one PTM was present in model peptides. This document does not teach the discrimination of PTM types when more than one PTM is present. [0012] Ensslen et al., J. Am. Chem. Soc. 2022, 144, 16060−16068, has shown the discrimination of peptides with either one, two or three PTM of a single type selected from acetylation and methylation, on different lysine residues of human histone H4. A mutant aerolysin pore is used. This study does not teach discrimination between acetylation and methylation. [0013] Huo et al., Proteomics 2022, 22:2100041, discloses the use of an aerolysin nanopore to discriminate between acetylation on one site of a peptide, phosphorylation on a different site of such peptide and the combination of these two PTMs. This study does not discriminate between different PTMs on the same amino acid. [0014] None of the prior art documents discloses discrimination of different PTM types at a given position of a peptide, polypeptide or protein. Such discrimination would however be of highest importance, for example in view of diagnosing adverse medical conditions associated with abnormal PTMs. Several disorders are associated with the replacement of a normal PTM by a PTM of a different type. For example, synucleinopathies (i.e. diseases associated with abnormal α-synuclein) can be caused by an erroneous PTM replacing a normal PTM at a given position of α-synuclein. [0015] Furthermore, the prior art achieved only the detection of PTMs from isolated/synthetic peptides. In order to use PTM detection of medical applications, it would be desirable to be able to perform PTM detection directly from a patient sample. However, the task of measuring biomarkers peptides in biological samples is challenging due to the intrinsic low sensitivity and selectivity of the techniques applied to bodily fluids. [0016] The present invention aims at solving these problems. Summary of the invention [0017] In a first aspect, the invention relates to the use of an aerolysin nanopore for the discrimination of at least two isoforms of a peptide, polypeptide, or protein, wherein the at least two isoforms differ from each other at least in that they bear different post-translational modifications at the same position of the peptide, polypeptide, or protein. [0018] In a second aspect, the invention relates to a method of discriminating between at least two isoforms of a peptide, polypeptide, or protein, wherein the at least two isoforms differ from each other at least in that they bear different post-translational modifications at the same position of the peptide, polypeptide, or protein, the method comprising: a. optionally contacting one or more peptide, polypeptide or protein isoform(s) with a protease such as to cleave one or more fragment(s) comprising one or more amino acids of interest; b. placing a sample comprising the peptide, polypeptide or protein isoform(s) or the fragment(s) thereof obtained in step a) in a first compartment of a chamber separated into a first and a second compartments by a membrane holding an aerolysin nanopore, wherein the aerolysin nanopore connects both compartments of the chamber; c. triggering translocation of the peptide, polypeptide or protein isoform(s) or of the fragment(s) thereof obtained in step a) through the aerolysin nanopore by applying a potential, preferably a voltage potential, across the aerolysin nanopore; d. measuring at least one characteristic indicative of the type(s), number and position(s) of any post-translational modification(s) of the peptide, polypeptide or protein isoform(s) or of the fragment(s) thereof obtained in step a) in the aerolysin nanopore, wherein said characteristic is preferably selected from dwell time, averaged relative current, relative current variation, skewness of relative current, kurtosis of relative current, minimum of relative current, maximum of relative current, peak to peak variation, integrated current and combinations thereof; and e. identifying the type(s), number and position(s) of any post- translational modification of the peptide, polypeptide or protein isoform(s) or of the fragment(s) thereof obtained in step a) present in the sample by comparing the at least one characteristic measured in step d) with characteristics previously measured for specific peptide, polypeptide or protein isoforms or fragments thereof for which the type(s), number and position(s) of post- translational modification(s) were known. [0019] In a third aspect, the invention relates to a method of detecting in a subject a disease or disorder associated with the presence of one or more PTM(s) of an aberrant type on one or more amino acid(s) of interest in a peptide, polypeptide or protein instead of one or more PTM(s) of a healthy type on the same amino acid(s), the method comprising: a. optionally contacting the peptide, polypeptide or protein from the subject with a protease such as to cleave one or more fragment(s) comprising one or more amino acids of interest; b. placing a sample comprising the peptide, polypeptide or protein from the subject or the fragment(s) thereof obtained in step a) in a first compartment of a chamber separated into a first and a second compartments by a membrane holding an aerolysin nanopore, wherein the aerolysin nanopore connects both compartments of the chamber; c. triggering translocation of the peptide, polypeptide or protein or of the fragment(s) thereof obtained in step a) through the aerolysin nanopore by applying a potential, preferably a voltage potential, across the aerolysin nanopore; d. measuring at least one characteristic indicative of the type(s), number and position(s) of any post-translational modification(s) of the peptide, polypeptide or protein or of the fragment(s) thereof obtained in step a) in the aerolysin nanopore, wherein said characteristic is preferably selected from dwell time, averaged relative current, relative current variation, skewness of relative current, kurtosis of relative current, minimum of relative current, maximum of relative current, peak to peak variation, integrated current and combinations thereof; and e. identifying the type(s), number and position(s) of any post- translational modification(s) of the peptide, polypeptide or protein or of the fragment(s) thereof obtained in step a) by comparing the at least one characteristic measured in step d) with characteristics previously measured for specific peptides, polypeptides or proteins or fragments thereof for which the type(s), number and position(s) of post-translational modification(s) was known. [0020] In a fourth aspect, the invention relates to a method of treating in a subject a disease or disorder associated with the presence of one or more PTM(s) of an aberrant type on one or more amino acid(s) of interest in a peptide, polypeptide or protein instead of one or more PTM(s) of a healthy type on the same amino acid(s), the method comprising: a. optionally contacting the peptide, polypeptide or protein from the subject with a protease such as to cleave one or more fragment(s) comprising the one or more amino acids of interest; b. placing a sample comprising the peptide, polypeptide or protein from the subject or the fragment thereof obtained in step a) in a first compartment of a chamber separated into a first and a second compartments by a membrane holding an aerolysin nanopore, wherein the aerolysin nanopore connects both compartments of the chamber; c. triggering translocation of the peptide, polypeptide or protein or of the fragment(s) thereof obtained in step a) through the aerolysin nanopore by applying a potential, preferably a voltage potential, across the aerolysin nanopore; d. measuring at least one characteristic indicative of the type(s), number and position(s) of any post-translational modifications of the peptide, polypeptide or protein or of the fragment(s) thereof obtained in step a) in the aerolysin nanopore, wherein said characteristic is preferably selected from dwell time, averaged relative current, relative current variation, skewness of relative current, kurtosis of relative current, minimum of relative current, maximum of relative current, peak to peak variation, integrated current and combinations thereof; e. identifying the type(s), number(s) and position(s) of any post- translational modification(s) of the peptide, polypeptide or protein of the fragment(s) thereof obtained in step a) present in the sample by comparing the at least one characteristic measured in step d) with characteristics previously measured for specific peptides, polypeptides or proteins or fragments thereof, for which the type(s), number and position(s) of post-translational modification(s) was known; and f. provided a PTM of an aberrant type associated with the occurrence of a disease or disorder was identified in step e), treating the subject with a treatment appropriate to treat such disease or disorder. [0021] In a fifth aspect, the invention relates to a method of detecting a synucleinopathy in a subject comprising: a. optionally contacting a sample comprising α-synuclein from the subject with a protease, such as to cleave one or more fragment(s) of the α-synuclein, preferably the C-terminal sequence of the α- synuclein; b. placing the sample from the subject or the sample obtained in the end of step a. in a first compartment of a chamber separated into a first and a second compartments by a membrane holding an aerolysin nanopore, wherein the aerolysin nanopore connects both compartments of the chamber; c. triggering translocation of α -synuclein or of the fragment(s) obtained in step a. through the aerolysin nanopore by applying a potential, preferably a voltage potential, across the aerolysin nanopore; d. measuring at least one characteristic indicative of the type(s), number and position(s) of any post-translational modifications of α -synuclein or of the fragment(s) obtained in step a) in the aerolysin nanopore, wherein said at least one characteristic is preferably selected from dwell time, averaged relative current, relative current variation, skewness of relative current, kurtosis of relative current, minimum of relative current, maximum of relative current, peak to peak variation, integrated current and combinations thereof; and e. identifying the type(s), number and position(s) of any post- translational modification of α-synuclein or of the fragment(s) obtained in step a. by comparing the characteristics measured in step c. with characteristics previously measured for specific α- synuclein isoforms or fragments thereof for which the type(s), number and position(s) of post-translational modification(s) were known. [0022] In a sixth aspect the invention relates to a method of treating a synucleinopathy in a subject comprising: a. optionally contacting a sample comprising α-synuclein from the subject with a protease, such as to cleave one or more fragment(s) of the α-synuclein, preferably the C-terminal sequence of the α- synuclein; b. placing the sample from the subject or the sample obtained in the end of step a. in a first compartment of a chamber separated into a first and a second compartments by a membrane holding an aerolysin nanopore, wherein the aerolysin nanopore connects both compartments of the chamber; c. triggering translocation of α -synuclein or of the fragment(s) obtained in step a. through the aerolysin nanopore by applying a potential, preferably a voltage potential, across the aerolysin nanopore; d. measuring at least one characteristic indicative of the type(s), number and position(s) of any post-translational modifications of α -synuclein or of the fragment(s) obtained in step a) in the aerolysin nanopore, wherein said at least one characteristic is preferably selected from dwell time, averaged relative current, relative current variation, skewness of relative current, kurtosis of relative current, minimum of relative current, maximum of relative current, peak to peak variation, integrated current and combinations thereof; e. identifying the type(s), number and position(s) of any post- translational modification of α-synuclein or of the fragment(s) obtained in step a. by comparing the characteristics measured in step c. with characteristics previously measured for specific α- synuclein isoforms or fragments thereof for which the type(s), number and position(s) of post-translational modification(s) were know; and f. provided one or more PTM(s) indicative of a synucleinopathy was identified in step e), treating the patient with a treatment appropriate to treat such synucleinopathy. [0023] In a seventh aspect, the invention relates to the use of an aerolysin nanopore for the identification of at least one post-translational modification born by a peptide, polypeptide or protein, wherein said at least one post-translational modification is a nitration or an oxidation. Description of the figures [0024] [Fig. 1]: Illustration of the single-channel recording setup used in the Examples, composed of two chambers separated by a lipid membrane that is formed across an orifice; the chambers are named cis and trans. Voltage is applied across the pore using two Ag/AgCl electrodes. [0025] [Fig. 2]: Nanopore single channel recording (raw current traces; typical events; contour plots, I/I0 percentage and dwell time histograms) of wt α-syn124-140 peptide in 1.0 M KCl solution buffered with 10 mM Tris, and 1.0 mM EDTA at pH=7.4 of Example 1. All data were obtained by applying a voltage of +100 mV. [0026] [Fig. 3]: Nanopore single channel recording (raw current traces; typical events; contour plots, I/I₀ percentage and dwell time histograms) of wt α-syn124-140 peptide in 1.0 M KCl, 8.0 M urea solution buffered with 10 mM Tris, and 1.0 mM EDTA at pH=7.4 of Example 1. All data were obtained by applying a voltage of +100 mV. [0027] [Fig. 4]: Typical ionic current signals of different PTM types and their spatial combinations. The scale shown in the left bottom panel applies to all the events of Example 1. [0028] [Fig. 5]: Deep learning platform used in Example 2: a LSTM recurrent neural network was used to read the events and then followed by a MLP to predict the peptides. [0029] [Fig. 6]: Confusion matrix of wt, pY129 and nY125 peptides obtained by deep learning approach in Example 2. [0030] [Fig.7]: Assignment percentage of different mixture samples of wt and pY125 at a concentration ratio of 1:1 (left), 1:3 (middle) and 3:2 (right) from Example 2. The theoretical accuracy is shown by the white columns while the predicted accuracy is represented by blue columns. [0031] [Fig. 8]: Selection percentage versus averaged accuracy obtained from deep learning approach of peptides in Example 2, including wt, pY125, nY125, pS129, pY125pS129, nY125nY133nY136 and nY125pS129. [0032] [Fig.9]: The confusion matrix of wt, pY125, nY125, pS129, pY125pS129, nY125nY133nY136 and nY125pS129 classification of Example 2. Columns represent actual peptides from the test set, while rows are the peptides that the deep learning algorithm assigned them to. All data were obtained using 1.0 M KCl, 10 mM Tris, and 1.0 mM EDTA buffer at pH 7.4 applying a voltage of +100 mV. [0033] [Fig. 10]: Raw current traces after addition of serial dilutions of RBCs (left) and plasma samples (right) in Example 3. [0034] [Fig. 11]: Raw current traces of RBCs samples (left) and crude plasma sample (right) in presence of 6 µM wt α-synuclein peptides of Example 3. The RBCs used here is 1:10 dilution while the crude plasma sample is 1:50 dilution. [0035] [Fig.12]: Raw current traces of Example 3 showing signals in symmetric salt (top, 1.0 M KCl) and in a gradient salt concentration (bottom, 0.15 M/3.0 M (cis/trans) KCl). [0036] [Fig.13]: Correlation between signal frequency f_sig and the concentration of wt α-synuclein in gradient salt conditions of Example 3 ranging from 100 pM to 200 nM peptide in the cis chamber under 100 mV applied voltage. [0037] [Fig.14]: Possible peptide fragments from cathepsin D (CtsD) digestion based on previous studies and MS characterization of CtsD digestion of full-length α-synuclein. [0038] [Fig.15]: Raw current trace of nanopore measurement of CtsD digested samples (Example 4). [0039] [Fig. 16]: Deep learning result from five independent experiments from Example 4. Detailed description of the invention [0040] The subject matter herein described will be clarified by means of the following description. It is however to be understood that the subject matter described in this specification is not limited to the aspects described herein and depicted in the drawings; to the contrary, the scope of the subject-matter herein described is defined by the claims. Moreover, it is to be understood that the specific conditions or parameters described and/or shown in the following are not limiting of the subject matter herein described, and that the terminology used herein is for the purpose of describing particular aspects by way of example only and is not intended to be limiting. [0041] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Further, unless otherwise required by the context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Further, for the sake of clarity, the use of the term “about” is herein intended to encompass a variation of +/‒ 10% of a given value. [0042] As used in the following and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Also, the use of "or" means "and/or" unless stated otherwise. Similarly, "comprise", "comprises", "comprising", "include", "includes" and "including" are interchangeable and not intended to be limiting. It is to be further understood that where for the description of various embodiments use is made of the term "comprising", those skilled in the art will understand that in some specific instances, an embodiment can be alternatively described using language "consisting essentially of" or "consisting of." [0043] Use of an aerolysin nanopore for the discrimination of at least two isoforms of a peptide, polypeptide, or protein [0044] The present invention provides for the use of an aerolysin nanopore for the discrimination of at least two isoforms of a peptide, polypeptide, or protein, wherein the at least two isoforms differ from each other at least in that they bear different post-translational modifications at the same position of the peptide, polypeptide, or protein. In other words, the aerolysin nanopore is used to distinguish between two alternative PTMs on the same amino acid. [0045] In a preferred aspect, the PTMs are selected from acetylation, acetylation, ubiquitination, oxidation, nitration, phosphorylation and combinations thereof, preferably oxidation, nitration, phosphorylation and combinations thereof, more preferably oxidation, nitration and combinations thereof. [0046] This is very useful, as numerous adverse health conditions are associated with aberrant PTMs on specific amino acids. For example, several neurodegenerative diseases including synucleinopathies such as Alzheimerʼs disease (AD) and Parkinsonʼs disease (PD) are caused by the accumulation of misfolded and aggregated proteins in the brain regions that are affected by the disease (e.g., amyloid-β in amyloid plaques, Tau in neurofibrillary tangles and α-synuclein in Lewy bodies and Lewy neurites). One shared characteristic among these proteins is that their aggregated forms are heavily modified. PTMs have emerged as key signatures of disease pathologies and are commonly used as the primary (bio)markers of disease progression and pathology formation, spreading and clearance in response to therapies. [0047] As another example, estrogen receptor- β can bear two alternative PTMs at Ser16: O-linked N-acetylglucosamination or O-linked phosphorylation. [0048] The peptide, polypeptide or protein is preferably any peptide, polypeptide or protein for which aberrant PTM(s) is/are associated with adverse health conditions, such as a disease or disorder. In a preferred aspect, the peptide polypeptide or protein is selected form the group consisting of amyloid-β, tau, α-synuclein, estrogen receptor-βand fragments thereof. Preferably it is selected from the group consisting of amyloid-β, tau, α-synuclein and fragments thereof. Most preferably it α-synuclein or a fragment thereof. [0049] Method of discriminating between at least two isoforms of a peptide, polypeptide, or protein; Method of detecting an adverse health condition; Method of detecting a synucleinopathy [0050] The detection is performed by contacting the peptide, polypeptide or protein with an aerolysin pore such that the target peptide, polypeptide or protein moves (translocates) through the pore. [0051] This is performed by placing the peptide, polypeptide or protein, or a fragment thereof in a first compartment of a chamber separated into a first and a second compartments by a membrane holding an aerolysin nanopore wherein the aerolysin nanopore connects both compartments of the chamber. [0052] Any membrane may be used in accordance with the invention. Suitable membranes are well-known in the art. The membrane is preferably an amphiphilic layer. An amphiphilic layer is a layer formed from amphiphilic molecules, such as phospholipids, which have both at least one hydrophilic portion and at least one lipophilic or hydrophobic portion. The amphiphilic molecules may be synthetic or naturally occurring. [0053] The amphiphilic layer may be a monolayer or a bilayer. The amphiphilic layer is typically a planar lipid bilayer or a supported bilayer. The amphiphilic layer is typically a lipid bilayer. Lipid bilayers are models of cell membranes and serve as excellent platforms for a range of experimental studies. For example, lipid bilayers can be used for in vitro investigation of membrane proteins by single-channel recording. Alternatively, lipid bilayers can be used as biosensors to detect the presence of a range of substances. The lipid bilayer may be any lipid bilayer. Suitable lipid bilayers include, but are not limited to, a planar lipid bilayer, a supported bilayer or a liposome. [0054] In another preferred embodiment, the membrane is a solid-state layer. A solid-state layer is not of biological origin. In other words, a solid-state layer is not derived from or isolated from a biological environment such as an organism or cell, or a synthetically manufactured version of a biologically available structure. Solid state layers can be formed from both organic and inorganic materials including, but not limited to, microelectronic materials, insulating materials, organic and inorganic polymers such as polyamide, plastics such as Teflon® or elastomers such as two-component addition-cure silicone rubber, and glasses, and provides therefore the advantage of a manufacturing process free of any biologically derived material. The solid-state layer may be formed from monatomic layers, such as graphene, or layers that are only a few atoms thick. [0055] The method is typically carried out using (i) an artificial amphiphilic layer comprising a pore, (ii) an isolated, naturally occurring lipid bilayer comprising a pore, or (iii) a cell having a pore inserted therein. The method is typically carried out using an artificial amphiphilic layer, such as an artificial lipid bilayer. The layer may comprise other transmembrane and/or intramembrane proteins as well as other molecules in addition to the pore. The methods of the invention are typically carried out in vitro. [0056] The peptide, polypeptide or protein may be coupled to the membrane. This may be done using any known method. If the membrane is an amphiphilic layer, such as a lipid bilayer (as discussed in detail above), the peptide, polypeptide or protein is preferably coupled to the membrane via a polypeptide present in the membrane or a hydrophobic anchor present in the membrane. The hydrophobic anchor is preferably a lipid, fatty acid, sterol, carbon nanotube or amino acid. The peptide, polypeptide or protein may be coupled directly to the membrane. The peptide, polypeptide or protein is preferably coupled to the membrane via a linker. Preferred linkers include, but are not limited to, polymers, such as polynucleotides, polyethylene glycols (PEGs) and polypeptides. [0057] The peptide, polypeptide or protein may be transiently coupled to an amphiphilic layer, such as a lipid bilayer using cholesterol or a fatty acyl chain. Any fatty acyl chain having a length of from 6 to 30 carbon atoms, such as hexadecanoic acid, may be used. In preferred embodiments, the peptide, polypeptide or protein is coupled to an amphiphilic layer. Coupling of the peptide, polypeptide or protein to synthetic lipid bilayers has been carried out previously with various different tethering strategies. [0058] The pore used in the methods of the invention is a pore such as described herein, i.e. an aerolysin nanopore or a mutant thereof, such as an aerolysin nanopore comprising at least one mutant monomer such as described herein or at least one construct , as detailed herein below. The pore may be chemically modified in any of the ways discussed above. The pore is preferably modified with a covalent adaptor that is capable of interacting with the target peptide, polypeptide or protein. [0059] The methods may be carried out using any apparatus that is suitable for transmembrane pore sensing. For example, the apparatus comprises a chamber comprising an aqueous solution and a barrier that separates the chamber into two sections. The barrier has an aperture in which the membrane containing the pore is formed. For instance, the methods may be carried out using the apparatus described in International Application WO 2008/102120. [0060] One or more characteristic(s) indicative of the type(s), number and position(s) of any post-translational modification(s) of the peptide, polypeptide or protein is/are then measured as the peptide, polypeptide or protein moves with respect to the pore using standard methods known in the art. One or more characteristics indicative of the type(s), number and position(s) of any post-translational modification(s) of the peptide, polypeptide or protein are preferably measured as the peptide, polypeptide or protein moves through the pore. [0061] The method comprises contacting the target peptide, polypeptide or protein with a pore such as described above, such that the target peptide, polypeptide or protein moves through the pore. One or more characteristics indicative of the type(s), number and position(s) of any post-translational modification(s) of the target peptide, polypeptide or protein are then measured as the peptide, polypeptide or protein moves with respect to the pore using standard methods known in the art. One or more characteristics indicative of the type(s), number and position(s) of any post-translational modification(s) of the target peptide, polypeptide or protein are preferably measured as the peptide, polypeptide or protein moves through the pore. Step c. is preferably carried out with a potential applied across the pore. [0062] The applied potential is preferably a voltage potential, preferably a voltage potential of 50 to 350 mV, more preferably 75 to 350 mV, even more preferably 100 to 350 mV. the detection limit can be lowered by using a voltage towards the top of the range. Therefore, in a particular aspect, the voltage is of 100 to 350mV, preferably 250 to 350 mV, more preferably 200 to 350 mV, more preferably 220 to 350 mV, 230 to 350 mV, 240 to 350 mV, 250 to 350 mV or 260 to 350 mV. The latter particular aspect is particularly useful when the peptide, polypeptide or protein is present in low concentrations. [0063] Alternatively, the applied potential may be a chemical potential. An example of this is using a salt gradient across an amphiphilic layer or any other suitable membrane. [0064] The method may comprise sensing and/or characterising any number of peptides, polypeptides or proteins, such as 1, 2, 5, 10, 15, 20, 30, 40, 50, 100 or more peptides, polypeptides or proteins. [0065] The measured characteristics are preferably selected from dwell time (dt), averaged relative current (arc), relative current variation (rcv), skewness of relative current (src), kurtosis of relative current (krc), minimum of relative current (minrc), maximum of relative current (maxrc), peak to peak variation (ppv) and integrated current (ic) and combinations thereof. The more characteristics are measured, the more accurate the discrimination between different PTMs will be. In other words, the presence, number, absence or type of PTM at one or more position on the peptide, polypeptide or protein are indirectly obtained by measuring at least one characteristic indicative of the type(s), number and position(s) of any post-translational modification(s) of the peptide, polypeptide or protein, or a fragment thereof, upon translocation through the aerolysin nanopore. As a way of example, the presence, number, absence or type of PTM at one or more position on the peptide, polypeptide or protein will change the dwell type of the analyte peptide, polypeptide or protein upon translocation through the aerolysin nanopore compared to a reference peptide, polypeptide or protein. [0066] The methods are typically carried out in the presence of a buffer. In the exemplary apparatus discussed above, the buffer is present in the aqueous solution in the chamber. Any suitable buffer including at least one electrolyte may be used in the methods of the invention. Typically, the buffer is HEPES. Another suitable buffer is Tris-HCl buffer. The methods are typically carried out at a pH of from 3.0 to 12.0, preferably about 7.5. [0067] The methods may be carried out at from 0 °C to 100 °C, from 15 °C to 95 °C, from 16 °C to 90 °C, from 17 °C to 85 °C, from 18 °C to 80 °C, 19 °C to 70 °C, or from 20 °C to 60 °C. The methods are typically carried out at room temperature (around 25 °C, such as between 20 and 30 °C). The methods are optionally carried out at a temperature that supports enzyme function, such as about 37 °C. [0068] The characteristics indicative of the type(s), number and position(s) of any post-translational modification(s) of a peptide, polypeptide or protein to be analysed are measured and compared with characteristics indicative of the type(s), number and position(s) of any post- translational modification(s) of peptides, polypeptides or proteins having a known type of PTM(s). Preferably, a databased is produced, with the characteristics indicative of the type(s), number and position(s) of any post-translational modification(s) of peptides, polypeptides or proteins having known PTM(s) on amino acid(s) of interest, including characteristics indicative of the type(s), number and position(s) of any post-translational modification(s) of peptides, polypeptides and proteins having each possible type of PTM on each amino acid of interest. The characteristics measured with the peptide, polypeptide or protein of interest are then compared to the database and the type of PTM at each amino acid of interest can be defined. [0069] In a particular aspect, the comparison of the characteristics indicative of the type(s), number and position(s) of any post-translational modification(s) of the peptide, polypeptide or protein of interest is compared to peptide(s), polypeptide(s) or protein(s) having known PTM(s) type(s) at amino acid(s) of interest using a deep learning algorithm. [0070] The essential steps of the method may be preceded by an optional step of cleaving a peptide, polypeptide or protein using a protease. Such protease can be selected by the person skilled in the art, depending on the fragment that is sought to be obtained. Typically, the protease is selected such that the peptide, polypeptide or protein is cleaved such as to obtain a fragment comprising the specific amino acid(s) of interest, i.e. the amino acid(s) that serve as biomarker of an adverse health condition when it/they bear aberrant PTM(s). [0071] When the protein is α-synuclein, the protease is preferably selected from cathepsin D (CtsD), trypsin and endoproteinase Glu-C, most preferably it is cathepsin D. These enzymes all have the capability to cleave the C-terminal section of α-synuclein, leading to one or more fragment(s) comprising all known disease-associated PTM sites (p/nY125, pS129, nY133, nY136). Cathepsin D is the most preferred protease, because its cleavage of α -synuclein results in a single, reasonably sized fragment containing all the desired PTM sites. [0072] Peptides, polypeptides and proteins bearing PTM to be discriminated [0073] The peptide, polypeptide or protein can be provided in purified form or in the form of a mixture of several peptides, polypeptides and/or proteins, such as for example as part of a biological on non-biological sample. The possibility to detect PTMs even when multiple peptides, polypeptides and/or proteins are present is particularly advantageous, as it allows to detect several biomarkers at the same time. [0074] The peptide, polypeptide or protein can be secreted from cells. Alternatively, the peptide, polypeptide or protein can be a peptide, polypeptide or protein that is present inside cells such that the peptide, polypeptide or protein must be extracted from the cells before the invention can be carried out. [0075] The peptide, polypeptide or protein can be provided as part of a biological sample from a subject, such as plasma or a sample comprising red blood cells, preferably wherein haemoglobin proteins have been removed. Indeed, aerolysin, preferably the aerolysin nanopores disclosed below are capable of sensing PTMs as described above even from a complex matrix such as a biological sample. Sensing accuracy is improved when haemoglobin proteins are removed from such sample. Methods for removing haemoglobin proteins are well-known to the person skilled in the art. [0076] The sample is preferably a biological sample. The invention can be carried out in vitro on a sample obtained from or extracted from any organism or microorganism. The organism or microorganism is typically archaean, prokaryotic or eukaryotic and typically belongs to one the five kingdoms: plantae, animalia, fungi, monera and protista. The invention cam be carried out in vitro on a sample obtained from or extracted from any virus. The sample is preferably a fluid sample. The sample preferably comprises a body fluid of the patient. The sample may be urine, lymph, saliva, mucus or amniotic fluid but is preferably blood, plasma or serum. Typically, the sample is human in origin, but alternatively it may be from another mammal animal such as from commercially farmed animals such as horses, cattle, sheep or pigs or may alternatively be pets such as cats or dogs. [0077] The sample is typically processed prior to being assayed, for example by centrifugation or by passage through a membrane that filters out unwanted molecules or cells. The sample may be measured immediately upon being taken. The sample may also be typically stored prior to assay, preferably below -70°C. [0078] In a preferred aspect, the peptide, polypeptide or protein is provided as part of a biological sample and the biological sample is diluted with a biologically compatible aqueous solvent at a biological sample to solvent ratio of 1:10 to 1:50. Such ratios are advantageous in that they ensure a stable baseline of the ionic current. [0079] In a particular aspect, the peptide, polypeptide or protein is a protein known to be associated with an adverse health condition, such as a disease or disorder, when it bears one or more aberrant PTM on selected amino acid(s). In a preferred aspect, the peptide, polypeptide or protein is selected from amyloid-β, Tau, α-synuclein, estrogen receptor-β, and fragments thereof, more preferably amyloid-β, Tau, α-synuclein and fragments thereof. [0080] Erroneous PTMs on specific amino acids of α-synuclein such as K1, K6, K12, K21, K23, K32, K34, Y39, K45, K58, T59, K60, K61, T64, T72, K80, T81, K95, K96, Y125, S129, Y133, Y136 and Y39, particularly Y125, S129, Y133, Y136 and Y39 and more particularly S129, have been found relevant as biomarkers of Alzheimer and Parkinson diseases. Therefore, in a preferred aspect, the peptide, polypeptide or protein is α-synuclein or a fragment thereof, preferably the C-terminal fragment, more preferably the fragment from residues 124 to 140 of α-synuclein. [0081] In a preferred aspect, the peptide, polypeptide or protein is in unfolded form. This is advantageous as it makes translocation of the peptide, polypeptide or protein easier and the sensing of the peptide, polypeptide or protein more accurate. In a more preferred aspect, the peptide, polypeptide or protein is in unfolded form and the aerolysin nanopore is in folded form. Unfolding of the peptide, polypeptide or protein can be achieved by any means known to the person skilled in the art. For example, the peptide, polypeptide or protein can be unfolded by adding urea, as known by the person skilled in the art, optionally urea together with KCl, Tris and EDTA at pH 7.3 to 7.5, such as pH 7.4. In a most preferred aspect, the peptide, polypeptide or protein is unfolded α- synuclein or a fragment thereof. Synuclein can be unfolded by using urea, as described above, most preferably using 7 to 9M urea, optionally with KCl, Tris and EDTA, such as with 0.5 to 1.5M KCl (preferably 1M KCl), 5 to 15mM Tris (preferably 10mM Tris) and 0.5 to 1.5mM EDTA (preferably 1mM EDTA) at pH7.3 to 7.5, such as pH 7.4. [0082] Aerolysin polypeptides or monomers and mutants thereof [0083] Alternative to the use of wild-type aerolysin-based nanopores, the present inventors have further found that aerolysin nanopore formed from specific mutant aerolysin monomers, such as defined herein below, were particularly advantageous for use in the present invention because they are characterized by high sensing accuracy. Such mutants are advantageous because they provide for better discrimination between different PTMs at a given position, as a result of the structure of the mutants. The high sensing accuracy is namely made possible because the mutants make it possible to use particularly high voltage. Such pores are also characterized by a highly efficient analyte translocation. [0084] In particular, such mutants are characterized by a constriction site having a small diameter and a small height. In preferred aspects, the aerolysin monomer also advantageously comprises only one single constriction site. These features enable an absolutely single amino acid resolution of the reading site. [0085] In a preferred aspect of the invention, specific mutant aerolysin polypeptides are used. Such mutant polypeptides are either mutants of any of SEQ ID NO:1 to 7, variants or fragments thereof, or mutants of any of SEQ ID NO:8 or 10 to 15, variants or fragments thereof. Mutants of SEQ ID NO:1 to 7 are non-mature polypeptides that cannot form pores. Mutants of SEQ ID NO:8 or 10 to 15 are the mature forms of the variants of SEQ ID NO:1 to 7, wherein the C-terminal propeptide has been removed. Such variants are monomer that retain the ability to form pores together with other aerolysin monomers. Variants of any one of SEQ ID NO:1 to 7 can be turned into monomers by maturation of the polypeptide. After maturation, such methods for confirming the ability of mutant monomers to form pores are well-known in the art. Variants of any one of SEQ ID NO:1 to 7 can be turned into monomers by maturation of the polypeptide (removal of the C-terminal propeptide), thus acquiring their ability to form monomers. [0086] In a preferred aspect, the mutant aerolysin polypeptides have an amino acid selected from A, S, G, Q, W, E, D and N, preferably A, S, G, or D, more preferably A, S or G, more preferably A or S, most preferably A in position 238 and an amino acid selected from W and A, most preferably A in position 242. [0087] In a more preferred aspect, the first amino acid substitution is selected from the group consisting of K238A, K238S, K238G and K238D or from the group consisting of V238A, V238S, V238G, and V238D, respectively. More preferably, it is selected from the group consisting of K238A, K238S and K238G, or from the group consisting of V238A, V238S and V238G, respectively. Even more preferably, it is selected from the group consisting of K238A and K238S or from the group consisting of V238A and V238S respectively. Most preferably it is K238A or V238A, respectively. [0088] In another preferred aspect, the second amino acid substitution is K242A, N242A or S242A, respectively. [0089] The first and the second mutation result in a narrower diameter of the constriction site in position R220. [0090] Preferred polypeptides for use in the present invention are polypeptides of SEQ ID NO:16, which is a polypeptide of SEQ ID NO:1, modified with the substitution K238A, and the polypeptide of SEQ ID NO:17, which is the polypeptide of SEQ ID NO:16, further modified with the substitution K242A. [0091] In another preferred aspect, the mutant aerolysin polypeptide or monomer comprises at least one additional mutation that contributes to the removal of other constriction sites in the monomer. Indeed, wild-type aerolysin comprises four constriction sites, at positions 282, 220, 238 and 242. The constriction site at position 220 is the constriction site having the narrowest diameter among the aerolysin constriction sites. It is thus the main constriction site of an aerolysin monomer for analytical purposes. As explained above, the mutant aerolysin peptides or monomers are engineered to further reduce the diameter of the constriction site at position 220 (due to the first and second amino acid substitution). In order to further improve sensing accuracy, preferred mutant aerolysin polypeptides or monomers are also engineered to increase the diameter or even eliminate the constriction sites in positions 282, 238 and 242. This results in improved accuracy, as it reduces the impact of secondary constriction sites on the signal. Thus, the mutant aerolysin polypeptide or monomer comprises at least one of - a third amino acid substitution selected from the group consisting of R282A, R282S, R282G, R282D, R282W, preferably selected from the group consisting of R282A and R282S, most preferably R282S when the sequence is selected from any one of SEQ ID NO:1,2, 4, 6 to 8, 10, 12, 14 and 15 or a variant or fragment thereof or a third amino acid substitution selected from P282A, P282S, P282G, P282D, P282W, preferably selected from the group consisting of P282A and P282S, most preferably P282S when the sequence is selected from SEQ ID NO:3 and 11 or a variant or fragment thereof; - a fourth amino acid substitution selected from the group consisting of D216A, D216S, D216G, D216D and D216W, preferably selected from the group consisting of D216A and D216S, most preferably D216S when the sequence is selected from any one of SEQ ID NO:1, 2, 4, 7, 8, 10, 12 and 15 or a variant or fragment thereof or a fourth amino acid substitution selected from selected from the group consisting of V216A, V216S, V216G, V216D and V216W, preferably selected from the group consisting of V216A and V216S, most preferably V216S when the sequence is selected from SEQ ID NO: 3, 6, 11 or 14 or a variant or fragment thereof or a fourth amino acid substitution selected from selected from the group consisting of G216A and G216S, most preferably V216S when the sequence is selected from SEQ ID NO: 5 and 13 or a variant or fragment thereof; - a fifth amino acid substitution selected from the group consisting of D222A, D222S, D222G and D222D, preferably selected from the group consisting of D222A and D222S, most preferably D222S when the sequence is selected from any one of SEQ ID NO:1,2, 4, 6 to 8, 10, 12, 14 and 15 or a variant or fragment thereof or a fifth amino acid substitution selected the group consisting of K222A, K222S, K222G and K222D, preferably selected from the group consisting of K222A and K222S, most preferably K222S when the sequence is selected from SEQ ID NO:3 or 11 or a variant or fragment thereof or a fifth amino acid substitution selected from the group consisting of N222A, N222S, N222G and N222D, preferably selected from the group consisting of N222A and N222S, most preferably N222S when the sequence is selected from any one of SEQ ID NO: 5 and 13 or a variant or fragment thereof; - a sixth amino acid substitution selected from the group consisting of E258A, E258S, E258G, E258D and E258W, preferably selected from the group consisting of E258A and E258S, most preferably E258S when the sequence is selected from any one of SEQ ID NO:1, 2, 4, 6 to 8, 10, 12, 14 and 15 or a variant or fragment thereof or a sixth amino acid substitution selected from the group consisting of I258A, I258S, I258G, I258D and I258W, preferably selected from the group consisting of I258A and I258S, most preferably I258S when the sequence is selected from any one of SEQ ID NO: 3 and 11 or a variant or fragment thereof; - a seventh amino acid substitution selected from the group consisting of E254A, E254S, E254G, E254D and E254W, preferably selected from the group consisting of E254A and E254S, most preferably E254S when the sequence is selected from any one of SEQ ID NO:1,2, 4, 5, 6 to 8, 10, 12, 13, 14 and 15 or a variant or fragment thereof or a seventh amino acid substitution selected from the group consisting of L254A, L254S, L254G, L254D and L254W, preferably selected from the group consisting of L254A and L254S, most preferably L254S when the sequence is selected from any one of SEQ ID NO: 3 and 11 or a variant or fragment thereof; - an eighth amino acid substitution selected from the group consisting of K244A, K244S, K244G, K244D and K244W, preferably selected from the group consisting of K244A and K244S, most preferably K244S when the sequence is selected from any one of SEQ ID NO:1, 2, 4, 5, 6 to 8, 10, 12, 13, 14 and 15 or a variant or fragment thereof or an eighth amino acid substitution selected from the group consisting of F244A, F244S, F244G, F244D and F244W, preferably selected from the group consisting of F244A and F244S, most preferably F244S when the sequence is selected from any one of SEQ ID NO: 3 and 11 or a variant or fragment thereof; - a ninth amino acid substitution selected from the group consisting of E252A, E252S, E252G, E252D and E252W, preferably selected from the group consisting of E252A and E252S, most preferably E252S when the sequence is selected from any one of SEQ ID NO:1, 2, 4, 6 to 8, 10, 12, 14 and 15 or a variant or fragment thereof or a ninth amino acid substitution selected from the group consisting of T252A, T252S, T252G, T252D and T252W, preferably selected from the group consisting of T252A and T252S, most preferably T252S when the sequence is selected from any one of SEQ ID NO: 3 and 11 or a variant or fragment thereof or a ninth amino acid substitution selected from the group consisting of V252A, V252S, V252G, V252D and V252W, preferably selected from the group consisting of V252A and V252S, most preferably V252S when the sequence is selected from any one of SEQ ID NO: 5 and 13 or a variant or fragment thereof; and - a tenth amino acid substitution selected from the group consisting of K246S, K246A K246G, K246D and K246W, preferably selected from the group consisting of K246A and K246S, most preferably K246S when the sequence is selected from any one of SEQ ID NO:1, 2, 4, 5, 7, 8, 10, 12, 13, and 15 or a variant or fragment thereof or a tenth amino acid substitution selected from the group consisting of W246S, W246A W246G, W246D and W246W, preferably selected from the group consisting of W246A and W246S, most preferably W246S when the sequence is selected from any one of SEQ ID NO: 3 and 11 or a variant or fragment thereof or a tenth amino acid substitution selected from the group consisting of Q246S, Q246A Q246G, Q246D and Q246W, preferably selected from the group consisting of Q246A and Q246S, most preferably Q246S when the sequence is selected from any one of SEQ ID NO: 6 and 14 or a variant or fragment thereof. [0092] In a preferred aspect, the polypeptides or monomers comprises at least a first, a second and a third amino acid substitution as described above; preferably it comprises exactly such mutations. In a more preferred aspect, the polypeptides or monomers comprises at least a first, a second, a third and a fourth amino acid substitution as described above; preferably it comprises exactly such mutations. In a more preferred aspect, the polypeptides or monomers comprises at least a first, a second, a third, a fourth and a fifth amino acid substitution as described above; preferably it comprises exactly such mutations. In a more preferred aspect, the polypeptides or monomers comprises at least a first, a second, a third, a fourth, a fifth and a sixth amino acid substitution as described above; preferably it comprises exactly such mutations. In a more preferred aspect, the polypeptides or monomers comprises at least a first, a second, a third, a fourth, a fifth, a sixth and a seventh amino acid substitution as described above; preferably it comprises exactly such mutations. In a more preferred aspect, the polypeptides or monomers comprises at least a first, a second, a third, a fourth, a fifth, a sixth, a seventh amino acid and an eighth amino acid substitution as described above; preferably it comprises exactly such mutations. In an even more preferred aspect, the polypeptides or monomers comprises at least a first, a second, a third, a fourth, a fifth, a sixth, a seventh, an eighth and a ninth amino acid substitution as described above; preferably it comprises exactly such mutations. In a most preferred aspect, the polypeptides or monomers comprises all of the amino acid substitution described above; preferably it comprises exactly such mutations. By the terms " it comprises exactly such mutations" it is meant that the polypeptide or monomer corresponds to any one of SEQ ID NO:1 to 8 or 10 to 15 (or a variant or fragment thereof), respectively, mutated to bear all of the recited mutations and only such recited mutations, in accordance with any of the above-described embodiments. [0093] The constriction site at position 220 is preferably characterized by a diameter of at most 1.2 nm, more preferably at most 1.1 nm, most preferably at most 1.0 nm. In another preferred aspect of the invention, the constriction site at position 220 is characterized by a height of at most 0.7 nm, preferably at most 0.6 nm, most preferably at most 0.5 nm. For the sake of clarity, diameter and height as referred herein cannot be less than 0.1 nm in size. [0094] The combination of the first and second mutations ultimately results in a narrowing of the 220 reading site enabling this site to identify with particular accuracy the type of PTM on a given amino acid.. Combination of all additional amino acid substitutions ultimately lead to a monomer having only one reading site. Thus, the accuracy is improved [0095] Such engineering of the monomer is also advantageous in that it makes it possible to use currents with high voltage when the monomers are used as described below, thus further increasing the accuracy of the sensing method. [0096] Furthermore, the above-mutations are tailored to improve the translocation of the analyte through the pore. [0097] The pores constructed from the aerolysin polypeptides or monomer or mutants thereof as described herein capture and efficiently translocate at optimal rate (i.e., few amino acids per millisecond, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids per millisecond) peptides, polypeptides or proteins. [0098] Importantly, the mutant aerolysin monomers such as described above retain their ability to form a pore. As it will be evident to a person skilled in the relevant art, the ability of the mutant aerolysin monomer to form a pore, as for many other similar pore-forming polypeptides, derives from its structure and the presence of suitable, e.g. physiological, homo- hetero oligomerization conditions. In particular, the aerolysin monomers, both in the wild-type and in the mutated form, undergo a maturation/folding process that foresees several passages. Aerolysin is produced as an inactive precursor, proaerolysin, which contains a C- terminal peptide (CTP) required for folding into its soluble form. Proteolysis in the loop that connects the CTP to the main body allows aerolysin to oligomerize in a heptameric ring-like complex that inserts into the target membrane to form the pore. It is therefore herein tacitly understood that, when referring to a formed pore, the mutant aerolysin monomer comprises, consists of or substantially consists of a polypeptide having the sequence shown in any one of SEQ ID NO: 8 or 10 to 15, i.e. the mature aerolysin monomer without an N-terminal signal peptide and without a C-terminal propeptide, and which substantially differs from the sequence shown in SEQ ID NO: 1 in the C-terminal domain. “Substantially” herein means that, upon alignment of SEQ ID NO: 1 with a sequence comprising SEQ ID NO: 8, no more than five consecutive amino acid residues in the CTP must be equal. [0099] The ability of the monomer to interact with a peptide, polypeptide or protein can be determined using methods that are well-known in the art. The monomer may interact with a peptide, polypeptide or protein in any way, e.g. by non-covalent interactions, such as hydrophobic interactions, hydrogen bonding, Van der Waal's forces or electrostatic forces. For instance, the ability of the region to bind to a peptide, polypeptide or protein can be measured using a conventional binding assay. [00100] Suitable assays include, but are not limited to, fluorescence-based binding assays, nuclear magnetic resonance (NMR), Isothermal Titration Calorimetry (ITC) or Electron spin resonance (ESR) spectroscopy. [00101] Modifications of protein nanopores that alter their ability to interact with a peptide, polypeptide or protein, in particular improve their ability to capture and/or recognise or discriminate peptides, polypeptides or proteins, are well documented in the art. For instance, such modifications are disclosed in WO 2010/034018 and WO 2010/055307. Similar modifications can be made to the aerolysin monomer described above.. [00102] In addition to the specific mutations discussed above, the variant may include other mutations. These mutations do not necessarily enhance the ability of the monomer to interact with the peptide, polypeptide or protein. The mutations may facilitate, for example, expression and/or purification. Over the entire length of the amino acid sequence of any one of SEQ ID NO: 1 to 8 and 10 to 15, a variant will preferably be at least 50% homologous to that sequence based on amino acid identity. More preferably, the variant may be at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of any one of SEQ ID NO: 1 to 8 and 10 to 15 over the entire sequence. There may be at least 80%, for example at least 85%, 90% or 95%, amino acid identity over a stretch of 100 or more, for example 125, 150, 175 or 200 or more, contiguous amino acids ("hard homology"). [00103] Standard methods in the art may be used to determine homology. For example, the UWGCG Package provides the BESTFIT program which can be used to calculate homology, for example used on its default settings (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent residues or corresponding sequences (typically on their default settings)). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). [00104] Amino acid substitutions may be made to any one of the amino acid sequence of SEQ ID NO: 1 to 8 or 10 to 15, in addition to those discussed above, for example up to 1, 2, 3, 4, 5, 10, 20, 30 or even more substitutions. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace. Alternatively, the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well-known in the art and may be selected in accordance with the properties of the 20 main amino acids as defined in Table 1 below. Where amino acids have similar polarity, this can also be determined by reference to the hydropathy scale for amino acid side chains in Table 2. [00105] Table 1 - Chemical properties of amino acids [00106] Table 2 - Hydropathy scale

[00107] A variant may comprise one or more substitutions beyond those specified above, in which amino acids are replaced with those at the corresponding position(s) in homologues and paralogues of aerolysin. [00108] One or more amino acid residues of the amino acid sequence of any one of SEQ ID NO: 1 to 8 and 10 to 15 may additionally be deleted from the variants described above. Up to 1, 2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more. [00109] Variants may include variants of fragments of any one of SEQ ID NO: 1 to 8 or 10 to 15. Such fragments retain pore forming activity. This may be assayed as described above. Fragments may be at least 50, 100, 150, 200 or 250 amino acids in length. Such fragments may be used to produce the pores. [00110] A fragment of any one of SEQ ID NO: 1 to 8 or 10 to 15 more preferably comprises the region from about position 216 to about position 282 of any one of SEQ ID NO: 1 to 8 or 10 to 15, preferably of SEQ ID NO:1 or SEQ ID NO:8. [00111] One or more amino acids may be alternatively or additionally added to the aerolysin polypeptide or monomer or mutant thereof described above. An extension may be provided at the amino terminal or carboxy terminal of the amino acid sequence of the variant of any one of SEQ ID NO: 1 to 8 or 10 to 15, preferably SEQ ID NO:1 or SEQ ID NO:8, including a fragment thereof. The extension may be quite short, for example from 1 to 10 amino acids in length. Alternatively, the extension may be longer, for example up to 50 or 100 amino acids. A carrier protein may be fused to an aerolysin polypeptide or monomer such as described above. [00112] In addition to the modifications described herein, a variant of any one of SEQ ID NO: 1 to 8 or 10 to 15, preferably SEQ ID NO:1 or 8, may include one or more additional modifications, such as substitutions, additions or deletions. These modifications are preferably located in the stretches in the variant that correspond to from about position 1 to about position 206 and from about position 291 to about position 493 of any one of SEQ ID NO: 1 to 8 or 10 to 15 (i.e. outside of the region modified as described herein). [00113] The aerolysin polypeptide or monomer or mutant thereof may be modified to assist their identification or purification, for example by the addition of histidine residues (a “his tag”), aspartic acid residues (an “asp tag”), a streptavidin tag or a flag tag, or by the addition of a signal sequence to promote their secretion from a cell where the polypeptide does not naturally contain such a sequence. An alternative to introducing a genetic tag is to chemically react a tag onto a native or engineered position on the pore. An example of this would be to react a gel-shift reagent to a cysteine engineered on the outside of the pore. This has been demonstrated in a similar context as a method for separating hemolysin hetero-oligomers (Chem Biol.1997 Jul;4(7):497-505). [00114] The aerolysin polypeptide or monomer or the mutant thereof may be labelled with a revealing label. The revealing label may be any suitable label which allows the pore to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, enzymes, antibodies, antigens, polynucleotides, polyethylene glycols (PEGs), peptides and ligands such as biotin. [00115] The mutant aerolysin polypeptide or monomer may also be produced using D-amino acids. For instance, the mutant aerolysin polypeptide or monomer may comprise a mixture of L-amino acids and D-amino acids. This is conventional in the art for producing such proteins or peptides. [00116] The mutant aerolysin polypeptide or monomer may contain one or more specific modifications to facilitate interaction with the peptide, polypeptide or protein to be sensed. The mutant aerolysin polypeptide or monomer may also contain other non-specific modifications as long as they do not interfere with pore formation. A number of non-specific side chain modifications are known in the art and may be made to the side chains of the mutant aerolysin monomer. [00117] Such modifications include, for example, reductive alkylation of amino acids by reaction with an aldehyde followed by reduction with NaBH4, amidination with methylacetimidate or acylation with acetic anhydride. [00118] The mutant aerolysin polypeptide or monomer can be produced using standard methods known in the art. The monomer may be made synthetically or by recombinant means. For example, the monomer may be synthesized by in vitro translation and transcription (IVTT). Suitable methods for producing pore monomers are discussed in International Applications WO 2010/004273, WO 2010/004265 or WO 2010/086603. Methods for inserting pores into membranes are discussed below. [00119] An aerolysin polypeptide or monomer or mutant thereof that can be used in the present invention may be isolated, substantially isolated, purified or substantially purified. A mutant aerolysin polypeptide or monomer that can be used in the present invention is isolated or purified if it is completely free of any other components, such as lipids. A aerolysin polypeptide or monomer or mutant thereof is substantially isolated if it is mixed with carriers or diluents which will not interfere with its intended use. For instance, an aerolysin polypeptide or monomer or mutant thereof is substantially isolated or substantially purified if it is present in a form that comprises less than 10%, less than 5%, less than 2% or less than 1% of other components, such as lipids. [00120] Polynucleotide sequences encoding an aerolysin polypeptide or monomer or a mutant thereof may be derived and replicated using standard methods in the art. Polynucleotide sequences encoding a aerolysin polypeptide or monomer or mutant thereof may be expressed in a bacterial host cell using standard techniques in the art. The aerolysin polypeptide or monomer or mutant thereof may be produced in a cell by in situ expression of the polypeptide from a recombinant expression vector. The expression vector optionally carries an inducible promoter to control the expression of the polypeptide. [00121] An aerolysin polypeptide or monomer or mutant thereof may be produced in large scale following purification by e.g. any protein liquid chromatography system from pore producing organisms or after recombinant expression as described below. Typical protein liquid chromatography systems include FPLC, AKTA systems, the Bio-Cad system, the Bio-Rad BioLogic system and the Gilson FIPLC system. [00122] In some embodiments, the aerolysin polypeptide or monomer or mutant thereof is chemically modified. The aerolysin polypeptide or monomer or mutant thereof can be chemically modified in any way and at any site. The aerolysin polypeptide or monomer or mutant thereof is preferably chemically modified by attachment of a molecule to one or more cysteines (cysteine linkage), attachment of a molecule to one or more lysines, attachment of a molecule to one or more non-natural amino acids, enzyme modification of an epitope or modification of a terminus. Suitable methods for carrying out such modifications are well- known in the art. Suitable non-natural amino acids include, but are not limited to, 4-azido-L-phenylalanine (Faz). [00123] The aerolysin polypeptide or monomer or mutant thereof may be chemically modified by the attachment of any molecule. For instance, the aerolysin polypeptide or monomer or mutant thereof may be chemically modified by attachment of a polyethylene glycol (PEG), a nucleic acid, such as DNA, a dye, a fluorophore or a chromophore. In some embodiments, the aerolysin polypeptide or monomer or mutant thereof is chemically modified with a molecular adaptor that facilitates the interaction between a pore comprising the polypeptide or monomer and a target peptide, polypeptide or protein to be sensed. The presence of the adaptor improves the host-guest chemistry of the pore and the peptide, polypeptide or protein and thereby improves the sequencing ability of pores formed from the mutant aerolysin polypeptide or monomer such as described herein. The principles of host-guest chemistry are well-known in the art. The adaptor has an effect on the physical or chemical properties of the pore that improves its interaction with the peptide, polypeptide or protein. The adaptor may alter the charge of the barrel or channel of the pore or specifically interact with or bind to the peptide, polypeptide or protein, thereby facilitating its interaction with the pore. [00124] Nanopores The nanopores preferably used in the invention are ideal for characterising peptide, polypeptide or protein analytes. Such nanopores are especially (but not exclusively) ideal for discriminating different PTMs on the same amino acid of interest with a high degree of sensitivity. The amino acid resolution of the pores is surprisingly high. [00125] Homo-oligomeric pores [00126] The pore can be a homo-oligomeric pore derived from aerolysin comprising identical monomers such as described above. The monomers are identical in terms of their amino acid sequence. Such homo- oligomeric pore is ideal for discriminating different PTM on a given amino acid. The homo-oligomeric pore may have any of the advantages discussed above. [00127] The homo-oligomeric pore may contain any number of monomers. The pore typically comprises two or more monomers. One or more of the monomers is preferably chemically modified as discussed above. In other words, one or more of the monomers being chemically modified (and the others not being chemically modified) does not prevent the pore from being homo-oligomeric as long as the amino acid sequence of each of the monomers is identical. [00128] Hetero-oligomeric pores [00129] The pore can be a hetero-oligomeric pore derived from aerolysin comprising at least one monomer such as described above, wherein at least one of the monomers differs from the others. The monomer differs from the others in terms of its amino acid sequence. The homo- oligomeric pore may have any of the advantages discussed above. [00130] The hetero-oligomeric pore contains sufficient monomers to form the pore. The monomers may be of any type. The pore typically comprises seven monomers. The pore may comprise at least one monomer comprising the sequence shown in any one of SEQ ID NO: 8 or 10 to 15 or a variant or fragment thereof, which does not necessarily have a mutation required by the mutant monomers as described above. In this embodiment, the remaining monomers are preferably mutant monomers as disclosed above. [00131] In some embodiments, the pore comprises (a) one aerolysin monomer as described herein and (b) a sufficient number of identical monomers to form the pore, wherein the mutant monomer in (a) is different from the identical monomers in (b). In a specific aspect, the aerolysin monomer in (a) is a mutant monomer such as described above an the aerolysin monomers in (b)preferably comprise the sequence shown in any one of SEQ ID NO: 8 or 10 to 15, a variant or a fragment thereof, which does not have a mutation required by the mutant monomers described above. [00132] A hetero-oligomeric pore may comprise only one mutant aerolysin monomer as described herein. In another embodiment, all of the monomers in the hetero-oligomeric pore are mutant monomers as described herein and at least one of them differs from the others. In any of the embodiments discussed above, one or more of the mutant monomers may be chemically modified as discussed above. The presence of a chemical modification on one monomer does not result in the pore being hetero-oligomeric. The amino acid sequence of at least one monomer must differ from the sequence(s) of the other monomers. Methods for making pores are discussed in more detail below. [00133] Producing pores [00134] Aerolysin nanopores that can be used in the present invention are typically formed by allowing at least one aerolysin monomer or a mutant thereof, such as described herein, to oligomerise with a sufficient number of aerolysin monomers or mutants thereof as described herein or monomers derived from aerolysin to form a pore. If the method concerns making a homo-oligomeric pore, all of the monomers are aerolysin monomers or mutants thereof having the same amino acid sequence. If the method concerns making a hetero-oligomeric pore, at least one of the monomers is different from the others. EXAMPLES Example 1: Characterization of the C-terminal region of α-synuclein with a nanopore [00135] The ability of nanopore to detect PTMs was examined by means of single-channel recording experiments as shown in [Fig. 1]. A lipid membrane separates the chamber into two compartments, cis and trans, and holds an aerolysin pore that connects them. An engineered variant of aerolysin is used here, namely K238A, where lysine residues at position 238 are substituted by alanine residues, providing a significantly enhanced resolution for biomolecular sensing compared to wt aerolysin. We focused on α-synuclein as a model system because of the strong links between its PTM profile and the brain pathology of several neurodegenerative diseases, most notable of Parkinson's Disease (PD). [00136] The C-terminal domain of α-synuclein, encompassing the residues 124-140, harbors several PTMs that are found in pathological α- synuclein aggregates in the brain of patients with PD and other neurodegenerative diseases (Phosphorylation/nitration on Y125, oxidation on M126, phosphorylation on S129, phosphorylation/nitration on Y133, phosphorylation/nitration on Y136). Many of these PTMs, including phosphorylation and nitration, have emerged as reliable markers of α-synuclein pathology formation in human brains and animal models of PD and related synucleinopathies. The C-terminal domain of α-synuclein is rich in proline residues, it is highly negatively charged and does not adopt a stable secondary structure in the monomeric state of the protein, which in this context is particularly convenient as these peptides can be easily driven to translocate through the nanopore by the potential applied across the lipid membrane. All peptides were prepared using Fmoc-based solid-phase peptide synthesis and purified as previously reported (see Materials and Methods). [00137] α-Synuclein peptides (C-terminal fragment with residues 124-140, named hereafter "α-syn124-140") were added into the cis compartment and when a positive voltage was applied to the trans compartment, clear and reproducible blockades of ionic current were obtained ([Fig.2] and [Fig.3]). For wt α-syn124-140, a peculiar and well recognizable 2-level blockade was observed and the signal often contained a higher residual current at the last fraction ([Fig. 2] bottom left). As a result, two populations were observed ([Fig.2] bottom right): one exhibited a relative current percentage (I/I0, see Materials and Methods) of 9.0 ±2.0, while the other 14.1 ±2.0 (named hereafter PI and PII, respectively). These values correspond to the mean and standard deviation derived from a Gaussian fit of the relative current histogram. [00138] To gain further insights into this peculiar two-level signal, we considered the structure of this α-synuclein fragment. Based on previous studies, there is a highly hydrophobic segment between residues 125-129 that can produce a structurally compact cluster likely responsible for the initial lower residual current. To test this hypothesis, we reasoned that destabilizing this conformation by the addition of urea could produce signals expected for a more extended peptide. We thus used 8.0 M urea (along with 1.0 M KCl, 10 mM Tris, 1.0 mM EDTA, pH 7.4) to induce complete unfolding of the peptide while aerolysin remained folded and functional. Under these conditions, the open pore current was lower (25 ±1.8 pA at 100 mV) compared to the same salt concentration without urea (72 ±1.5 pA at 100 mV). This is because the additional urea significantly decreased the mobility of the ions. However, in these conditions, wt α-syn124-140 translocated with a simple one- level signal as evidenced by one population in the scatter plot and the I/I0 histograms ([Fig. 3]). These one-level current signals suggest that the addition of urea results in a more extended conformation of the polypeptide chain, which facilitates its translocation through the pore. In addition, due to the decrease of ionic mobility, the dwell time became much longer, 8.6 ±0.24 ms, approximately 4 times longer than in normal conditions. These results demonstrate that the conformation of peptides can be monitored at the single-molecule level. Using a nanopore, while subtle conformational changes in the highly dynamic regions of proteins, such as the C-terminus of α-synuclein, are difficult to detect by other techniques such as circular dichroism spectroscopy. Example 2: Detection of different PTMs and their spatial combinations using a nanopore and discrimination of them using deep learning [00139] We assessed the feasibility of detecting PTMs of α-syn124-140 peptide. First, we investigated the phosphorylation at Ser129 (pS129), which represents the most dominant PTM in pathological α-synuclein aggregates, approximately 90% of α-synuclein deposited in Lewy bodies presents pS12949, and has been reported to be elevated in biological fluids of PD patient. Second, different types of modifications at the same amino acid (pY125 and nY125) were measured. For this category, we also investigated the peptides bearing multiple PTMs, including the same type of PTM located at different position (pS129 and nY136), multiple PTMs (pY125pS129 and nY125nY133nY136); and different PTM combinations (nY125pS129). [00140] As demonstrated in the [Fig.4], the nanopore current signatures of the different PTMs we investigated appeared to have distinct characteristics. Compared to the unmodified peptide, both phosphorylation and nitration at Y125 presented only one single level and their relative current were lower than for the wt peptide. This decrease in the relative current value is consistent with a deeper blockade induced by the additional volume due to the presence of the PTMs. Moreover, the dwell time varied significantly with different types of PTMs, the fitted values being 2.58 ±0.4 ms for wt, 0.55 ±0.08 ms for pY125 and 4.51 ±0.5 ms for nY125, indicating phosphorylation speeds up the translocation process, while nitration significantly slows it down. The faster translocation of pY125 could be induced by the additional negatively charged phosphate group. In addition, we investigated the effect of oxidation of methionine at position 127 of α-syn124-140 (oM127, data not shown). Similar to pY125 and nY125, oM127 also showed only one single level. [00141] For the same PTM, when it located at different positions of peptide sequence, the ionic current and dwell time were modulated differently. The dwell time of pS129 (3.62 ±0.2 ms) is 6.5 times longer than pY125 (0.55 ±0.08 ms). We hypothesize that when the hydrophobic cluster of α-syn124-140 is disrupted by phosphorylation (pY125), all amino acids become exposed and translocate the pore in a linear form. In this case, the additional phosphate group of pY125 contributed to the faster translocation under the applied voltages, which was not the case for pS129 since the α-synuclein hydrophobic cluster was conserved. Similar results were obtained when we compared nY125 and nY136 α- syn124-140 (data not shown). Unlike nY125, which only showed one population, the relative current of nY136 was less pronounced compared to wt and showed two populations like wt α-syn124-140. This suggests that disrupting the local structure is beneficial to better detect PTMs in proteins by an aerolysin pore. This could be achieved by using chemical denaturants such as urea or guanidinium chloride. [00142] To assess the possibility of using the nanopore for the detection of multiple PTMs occurring simultaneously on the same peptide, we measured the ionic current response of double-phosphorylated α- syn124-140, pY125pS129, and triple nitrated α-syn124-140, nY125nY133nY136. Here, nanopore results showed that compared to the unmodified peptide (wt) and the singly phosphorylated peptides (i.e., pY125 and pS129), the translocation speed of pY125pS129 (0.45 ±0.02 ms) was much faster, likely due to its increased negative charges. While the dwell time of nY125nY133nY136 was around 2-fold longer (5.18 ± 0.23 ms) compared to wt and nY136, and slightly longer than nY125, its relative current was the lowest among all peptides (7.2 ±1.0) since the three modifications contribute to increase the overall volume of the peptide and therefore induce a deeper blockade of ionic current. In the case of the peptides containing two types of PTMs at different positions, as nitration at Y125 and phosphorylation at S129 (nY125pS129, data not shown), only one population was observed. The relative current of nY125pS129 was between the values of the single modification, nY125 and pS129, as observed also for the width of the relative current distribution. The dwell time of nY125pS129 was slightly longer than pS129, but identical to nY125. For all these peptides, the dwell time decreases as the voltage increases, indicating that the collected signals are indeed induced by peptides translocating through the nanopore. Altogether, these observations demonstrate that the engineered K238A aerolysin is able to capture the diversity of PTMs. [00143] To classify these PTMs in a more precise, unbiased and automatic procedure that allows translation to clinical applications in the future. we developed a tailored deep learning approach for processing the nanopore current readouts. We first extracted the statistical performance of the core features (mean residual current, standard deviation, dwell time, frequency and local extrema) of recorded events using a current threshold at 3σ from the open pore current distribution. For each single peptide, we randomly used 80% of the single peptide data to fed the deep learning algorithm as the training set for the model, and the rest 20% as the validation of the model to get the accuracy of the ML model for each single peptide. Then, a long short-term memory (LSTM) recurrent neural network was integrated to read the core features of events followed by a multilayer perceptron (MLP) to classify the peptides ([Fig.5]), similar to our previous analysis for informational polymers reading (see details in Materials and Methods). [00144] As shown in [Fig.6], this approach allows for differentiating wt α- syn124-140 from α-syn124-140 containing different modifications at the same position Y125 (pY125, nY125) with an accuracy of 94%. Control experiments with a mixture of wt and pY125 at different ratios of concentration were performed to further test the deep learning approach [Fig. 7]. First, a mixture of an equimolar ratio (1:1) was measured in nanopore experiments and the percentage of assignment of wt and pY125 was 52.8 ± 3.5% and 43.2 ± 3.1%, respectively. This is in line with the theoretical predictions. When the ratio of wt:pY125 was changed to 1:3, the percentage of wt assignment decreased to 19.0 ± 0.3% while pY125 increased to 78.2 ± 0.7%. Finally, a mixture ratio of 3:2 (wt:pY125) was tested. As shown in [Fig. 8], the assignment of wt and pY125 was as expected 61.5 ± 0.1% and 35.0 ± 0.2%, respectively. Based on this approach, the expectation is that any sequence in the library of peptides (wt, pY125, or nY125) can be identified directly with a confidence level of 94%. [00145] Data from wt and the 7 tested α-syn124-140 PTM variants (pY125, nY125, pS129, nY136, pY125pS129, nY125nY133nY136 and nY125pS129) have been used as training set for the same deep learning model, with a resulting accuracy of 78.2% using a selection percentage of 25% (i.e., accuracy dependence on selection percentage as shown in [Fig. 8]). The selection percentage was translated into a confidence threshold for the predictions. If the event is too noisy or doesnʼt contain enough information as in the case of short events, the prediction confidence would be lower. The selection percentage allows for a trade- off between accuracy and filtering without requiring heavy filtering during the preprocessing of the events. As illustrated in [Fig. 9], 13% of pY125 was confused with pY25pS129, because the signals of both pY125 and pY25pS129 showed very short dwell times. This could be further improved if higher bandwidth instruments have been employed. Additionally, 6% of nY125 was confused with nY125nY133nY136, while the other 16% of this peptide was confused with nY125pS129, 10% nY136 confused with pS129 and the other 9% confused with wt. We think this prediction confusion was caused by the similar dwell time between these modified peptides. Nonetheless, the deep learning approach we developed here provides high reading accuracy for all the tested PTMs peptides, particularly for pS129 which is one of the most relevant biomarkers for synucleinopathies. Example 3: Detection of α-synuclein peptides within clinical conditions [00146] We explored if K238A aerolysin was able to detect α-synuclein and related PTMs peptides in clinically relevant conditions, including plasma and red blood cells (RBCs) extracts, devoid of the haemoglobin proteins. We first determined the dilution necessary to ensure a stable baseline of the ionic current through serial dilutions of both RBCs and plasma samples. While there is no report to date about direct nanopore detection of plasma samples, in the case of RBCs no particular instabilities of the lipid bilayer were observed except when dilutions were lower than 1:50 or 1:30. In another study, it was reported that the optimal condition for serum samples was a dilution to 1:2061. Based on these previous reports, we scanned a range of dilutions up to 1:100 ([Fig. 10]). After addition of 3 μl of haemoglobin-depleted RBCs extracts into the cis chamber, we observed no obvious effects on the membrane stability; the same behavior was observed for crude plasma samples. During the experiments with both RBCs or plasma samples, we could continually collect data for up to three hours, indicating that the aerolysin pore remained invariantly intact likely due to the ultra-stability provided by its unique double β-barrel structure. When testing plasma samples, we observed sporadic current signals when the dilution ratio was lower than 1:50, which may have resulted from translocation of other short peptides possibly contained in plasma. On the other hand, when the concentration of RBCs sample increased in the chamber, occasional signals were also observed (data not shown). Notably, all raw current traces shown in [Fig. 10] to [Fig.14] were collected 30 min after clinical samples were added. [00147] Next, we sought to quantify the presence of α-synuclein wt peptides within the RBCs and plasma samples, with the aim of mimicking realistic complexity of pathological conditions within a clinical setting. Based on the previous tests, dilutions of 1:10 and 1:50 were chosen as the background for RBCs and crude plasma samples, respectively. As illustrated in [Fig.11], after the addition of wt α-syn124-140 typical two- level signals were recorded. [00148] While these results already showed a promising potential of aerolysin pores to detect C-terminal α-synuclein peptides directly from clinical samples, another prerequisite towards the development of aerolysin pores into an efficient single-molecule proteomic device for biomarkers detection is the ability to detect molecules in low abundance, as often found in the clinical setting. In previous studies, the frequency of signals has been used to quantify the detected molecule. Therefore, we used the equation f_sig = k_on [PTMs]₀ to explore the theoretical detection limit of aerolysin nanopores for sensing α-synuclein peptides. We verified that the frequency of blockade events, f_sig, was proportional to the concentration of α-synuclein ranging from 120 nM to 24 μM, which was applying 100 mV in a symmetric buffer concentration of 1.0M KCl. The physiological concentration of α-synuclein in RBCs was however much lower than these tested conditions, namely 26.2 ± 3.0 µg/ml, which corresponds to 35 nM if 3µl of the original sample was added into the nanopore system. Therefore, to enhance the detection limit of our setting, we measured peptides in a gradient of salt concentration, which previously proved an efficient way to increase the capture rate for ssDNA. An asymmetrical KCl buffer solution consisting of 0.15 M in the cis chamber and 3.0 M in the trans was thus used ([Fig. 12]), which enabled the detection of wt α-synuclein peptides at far lower concentrations (i.e., 100 pM, [Fig.13]) than when using canonically symmetric buffer conditions. [00149] Altogether, these results demonstrate that K238A aerolysin nanopores are able to directly detect and discriminate α-synuclein peptides holding a broad variety of PTMs. As these modifications located at the C-terminal of α-synuclein have been identified as potential biomarkers for the diagnosis of synucleinopathies, this nanopore approach has the potential to detect them at picomolar concentrations, that is overlapping with the concentrations at which they are found in a typical clinical setting. Considering that an even lower detection limit could be achieved by applying higher voltages, minimizing the volume of the chambers or optimizing the pore variants, the possibility to develop an effective nanopore-based tool for the diagnosis of synucleinopathies appears within reach. Example 4: Generating the target peptides by proteolytic digestion of full length α-synuclein and its detection by nanopore [00150] To generate C-terminal peptide fragments from full length α- synuclein protein, we chose to use cathepsin D (CtsD). Previous studies showed that CtsD-mediated digestion of α-synuclein results in the generation of a C-terminal fragment spanning residues 125-140, in addition to four other α-synuclein fragments of various length ([Fig. 14]). The C-terminal fragment contains all known disease-associated PTM sites (p/nY125, pS129, nY133, nY136) and the ones we investigated in the examples above. [00151] We incubated CtsD with full-length α-synuclein (see details in Methods), and detected by nanopore afterwards. In parallel, the same samples have been characterized by MS. As shown in [Fig.14], with CtsD digestion, we have identified three main peptides fragments, 5-38, 95- 124, and 125-140 fragments. After adding the CtsD digested α- synuclein sample into the nanopore system, a lot of signals were recorded ([Fig. 15]). As illustrated in [Fig. 16], under these conditions, the deep learning model was able to identify the C-terminal peptides with an accuracy of 81.4 ± 1.1% in five independent experiments, which means the other four proteolytic fragments did not interfere with the measurement. As 5-38 peptide is positively charged, we donʼt expect it to be captured in nanopore system when using +100 mV voltage. Therefore, the only possible fragment that may interfere with the measurement is the 95-124 peptide since it is negatively charged and could also be captured by the nanopore system. However, it should be noted that the length of 95-124 peptide is two times longer than 125-140 fragment which may lead to a low capture in the conditions we used here. Indeed, for DNA, the capture of long ssDNA is dramatically reduced in the aerolysin nanopores. Conclusion [00152] The present examples demonstrate that an engineered aerolysin nanopore can detect and distinguish between peptides carrying different types and number of PTMs, PTMs occurring at different residues and alternative PTMs on the same residue. It can capture subtle structural features that are challenging to be characterized by other biophysical methods without fluorescently labeling or modifying the peptides/proteins. Using deep learning for signal processing, all investigated PTMs could be automatically identified in a supervised context, which means this approach can be scaled up to identify more PTMs or scaled down to fit a specific application. Importantly, this nanopore approach can reach a detection limit as low as picomolar concentration and is amenable for high-throughput applications, which are challenging for other techniques such as MS. Finally, one major advantage of the nanopore-based approach is that it enables simultaneous detection of several protein PTMs which is difficult to achieve using immunoassay/antibody-based methods. This is because the presence of multiple PTMs alters the biochemical properties of the antibody targeting epitopes. Altogether, the present examples demonstrate that the present nanopore detection of PTMs is efficient as a single-molecule proteomic device and diagnostic tool, including direct detection of circulating peptides. Disease-relevant proteins, such as α- synuclein, can be digested into smaller peptides and subsequently detected by the present methods and uses, even directly from a biological sample. [00153] The ability of the aerolysin pore to detect and distinguish between long peptides bearing multiple PTMs also paves the way for more precise remapping of PTMs in proteins at the single-molecule level, which remains a challenge for MS and other methods. In conclusion, the nanopore-based technology presented here, besides the natural advantages of being fast, cheap, label-free, and high-throughput, provides the possibility to be developed into a portable diagnostic device of medical and commercial potential. Materials and Methods Synthesis of C-terminal α-synuclein peptides and its PTMs [00154] The majority of peptides used in nanopore experiments were produced as described in J. Am. Chem. Soc. 134, 5196‒5210 (2012), Chem. Commun. 49, 9254‒9256 (2013) and Proc. Natl. Acad. Sci. 110, 17726‒17731 (2013). Few peptides (oM127 and wt) were provided by GenicBio Limited. All peptides were characterized by Liquid chromatography-mass spectrometry (LC-MS) as previously described in Neurobiol. Dis. 146, 105086 (2020). Their purity was also assessed by UPLC analysis, on a Waters Acquity H-Class system using a C18 column (with UV detection at 214 nm and run time of 4 min (gradient 10% to 90% acetonitrile) with 0.6 mL/min flow rate. [00155] RBCs were prepared similarly as described in Sci. Rep. 7, 13713 (2017) using the Hemovoid kit (Biotech Support Group), aiming to remove haemoglobin (the most abundant protein in RBCs) but also to enrich low abundant proteins such as α-synuclein. In the case of plasma samples, we tested crude/raw plasma. Importantly, all biological fluids used in this study are derived from healthy controls. [00156] CtsD digestion experiments were performed in a total volume of 100 µl, 2 µl of 500 µM CtsD (Sigma-Aldrich Chemie GmbH, Buchs, SG Switzerland), 3 µl of 100µM full length ^-synuclein and 95 µl buffer (40mM sodium acetate, 50mM NaCl and 5mM DTT, pH=5) incubated at 37°C for 20h at 300 rpm in a ThermoMixer C (Eppendorf). Aerolysin productions [00157] The recombinant K238A aerolysin proteins were generated from the aerolysin gene in the pET22b vector with a C-terminal hexa-histidine tag as described in Nat. Commun. 10, 4918 (2019) and Biomed. J. S2319417021001827 (2021), and then expressed and purified from BL21 DE3 pLys E. coli cells. Cells were grown to an optical density of 0.6-0.7 in Luria-Bertani (LB) media. Protein expression was induced by the addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and subsequent growth overnight at 20°C. Cell pellets were resuspended in lysis buffer (20 mM Sodium phosphate pH 7.4, 500 mM NaCl) mixed with cOmplete™ Protease Inhibitor Cocktail (Roche) and then lysed by sonication. The resulting suspensions were centrifuged (12.000 rpm for 35 min at 4°C) and the supernatants were purified through a HisTrap HP column (GE Healthcare) previously equilibrated with lysis buffer. The protein was eluted with a gradient over 40 column volumes of elution buffer (20 mM Sodium phosphate pH 7.4, 500 mM NaCl, 500 mM Imidazole), and buffer exchanged into a final buffer (20 mM Tris, pH 7.4, 500 mM NaCl) using a HiPrep Desalting column (GE Healthcare). The purified protein was flash-frozen in liquid nitrogen and stored at -20°C. Single-channel recording experiments [00158] Phospholipids of 1,2-Diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) powder (Avanti Polar Lipids, Alabaster, USA) were dissolved in octane (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) for a final concentration of 1.0 mg per 100 µl. Purified protein was diluted to the concentration of 0.2 µg/ml and then incubated with Trypsin-agarose (Sigma-Aldrich Chemie GmbH, Buchs, SG Switzerland) for 2 h under 4°C temperature. The solution was finally centrifuged to remove trypsin. [00159] Nanopore single-channel recording experiments were performed on Orbit Mini equipment (Nanion, Munich, Germany) and an Axopatch 200B Amplifier system (Molecular Devices, San Jose, USA). In the Orbit Mini set-up, DPhPC membranes were formed across a MECA 4 recording chip that contains a 2 x 2 array of cylindrical 50 µm diameter in a highly inert polymer. Each of the four cavity contains an individual integrated Ag/AgCl-microelectrode and sustains one DPhPC bilayer. If not indicated otherwise, the measurement chamber temperature was set to 20°C. Data was collected at 10 kHz sampling rate with a 5 kHz low-pass filter. [00160] Ionic strength gradient experiments were carried out as follows. Teflon films with 50 µm apertures were mounted in Teflon chambers using high-vacuum grease (Dow Corning Corporation, Midland, MI, USA). The films separated two compartments (cis/trans) only connected through the Teflon film aperture, with one Ag/AgCl electrode in each compartment. Apertures were pretreated with 1 µL 2 % (v/v) hexadecane in hexane on both sides using a standard pipette and the chamber was mounted in the recording setup. DPhPC bilayers were formed by folding as described in Proc. Natl. Acad. Sci.69, 3561‒3566 (1972) and Biophys. J. 85, 2684‒2695 (2003). Briefly, electrolyte solution was added to both sides taking care that the level stayed below the aperture, lipids (10 mg/mL in pentane) were added onto the electrolyte surface in both compartments. After the pentane evaporated, the electrolyte level was raised above the aperture and a lipid bilayer was formed. The quality of the lipid bilayer was monitored through its capacitance and its stability was verified through the application of 150 mV over the course of at least 5 minutes. After peptide addition, the cis chamber was carefully mixed by pipetting up and down. Currents were sampled at 200 kHz and low- pass filtered at 100 kHz with the Axopatch 200B (Molecular Devices, LLC., San Jose, CA, USA). [00161] Peptides (lyophilized powder) were pre-diluted in 10 mM Tris and 1.0 mM EDTA solution (pH=7.4) to a stock concentration of 500 µM and added to the cis side of the chamber in 1.0 M KCl solution buffered with 10 mM Tris and 1.0 mM EDTA (pH=7.4) to the final concentration indicated in the figure caption. All experiments shown here were repeated with at least 4 different pores. Signals processing and classifications using deep learning [00162] The signal processing was done as described in Sci. Adv. 6, eabc2661 (2020). The open pore current distribution is measured by fitting a Gaussian function on the peak distribution of current with the highest mean current. The signal segments with an open pore current between 55 to 85 pA and standard deviation between 1.5 to 5 pA are kept. The events are extracted using a current threshold at 3σ from the open pore current distribution. The relative current percentage (I/I₀) is computed from the mean open pore current (I₀) and the mean residual current (I). The dwell time, average relative current, relative standard deviation of the current σ_rel=σ/σ₀ (σ₀ is the value of the open pore current standard deviation and σ is the residual current standard deviation) and local extrema are computed. The events are selected on the basis of the dwell time (0.2 to 100.0 ms) and the average relative current (0 to 40%) discarding the events that are too short, too long or that do not block the current sufficiently. [00163] The machine learning pipeline is composed of two steps. The first one is the classification of every event and the second is the assessment of the quality of the prediction of the classifier. The neural network architecture for both the classification and the assessment is a long short-term memory (LSTM) neural network followed by a multilayer perceptron (MLP) using the position in time and relative current of the local extrema for each event as input features. The features are rescaled by a fixed factor to decrease the training time. The classifier is composed of a LSTM with state size 128 without any activation function followed by 6 fully connected hidden layers of size 256 with rectified linear unit (ReLU) as activation functions and finally an output layer of size 8 with softmax activation function. The assessment is done with a scaled down version of the classifier with a LSTM with a state of size 32, 3 fully connected hidden layers of size 64 with hyperbolic tangent activation functions and an output layer of size 1 with sigmoid activation function. The neural networks for the classification and assessment are trained together using a 3-parts loss function. The first part is the full classification cross-entropy loss of the predictions from the classifier and the peptides label. The second part is the assessment of cross- entropy loss between the predicted and actual prediction validity from the classifier. The third part is the reinforcement classification loss which is the full classification cross-entropy loss scaled by the assessment prediction. [00164] SEQUENCE LISTING [00165] SEQ ID NO: 1 >sp|P09167|AERA_AERHY Aerolysin OS=Aeromonas hydrophila GN=aerA AEPVYPDQLRLFSLGQGVCGDKYRPVNREEAQSVKSNIVGMMGQWQISG LANGWVIMGPGYNGEIKPGTASNTWCYPTNPVTGEIPTLSALDIPDGDEV DVQWRLVHDSANFIKPTSYLAHYLGYAWVGGNHSQYVGEDMDVTRDGD GWVIRGNNDGGCDGYRCGDKTAIKVSNFAYNLDPDSFKHGDVTQSDRQ LVKTVVGWAVNDSDTPQSGYDVTLRYDTATNWSKTNTYGLSEKVTTKN KFKWPLVGETELSIEIAANQSWASQNGGSTTTSLSQSVRPTVPARSKIPVK IELYKADISYPYEFKADVSYDLTLSGFLRWGGNAWYTHPDNRPNWNHTF VIGPYKDKASSIRYQWDKRYIPGEVKWWDWNWTIQQNGLSTMQNNLAR VLRPVRAGITGDFSAESQFAGNIEIGAPVPLAADSKVRRARSVDGAGQGLR LEIPLDAQELSGLGFNNVSLSVTPAANQ [00166] SEQ ID NO: 2 >sp|Q06306|AER5_AERHY Aerolysin-5 OS=Aeromonas hydrophila GN=ahh5 AEPVYPDQLRLFSLGQEVCGDKYRPITREEAQSVKSNIVNMMGQWQISGL ANGWVIMGPVYNGEIKPGSASNTWCYPVNPVTGEIPTLSALDIPDGDEVD VQWRLVHDSANFIKPTSYLAHYLGYAWVGGNHSQYVGEDMDVTRDGDG WVIRGNNDGGCEGYRCGEKTAIKVSNFAYNLDPDSFKHGDVTQSDRQLV KTVVGWAINDSYTPQSAYDVTLRYDTATNWSKTNTYGLSEKVTTKNKFK WPLVGETELSIEIAANQSWASQNGGSTTTSLSQSVRPTVPARSKIPVKIEL YKADISYPYEFKADVSYDLTLSGFLRWGGNAWYTHPDNRPNWNHTFVIG PYKDKASSIRYQWDKRYIPGEVKWWDWNWTIQQNGLSTMQNNLARVLR PVRAGITGDFSAESQFAGNIEIGAPVPLAADGKAPRALSARRGEQGLRLAIP LECRKSSPGLASATSA [00167] SEQ ID NO: 3 >sp|Q06304|AERA_AERSO Aerolysin OS=Aeromonas sobria GN=asa1 AEPVYPDQVKWAGLGTGVCASGYRPLTRDEAMSIKGNLVSRMGQWQIT GLADRWVIMGPGYNGEIKQGTAGETWCYPNSPVSGEIPTLSDWNIPAGD EVDVQWRLVHDNDYFIKPVSYLAHYLGYAWVGGNHSPYVGEDMDVTRV GDGWLIKGNNDGGCSGYRCGEKSSIKVSNFSYTLEPDSFSHGQVTESGK QLVKTITANATNYTDLPQQVVVTLKYDKATNWSKTDTYSLSEKVTTKNK FQWPLVGETELAIEIAASQSWASQKGGSTTETVSVEARPTVPPHSSLPVRV ALYKSNISYPYEFKAEVNYDLTMKGFLRWGGNAWYTHPDNRPTWEHTL LLGPFRGQGEQHPLPVDKRYIPGEVKWWDWNWTISEYGLSTMQNNLGR VLRPIRSAVTGDFYAESQFAGDIEIGQPQTRSAKAAQLRSASAEEVALTSV DLDSEALANEGFGNVSLTIVPVQ [00168] SEQ ID NO: 4 >sp|Q06305|AER3_AERHY Aerolysin-3 OS=Aeromonas hydrophila GN=ahh3 AEPVYPDQLRLFSLGQEVCGDKYRPVNREEAQSVKSNIVGMMGQWQISG LANGWVIMGPGYNGEIKPGSASSTWCYPTNPATGEIPTLSALDIPDGDEV DVQWRLVHDSANFIKPTSYLAHYLGYAWVGGNHSQYVGEDMDVTRDGD GWVIRGNNDGGCDGYRCGDKTSIKVSNFAYNLDPDSFKHGDVTQSDRQ LVKTVVGWAINDSDTPQSGYDVTLRYDTATNWSKTNTYGLSEKVTTKNK FKWPLVGETELSIEIAANQSWASQNGGSPTTSLSQSVRPTVPAHSKIPVKI ELYKADISYPYEFKADVSYDLTLSGFLRWGGNAWYTHPDNRPNWNHTF VIGPYKDKASSIRYQWDKRYIPGEVKWWDWNWTIQQNGLPTMQNNLAK VLRPVRAGITGDFSAESQFAGNIEIGAPVPVAAASHSSRARNLSAGQGLRL EIPLDAQELSGLGFNNVSLSVTPAANQ [00169] SEQ ID NO: 5 >sp|P09166|AERA_AEREN Aerolysin OS=Aeromonas enteropelogenes GN=aerA NAAEPIYPDQLRLFSLGEDVCGTDYRPINREEAQSVRNNIVAMMGQWQIS GLANNWVILGPGYNGEIKPGKASTTWCYPTRPATAEIPVLPAFNIPDGDA VDVQWRMVHDSANFIKPVSYLAHYLGYAWVGGDHSQFVGDDMDVIQEG DDWVLRGNDGGKCDGYRCNEKSSIRVSNFAYTLDPGSFSHGDVTQSERT LVHTVVGWATNISDTPQSGYDVTLNYTTMSNWSKTNTYGLSEKVSTKNK FKWPLVGETEVSIEIAANQSWASQNGGAVTTALSQSVRPVVPARSRVPVKI ELYKANISYPYEFKADMSYDLTFNGFLRWGGNAWHTHPEDRPTLSHTFA IGPFKDKASSIRYQWDKRYLPGEMKWWDWNWAIQQNGLATMQDSLAR VLRPVRASITGDFRAESQFAGNIEIGTPVPLGSDSKVRRTRSVDGANTGLK LDIPLDAQELAELGFENVTLSVTPARN [00170] SEQ ID NO: 6 >sp|Q08676|AERA_AERSA Aerolysin OS=Aeromonas salmonicida GN=ash3 WHEPVYPDQVKWAGLGTGVCASGYRPLTRDEAMSIKGNLVSRMGQWQI TGLADRWVIMGPGYNGEIKQGTAGETWCYPNSPVSGEIPTLSDWNIPAG DEVDVQWRLVHDNDYFIKPVSYLAHYLGYAWVGGNHSPYVGEDMDVTR VGDGWLIKGNNDGGCSGYRCGEKSSIKVSNFSYTLEPDSFSHGQVTESG KQLVKTITANATNYTDLPQQVVVTLKYDKATNWSKTDTYSLSEKVTTKN KFQWPLVGETELAIEIAASQSWASQKGGSTTETVSVEARPTVPPHSSLPV RVALYKSNISYPYEFKAEVNYDLTMKGFLRWGGNAWYTHPDNRPTWEH TFRLGPFRGQGEQHPLPVDKRYIPGEVKWWDWNWTISEYGLSTMQNNL GRVLRPIRSAVTGDFYAESQFAGDIEIGQPQTRSAKAAQLRSASAEEVALT SVDLDSEALANEGFGNVSLTIVPVQ [00171] SEQ ID NO: 7 >sp|Q06303|AER4_AERHY Aerolysin-4 OS=Aeromonas hydrophila GN=ahh4 AEPVYPDQLRLFSLGQEVCGDKYRPVNREEAQSIKSNIVGMMGQWQISG LANGWVIMGPGYNGEIKPGSASSTWCYPTNPATGEIPTLSALDIPDGDEV DVQWRLVHDSANFIKPTSYLAHYLGYAWVGGNHSQYVGEDMDVTRDGD GWVIRGNNDGGCDGYRCGDKTSIKVSNFAYNLDPDSFKHGDVTQSDRQ LVKTVVGWAINDSDTPQSGYDVTLRYDTATNWSKTNTYGLSEKVTTKNK FKWPLVGETELSIEIAANQSWASQNGGSTTTSLSQSVRPTVPAHSKIPVKI ELYKADISYPYEFKADVSYDLTLSGFLRWGGNAWYTHPDNRPNWNHTF VIGPYKDKASSIRYQWDKRYIPGEVKWWDWNWTIQQNGLPTMQNNLAK VLRPVRAGITGDFSAESQFAGNIEIGAPVPVAAASHSSRARNLSAGQGLRL EIPLDAQELSGLGFNNVSLSVTPAANQ [00172] SEQ ID NO: 8 >sp|P09167|AERA_AERHY Aerolysin OS=Aeromonas hydrophila GN=aerA without C-terminal propeptide AEPVYPDQLRLFSLGQGVCGDKYRPVNREEAQSVKSNIVGMMGQWQISG LANGWVIMGPGYNGEIKPGTASNTWCYPTNPVTGEIPTLSALDIPDGDEV DVQWRLVHDSANFIKPTSYLAHYLGYAWVGGNHSQYVGEDMDVTRDGD GWVIRGNNDGGCDGYRCGDKTAIKVSNFAYNLDPDSFKHGDVTQSDRQ LVKTVVGWAVNDSDTPQSGYDVTLRYDTATNWSKTNTYGLSEKVTTKN KFKWPLVGETELSIEIAANQSWASQNGGSTTTSLSQSVRPTVPARSKIPVK IELYKADISYPYEFKADVSYDLTLSGFLRWGGNAWYTHPDNRPNWNHTF VIGPYKDKASSIRYQWDKRYIPGEVKWWDWNWTIQQNGLSTMQNNLAR VLRPVRAGITGDFSAESQFAGNIEIGAPVPL [00173] SEQ ID NO: 9 >cds for P09167|AERA_AERHY Aerolysin OS=Aeromonas hydrophila GN=aerA (including C-terminal peptide) gcagagcccg tctatccaga ccagcttcgc ttgttttcat tgggccaagg ggtctgtggc gacaagtatc gccccgtcaa tcgagaagaa gcccaaagcg ttaaaagcaa tattgtcggc atgatggggc aatggcaaat aagcgggctg gccaacggct gggtcattat ggggccgggt tataacggtg aaataaaacc agggacagcg tccaatacct ggtgttatcc gaccaatcct gttaccggtg aaataccgac actgtctgcc ctggatattc cagatggtga cgaagtcgat gtgcagtggc gactggtaca tgacagtgcg aatttcatca aaccaaccag ctatctggcc cattacctcg gttatgcctg ggtgggcggc aatcacagcc aatatgtcgg cgaagacatg gatgtgaccc gtgatggcga cggctgggtg atccgtggca acaatgacgg cggctgtgac ggctatcgct gtggtgacaa gacggccatc aaggtcagca acttcgccta taacctggat cccgacagct tcaagcatgg cgatgtcacc cagtccgacc gccagctggt caagactgtg gtgggctggg cggtcaacga cagcgacacc ccccaatccg gctatgacgt caccctgcgc tacgacacag ccaccaactg gtccaagacc aacacctatg gcctgagcga gaaggtgacc accaagaaca agttcaagtg gccactggtg ggggaaaccc aactctccat cgagattcgt gccaatcagt cctgggcgtc ccagaacggg ggctcgacca ccacctccct gtctcagtcc gtgcgaccga ctgtgccggc ccgctccaag atcccggtga agatagagct ctacaaggcc gacatctcct atccctatga gttcaaggcc gatgtcagct atgacctgac cctgagcggc ttcctgcgct ggggcggcaa cgcctggtat acccacccgg acaaccgtcc gaactggaac cacaccttcg tcataggtcc gtacaaggac aaggcgagca gcattcgcta ccagtgggac aagcgttaca tcccgggtga agtgaagtgg tgggactgga actggaccat acagcagaac ggtctgtcta ccatgcagaa caacctggcc agagtgctgc gcccggtgcg ggcggggatc accggtgatt tcagtgccga gagccagttt gccggcaaca tagagatcgg tgctcccgtg ccgctcgcgg ctgacagcaa ggtgcgtcgt gctcgcagtg tggacggcgc tggtcaaggc ctgaggctgg agatcccgct cgatcgcgaa gagctctccg ggcttggctt caacaagtca gcctcagcgt ga [00174] SEQ ID NO: 10 >sp|Q06306|AER5_AERHY Aerolysin-5 OS=Aeromonas hydrophila GN=ahh5 without C-terminal propeptide [00175] AEPVYPDQLRLFSLGQEVCGDKYRPITREEAQSVKSNIVNMMGQW QISGLANGWVIMGPVYNGEIKPGSASNTWCYPVNPVTGEIPTLSALDIPD GDEVDVQWRLVHDSANFIKPTSYLAHYLGYAWVGGNHSQYVGEDMDVT RDGDGWVIRGNNDGGCEGYRCGEKTAIKVSNFAYNLDPDSFKHGDVTQ SDRQLVKTVVGWAINDSYTPQSAYDVTLRYDTATNWSKTNTYGLSEKVT TKNKFKWPLVGETELSIEIAANQSWASQNGGSTTTSLSQSVRPTVPARSKI PVKIELYKADISYPYEFKADVSYDLTLSGFLRWGGNAWYTHPDNRPNWN HTFVIGPYKDKASSIRYQWDKRYIPGEVKWWDWNWTIQQNGLSTMQNN LARVLRPVRAGITGDFSAESQFAGNIEIGAPVPL [00176] SEQ ID NO: 11 >sp|Q06304|AERA_AERSO Aerolysin OS=Aeromonas sobria GN=asa1 without C-terminal propeptide AEPVYPDQVKWAGLGTGVCASGYRPLTRDEAMSIKGNLVSRMGQWQIT GLADRWVIMGPGYNGEIKQGTAGETWCYPNSPVSGEIPTLSDWNIPAGD EVDVQWRLVHDNDYFIKPVSYLAHYLGYAWVGGNHSPYVGEDMDVTRV GDGWLIKGNNDGGCSGYRCGEKSSIKVSNFSYTLEPDSFSHGQVTESGK QLVKTITANATNYTDLPQQVVVTLKYDKATNWSKTDTYSLSEKVTTKNK FQWPLVGETELAIEIAASQSWASQKGGSTTETVSVEARPTVPPHSSLPVRV ALYKSNISYPYEFKAEVNYDLTMKGFLRWGGNAWYTHPDNRPTWEHTL LLGPFRGQGEQHPLPVDKRYIPGEVKWWDWNWTISEYGLSTMQNNLGR VLRPIRSAVTGDFYAESQFAGDIEIGQPQ [00177] SEQ ID NO: 12 >sp|Q06305|AER3_AERHY Aerolysin-3 OS=Aeromonas hydrophila GN=ahh3 without C-terminal propeptide AEPVYPDQLRLFSLGQEVCGDKYRPVNREEAQSVKSNIVGMMGQWQISG LANGWVIMGPGYNGEIKPGSASSTWCYPTNPATGEIPTLSALDIPDGDEV DVQWRLVHDSANFIKPTSYLAHYLGYAWVGGNHSQYVGEDMDVTRDGD GWVIRGNNDGGCDGYRCGDKTSIKVSNFAYNLDPDSFKHGDVTQSDRQ LVKTVVGWAINDSDTPQSGYDVTLRYDTATNWSKTNTYGLSEKVTTKNK FKWPLVGETELSIEIAANQSWASQNGGSPTTSLSQSVRPTVPAHSKIPVKI ELYKADISYPYEFKADVSYDLTLSGFLRWGGNAWYTHPDNRPNWNHTF VIGPYKDKASSIRYQWDKRYIPGEVKWWDWNWTIQQNGLPTMQNNLAK VLRPVRAGITGDFSAESQFAGNIEIGAPVPV [00178] SEQ ID NO: 13 >sp|P09166|AERA_AEREN Aerolysin OS=Aeromonas enteropelogenes GN=aerA without C-terminal propeptide NAAEPIYPDQLRLFSLGEDVCGTDYRPINREEAQSVRNNIVAMMGQWQIS GLANNWVILGPGYNGEIKPGKASTTWCYPTRPATAEIPVLPAFNIPDGDA VDVQWRMVHDSANFIKPVSYLAHYLGYAWVGGDHSQFVGDDMDVIQEG DDWVLRGNDGGKCDGYRCNEKSSIRVSNFAYTLDPGSFSHGDVTQSERT LVHTVVGWATNISDTPQSGYDVTLNYTTMSNWSKTNTYGLSEKVSTKNK FKWPLVGETEVSIEIAANQSWASQNGGAVTTALSQSVRPVVPARSRVPVKI ELYKANISYPYEFKADMSYDLTFNGFLRWGGNAWHTHPEDRPTLSHTFA IGPFKDKASSIRYQWDKRYLPGEMKWWDWNWAIQQNGLATMQDSLAR VLRPVRASITGDFRAESQFAGNIEIGTPVPL [00179] SEQ ID NO: 14 >sp|Q08676|AERA_AERSA Aerolysin OS=Aeromonas salmonicida GN=ash3 without C-terminal propeptide WHEPVYPDQVKWAGLGTGVCASGYRPLTRDEAMSIKGNLVSRMGQWQI TGLADRWVIMGPGYNGEIKQGTAGETWCYPNSPVSGEIPTLSDWNIPAG DEVDVQWRLVHDNDYFIKPVSYLAHYLGYAWVGGNHSPYVGEDMDVTR VGDGWLIKGNNDGGCSGYRCGEKSSIKVSNFSYTLEPDSFSHGQVTESG KQLVKTITANATNYTDLPQQVVVTLKYDKATNWSKTDTYSLSEKVTTKN KFQWPLVGETELAIEIAASQSWASQKGGSTTETVSVEARPTVPPHSSLPV RVALYKSNISYPYEFKAEVNYDLTMKGFLRWGGNAWYTHPDNRPTWEH TFRLGPFRGQGEQHPLPVDKRYIPGEVKWWDWNWTISEYGLSTMQNNL GRVLRPIRSAVTGDFYAESQFAGDIEIGQPQ [00180] SEQ ID NO: 15 >sp|Q06303|AER4_AERHY Aerolysin-4 OS=Aeromonas hydrophila GN=ahh4 without C-terminal propeptide AEPVYPDQLRLFSLGQEVCGDKYRPVNREEAQSIKSNIVGMMGQWQISG LANGWVIMGPGYNGEIKPGSASSTWCYPTNPATGEIPTLSALDIPDGDEV DVQWRLVHDSANFIKPTSYLAHYLGYAWVGGNHSQYVGEDMDVTRDGD GWVIRGNNDGGCDGYRCGDKTSIKVSNFAYNLDPDSFKHGDVTQSDRQ LVKTVVGWAINDSDTPQSGYDVTLRYDTATNWSKTNTYGLSEKVTTKNK FKWPLVGETELSIEIAANQSWASQNGGSTTTSLSQSVRPTVPAHSKIPVKI ELYKADISYPYEFKADVSYDLTLSGFLRWGGNAWYTHPDNRPNWNHTF VIGPYKDKASSIRYQWDKRYIPGEVKWWDWNWTIQQNGLPTMQNNLAK VLRPVRAGITGDFSAESQFAGNIEIGAPVPV [00181] SEQ ID NO: 16 Aerolysin mutant monomer K238A AEPVYPDQLRLFSLGQGVCGDKYRPVNREEAQSVKSNIVGMMGQWQISG LANGWVIMGPGYNGEIKPGTASNTWCYPTNPVTGEIPTLSALDIPDGDEV DVQWRLVHDSANFIKPTSYLAHYLGYAWVGGNHSQYVGEDMDVTRDGD GWVIRGNNDGGCDGYRCGDKTAIKVSNFAYNLDPDSFKHGDVTQSDRQ LVKTVVGWAVNDSDTPQSGYDVTLRYDTATNWSKTNTYGLSEAVTTKN KFKWPLVGETELSIEIAANQSWASQNGGSTTTSLSQSVRPTVPARSKIPVK IELYKADISYPYEFKADVSYDLTLSGFLRWGGNAWYTHPDNRPNWNHTF VIGPYKDKASSIRYQWDKRYIPGEVKWWDWNWTIQQNGLSTMQNNLAR VLRPVRAGITGDFSAESQFAGNIEIGAPVPLAADSKVRRARSVDGAGQGLR LEIPLDAQELSGLGFNNVSLSVTPAANQ [00182] SEQ ID NO: 17 Aerolysin mutant monomer K238A&K242A AEPVYPDQLRLFSLGQGVCGDKYRPVNREEAQSVKSNIVGMMGQWQISG LANGWVIMGPGYNGEIKPGTASNTWCYPTNPVTGEIPTLSALDIPDGDEV DVQWRLVHDSANFIKPTSYLAHYLGYAWVGGNHSQYVGEDMDVTRDGD GWVIRGNNDGGCDGYRCGDKTAIKVSNFAYNLDPDSFKHGDVTQSDRQ LVKTVVGWAVNDSDTPQSGYDVTLRYDTATNWSKTNTYGLSEAVTTAN KFKWPLVGETELSIEIAANQSWASQNGGSTTTSLSQSVRPTVPARSKIPVK IELYKADISYPYEFKADVSYDLTLSGFLRWGGNAWYTHPDNRPNWNHTF VIGPYKDKASSIRYQWDKRYIPGEVKWWDWNWTIQQNGLSTMQNNLAR VLRPVRAGITGDFSAESQFAGNIEIGAPVPLAADSKVRRARSVDGAGQGLR LEIPLDAQELSGLGFNNVSLSVTPAANQ [00183] SEQ ID NO: 18 nucleotide sequence encoding for the polypeptide of SEQ ID NO:16 gcagagcccg tctatccaga ccagcttcgc ttgttttcat tgggccaagg ggtctgtggc gacaagtatc gccccgtcaa tcgagaagaa gcccaaagcg ttaaaagcaa tattgtcggc atgatggggc aatggcaaat aagcgggctg gccaacggct gggtcattat ggggccgggt tataacggtg aaataaaacc agggacagcg tccaatacct ggtgttatcc gaccaatcct gttaccggtg aaataccgac actgtctgcc ctggatattc cagatggtga cgaagtcgat gtgcagtggc gactggtaca tgacagtgcg aatttcatca aaccaaccag ctatctggcc cattacctcg gttatgcctg ggtgggcggc aatcacagcc aatatgtcgg cgaagacatg gatgtgaccc gtgatggcga cggctgggtg atccgtggca acaatgacgg cggctgtgac ggctatcgct gtggtgacaa gacggccatc aaggtcagca acttcgccta taacctggat cccgacagct tcaagcatgg cgatgtcacc cagtccgacc gccagctggt caagactgtg gtgggctggg cggtcaacga cagcgacacc ccccaatccg gctatgacgt caccctgcgc tacgacacag ccaccaactg gtccaagacc aacacctatg gcctgagcga ggccgtgacc accaagaaca agttcaagtg gccactggtg ggggaaaccc aactctccat cgagattcgt gccaatcagt cctgggcgtc ccagaacggg ggctcgacca ccacctccct gtctcagtcc gtgcgaccga ctgtgccggc ccgctccaag atcccggtga agatagagct ctacaaggcc gacatctcct atccctatga gttcaaggcc gatgtcagct atgacctgac cctgagcggc ttcctgcgct ggggcggcaa cgcctggtat acccacccgg acaaccgtcc gaactggaac cacaccttcg tcataggtcc gtacaaggac aaggcgagca gcattcgcta ccagtgggac aagcgttaca tcccgggtga agtgaagtgg tgggactgga actggaccat acagcagaac ggtctgtcta ccatgcagaa caacctggcc agagtgctgc gcccggtgcg ggcggggatc accggtgatt tcagtgccga gagccagttt gccggcaaca tagagatcgg tgctcccgtg ccgctcgcgg ctgacagcaa ggtgcgtcgt gctcgcagtg tggacggcgc tggtcaaggc ctgaggctgg agatcccgct cgatcgcgaa gagctctccg ggcttggctt caacaagtca gcctcagcgt ga [00184] SEQ ID NO: 19 nucleotide sequence encoding for the polypeptide of SEQ ID NO:17 gcagagcccg tctatccaga ccagcttcgc ttgttttcat tgggccaagg ggtctgtggc gacaagtatc gccccgtcaa tcgagaagaa gcccaaagcg ttaaaagcaa tattgtcggc atgatggggc aatggcaaat aagcgggctg gccaacggct gggtcattat ggggccgggt tataacggtg aaataaaacc agggacagcg tccaatacct ggtgttatcc gaccaatcct gttaccggtg aaataccgac actgtctgcc ctggatattc cagatggtga cgaagtcgat gtgcagtggc gactggtaca tgacagtgcg aatttcatca aaccaaccag ctatctggcc cattacctcg gttatgcctg ggtgggcggc aatcacagcc aatatgtcgg cgaagacatg gatgtgaccc gtgatggcga cggctgggtg atccgtggca acaatgacgg cggctgtgac ggctatcgct gtggtgacaa gacggccatc aaggtcagca acttcgccta taacctggat cccgacagct tcaagcatgg cgatgtcacc cagtccgacc gccagctggt caagactgtg gtgggctggg cggtcaacga cagcgacacc ccccaatccg gctatgacgt caccctgcgc tacgacacag ccaccaactg gtccaagacc aacacctatg gcctgagcga ggccgtgacc accgccaaca agttcaagtg gccactggtg ggggaaaccc aactctccat cgagattcgt gccaatcagt cctgggcgtc ccagaacggg ggctcgacca ccacctccct gtctcagtcc Ķķ gtgcgaccga ctgtgccggc ccgctccaag atcccggtga agatagagct ctacaaggcc gacatctcct atccctatga gttcaaggcc gatgtcagct atgacctgac cctgagcggc ttcctgcgct ggggcggcaa cgcctggtat acccacccgg acaaccgtcc gaactggaac cacaccttcg tcataggtcc gtacaaggac aaggcgagca gcattcgcta ccagtgggac aagcgttaca tcccgggtga agtgaagtgg tgggactgga actggaccat acagcagaac ggtctgtcta ccatgcagaa caacctggcc agagtgctgc gcccggtgcg ggcggggatc accggtgatt tcagtgccga gagccagttt gccggcaaca tagagatcgg tgctcccgtg ccgctcgcgg ctgacagcaa ggtgcgtcgt gctcgcagtg tggacggcgc tggtcaaggc ctgaggctgg agatcccgct cgatcgcgaa gagctctccg ggcttggctt caacaagtca gcctcagcgt ga

Claims

Claims 1. Use of an aerolysin nanopore for the discrimination of at least two isoforms of a peptide, polypeptide, or protein, wherein the at least two isoforms differ from each other at least in that they bear different post-translational modifications at the same position of the peptide, polypeptide, or protein.

2. Use according to claim 1 wherein at least one of the said post-translational modifications is a phosphorylation, a nitration or an oxidation, preferably a nitration or an oxidation.

3. Use according to claim 1 or 2, wherein the said peptide, polypeptide or protein is α-synuclein or a fragment thereof, preferably the C-terminal domain of α -synuclein, more preferably the residues 124 to 140 of α-synuclein.

4. Use according to claim 3, wherein the peptide, polypeptide or protein bears multiple PTMs at multiple residues, wherein the PTMs are selected from oxidation, nitration, and mixtures thereof.

5. A method of discriminating between at least two isoforms of a peptide, polypeptide, or protein, wherein the at least two isoforms differ from each other at least in that they bear different post-translational modifications at the same position of the peptide, polypeptide, or protein, the method comprising: a. optionally contacting one or more peptide, polypeptide or protein isoform(s) with a protease such as to cleave one or more fragment(s) comprising one or more amino acids of interest; b. placing a sample comprising the peptide, polypeptide or protein isoform(s) or the fragment(s) thereof obtained in step a) in a first compartment of a chamber separated into a first and a second compartments by a membrane holding an aerolysin nanopore, wherein the aerolysin nanopore connects both compartments of the chamber; c. triggering translocation of the peptide, polypeptide or protein isoform(s) or of the fragment(s) thereof obtained in step a) through the aerolysin nanopore by applying a potential, preferably a voltage potential, across the aerolysin nanopore; d. measuring at least one characteristic indicative of the type(s), number and position(s) of any post-translational modification(s) of the peptide, polypeptide or protein isoform(s) or of the fragment(s) thereof obtained in step a) in the aerolysin nanopore, wherein said characteristic is preferably selected from dwell time, averaged relative current, relative current variation, skewness of relative current, kurtosis of relative current, minimum of relative current, maximum of relative current, peak to peak variation, integrated current and combinations thereof; and e. identifying the type(s), number and position(s) of any post-translational modification of the peptide, polypeptide or protein isoform(s) or of the fragment(s) thereof obtained in step a) present in the sample by comparing the at least one characteristic measured in step d) with characteristics previously measured for specific peptide, polypeptide or protein isoforms or fragments thereof for which the type(s), number and position(s) of post-translational modification(s) were known.

6. A method of detecting in a subject a disease or disorder associated with the presence of one or more PTM(s) of an aberrant type on one or more amino acid(s) of interest in a peptide, polypeptide or protein instead of one or more PTM(s) of a healthy type on the same amino acid(s), the method comprising: a. optionally contacting the peptide, polypeptide or protein from the subject with a protease such as to cleave one or more fragment(s) comprising one or more amino acids of interest; b. placing a sample comprising the peptide, polypeptide or protein from the subject or the fragment(s) thereof obtained in step a) in a first compartment of a chamber separated into a first and a second compartments by a membrane holding an aerolysin nanopore, wherein the aerolysin nanopore connects both compartments of the chamber; c. triggering translocation of the peptide, polypeptide or protein or of the fragment(s) thereof obtained in step a) through the aerolysin nanopore by applying a potential, preferably a voltage potential, across the aerolysin nanopore; d. measuring at least one characteristic indicative of the type(s), number and position(s) of any post-translational modification(s) of the peptide, polypeptide or protein or of the fragment(s) thereof obtained in step a) in the aerolysin nanopore, wherein said characteristic is preferably selected from dwell time, averaged relative current, relative current variation, skewness of relative current, kurtosis of relative current, minimum of relative current, maximum of relative current, peak to peak variation, integrated current and combinations thereof; and e. identifying the type(s), number and position(s) of any post-translational modification(s) of the peptide, polypeptide or protein or of the fragment(s) thereof obtained in step a) by comparing the at least one characteristic measured in step d) with characteristics previously measured for specific peptides, polypeptides or proteins or fragments thereof for which the type(s), number and position(s) of post-translational modification(s) was known.

7. The method of claim 5 or 6, wherein the peptide, polypeptide or protein is α- synuclein or a fragment thereof.

8. The method of claim 7, wherein the α-synuclein fragment is a C-terminal fragment of α-synuclein, preferably comprising the residues 124 to 140 of α -synuclein.

9. The method of any one of claims 5 to 8, wherein step e. is performed using a deep learning algorithm trained with a database comprising characteristics previously measured for specific isoforms for which the type(s), number and position(s) of post-translational modification(s) was known.

10.The method according to any one of claims 6 to 9, wherein the disorder associated with the presence of a PTM of an aberrant type on one or more amino acid(s) of interest in a peptide, polypeptide or protein instead of a PTM(s) of a healthy type on the same amino acid is a neurodegenerative disease, preferably a synucleinopathy, more preferably selected from Alzheimer disease and Parkinson diseases.

11.The use according to any one of claims 1 to 4 or the method according to any one of claims 5 to 10, wherein the peptide, polypeptide or protein or the α- synuclein or fragment thereof is in unfolded form, and wherein the aerolysin nanopore is in folded form.

12. The use according to any one of claims 1 to 4 or the method according to any one of claims 5 to 11, wherein the peptide, polypeptide, or protein or α- synuclein or the fragment thereof is provided in a biological sample, preferably plasma or a sample comprising red blood cells, preferably wherein haemoglobin proteins have been removed.

13.The use according to any one of claims 1 to 4 or the method according to any one of claims 5 to 12, wherein the peptide, polypeptide, or protein or α- synuclein or the fragment thereof is provided in a biological sample diluted with a biologically compatible aqueous solvent at a biological sample to solvent ratio of 1:10 to 1:50. The use according to any one of claims 1 to 4 or the method according to any one of claims 5 to 13, wherein the aerolysin nanopore comprises a monomer consisting of a mutant polypeptide selected from a. a polypeptide comprising a first amino acid substitution selected from the group consisting of K238A, K238S, K238G and K238D of the amino acid sequence of any one of SEQ ID NO:1, 2, 4, 6, 7, 8, 10, 12, 14 or 15 or a variant or fragment thereof; and b. a polypeptide comprising a first amino acid substitution selected from the group consisting of V238A, V238S, V238G and V238D of the amino acid sequence of any one of SEQ ID NO:3 or 11 or a variant or fragment thereof. The use or the method according to claim 14, wherein the aerolysin monomer further comprises at least one of - a second amino acid substitution selected from the group consisting of K242W and K242A, preferably K242A when the sequence is selected from any one of SEQ ID NO:1, 2, 4, 6, 7, 8, 10, 12, 14 and 15 of variants or fragments thereof or a second amino acid substitution selected from the group consisting of N242W and N242A, preferably N242A, when the sequence is selected from any one of SEQ ID NO:3 or 11 or a variant or fragment thereof or a second amino acid substitution selected from the group consisting of S242W and S242A, preferably S242A, when the sequence is selected from any one of SEQ ID NO:5 or 13 or a variant or fragment thereof. - a third amino acid substitution selected from the group consisting of R282A, R282S, R282G, R282D, R282W, preferably selected from the group consisting of R282A and R282S, most preferably R282S when the sequence is selected from any one of SEQ ID NO:1,2, 4, 6 to 8, 10, 12, 14 and 15 or a variant or fragment thereof or a third amino acid substitution selected from P282A, P282S, P282G, P282D, P282W, preferably selected from the group consisting of P282A and P282S, most preferably P282S when the sequence is selected from SEQ ID NO:3 and 11 or a variant or fragment thereof; - a fourth amino acid substitution selected from the group consisting of D216A, D216S, D216G, D216D and D216W, preferably selected from the group consisting of D216A and D216S, most preferably D216S when the sequence is selected from any one of SEQ ID NO:1, 2, 4, 7, 8, 10, 12 and 15 or a variant or fragment thereof or a fourth amino acid substitution selected from selected from the group consisting of V216A, V216S, V216G, V216D and V216W, preferably selected from the group consisting of V216A and V216S, most preferably V216S when the sequence is selected from SEQ ID NO: 3, 6, 11 or 14 or a variant or fragment thereof or a fourth amino acid substitution selected from selected from the group consisting of G216A and G216S, most preferably V216S when the sequence is selected from SEQ ID NO: 5 and 13; - a fifth amino acid substitution selected from the group consisting of D222A, D222S, D222G and D222D, preferably selected from the group consisting of D222A and D222S, most preferably D222S when the sequence is selected from any one of SEQ ID NO:1,2, 4, 6 to 8, 10, 12, 14 and 15 or a variant or fragment thereof or a fifth amino acid substitution selected the group consisting of K222A, K222S, K222G and K222D, preferably selected from the group consisting of K222A and K222S, most preferably K222S when the sequence is selected from SEQ ID NO:3 or 11 or a variant or fragment thereof or a fifth amino acid substitution selected from the group consisting of N222A, N222S, N222G and N222D, preferably selected from the group consisting of N222A and N222S, most preferably N222S when the sequence is selected from any one of SEQ ID NO: 5 and 13; - a sixth amino acid substitution selected from the group consisting of E258A, E258S, E258G, E258D and E258W, preferably selected from the group consisting of E258A and E258S, most preferably E258S when the sequence is selected from any one of SEQ ID NO:1, 2, 4, 6 to 8, 10, 12, 14 and 15 or a variant or fragment thereof or a sixth amino acid substitution selected from the group consisting of I258A, I258S, I258G, I258D and I258W, preferably selected from the group consisting of I258A and I258S, most preferably I258S when the sequence is selected from any one of SEQ ID NO: 3 and 11 or a variant or fragment thereof; - a seventh amino acid substitution selected from the group consisting of E254A, E254S, E254G, E254D and E254W, preferably selected from the group consisting of E254A and E254S, most preferably E254S when the sequence is selected from any one of SEQ ID NO:1,2, 4, 5, 6 to 8, 10, 12, 14 and 15 or a variant or fragment thereof or a seventh amino acid substitution selected from the group consisting of L254A, L254S, L254G, L254D and L254W, preferably selected from the group consisting of L254A and L254S, most preferably L254S when the sequence is selected from any one of SEQ ID NO: 3 and 11 or a variant or fragment thereof; - an eighth amino acid substitution selected from the group consisting of K244A, K244S, K244G, K244D and K244W, preferably selected from the group consisting of K244A and K244S, most preferably K244S when the sequence is selected from any one of SEQ ID NO:1, 2, 4, 5, 6 to 8, 10, 12, 14 and 15 or a variant or fragment thereof or an eighth amino acid substitution selected from the group consisting of F244A, F244S, K244G, F244D and F244W, preferably selected from the group consisting of F244A and F244S, most preferably F244S when the sequence is selected from any one of SEQ ID NO: 3 and 11 or a variant or fragment thereof; - a ninth amino acid substitution selected from the group consisting of E252A, E252S, E252G, E252D and E252W, preferably selected from the group consisting of E252A and E252S, most preferably E252S when the sequence is selected from any one of SEQ ID NO:1, 2, 4, 6 to 8, 10, 12, 14 and 15 or a variant or fragment thereof or a ninth amino acid substitution selected from the group consisting of T252A, T252S, T252G, T252D and T252W, preferably selected from the group consisting of T252A and T252S, most preferably T252S when the sequence is selected from any one of SEQ ID NO: 3 and 11 or a variant or fragment thereof or a ninth amino acid substitution selected from the group consisting of V252A, V252S, V252G, V252D and V252W, preferably selected from the group consisting of V252A and V252S, most preferably V252S when the sequence is selected from any one of SEQ ID NO: 5 and 13 or a variant or fragment thereof; and - a tenth amino acid substitution selected from the group consisting of K246S, K246A K246G, K246D and K246W, preferably selected from the group consisting of K246A and K246S, most preferably K246S when the sequence is selected from any one of SEQ ID NO:1, 2, 4, 5, 7, 8, 10, 12, 13, and 15 or a variant or fragment thereof or a tenth amino acid substitution selected from the group consisting of W246S, W246A W246G, W246D and W246W, preferably selected from the group consisting of W246A and W246S, most preferably W246S when the sequence is selected from any one of SEQ ID NO: 3 and 11 or a variant or fragment thereof or a tenth amino acid substitution selected from the group consisting of Q246S, Q246A Q246G, Q246D and Q246W, preferably selected from the group consisting of Q246A and Q246S, most preferably Q246S when the sequence is selected from any one of SEQ ID NO: 6 and 14 or a variant or fragment thereof.