NL2023062B1 - Protein current trace signal acquisition using a nanopore - Google Patents

Abstract

The invention provides a. method for acquiring a characteristic signal (400) of a protein (300) passing a membrane nanopore (100) comprised by a membrane (200), wherein the membrane nanopore (100) comprises a nanopore constriction space having a constriction length Lc £ 20 nm and a circular equivalent constriction diameter dc £ 20 nm, wherein the protein (300) is linear, and wherein the protein (300) comprises a C-terminal first charged group (310) and an N—terrninal second charged group (320), wherein the first charged group (310) and the second charge group (320) have charges of opposite sign, and wherein the protein (300) comprises a modified amino acid (330), Wherein the modified amino acid (330) comprises a tag (332) and/or a post-translational modification (334), and Wherein the method comprises a nanopore passage stage (20) comprising passing the protein (300) through the membrane nanopore (100), Wherein a voltage V is applied across the membrane (200), and Wherein an ionic current is measured over the membrane (200) thereby providing the characteristic signal (400).

Description

P1600107NL00 Protein current trace signal acquisition using a nanopore

FIELD OF THE INVENTION The invention relates to a method for acquiring a characteristic signal of a protein passing a nanopore. The invention further relates to a method for identifying a protein. The invention further relates to an analysis system. The invention further relates to a computer program product.

BACKGROUND OF THE INVENTION Methods for acquiring a characteristic signal of a protein using a nanopore are known in the art. For example, WO2018012963 relates generally to the field of nanopores and the use thereof in various applications, such as analysis of biopolymers and macromolecules, typically by making electrical measurements during translocation through a nanopore. Provided is a system comprising a funnel-shaped proteinaceous nanopore comprising an a-helical pore-forming toxin that is a member from the actinoporin protein family, more in particular Fragaceatoxin C (FraC), a mutant FraC, a FraC paralog, or a FraC homolog.

SUMMARY OF THE INVENTION Proteins may be considered the workhorses in all living cells. Thousands of different proteins may sustain cellular functions, from copying DNA and catalyzing basic metabolism to providing cellular motion. Protein identification and analysis may therefore provide key information for the understanding of biological processes, including diseases. Recent technological developments may have mostly focused on the study of genomes, making DNA sequencing relatively fast, cheap, and ubiquitous. The study of other -omics, especially proteomics, however, may still remain costly and time-consuming. Despite the importance of protein analysis, for example in the context of biomarkers, the identification and characterization of proteins in complex samples may be rather limited.

Mass spectrometry may be the current gold standard method for proteomics, but may be relatively expensive and time-consuming. Further, mass spectrometry may rely on ionization and fragmentation of the proteins, and subsequent detection of the masses of the protein fragments. Based on the detected masses and their frequencies, the identity and abundance of proteins may be inferred based on masses corresponding to protein sequences predicted from genetic information. However, the process may be limited due to (1) different potential fragmentation patterns for a protein, (ii) post-translational modifications of the protein, particularly as an exhaustive overview of potential post-translational modifications for a given protein may typically not be available, (iii) low abundance proteins may be hard to detect, and/or (iv) there may be an information loss due to protein fragmentation, as similar proteins may provide (some) identical protein fragments, which may be particularly problematic with regards to protein isoforms.

Cost effective methods such as immunoassays may, on the other hand, only be capable of analyzing relatively few proteins in a sample. Further, immunoassays may be time- intensive to adapt and may be limited by the availability of antibodies, which may also often present specificity issues. Yet further, immunoassays may be limited in their ability to distinguish between isoforms of a protein.

Hence, one of the main challenges in proteomics may be the lack of sensitive techniques that allow for detection of proteins present in low abundance. As, unlike for DNA, there may be no biochemical method to amplify proteins present in a sample.

In a nanopore experiment, an insulating membrane containing a nanometer- sized pore may be placed between two electrolyte-filled compartments. When a voltage is applied to the compartments, an ionic current may flow through the nanopore. As a molecule translocates through the nanopore, a modulation of the ionic currents may be observed, which may provide structural information about the molecule. Nanopore-based protein identification may rely on a current trace signal: a measurement of changes in an ionic current across a nanopore as a (single) protein approaches, departs and/or passes through the nanopore. The current trace signal may then in principle be used to identify the protein. However, it may be difficult to distinguish proteins based on their current trace signals, among others due to background noise, particularly with regards to complex samples comprising many proteins and/or similar proteins. In particular, it may be difficult to differentiate between the signal of the different (proteinogenic) amino acids as they pass through the pore.

Hence, it is an aspect of the invention to provide an alternative method for acquiring a characteristic signal of a protein, which preferably further at least partly obviates one or more of above-described drawbacks. The present invention may have as object to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

Therefore, in a first aspect, the invention provides a method for acquiring a characteristic signal, especially a current trace signal, of a protein passing a membrane nanopore (also: “nanopore”) comprised by a membrane. The membrane nanopore may comprise a nanopore constriction space. In embodiments, the nanopore constriction space may have a constriction length Le < 20 nm. In further embodiments, the nanopore constriction space may have a circular equivalent constriction diameter de < 20 nm. In embodiments, the protein may be linear. In further embodiments, the protein may comprise a C-terminal first charged group and an N-terminal second charged group, especially wherein the first charged group and the second charge group have charges of opposite sign (during operation conditions). In further embodiments, the protein may comprise a modified amino acid, especially wherein the modified amino acid comprises a tag and/or a post-translational modification. The method may comprise: a nanopore passage stage comprising passing the protein through the membrane nanopore, especially wherein a voltage V is applied across the membrane nanopore, more especially across the membrane comprising the membrane nanopore, and especially wherein an ionic current is measured over the membrane nanopore, more especially over the membrane, thereby providing the characteristic signal.

The present invention may provide the benefit that the protein provides a more distinctive characteristic signal, as the protein passes through the nanopore more slowly and with more distinctive effects on the ionic current. Specifically, the relatively confined nanopore constriction space may provide a focused measurement site, the C-terminal and N- terminal charged groups may provide an increased passage time of the protein through the nanopore, and the modified amino acid may provide a more distinctive change in the current charge (density) as the corresponding section of the (linear) protein passes through the nanopore (constriction space).

In specific embodiments, the invention may provide a method for acquiring a characteristic signal of a protein passing (through) a nanopore arranged in a membrane, wherein the nanopore comprises a nanopore constriction space having a constriction length

Le < 20 nm and a circular equivalent constriction diameter de < 20 nm, wherein the protein is linear, and wherein the protein comprises a C-terminal first charged group and an N-terminal second charged group, wherein the first charged group and the second charge group have charges of opposite sign, and wherein the protein comprises a modified amino acid, wherein the modified amino acid comprises a tag and/or a post-translational modification, and wherein the method comprises: a nanopore passage stage comprising passing the protein through the nanopore, wherein a voltage V is applied across the membrane, and wherein an ionic current is measured over the membrane thereby providing a characteristic signal.

Hence, the method may be used for acquiring a characteristic signal of a protein, especially a protein passing a membrane nanopore, more especially a membrane nanopore comprised by a membrane.

The term “protein” may refer herein to a chain of amino acids of any length, especially a chain of at least two amino acids, more especially a chain of ten or more amino acids. The term protein may thus also refer to a peptide, oligopeptide, and polypeptide. In embodiments, the protein may comprise a peptide, especially wherein the peptide comprises a number of amino acids selected from the range of 2 — 50.

A membrane nanopore is a pore with an (equivalent circular) diameter in the nanometer range, for example with a (smallest) circular equivalent diameter selected from the range of 0.01 — 100 nm. The nanopore may be comprised by or may be formed by a pore- forming protein and/or a solid state nanopore (a hole in a synthetic material). The membrane nanopore may especially comprise a through-hole through the membrane comprising the membrane nanopore. The term “membrane nanopore” may herein also be referred to as “nanopore”.

Hence, in embodiments, the nanopore may be comprised by or may be formed by a pore-forming protein. Especially, the membrane may comprise a pore-forming protein providing the nanopore. The pore-forming protein may comprise a channel through which a molecule, such as a protein, could pass. In further embodiments, the nanopore may be arranged in a membrane, especially such that a molecule, such as the protein, could pass from a first side of the membrane via a channel in the nanopore to a second side of the membrane.

In embodiments, the membrane may comprise a lipid membrane, especially a lipid bilayer.

In specific embodiments, the membrane may be provided from 1,2- diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), i.e., the membrane may comprise 1,2-

diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). Especially, the membrane may essentially consist of 1,2-diphytanoyl-sn-glycero-3-phosphocholine. In further embodiments, the nanopore may comprise a solid state nanopore, especially a solid state nanopore in a synthetic membrane. Essentially, the solid state nanopore 5 may be formed by providing a hole in the synthetic membrane, via, for example, one or more of ion beam sculpting and/or transmission electron microscopy. In embodiments, the synthetic membrane may comprise a material selected from the group comprising silicon nitride (SiN), graphene, especially box-shaped graphene, molybdenum disulfide (MoS2), boron nitride (BN), and silicon dioxide (SiOz).

The term “characteristic signal” herein refers to a measurement of an interaction between the protein and the nanopore. In general, the characteristic signal may comprise a current trace signal, i.e., a measurement over time of the ionic current over the membrane, wherein the interaction between the protein and the nanopore results in modulation of the ionic current. For example, the protein may pass through (also: “translocate”) the nanopore, resulting in a modulation of the ionic current as the protein moves. Hence, in embodiments, the characteristic signal may comprise a current trace signal.

In embodiments, the nanopore may comprise a nanopore constriction space. The nanopore constriction space may be the space within the nanopore with the smallest (also: narrowest) circular equivalent constriction diameter. This may result in the largest modulations to the ionic current occurring due to the presence of the part of the protein in or nearest to the nanopore constriction space. For example, it may be ideal if the nanopore constriction space is sized such that as a linear protein moves through the nanopore, only a single amino acid of the protein (essentially) modulates the ionic current.

In further embodiments, the nanopore constriction space may have a constriction length Le and a circular equivalent constriction diameter d.. Especially, the constriction length L. may essentially be parallel to a channel in the nanopore and the circular equivalent constriction diameter d. may essentially be perpendicular to the constriction length Lc. Hence, the constriction length Lc and the circular equivalent constriction diameter de may together define a cylinder approximating the space within the nanopore with the smallest circular equivalent constriction diameter.

In embodiments, the constriction length Lc may be < 30 nm, such as <25 nm, especially < 20 nm, such as < 15 nm, especially < 10 nm, such as < 8 nm, especially < 6 nm,

such as < 5 nm, especially < 4 nm, such as <3 nm, especially < 2 nm, such as < 1 nm. In further embodiments, the constriction length L« may be > 0.5 nm, especially > 1 nm, such as > 2 nm, especially > 3 nm.

In further embodiments, the constriction length Lc may be essentially parallel to the pore of the nanopore. In further embodiments, the constriction length Le may be essentially perpendicular to the (plane of the) membrane.

In further embodiments, the circular equivalent constriction diameter de may be < 20 nm, such as < 15 nm, especially < 10 nm, such as < 8 nm, especially < 6 nm, such as <5 nm, especially <4 nm, such as < 3.5 nm, especially <3 nm, such as < 2.5 nm, especially <2 nm, such as < 1.5 nm, especially < 1 nm. In further embodiments, the constriction length Le may be > 1nm, especially > 0.5 nm, such as > 1 nm, especially > 1.5 nm, such as > 2 nm.

In specific embodiments, the membrane nanopore may be comprised by or may be formed by a biological nanopore (forming protein) selected from the group comprising FraC, MspA, alpha-hemolysin, aerolysin, OmpG, ClyA, Phi29, FhuA, and SPI, especially the membrane nanopore may be comprised by or may be formed by a pore-forming protein, wherein the pore-forming protein is selected from the group comprising FraC, MspA, alpha-hemolysin and Phi29. Especially, the membrane nanopore may be comprised by or may be formed by FraC, i.e., the membrane nanopore may be comprised by or may be formed by a pore-forming protein, wherein the pore-forming protein comprises FraC.

In further embodiments, the nanopore may be comprised by or may be formed by a mutant of a biological nanopore (forming protein) selected from the group comprising FraC, MspA, alpha-hemolysin, aerolysin, OmpG, ClyA, Phi29, FhuA, and SP1, especially the nanopore may be comprised by or may be formed by a pore-forming protein, wherein the pore-forming protein is a mutant protein of one or more pore-forming proteins selected from the group comprising FraC, MspA, alpha-hemolysin, aerolysin, OmpG, ClyA, Phi29, FhuA, and SP1.

In further embodiments, the protein may be linear. A linear protein may pass (through) the nanopore in a linear fashion, i.e., the linear protein may pass through the nanopore in an outstretched chain.

If the nanopore constriction space is sufficiently small, then the modulation of the ionic current by a linear protein may be essentially determined by a few amino acids, or even by a single amino acid, at a time. Hence, a small nanopore constriction space may provide an improved spatial resolution in the acquisition of the characteristic signal. Distinguishing between the modulation caused by subsequent amino acids in the protein may, however, be challenging if the protein passes through the nanopore quickly. Hence, in embodiments, the protein may comprise a C-terminal first charged group and an N- terminal second charged group, especially wherein the first charged group and the second charged group have charges of opposite sign. For example, the first charged group may have a net positive charge and the second charge group may have a net negative charge, or vice versa. As the (linear) protein passes through the nanopore, the terminal charged groups may be arranged on different sides of the nanopore in (different) electrolytes (see further below), providing a sort of “tug of war” between the terminal ends of the protein, which may result in a substantially prolonged passage (also: “translocation time” or “dwell time”) of the protein through the nanopore. Hence, the terminal charged groups may essentially provide an improved temporal resolution in the acquisition of the characteristic signal.

The combination of a relatively small nanopore constriction space and a relatively long translocation time may provide the benefit that the modulation caused by different amino acids may be better distinguished.

Typically, organisms may produce proteins comprising up to 22 different (proteinogenic) amino acids. These different amino acids may have different (chemical) properties, including, among others, different masses, sizes, isoelectric points, hydrophobicities, and polarities. Thereby, the different amino acids may provide different modulations to the ionic current as they pass (through) the nanopore. However, these relative differences (between at least some amino acids) may be hard to distinguish, which may at least in part be due to the size of their relative differences in modulation in relation to background noise. For example, it may be difficult to distinguish between amino acids with similar chemical features, such as leucine and isoleucine, and such as serine and cysteine. However, a modified amino acid may provide a distinct modulation.

Hence, in embodiments, the protein may comprise a modified amino acid, especially wherein the modified amino acid comprises a (side chain) tag and/or a post- translational modification. The term “modified amino acid” may also refer to a plurality of modified amino acids.

The term “tag” (also: “label”’) may herein refer to a non-natural modification, ie, a modification that was deliberately applied to the protein for measurement.

In embodiments, an amino acid of a specific type, such as a cysteine, in the protein may be deliberately tagged.

In further embodiments, especially (essentially) each amino acid of a specific type, such as each cysteine, may be tagged.

In such an embodiment, it may be possible to identify the positions of the tagged type of amino acid in the protein as the protein passes (through) the nanopore, which may substantially facilitate protein identification.

In embodiments, the modified amino acid may comprise a tag.

The tag may require a minimal size to provide sufficient distinguishing modulation.

Further, the tag may have a maximal size to allow the protein to translocate the nanopore (also dependent on pore size). Hence, in specific embodiments, the tag may have a molecular weight of at least 7 Dalton, such as at least 14 Dalton, especially at least 20 Dalton, such as at least 50 Dalton, especially at least 100 Dalton, such as at least 200 Dalton, especially at least 400 Dalton.

In further embodiments, the tag may have a molecular weight of at most 5000 Dalton, such as at most 4000 Dalton, especially at most 3500 Dalton, such as at most 3000 Dalton, especially at most 2500 Dalton, such as at most 2000 Dalton, especially at most 1500 Dalton.

In specific embodiments, the tag may have a molecular weight selected from the range of 14 - 3000 Dalton.

In further embodiments, the tag may have a net charge.

The net charge may have a maximal net charge to limit the influence on the “tug of war” during protein translocation.

Hence, in further embodiments, the tag may have a net charge selected from the range of -8 to 8, especially from the range of -7 to 7, such as from the range of -6 to 6, especially from the range of -5 to 5, such as from the range of -4 to 4. In specific embodiments, the tag may be selected from the group comprising fluorescein-5-maleimide, Texas Red C2 maleimide, Alexa Fluor 633 C5 maleimide, EZ-link maleimide-PEG1 1-biotin, Histidine6 maleimide, and 5’maleimide 3PolyA.

The term “tag” may also refer to a plurality of (different) tags.

Especially, different (types of) amino acids in the protein may be tagged with different tags.

The term “post-translational modification” (also: PTM) may herein refer to a natural modification, i.e., a modification that was provided to the protein in its biological environment.

In principle, the same modification could be provided via either a tag or a PTM.

In specific embodiments, the modified amino acid comprise the post- translational modification, wherein the post-translational modification is obtainable by one or more of phosphorylation, O-linked glycosylation, acetylation, methylation, nitration, farnesylation, palmitoylation, myristoylation, and S-nitrosylation, especially via one or more of phosphorylation and O-linked glycosylation, 7.e., the PTM may be selected from the group comprising a phosphoryl group, an O-glycan, an acetyl group, a methyl group, a nitro group, a farnesyl group, a palmitoyl group, a myristoyl group, and an S-nitrothiol group, especially from the group comprising a phosphoryl group and an O-glycan. The term “PTM” may also refer to a plurality of (different) PTMs.

In embodiments, the modified amino acid may comprise both a PTM and a tag. Especially, the PTM may be tagged, i.e., the tag may be provided to the PTM.

In further embodiments, the protein may comprise a plurality of modified amino acids, wherein each of the plurality of modified amino acids comprises a tag or a PTM, especially wherein at least one of the plurality of modified amino acids comprises a tag and at least one of the plurality of modified amino acids comprises a PTM.

The modified amino acid may provide a distinct modulation of the ionic current relative to other (nonmodified) amino acids in the protein. Hence, the modified amino acid may provide distinctive modulation in the acquisition of the characteristic signal.

In embodiments, the method may comprise a nanopore passage stage, the nanopore passage stage comprising passing the protein through the membrane nanopore, especially wherein a voltage V is applied across the membrane nanopore, more especially across a membrane in which the nanopore is arranged, and especially wherein an ionic current is measured over the nanopore, more especially over the membrane, thereby providing a characteristic signal.

In embodiments, the nanopore may be arranged in a membrane. In further embodiments, the nanopore may be arranged between a first electrolyte and a second electrolyte, especially wherein the first electrolyte and the second electrolyte are in fluid communication via the nanopore.

In embodiments, the method may comprise applying a voltage V over the membrane, especially applying a voltage V over the membrane such that the protein passes through the nanopore. In specific embodiments, an apparatus is applied which may apply a vanable voltage V. Hence, the voltage V may in embodiments be tunable. The term “voltage V” especially refers to a potential difference.

In embodiments, the method may comprise subjecting the nanopore to an electric field to provide the voltage V across the membrane.

In further embodiments, the voltage V may be selected from the range of -500 — 500 mV, especially from the range of -300 — 300 mV, such as from the range of -250 — 250 mV, especially from the range of -200 — 200 mV, such as from the range of -150 — 150 mV, especially from the range of -100 — 100 mV.

During the nanopore passage stage, the (linear) protein may pass through the nanopore, during which an ionic current is measured. The ionic current may be modulated by (modified) amino acids in the protein, such that a characteristic signal is obtained of the protein as it moves through the nanopore. Hence, the characteristic signal may comprise a current trace signal.

In embodiments, the membrane nanopore may be arranged in a liquid solution (also: “buffer”). In further embodiments, the solution may comprise a salt concentration selected from the range of 1 mM — 10 M, especially from the range of 200 mM to 3 M. In further embodiments, the solution may have a pH selected from the range of 3 — 9, especially from the range of 6 — 8.5.

In further embodiments, the solution may comprise a first electrolyte and a second electrolyte, wherein the first electrolyte and the second electrolyte are arranged on opposite sides of the membrane, and wherein the first electrolyte and the second electrolyte are in fluid communication via the nanopore. In further embodiments, the first electrolyte and the second electrolyte may comprise a salt concentration independently selected from the range of 1 mM — 10 M, especially from the range of 200 mM to 3 M. In further embodiments, the first electrolyte and the second electrolyte may have a pH independently selected from the range of 3 — 9, especially from the range of 6 — 8.5.

During the nanopore passage stage, the protein may thus (1) be linear, (i1) comprise a modified amino acid, and/or (1ii) comprise terminal charged groups. Especially, the protein is linear, comprises a modified amino acid, and comprises terminal charged groups. Such a protein may facilitate distinguishing between the effects on ionic current of adjacent amino acids, may provide a distinctive modulation of the ionic current via the modified amino acid, and may provide a prolonged measurement time by passing through the nanopore relatively slowly. These benefits may be particularly synergistic as the combination of improved spatial resolution, improved temporal resolution and distinct modulation may provide a substantially more distinct characteristic signal than with two out of three aforementioned benefits.

A protein found in a natural sample may, however, typically not have (all) of these features.

Hence in embodiments, the method may further comprise a pretreatment stage for providing the protein from an initial protein, especially wherein the method comprises executing the pretreatment stage prior to the nanopore passage stage.

The term “initial protein” herein refers to a (natural) protein prior to the pretreatment stage.

In embodiments, the pretreatment stage may comprise one or more of: (i) denaturation of the initial protein, (ii) providing a tag to an amino acid in the initial protein, and (ii1) providing the first charged group to the C-terminal end of the initial protein and the second charged group to the N-terminal end of the initial protein; especially thereby providing the protein.

In further embodiments, the pretreatment stage comprises denaturation of the protein.

Denaturation is a process wherein the protein unfolds to its primary structure.

Herein, the term “denaturation” refers to both unfolding of the protein and disulfide bond reduction.

Methods for denaturation of proteins will be known to the person skilled in the art.

In embodiments, the denaturation of the initial protein may comprise exposing the initial protein to one or more denaturants, especially one or more denaturants selected from the group comprising urea, SDS, guanidinium chloride, high salt concentration, and lithium perchlorate.

In further embodiments, the pretreatment stage may comprise providing a tag to an amino acid in the initial protein, especially to provide the modified amino acid.

In further embodiments, the pretreatment stage may comprise providing a tag to the modified amino acid, especially wherein the modified amino acid comprises a post-translational modification.

In principle the modified amino acid may comprise any amino acid.

Especially, a modified amino acid comprising a PTM may comprise any amino acid a cell may modify post-translationally.

Similarly, a modified amino acid comprising a tag may especially comprise any amino acid that may be (chemically) tagged.

In embodiments, the modified amino acid may comprise an amino acid selected from the group consisting of lysine, cysteine, methionine, threonine, serine, and tyrosine, especially from the group consisting of lysine, cysteine, threonine, serine and tyrosine.

In further embodiments, the modified amino acid may comprise an N-terminal or C-

terminal amino acid, i.e., the modified amino acid may comprise a terminal amino acid, wherein the terminal amino acid is bound to the first charged group or to the second charged group. The terminal amino acid was thus especially the C-terminal amino acid or the N- terminal amino acid of the protein prior to providing the charged groups to the protein.

In further embodiments, the pretreatment stage may comprise providing the first charged group to the C-terminal end of the protein and the second charged group to the N-terminal end of the protein.

In further embodiments, either: (i) the first charged group may comprise nl positive charges, and the second charged group may comprise n2 negative charges; or (ii) the first charged group may comprise nl negative charges, and the second charged group may comprise n2 positive charges; especially wherein nl and n2 are independently selected from the range of 3 — 30. The first charged group and the second charged group may comprise any type of charged group suitable for terminal attachment to a protein. In specific embodiments, the first charged group and/or the second charged group may comprise a charged polymer, especially an amino acid chain and/or DNA.

It will be clear to the person skilled in the art that the number of positive and/or negative charges in the charged groups will depend on the pH the protein is exposed to. For example, in embodiments wherein the first charged group and/or the second charged group comprises an amino acid chain, a lysine residue may have a pKa of about 10, and may have a positive charge at about pH 7, while having a neutral charge at about pH 13; similarly, glutamate may have a pKa of about 4 and may have a neutral charge at about pH 2, while having a negative charge at about pH 7. Hence, herein the indicated number of charged groups may specifically relate to the pH the protein will be exposed to during the nanopore passage stage.

The terminal charged groups may prolong the passage through the nanopore by pulling towards different, especially opposite, directions (also: “tug of war”) in the imposed electric field. It may be preferable that the forces pulling in the difterent directions are similar. Hence, in embodiments, nl and n2 may be tailored for a specific protein, as the protein may natively have (terminal) charges. In further embodiments, nl and n2 may be similar, such as [nl-n2| < 5, such as <4, especially < 3, such as < 2, especially < 1, including

0. In specific embodiments, nl and n2 may be the same.

The terminal charged groups may require a sufficient number of charges to pull the protein in the different directions. Hence, in embodiments, nl and n2 may (both) be at least 3, especially at least 4 such as at least 5, especially at least 6, such as at least 7, especially at least 8, such as at least 9, especially at least 10.

An increase in the number of charges in the terminal charged groups may result in a longer translocation time. Hence, the number of charges in the terminal charged groups may be selected to avoid impractically long translocation times. In further embodiments, nl and n2 may (both) be at most 40, such as at most 30, especially at most 25, such as at most 20, especially at most 15, such as at most 13, especially at most 10.

Hence, in specific embodiments, nl and n2 may be independently selected from the range of 8-15.

In embodiments, the denaturation of the initial protein may (typically) occur prior to providing a tag to an amino acid and prior to providing the first charged group and the second charged group. In further embodiments, providing a tag to an amino acid may occur prior to or after providing the first charged group and the second charged group.

The method of the invention may be particularly suitable for relatively short proteins. Hence, in embodiments, the protein may comprise < 200 amino acids, especially < 100 amino acids, such as < 70 amino acids, especially < 50 amino acids, such as <40 amino acids, especially < 30 amino acids, especially excluding any amino acids that may be present in the first charged group and the second charged group.

In further embodiments, the protein may comprise > 1 amino acid, such as > 2 amino acids, especially > 3 amino acids, such as > 4 amino acids, especially 5 amino acids, such as > 8 amino acids, especially > 10 amino acids, such as > 15 amino acids, especially > 20 amino acids, especially excluding any amino acids that may be present in the first charged group and the second charged group.

In further embodiments, the method may comprise a proteolysis stage to provide the (initial) protein by proteolysis of a large protein. Especially, the proteolysis stage may provide a plurality of (initial) proteins by proteolysis of the large protein. Methods for proteolysis will be known to the person skilled in the art, including, among others, the use of a protease such as trypsin, which may selectively hydrolyze at the carboxyl side of the amino acids lysine and arginine, as well as the use of a low pH and/or a high temperature.

In further embodiments, the proteolysis stage may comprise exposing the large protein to a protease, especially a protease selected from the group comprising trypsin, chymotrypsin, pepsin, papain, elastase, LysC, LysN, ArgC, GluC and AspN. The term “protease” may also refer to a plurality of (different) proteases.

In general, it may be preferable to obtain relatively large (initial) protein fragments from the proteolysis stage, as these may be more informative, especially with regards to isoform analysis. Hence, in specific embodiments, the proteolysis stage may comprise exposing the large protein to a protease such that the proteolysis provides an (initial) protein comprising > 5 amino acids, such as > 10 amino acids, especially > 15 amino acids, especially > 20 amino acids, such as > 25 amino acids, especially > 30 amino acids.

The term “stage” used herein refers to a (time) period (also “phase”) of the method. The different stages may (partially) overlap (in time). For example, the proteolysis stage may, in general, be initiated prior to the pretreatment stage, and may especially continue during the pretreatment stage. It will be clear to the person skilled in the art how the stages may be beneficially arranged in time. In embodiments, the proteolysis stage may be initiated, especially arranged, prior to the pretreatment stage.

In a second aspect the invention may further provide an identification method for identifying a protein, wherein the identification method may comprise the method for acquiring the characteristic signal, and wherein the identification method may further comprise an identification stage. The identification stage may comprise identifying the protein, especially by comparing information regarding the protein to protein-related information in reference data, wherein the information regarding the protein is (at least partially) based on the characteristic signal.

In embodiments, the identification method may be “blind”, ie., the identification method may comprise identifying the protein solely based on the characteristic signal. In further embodiments, the identification method may comprise identifying a protein based on the characteristic signal and information on all known proteins (in a database).

In further embodiments, the identification method may be (somewhat) “targeted”, i.e., the identification method may comprise identifying the protein based on the characteristic signal and information on a subset of all known proteins (in a database), such as all human proteins.

In further embodiments, it may be known beforehand that the protein is one of a relatively limited set of options. For example, a sample may be enriched, for example via antibodies, for a particular type of protein. The identification stage may then comprise identifying which of the alternatives interacted with the nanopore. For example, the identification stage may comprise an isoform analysis, i.e., the sample may have been enriched for specific protein isoforms, and the identification stage may comprise identifying the measured isoform. Alternatively or additionally, the sample may have been enriched for a protein that is optionally post-translationally modified, and the identification stage may comprise identifying the post-translational modification(s). Hence, the identification stage may comprise identifying the protein based on the characteristic signal and information on a selected subset of proteins.

The identification stage may comprise identifying the protein based on the characteristic signal obtained in the nanopore passage stage. In embodiments, the identification stage may comprise comparing the characteristic signal to characteristic signals of known proteins (in a database), especially wherein the characteristic signal comprises a current trace signal. Hence, in embodiments, the information regarding the protein comprises the characteristic signal, and wherein the identification stage comprises comparing the characteristic signal to predetermined characteristic signals in the reference data.

In further embodiments, the identification stage may comprise an analysis of the characteristic signal to determine protein features that may be compared to protein features of known proteins (in a database). For example, based on measured tags and/or PTMs, there may be a (spatial) pattern of well-determined amino acids which can be compared and matched to amino acid sequences of known proteins.

In further embodiments, the identification stage may comprise protein profiling (also: protein fingerprinting).

In further embodiments, the identification stage may comprise an analysis by an identification system trained using a machine learning algorithm. Especially, the identification stage may comprise protein profiling and/or protein fingerprinting using an identification system trained using a machine learning algorithm.

In specific embodiments, the identification method may comprise: a sequence deduction stage comprising deducing at least part of the amino acid sequence of the protein based on the characteristic signal, wherein the information regarding the protein comprises the deduced part of the amino acid sequence; and wherein the identification stage comprises comparing the deduced part of the amino acid sequence to translated nucleotide sequences and/or amino acid sequences in the reference data.

In a further aspect, the invention may further provide an analysis system, comprising (i) a device configured to execute the method according to the invention, and especially comprising (ii) a control system configured to (have the analysis system) execute in an operation mode the identification method according to the invention.

In embodiments, the device may comprise the membrane comprising a membrane nanopore. Especially, the device may comprise an (electrochemical) cell comprising the membrane comprising the membrane nanopore. In further embodiments, the device may comprise an voltage difference applicator apparatus configured for applying a voltage over the membrane, especially the device may comprise a first electrode on a first side of the membrane and a second electrode on the second side of the membrane, wherein the first electrode and the second electrode are configured for applying a voltage over the membrane. In further embodiments, the first electrode and the second electrode may be arranged in the (electrochemical) cell. In further embodiments, the first electrode and the second electrode may (both) be arranged at a distance from the nanopore, wherein the distance is longer than the length of the (linear) protein. In further embodiments, the first electrode and the second electrode may (both) be arranged at a distance from the nanopore, wherein the distance is at least 3,5 nm, such as at least 7 nm, especially at least 10 nm, such as at least 20 nm, especially at least 30 nm, such as at least 50 nm, especially at least 100 nm.

In further embodiments, the device may comprise a readout apparatus configured to obtain the characteristic signal (during operation of the analysis system). In specific embodiments, the readout apparatus may comprise an ionic current sensing apparatus. In further specific embodiments, the readout apparatus may comprise a patch- clamp amplifier.

In further embodiments, the voltage difference applicator apparatus and the readout apparatus may be the same apparatus.

In further embodiments, the control system may be configured to (have the analysis system) execute in an operation mode the identification stage of the identification method according to the invention, especially execute in an operation the identification method according to the invention. In further embodiments, the control system may be configured to communicate with an external database to acquire reference data. In further embodiments, the control system may be configured to execute a protein fingerprinting analysis and/or a sequence deduction analysis. In a further aspect, the invention may further provide a computer program product comprising instructions which, when the program is executed by a computer comprised by the analysis system according to the invention, cause the computer to execute the identification stage of the identification method according to the invention, especially cause the computer to execute the identification method according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which: Fig. 1 schematically depicts an embodiment of the method and an embodiment of the analysis system 10 of the invention; Fig. 2 depicts experimental data obtained using an embodiment of the method related to the tagging of different amino acid residues in the protein 300; Fig. 3A-C depict experimental data obtained using an embodiment of the method related to different tags 332; and Fig. 4A-C depict experimental data obtained using an embodiment of the method related to different post-translational modifications 334. The schematic drawings are not necessarily on scale.

DETAILED DESCRIPTION OF THE EMBODIMENTS Fig. 1 schematically depicts an embodiment of the method for acquiring a characteristic signal 400 of a protein 300 passing a membrane nanopore 100 comprised by a membrane 200. The membrane has a first membrane side 201 and a second membrane side 202, wherein the first membrane side 201 (also: the “cis side”) and the second membrane side 202 (also: the “trans side”) are in fluid communication via a channel 110 in the nanopore 100. In the depicted embodiment the nanopore 100 1s comprised by a pore-forming protein. The first membrane side (the cis side) may be the side facing the nanopore vestibule, as in the depicted embodiment. The membrane nanopore 100 comprises a nanopore constriction space.

The nanopore constriction space has a constriction length Lc <20 nm and a circular equivalent constriction diameter dc < 20 nm.

In the depicted embodiment, the protein 300 is linear, and comprises a C- terminal first charged group 310 and an N-terminal second charged group 320, wherein the first charged group 310 and the second charge group 320 have charges of opposite sign. In particular, in the depicted embodiment, the first charged group has positive charges due to the arginine residues and the second charged group has negative charges due to the aspartate residues.

On the left side of Fig. 1, the protein 300 comprises an unmodified protein Up, i.e. a protein without a modified amino acid 330. On the right side of Fig. 1, the protein 300 comprises a modified amino acid 330, wherein the modified amino acid 330 comprises a tag 332 and/or a post-translational modification 334; specifically the protein 300 is a tagged protein Tp comprising a modified amino acid 330 comprising a tag 332 corresponding to fluorescein Ti.

The depicted method comprises a nanopore passage stage 20 comprising passing the protein 300 through the membrane nanopore 100, especially passing the protein 300 from the first membrane side 201 to the second membrane side 202 through the nanopore

100. The nanopore passage stage further comprises applying a voltage V across the membrane 200, and comprises measuring an ionic current over the membrane 200 thereby providing the characteristic signal 400. The left characteristic signal 400 corresponds to measurements of the unmodified protein U,, whereas the right characteristic signal 400 corresponds to measurements of the tagged protein Tp.

In embodiments, the membrane nanopore may be comprised by or may be formed by a biological nanopore (forming protein) selected from the group comprising FraC, MspA, alpha-hemolysin, aerolysin, OmpG, ClyA, Phi29, FhuA, and SPI. In the depicted embodiment, the nanopore is comprised by or a pore forming protein comprising FraC.

In embodiments, the modified amino acid may comprise an amino acid selected from the group consisting of lysine, cysteine, threonine, serine, and tyrosine and/or wherein the modified amino acid comprises an N-terminal or C-terminal amino acid. In the depicted embodiment, the modified amino acid comprises cysteine.

In embodiments, either: the first charged group comprises n1 positive charges, and the second charged group comprises n2 negative charges; or the first charged group comprises n; negative charges, and the second charged group comprises n2 positive charges, wherein n; and n2 are independently selected from the range of 3 — 30, especially from the range of 8-15. In the depicted embodiment, the first charged group comprises nl positive charges and the second charged group comprises n2 negative charges, wherein nl and n2 may both essentially be 10.

In embodiments, the protein 300 may comprise < 200 amino acids, especially < 50 amino acids, especially excluding any amino acids from the terminal charged groups, ie. from the first charged group 310 and the second charged group 320. In the depicted embodiment the protein 300 comprises 30 amino acids, or 10 amino acids excluding the amino acids from the terminal charged groups.

Fig. 1 further schematically depicts an embodiment of the analysis system 10, comprising (i) a device 500 configured to carry out the method according to the invention, and (i1) a control system 600 configured to execute in an operation mode the identification method according to the invention.

EXPERIMENTS. Experiments 1 — 4 were performed using buffer containing IM NaCl, 10mM TRIS, and 1mM EDTA at pH 7.5, unless specified otherwise. The nanopore 100 comprises a wild-type FraC nanopore. The protein 300 comprises a 30 amino acid long peptide, containing 10 glutamates at the N-terminal second charged group 320, and 10 arginines at the C-terminal first charged group, which at neutral pH may feature a strongly negatively charged N-terminus and a strongly positively charged C-terminus. The protein is added to the cis compartment at concentrations between 0.1 pM and 0.5 uM. Negative voltages are applied to the trans compartment, especially to avoid gating that may be observed in FraC under positive bias. Experiments | - 4 were performed at a voltage bias of -90mV, unless stated otherwise.

EXPERIMENT 1 — unmodified and tagged protein. Upon applying a negative bias to the second side 202 of the nanopore set-up (Fig. 1), the protein 300 is dragged into the nanopore 100 with the first charged group 310 entering first. When the second charged group 320 subsequently enters the nanopore 100, the electrophoretic force pulls the second charged group 320 of the peptide in the opposite direction, thus stretching the protein 300 and stalling the protein 300 at the point where the forces in both directions equilibrate. The “tug-of-war” created thus allows for long observation times where the protein is probed at the nanopore constriction space. Well-defined translocation events are consistently observed when the protein 300 is present on the cis side. Notably, the relative current blockade may be well reproducible from nanopore to nanopore with an average relative blockade of 0.47 + 0.03 pA at -90mV, as measured in three independent experiments, wherein the relative blockade Ir is defined as the ratio between the current blockade (AI= lopenpore - IBtoekade) and the open nanopore current (lopenpor). Long translocation times of 4.2 + 0.6 ms are observed at -90mV. To probe the effect of an added tag 332 to the protein 300, the cysteine in position C11, i.e. near the N-terminus, was labelled with a fluorescein T1 dye using maleimide chemistry (right side of Fig. 1). Fluorescein maleimide may be a small molecule of molecular weight 427 Da. The tagged proteins were HPLC-purified and the tagging was verified using mass spectrometry (MALDI-TOF) with nearly 100% or the samples tagged. The unmodified proteins Up and the tagged proteins T, were measured separately, as depicted in Fig. 1 on respectively the left side and the right side of Fig. 1.

As can be clearly seen from the graphs, the current levels from the unmodified and tagged proteins are clearly separated. The unmodified protein produced a relative blockade of 0.47 + 0.01 while the tagged protein produced a relative blockade of 0.57 + 0.01. The values presented here correspond to the mean and standard deviation derived from a Gaussian fit of the relative blockade histograms. The increase in the relative blockade Ir upon tagging may be expected due to the additional volume provided by the presence of the fluorescein T} tag. Hence, the fluorescein-labelled proteins may be readily distinguished from their unmodified counterparts. The difference in the blockade levels may be so clear and reproducible that, while nanopore data typically rely on data comparison in stochastic scatter plots of hundreds of events, the tagged and unmodified proteins may be distinguished using the method of the invention on an individual basis based on their characteristic signal 400, especially their current levels in individual events.

Fig. 2 — EXPERIMENT 2 — Different tagging locations in a protein. During the stalling of the protein 300 in the nanopore 100, a particular portion of the protein 300 may be closest to the nanopore constriction space at the applied voltage of -90mV. This portion of the protein 300 may comprise the most sensitive sensing region of the method, and may facilitate protein fingerprinting. Hence, three different variants of the protein 300 of experiment 1 were tested, in which the cysteine was placed at different positions along the central part of the protein, namely, at position 11, 15, or 20 from the N-terminus (Fig. 2). The three different protein variants were tagged with fluorescein maleimide, HPLC-purified, and mass spectrometry-verified. The graphs on the right depict the number of counts N for different (binned) values of relative blockade Ir. The relative blockade Ir observed for an unmodified protein Up is displayed in Fig. 2 as a reference. The unmodified protein Up produced a relative blockade Ir of 0.47 + 0.01. A larger relative blockade Ir of 0.57 + 0.01 was observed for the protein tagged with fluorescein T in position C11; a consistent increase in the relative blockade Ir was detected with a well-defined current blockade level. With the protein 300 labelled with fluorescein Ty in position C15, in contrast, current fluctuations are observed and events may contain a lower current level in the later part of the event; as a consequence, a broad population was observed in the relative blockade Ig histogram with a mean of 0.54 and a larger standard deviation of 0.05. Finally, when the label was placed in position C20 a relative blockade Ir of 0.48 + 0.02 was observed.

Fig. 3A-C — EXPERIMENT 3 — Different tags on a protein. Protein fingerprinting may benefit from multiple different tags in a protein, especially wherein they tag different amino acids. Here, the peptide EEEEEEEEEECGSGSGSKGSRRRRRRRRRR was tagged separately with six different tags 332 at the cysteine.

Fig. 3A depicts in the top part the six different tags 332 in ascending order according to their molecular weight: fluorescein Ty (427 Da), Texas Red T: (728 Da), PEG11- biotin T4 (922 Da), His6 Ts (992 Da), Alexa633 Ts (1089 Da), and 3polyA Ts (1275 Da). These tags may be relatively large compared to amino acids, about 2-7 times larger than the largest amino acid (tryptophan, 204 Da), with different physicochemical properties. In the bottom part of Fig. 3A, a blockade histogram is shown for each tag 332 corresponding to the protein tagged with the tag 332. For each of these measurements, tagged and unmodified proteins were measured as a mixture, where the unmodified protein was acting as a reference. Two peaks may be clearly discernible in each of the histograms; the first peak near 0.45-0.50 may correspond to the unmodified protein Uy, and the second peak, near 0.56-0.66 may correspond to the tagged protein Tp for each of the different tags 332. The difference between the peaks is the change in relative blockade Al, which may vary for different tags 332 as depicted in Fig. 3B.

Interestingly, the increase in relative blockade produced by a particular tag 332 does not appear to correlate well with its molecular weight (R?2 = 0.16; graph not shown). For example, while Texas Red T2 has a lower molecular weight than PEG11-biotin T4 (728 Da vs. 922 Da, respectively), it generated a larger blockade. The poor correlation may be related to their different geometry. While PEGI 1-biotin T4 may have a linear flexible structure, Texas Red T: may comprise multiple aromatic rings packed tightly one next to another. Thereby, Texas red may block a larger number of ions while in the nanopore constriction space compared to the linear structure of PEG11-biotin T4. An alternative characteristic that takes into consideration both the molecular weight and shape may be the phenomenological parameter P = S x M, where M is the molecular weight of the tag and S is a shape factor defined by the ratio between the width Wt of the tag and its length Ls (P = W/L{*M), wherein the length of the tag is defined as the end-to-end distance of the tag following the direction of the linker attached to the nitrogen of the maleimide group, and the distance measured in the perpendicular axis is the width, as depicted in Fig. 3C. Fig. 3C further depicts the increase in relative blockade Ir measured for each tag vs. the calculated P value. A fair correlation (R* =

0.84) may be observed between parameter P and the increase in relative blockade Ir supporting the notion that not only the molecular weight of the tag, but also its geometry affects the amount of current blocked by a tag.

In specific embodiments, the tag may have a P-value, wherein the P-value is defined by P=WyL:*M, wherein W; is the width of the tag, Ls is the length of the tag, and M is the molecular weight of the tag. In further embodiments, the P-value (of the tag) may be > 5, such as > 10, especially > 20, such as > 50, especially > 100. In further embodiments, the P-value (of the tag) may be < 2000, such as < 1500, especially < 1000, such as < 750, especially < 500.

The different tags 332 were further found to have different influences on the translocation time of the protein 300, as depicted in Fig. 3D. Panels a and b of Fig. 3D depict scatter plots of relative blockade Ir vs. translocation time Ta (in ms) for His-6-tagged protein Tsp and Alexa633-tagged-protein Tsp. The translocation time Ty is significantly different with a mean translocation time of 29 ms for Ts, versus 0.22 ms for Tsp at a bias of -90 mV. The observations suggest that two properties of the tags 332 primarily affect the translocation time: charge and hydrophobicity. Charges present in the tags 332 may act together with the first charged group 310 and the second charged group 320 of the protein 300, thereby increasing the electrophoretic force pulling towards the cis or the trans opening of the nanopore 100, thus increasing or decreasing the translocation time respectively. Fig. 3D panel c shows a scatter plot of observed mean dwell time Ta (in ms) vs. net charge C for each of the tags (R? = 0.70).

Fig. 4A-C — EXPERIMENT 4 — different post-translational modifications 334 on a protein 300. The protein 300 N’-EEEEEEEEEESGSGSGSKGSRRRRRRRRRR- C° and variants of the protein 300 comprising a modified amino acid 330 (serine at position 11 from the N-terminal side) comprising a post-translational modification 334, wherein the PTM 334 comprises either a phosphoryl group or an O-GlcNAc (N-acetylglucosamine).

Fig. 4A depicts on the left a scatterplot of measurements of unmodified proteins Up containing no PTM 334, and on the right a scatterplot of measurements of a 1 to 1 mixture of unmodified proteins Up and phosphorylated proteins Ppp, both depicting relative blockade Ig versus the translocation time Ta (in ms) as well as corresponding histograms for the relative blockade Ir values. The unmodified protein Up was found to have a relative blockade Ir of 0.48 + 0.02, while a larger relative blockade of 0.52 + 0.01 was found for the phosphorylated protein Ppp. This indicates that the post-translational modification 334 phosphorylation can be clearly detected with the method according to the invention. The well- defined difference in relative blockade Ir may also be apparent in the right histogram which shows two clearly separated peaks.

Fig. 4B depicts on the left a scatterplot of measurements of unmodified proteins Up containing no PTM, and on the right a scatterplot of measurements of a 1 to 1 mixture of unmodified proteins Up and glycosylated proteins Pg, both depicting relative blockade Ig versus the translocation time T4 (in ms) as well as corresponding histograms for the relative blockade Ir values. The unmodified protein Up was found to have a relative blockade Ir of 0.48 + 0.02, while a larger relative blockade of 0.52 + 0.01 was found for the glycosylated protein Psp. This indicates that the post-translational modification glucosylation can be clearly detected with the method according to the invention. The well-defined difference in relative blockade Ir may also be apparent in the right histogram which shows two clearly separated peaks.

The observed increase in relative blockade Ir observed by the phosphorylated and glycosylated protein variants are almost identical (8.7% vs 8.3% ) despite their molecular weight difference (80 Da for phosphorylation versus 203 Da for glycosylation). Without being bound by theory, the inventors hypothesize that the negative charge of the phosphorylation group may create an Electrical double layer (EDL) of counter-ions that may lead to an increased effective volume of the phosphoryl group.

Unlike the O-GlcNAc group, which is essentially uncharged, the phosphoryl group may contain two negative charges at pH=7.5. Hence, the ionic strength of the buffer may therefore particularly affect the screening of the phosphoryl group, altering the extent of its electrical double layer (EDL) and the current blockade.

Hence, a mixture containing the three protein variants (the phosphorylated protein Ppp, the glycosylated protein Pg, and the unmodified protein Up) in equimolar concentrations in a buffer containing a lower salt concentration of 0.8M NaCl was measured at pH 7.5. Fig. 4C panel a depicts the current trace signal obtained for the mixture of the three protein variants. Reference Ow indicates the open pore level, Ppp, Psp, and U, indicate the rough positions of the typical current measurements for the three protein variants. As shown in Fig. 4C panel b, three different populations may be clearly discerned in the scatter plot and histogram.

The term “plurality” refers to two or more. Furthermore, the terms “a plurality of” and “a number of” may be used interchangeably.

The terms “substantially” or “essentially” herein, and similar terms, will be understood by the person skilled in the art. The terms “substantially” or “essentially” may also include embodiments with “entirely”, “completely”, “all”, etc. Hence, in embodiments the adjective substantially or essentially may also be removed. Where applicable, the term “substantially” or the term “essentially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. Moreover, the terms “about” and “approximately” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. For numerical values it is to be understood that the terms “substantially”, “essentially”, “about”, and “approximately” may also relate to the range of 90% - 110%, such as 95%- 105%, especially 99%-101% of the values(s) it refers to.

The term “comprise” includes also embodiments wherein the term “comprises” means “consists of”.

The term “and/or” especially relates to one or more of the items mentioned before and after “and/or”. For instance, a phrase “item 1 and/or item 2” and similar phrases may relate to one or more of item 1 and item 2. The term "comprising" may in an embodiment refer to "consisting of" but may in another embodiment also refer to "containing at least the defined species and optionally one or more other species”.

Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. The term “further embodiments” may refer to embodiments comprising the features of the previously discussed embodiments, but may also refer to an alternative embodiments.

The devices, apparatus, or systems may herein amongst others be described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation, or devices, apparatus, or systems in operation.

It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims.

In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.

Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, “include”, “including”, “contain”, “containing” and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.

The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In a device claim, or an apparatus claim, or a system claim, enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

The invention also provides a control system that may control the device, apparatus, or system, or that may execute the herein described method or process. Yet further,

the invention also provides a computer program product, when running on a computer which 1s functionally coupled to or comprised by the device, apparatus, or system, controls one or more controllable elements of such device, apparatus, or system.

The invention further applies to a device, apparatus, or system comprising one or more of the characterizing features described in the description and/or shown in the attached drawings. The invention further pertains to a method or process comprising one or more of the characterizing features described in the description and/or shown in the attached drawings. Moreover, if a method or an embodiment of the method is described being executed in a device, apparatus, or system, it will be understood that the device, apparatus, or system 1s suitable for or configured for (executing) the method or the embodiment of the method respectively.

The various aspects discussed in this patent can be combined in order to provide additional advantages. Further, the person skilled in the art will understand that embodiments can be combined, and that also more than two embodiments can be combined.

Furthermore, some of the features can form the basis for one or more divisional applications.

Claims (19)

Conclusions A method of obtaining a characteristic signal (400) from a protein (300) comprising a membrane nanopore (100), passing through a membrane (200), wherein the membrane nanopore (100) comprises a nanopore constriction space having a constriction length Le <20 nm and has a circular equivalent constriction diameter of <20 nm, wherein the protein (300) is linear, and wherein the protein (300) comprises a C-terminal first charged group (310) and an N-terminal second charged group (320) wherein the first charged group (310) and the second charged group (320) have charges of opposite sign, and wherein the protein (300) comprises a modified amino acid (330), the modified amino acid (330) comprising a tag (332 ) and / or a post-translational modification (334), the method comprising a nanopore passage stage (20) comprising passing the protein (300) through the membrane nanopore (100), applying a voltage V across the membrane (200) , and measuring an ion current across the membrane (200), thereby providing the characteristic signal (400). The method of claim 1, wherein the method comprises a pretreatment stage for providing the protein (100) from an initial protein, the pretreatment stage having one or more of: (i) denaturation of the initial protein, (ii) providing of a tag (332) on an amino acid in the initial protein, and (iii) providing the first charged group (310) at the C-terminal end of the initial protein and the second charged group (320) at the N- terminal end of the initial protein; and wherein the method comprises performing the pretreatment stage prior to the nanopore passage stage. The method of claim 2, wherein the pretreatment stage comprises denaturation of the initial protein. The method according to any of the preceding claims 2-3, wherein the pretreatment stage comprises providing a tag (332) to an amino acid in the initial protein to provide the modified amino acid (330). The method of any one of the preceding claims 2-4, wherein the pretreatment stage comprises providing the first charged group (310) at the C-terminal end of the protein and the second charged group (320) at the N-terminal end. of the protein (300). The method according to any of the preceding claims, wherein the constriction length Le <10 nm, and wherein the circular equivalent constriction diameter is the ≤ 5 nm. The method of any preceding claim, wherein the modified amino acid (330) comprises the post-translational modification (334), wherein the post-translational modification (334) is obtainable by one or more of phosphorylation, O-linked glycosylation , acetylation, methylation, nitration, farnesylation, palmitoylation, myristoylation and S-nitrosylation. The method of any preceding claim, wherein the membrane nanopore (100) comprises a biological nanopore selected from the group consisting of FraC, MspA, alpha-hemolysin, aerolysin, OmpG, ClyA, Phi29, FhuA and SP1. The method of any one of the preceding claims, wherein the voltage V is selected from the range -500-500 mV. The method of any preceding claim, wherein the modified amino acid (330) comprises an amino acid selected from the group consisting of lysine, cysteine, threonine, serine and tyrosine and / or wherein the modified amino acid (330) has an N-terminal or C-terminal amino acid. The method of any preceding claim, wherein either (1) the first charged group (310) includes no positive charges, and the second charged group (320) includes no negative charges; or (ii) the first charged group (310) includes n1 negative charges and the second charged group (320) includes n2 positive charges; where n; and nz are independently selected from the range of 3 to 30. The method of claim 11, wherein n: and n2 are independently selected from the range of 8-15. The method of any preceding claim, wherein the protein comprises (300) <50 amino acids. The method of any one of the preceding claims, wherein the membrane nanopore (100) is arranged in a liquid solution, wherein the solution comprises a salt concentration selected from the range 1 mM - 10 M, and wherein the solution has a pH selected from the range of 3 - 9. An identification method for identifying a protein (300), the identification method comprising the method of any of the preceding claims 1-14, and the identification method further comprising: - an identification stage comprising identifying the protein (300) by comparing information about the protein (300) with protein related information in reference data, wherein the information about the protein (300) is based on the characteristic signal (400). The identification method of claim 15, wherein the information about the protein (300) includes the characteristic signal (400), and the identification stage comprises comparing the characteristic signal (400) with predetermined characteristic signals in the reference data. The identification method according to any of the preceding claims 15-16, wherein the identification method comprises a sequence derivation step comprising deriving at least a portion of the amino acid sequence of the protein (300) based on the characteristic signal (400), wherein the information regarding the protein (300) includes the deduced portion of the amino acid sequence; wherein the identifying step comprises comparing the deduced portion of the amino acid sequence with translated nucleotide and / or amino acid sequences in the reference data. An analysis system (10), comprising (1) a device (500) configured to perform the method of any one of the preceding claims 1-14, and (ii) a control system (600) configured to operate the analysis system (10) to have the identification method according to any one of the preceding claims 15-17 carried out in an operating mode. A computer program product comprising instructions that, when the program contained by a computer is executed by the analysis system (10) of claim 18, cause the computer to perform the identification method of any of claims 15-17.