Proximity labelling and fret-based protein fingerprinting

Abstract

The invention provides a method for characterization of a protein (10), wherein the method comprises: a labelling stage comprising exposing the protein (10) to a first proximity labelling enzyme (71), wherein the first proximity labelling enzyme (71) is configured to label amino acids of a first amino acid type with a first label (81); and an exposure stage comprising: (i) exposing the protein (10) to a first probe (31), (ii) providing excitation radiation (50) to the protein (10), and (iii) measuring emission radiation (60) to provide an emission signal (61); wherein: the first probe (31) comprises an affinity-based probe (35) selected from the group comprising an aptamer, an antibody, a nanobody, a peptide, and an organic ligand; the first probe (31) is configured to transiently bind the protein (10) at a first binding site (11), wherein the first label (81) provides at least part of the first binding site (11), and wherein the first probe (31) comprises a first chromophore (21); and the first chromophore (21) has an excitation radiation range and an emission radiation range, wherein the radiation (50) comprises a wavelength selected from the excitation radiation range, and wherein the emission (60) is measured in the emission radiation range.

Description

[0001] Proximity labelling and FRET -based protein fingerprinting

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a method for characterization of a protein. Further, the invention relates to a system for characterization of a structure of a protein. Further yet, the invention relates to a data carrier having stored thereon program instructions.

[0004] BACKGROUND OF THE INVENTION

[0005] Methods for characterization of a protein are known in the art. For instance, WO2018102759A1 relates to methods and systems for identifying a protein within a sample. A panel of antibodies are acquired, none of which are specific for a single protein or family of proteins. Additionally, the binding properties of the antibodies in the panel are determined. Further, the protein is iteratively exposed to a panel of antibodies. Additionally, a set of antibodies which bind the protein are determined. The identity of the protein is determined using one or more deconvolution methods based on the known binding properties of the antibodies to match the set of antibodies to a sequence of a protein.

[0006] SUMMARY OF THE INVENTION

[0007] Proteins are biochemical workhorses in all living cells. For understanding of biological processes and their (dys)regulation, including diseases, it may be critical to identify and monitor the protein composition of cells by sequencing (i.e. determination of the amino acid sequence of proteins) and / or structural analysis (i.e. determination of the three- dimensional structure of proteins).

[0008] The number of protein encoding genes in the human genome may be estimated to be about 20,000. If all protein encoding genes result in a single protein, then the number of different proteins in the human proteome may also be about 20,000. However, a process known as alternative splicing may increase the number of transcripts to -80,000. Recently, a term “proteoform” has been defined. It refers to each individual molecular form of a protein that is derived from a single protein-encoding gene. There are several layers of cellular processes that can result in a large number of proteoforms transcribed from a single gene. The complexity of the proteome further increases due to post-translational modification (PTMs). The PTMs may result in many hundreds of thousands of additional protein variants. The large variety and number of PTMs may affect the protein function. Subtle differences in highly similar proteoforms may have profound effects on health. Moreover, the concentration range at which different proteoforms exist in the cell spans several orders of magnitude, with some of them being present with just a few copies per cell. To address these challenges, a method that can detect and discriminate proteoforms with single-molecule sensitivity may be needed. However, protein sequencing and structural analysis remains a challenge, especially when only a small sample of the protein is available.

[0009] Modern protein analysis commonly utilizes mass spectrometry-based identification techniques - determining the precise mass of a protein. Such current methods may suffer from limitations. First, they can analyze only fragments of proteins. Information on those fragments is then used to reconstruct the full-length amino acid sequence, but this often fails due to the combinatorial complexity. Second, they often fail to recognize minor protein species among highly abundant protein species, since sequence prediction is made through analysis of complex spectral peaks. As many important cellular proteins such as signaling proteins exist in very low abundance, it can be difficult to obtain comprehensive proteomic information. Early detection of diseases may also rely on detection of low concentrations of protein biomarkers and thereby forms a demand for protein sequencing and structural analysis techniques capable of working at the single-molecule scale. In addition, it may be particularly challenging to analyze proteoforms of a protein using mass spectrometry approaches.

[0010] Single-molecule techniques are cutting-edge detection tools to study biological processes at the nanoscale and may be suitable for samples containing target molecules (such as proteins) with low copy numbers. Potential single-molecule protein sequencing approaches have recently been explored. For example, nanopores based on a-hemolysin have been used to distinguish between non-phosphorylated and phosphorylated proteins. Further, a comparable biological nanopore in combination with a motor protein complex has been used to control protein translocation through a nanopore. Edman degradation has been used for fluorescence detection of single peptides and single-molecule fluorescence fingerprinting of peptides and cellular proteins has been demonstrated using a motor protein complex. However, it may remain challenging to identify the amino acid sequence of a protein using single-molecule techniques.

[0011] Prior art methods for protein characterization and identification may require linearization and fragmentation of the protein, hence losing the folded conformation and structure of the whole protein. Further, such methods may require the use of enzymes for translocation, linearization, and fragmentation of the protein, all of which may alter the protein structure via enzymatic activity. Prior art methods may require (covalently) attaching linkers to specific amino acid residues of a protein using techniques which may be time-consuming, labor-intensive, wasteful with regards to reagents (and thus costly), and may result in incomplete or off-target labelling. In addition, the attachment of such linkers may complicate the procedure, may hamper flexibly analyzing the protein, and may result in changes to the structure of the protein, such as to the secondary, tertiary and / or quaternary structure of the protein. Further, prior art methods may require labeling in an in vitro environment, and may be unsuitable to characterize the interaction partners of a protein.

[0012] Hence, it is an aspect of the invention to provide an alternative method for the characterization 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.

[0013] In a first aspect, the invention may provide a method for characterization of a protein. The method may comprise a labelling stage. The labelling stage may comprise exposing the protein to a first proximity labelling enzyme. Especially, the first proximity labelling enzyme may be configured to label amino acids of a first amino acid type with a first label. Further, the method may comprise an exposure stage comprising: (i) exposing the protein to a first probe, (ii) providing excitation radiation to the protein, and (iii) measuring emission radiation to provide an emission signal. The first probe may be configured to transiently bind the protein at a first binding site. Especially, the first label may provide at least part of the first binding site. Further, the first probe may comprise a first chromophore. The first chromophore may especially have an excitation radiation range and an emission radiation range. In such embodiments, the (excitation) radiation may comprise a wavelength selected from the excitation radiation range. Further, the emission (radiation) may be measured in the emission radiation range. The first probe may especially comprise an affinity-based probe. Affinitybased probes may be selected from the group comprising an aptamer, an antibody, a nanobody, a peptide, and an organic ligand. In embodiments, the first probe may thus comprise an affinitybased probe selected from the group comprising an aptamer, an antibody, a nanobody, a peptide, and an organic ligand.

[0014] Hence, in specific embodiments, the invention may provide a method for characterization of a protein, wherein the method may comprise (a) a labelling stage comprising exposing the protein to a first proximity labelling enzyme, wherein the first proximity labelling enzyme may be configured to label amino acids of a first amino acid type with a first label; and (b) an exposure stage comprising: (i) exposing the protein to a first probe, (ii) providing excitation radiation to the protein, and (iii) measuring emission radiation to provide an emission signal; wherein: (A) the first probe may comprise an affinity-based probe selected from the group comprising an aptamer, an antibody, a nanobody, a peptide, and an organic ligand; (B) the first probe may be configured to transiently bind the protein at a first binding site, wherein the first label may provide at least part of the first binding site, and wherein the first probe may comprise a first chromophore; and (C) the first chromophore may have an excitation radiation range and an emission radiation range, wherein the radiation may comprise a wavelength selected from the excitation radiation range, and wherein the emission may be measured in the emission radiation range.

[0015] In embodiments, the first probe may be especially be configured to transiently bind the protein at the first binding site with a first (binding) frequency. The first frequency may be dependent on one or more of the concentration of the first probe and the concentration of the first binding site. In embodiments, the method may thus comprise determining a concentration of the first binding site based on a first frequency. Optionally, the method may further comprise determining a number of amino acids (of the first amino acid type) in the protein based on the first frequency. Hence, in specific embodiments, the first probe may be configured to transiently bind the protein at the first binding site with a first frequency, wherein the first frequency may be dependent on one or more of the concentration of the first probe and the concentration of the first binding site; wherein the method may comprise determining (i) a concentration of the first binding site based on the first frequency, and / or a number of amino acids of the first amino acid type in the protein based on the first frequency.

[0016] Optionally, the method may further comprise exposing the protein to a second probe. Hence, in a further aspect, the invention may provide a method for characterization of a protein. The method may comprise a labelling stage, wherein the labelling stage may comprise exposing the protein to a proximity labelling enzyme. In embodiments, the proximity labelling enzyme may be configured to label amino acids of a first amino acid type with a first label (by catalyzing the generation of a short-lived reactive species in the vicinity of the protein). Further, the method may comprise an exposure stage. In embodiments, the exposure stage may comprise exposing the protein to a first probe and a second probe. Additionally, the exposure stage may comprise providing excitation radiation to the protein. Further, the exposure stage may comprise measuring emission (radiation) to provide an emission signal. In embodiments, the first probe may be configured to transiently bind the protein at a first binding site. Especially, the first label may provide at least part of the first binding site. Further, the first probe may comprise a first chromophore. Similarly, the second probe may comprise a second chromophore. In embodiments, the second probe may be configured to transiently bind the protein at the first binding site. Alternatively, the second probe may be configured to transiently bind the protein at a second binding site. In embodiments, the first chromophore and the second chromophore may form a FRET pair having a donor excitation range, a donor emission radiation range and an acceptor emission radiation range. In embodiments, the radiation may comprise a wavelength selected from the donor excitation radiation range. Further, the emission may be measured in the donor emission radiation range and in the acceptor emission radiation range.

[0017] The first probe and the second probe may especially comprise affinity-based probes. Affinity-based probes may be selected from the group comprising an aptamer, an antibody, a nanobody, a peptide, and an organic ligand. In embodiments, the first probe and the second probe may thus comprise affinity-based probes selected from the group comprising an aptamer, an antibody, a nanobody, a peptide, and an organic ligand.

[0018] Hence, in specific embodiments, the invention may provide a method for characterization of a protein, wherein the method comprises: (a) a labelling stage comprising exposing the protein to a proximity labelling enzyme, wherein the proximity labelling enzyme is configured to label amino acids of a first amino acid type with a first label; and (b) an exposure stage comprising: (i) exposing the protein to a first probe and a second probe, (ii) providing excitation radiation to the protein, and (iii) measuring emission radiation to provide an emission signal; wherein: (A) the first probe is configured to transiently bind the protein at a first binding site, wherein the first label provides at least part of the first binding site, and wherein the first probe comprises a first chromophore; (B) the second probe comprises a second chromophore, wherein the second probe is configured to transiently bind the protein at (i) the first binding site or (ii) a second binding site; and (C) the first chromophore and the second chromophore form a FRET pair having a donor excitation range, a donor emission radiation range and an acceptor emission radiation range, and wherein the radiation comprises a wavelength selected from the donor excitation radiation range, and wherein the emission is measured in the donor emission radiation range and in the acceptor emission radiation range.

[0019] The method of the invention may thus comprise exposing a protein to a first probe and a second probe both comprising FRET chromophores, wherein each probe associates with, especially (transiently) binds to, the protein at a respective binding site. The term “chromophore” may refer to a region in the molecule where the energy difference between two separate molecular orbitals falls within the range of the visible spectrum, i.e., the chromophore may be the part of a molecule responsible for its color. As a FRET donor may (upon excitation with radiation in the donor excitation radiation range) provide donor emission with an intensity highly dependent on the distance of said donor to a FRET acceptor, measuring the emission radiation provided by the donor and the acceptor may provide accurate information regarding the distance between the respective binding sites of said FRET donor and acceptor. In particular, by measuring a plurality of emission signals, and especially by determining a plurality of (pairwise) distances, such as between a first of a plurality of first binding sites and a second of a plurality of first binding sites, or such as between (pairwise) combinations of first and second binding sites, a protein fingerprint may be obtained, and especially a protein structure may be determined.

[0020] In the present invention, proximity labelling enzymes are used to label amino acids of a specific amino acid type. In embodiments, reactions with proximity labelling enzymes may be faster than chemical labelling methods described in prior art. Further, labelling reactions employing proximity labelling enzymes may be specific and demonstrate high labeling efficiencies. Hence, incomplete and / or non-specific labeling of amino acids may be reduced. In addition, the relatively fast reaction times observed for proximity labelling enzymes may provide a high-throughput method for the characterization of proteins.

[0021] With the present invention, the method for characterization of a structure of a protein using a first probe and a second probe may provide a single-molecule protein profiling platform that may facilitate the identification of proteins based on a unique “protein fingerprint” by combining single-molecule FRET technique with (transient) affinity-based probes. Such a protein fingerprint may be obtained by probing the target proteins with a large number of fluorescently labeled probes that provide a signal on the distance of their binding site to a binding site of another probe. Hence, this method may provide a sequencing technique for examining the protein structural profile and may consequently allow identification of the protein sequence. This method of single-molecule FRET detection, hereafter also called “protein fingerprinting”, may also facilitate determining and / or predicting protein structures.

[0022] Unlike mass spectrometry-based methods, protein fingerprinting may directly probe full-length proteins, thereby improving the accuracy of protein identification. By identifying single molecules, protein fingerprinting may provide a sensitivity of one molecule and may therefore facilitate sequencing proteins from an amount of samples 3-5 orders of magnitude smaller than needed for mass spectrometry. Protein fingerprinting may further facilitate technologies such as single-cell proteomics and real-time screening for on-site medical diagnostics and early-stage disease diagnosis. Further, the use of probes configured to transiently (rather than covalently) bind the protein may facilitate probing of the same binding site multiple times, wherein especially a different probe (of the same type) may bind the binding site each time. In other words, in embodiments, the method may ensure a high turnover of the (transiently bound) probes at the binding sites. Hence, photobleaching of the probes (at the binding site) may be mitigated, extending the time over which useful information regarding the emission radiation may be obtained. The term “photobleaching” is known to a person skilled in the art, and may refer to the photochemical alteration of a dye or a fluorophore molecule such that it is (permanently) unable to fluoresce. Such photochemical alteration may e.g. occur upon long-term exposure to light and / or upon (short-term) exposure to high-intensity light, such as during fluorescence imaging.

[0023] In embodiments, the invention may provide a method for characterization of a protein. The term “protein” may herein refer 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. Similarly, the term “peptide” may also refer to a (folded) protein (i.e. a protein having a secondary or tertiary structure), especially wherein the peptide comprises a number of amino acids selected from the range of > 2. Further, the term “peptide” or “protein” may refer to a (multi)protein complex comprising a plurality of proteins, such as a homomeric and / or heteromeric complex of proteins. Yet, 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. In embodiments, the protein may comprise at least 30 amino acids, such as at least 50 amino acids, especially at least 100 amino acids. The method may involve analyzing the protein to determine a protein characteristic. In particular, the term “characterization of a protein” may herein refer to one or more of identifying the protein, especially via sequencing, or determining (at least part of) the 3D protein structure of the protein.

[0024] As indicated, the invention may provide a method for characterization of a structure of a protein using a first probe and a second probe. Proteins may have up to four distinct levels of structural organization. The primary protein structure relates to the linear sequence of covalently bound amino acids that comprise the protein, beginning on one end of the amino acid sequence at an amino-terminal (or: “N-terminal”) amino acid and ending on the other end of the amino acid sequence at a carboxyl-terminal (or: “C-terminal”) amino acid. Hence, the primary protein structure begins at an N-terminal end and ends at a C-terminal end. The secondary protein structure relates to the highly regular structural formations localized to the backbone chain formed by the amino acid sequence. The tertiary protein structure relates to the total three-dimensional structure created by the single amino acid sequence of a single protein. The tertiary protein structure of a single protein may be subdivided into parts called protein domains. The quaternary protein structure relates to the total three-dimensional structure created by the aggregation of two or more amino acid sequences in multiprotein complexes. Hence, single proteins consisting of a single amino acid sequence may not comprise a quaternary protein structure. The protein fingerprinting method may provide for the characterization of one or more protein structure levels.

[0025] Hence, in embodiments, the method may be for characterization of a primary protein structure. In further embodiments, the method may be for characterization of a secondary protein structure. In further embodiments, the method may be for characterization of a tertiary protein structure. In further embodiments, the method may be for characterization of a quaternary protein structure.

[0026] In embodiments, the method may comprise a labelling stage. The labelling stage may comprise exposing the protein to a first proximity labelling enzyme. In embodiments, the first proximity labelling enzyme may be configured to label amino acids of a first amino acid type with a first label.

[0027] Further, in embodiments, the first proximity labelling enzyme may be configured to label (with a first label) a first type of post-translational modification (PTM) (comprised by a side chain of an amino acid). The term “post-translational modification” is known to a person skilled in the art, and may refer to an amino acid side chain modification established after biosynthesis of the protein. An amino acid may, for instance, be post- translationally modified via one or more of phosphorylation, O-linked glycosylation, acetylation, methylation, nitration, famesylation, palmitoylation, myristoylation, and S- nitrosylation. That is, 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 famesyl group, a palmitoyl group, a myristoyl group, and an S-nitrothiol group. The term “PTM” may also refer to a plurality of (different) PTMs. Furthermore, a PTM may also refer to proteolytic processing, which occurs when a protease cleaves one or more bonds in a target protein to modify its activity.

[0028] In embodiments, a proximity labelling enzyme may label amino acids (or PTMs) by catalyzing the generation of a short-lived reactive species in the vicinity of the protein. Hence, the proximity labelling enzyme may activate a label (“substrate”) (i.e. generate a short-lived reactive species), especially in its active center, which label may preferentially or selectively (covalently) bind to the targeted amino acid type (comprised by the protein). As an example to illustrate the function of the proximity labelling enzyme, the labelling of lysine is hereunder described. Note however that many (different) amino acid types may be labelled (by many (different) proximity labelling enzymes).

[0029] For example, the proximity labelling enzyme BioID may be mixed with a solution comprising the protein, biotin, and adenosine triphosphate (ATP). BioID may catalyze (in its reactive center) the conversion of biotin and ATP into the reactive species biotinadenosine monophosphate (biotin-5 ’-AMP). Due to a low(er) affinity of BioID towards biotins’ -AMP, the reactive species may diffuse out of the active center. Once the reactive species has diffused out of the reactive center, it may (selectively) react with amines (such as lysine) in the vicinity of BioID. Upon reaction with, e.g., lysine, the biotin moiety may be covalently bound to the lysine, thereby providing a label for binding of a (first) probe specifically at the lysine residue(s). In should be noted that, due to the reactivity of the reactive species biotin-5 ’- AMP, the species may generally only diffuse ~10 nm away from the enzyme BioID before the reactive species is lost (e.g. through degradation). As such, the enzyme BioID may only label (amine-comprising) amino acids in close (within ~10 nm) proximity to the enzyme. Thus, BioID may function as a proximity labelling enzyme.

[0030] In embodiments, the first proximity labelling enzyme may be selected from the group comprising HRP, APEX, APEX2, APEX2C32S, BioID, BIOID2, BASU, TurboID, miniTurbo, AirlD, microID, ultralD, miniSOG, Tyrosinase, mgSrtA, Lipoic Acid Ligase, and Laccase.

[0031] In embodiments, the labelling stage may comprise providing a labelling activation stimulus to the proximity labelling enzyme, wherein the labelling activation stimulus facilitates the generation of the short-lived reactive species by the proximity labelling enzyme. In embodiments, the labelling activation stimulus may be selected from the group comprising radiation and a chemical compound. Further, in embodiments, the labelling stage may comprise providing the label (or “substrate”) to the proximity labelling enzyme and / or to the protein. Hence, in embodiments, the labelling stage may comprise providing the protein, (first) proximity labelling enzyme, (first) label, and optionally a (first) labelling activation stimulus. Especially, in embodiments, the labelling stage may comprise combining (in a labelling medium) the protein, (first) proximity labelling enzyme, (first) label, and optionally a (first) labelling activation stimulus. In embodiments, the labelling medium may comprise an aqueous solution.

[0032] In embodiments, the labelling stage may comprise a chemical labelling step. Hence, in embodiments, the labelling stage may comprise exposing the protein to one or more of a proximity labelling enzyme and a chemical labelling agent, such as to (both) a proximity labelling enzyme and a chemical labelling agent. Further, in embodiments, the method of the present invention may be combined with a chemical labelling method, as known to a person skilled in the art. Chemical labelling may in embodiments especially be performed for PTMs. Further, in embodiments, the chemical labelling step may comprise performing one or more chemical labelling reactions selected from the group comprising P-elimination / Michael addition (BEMA), phosphoramidite chemistry (PAC), enzymatic removal of the PTM followed by BEMA, mild periodate oxidation, oxime ligation, and hydrazone ligation.

[0033] In embodiments, the labelling stage may comprise labelling at least one amino acid type using a proximity labelling enzyme. Additionally or alternatively, in embodiments, the labelling stage may comprise (i) labelling at least one amino acid type using a proximity labelling enzyme, and (ii) labelling at least one amino acid type using a chemical labelling agent. Yet, in embodiments, the labelling stage may comprise labelling all (of the to-be- labelled) amino acid types using a proximity labelling enzyme. Hence, in embodiments, the labelling stage may not comprise exposing the protein to a chemical labelling agent, i.e., the labelling stage may be executed in the absence of a chemical labelling agent.

[0034] In embodiments, the second binding site may be at least partially provided by a second label. In such embodiments, the second label may especially be provided by labelling of a second amino acid type by a second proximity labelling enzyme. Alternatively, the second label may be provided by labelling of a second type of post-translational modification by a second proximity labelling enzyme. In embodiments, the second proximity labelling enzyme may be a different proximity labelling enzyme than the first proximity labelling enzyme. Further, in embodiment, the second proximity labelling enzyme may be selected from the group comprising HRP, APEX, APEX2, APEX2C32S, BioID, BIOID2, BASU, TurboID, miniTurbo, AirlD, microID, ultralD, miniSOG, Tyrosinase, mgSrtA, Lipoic Acid Ligase, and Laccase.

[0035] Hence, in specific embodiments, the first proximity labelling enzyme and the second proximity labelling enzyme may comprise different enzymes selected from the group comprising HRP, APEX, APEX2, APEX2C32S, BioID, BIOID2, BASU, TurboID, miniTurbo, AirlD, microID, ultralD, miniSOG, Tyrosinase, mgSrtA, Lipoic Acid Ligase, and Laccase. Labelling an amino acid (type) (or a post-translational modification) using proximity labelling enzymes may be faster than labelling the same amino acid (type) (or post-translational modification) using conventional chemical methods. Further, proximity labelling enzymes may operate under (near-)physiological conditions, thus reducing the chance of denaturation of the protein, and further facilitating in vivo labelling in order to analyze protein co- localization and / or protein-protein interactions. The selection of a suitable proximity labelling enzyme may depend on the type of amino acid (or PTM) to be labelled, the type of label, and the activation conditions of the enzyme. In embodiments, proximity labelling enzyme other than the ones indicated above may be used. The selection of a suitable proximity labelling enzyme will be apparent to a person skilled in the art.

[0036] In embodiments, the first label (and / or the second label) may be selected from the group comprising chromophores (such as fluorophores), DNA segments, peptides (such as proteins, heteromeric protein complexes and homomeric protein complexes, e.g. streptavidin), RNA, metal ions, and organic ligands such as selected from the group comprising biotin (conjugates), carbohydrates, and lipids. In embodiments wherein the first label (and / or the second label) is selected from the group of a protein, a homomeric protein complex, and a heteromeric protein complex, the first label (and / or the second label) may especially have a first length h (and / or a second length h) between the labelled amino acid and the binding site for the first probe (and / or the second probe). In embodiments, the first length h (and / or the second length h) may be selected from the range of 2-20 nm, such as from the range of 3-15 nm, especially from the range of 3-10 nm. In embodiments, the first label (and / or the second label) may be small enough to penetrate the tertiary structure of the protein, thereby labelling amino acids on the inside of the protein. Yet, in embodiments, amino acids on the inside of the protein may be labelled inefficiently, e.g. in embodiments wherein the label is relatively bulky. Hence, in embodiments, the protein may be denatured prior to the labelling stage. The term “denatured” and conjugations thereof are known to a person skilled in the art, and may refer to a process of reducing a (native) protein to its primary structure. In embodiments, denaturation of the protein may be achieved by exposing the protein to one or more of heat, sodium dodecyl sulfate (SDS), and reducing agents (e.g. tris(2-carboxyethyl)phosphine (TCEP)). Protein denaturation may particularly facilitate determining the primary structure of a protein.

[0037] For the determination of the secondary, tertiary or quaternary structure of a protein a non-denatured (folded) protein may be required. Hence, in further embodiments, the protein may be a folded (non-denatured) protein, especially during the exposure stage.

[0038] In embodiments, the method may employ a first probe and (optionally) a second probe, configured to bind to a binding site. The first probe (and / or second probe) may interact with a localized protein substructure, such as a protein domain, protein sequence, amino acid, or post-translational modification, optionally in a highly specific manner. Further, the first probe (and / or second probe) may especially interact with a label connected to or comprised by an amino acid (type). Yet, in embodiments, the first probe (and / or the second probe) may interact with a (type of) post-translational modification (PTM) on the protein, especially on a specific amino acid type comprised by the protein. Thus, the binding of the first probe and / or the second probe may be a reliable indicator for the presence of a particular localized protein substructure, especially of a particular amino acid (type) (or a particular PTM). Examining the binding of the first probe and / or the second probe, such as examining the binding (in relation to each other) of a first probe and a (different) second probe, to a protein or protein substrate may provide information about the presence and relative locations of the amino acids (or PTMs) of a specific amino acid type (or PTM type) that may be present in the protein. This information may facilitate characterizing, such as identifying, the protein.

[0039] In embodiments, the method may comprise exposing the protein to a first probe. The first probe may especially comprise a chromophore, configured to generate especially (acceptor) emission radiation upon irradiation with excitation radiation. In embodiments, the first probe may be configured to transiently bind the protein at the first binding site with a first (binding) frequency. In embodiments, the first frequency may be dependent on one or more of the concentration of the first probe and the concentration of the first binding site (on the protein, and the concentration of the protein). Especially, at a first concentration of the first probe, the first frequency may in embodiments scale linearly with the concentration of the first binding sites (and thus the number of amino acids of the first amino acid type). Hence, in embodiments, measuring the first frequency may facilitate determining a concentration (or number) of the first binding sites (on the protein). Hence, in embodiments, the method may comprise determining the concentration (or amount) of the first binding site based on the first frequency and a reference frequency, wherein the reference frequency corresponds to the first probe interacting with a protein with a known number of first binding sites.

[0040] In embodiments, the reference frequency may be comprised by a standard curve, i.e., a standard curve may comprise a plurality of reference frequencies for the first probe interacting with the first binding site. Especially, in embodiments, the standard curve may be a curve depicting the first frequency in a (standard) system wherein the protein comprises (only) one first binding site. In embodiments, as the first frequency may scale linearly with the number of first binding sites (i.e., the concentration of the first binding site), such a reference frequency, especially such a standard curve, may be used to determine the concentration of first binding sites on a protein with an unknown number of first binding sites. In further embodiments, the method may further comprise obtaining a reference frequency, especially a standard curve, for the first probe. Further, measuring the first frequency may thus facilitate determining a concentration of a first amino acid type, especially in the protein. In embodiments, the binding of a first probe to an (immobilized) protein may be measured using a fluorescence microscope operated in total internal reflection, whereby a sharp increase in (acceptor) emission radiation intensity is observed upon binding of the first probe at a binding site on an immobilized protein (in the focal area of the microscope). Further, the method may comprise (subsequently) exposing the protein to a second probe. Hence, in embodiments, the method may comprise determining the concentration (or amount) of the second binding site based on the second frequency and a second reference frequency, wherein the second reference frequency corresponds to the second probe interacting with a protein with a known number of second binding sites. Hence, the method may (also) comprise obtaining a second reference frequency, especially a second standard curve, for the second probe. Especially, the method may comprise exposing the protein to a first probe, measuring the first frequency, removing the first probe, and subsequently exposing the protein to a second probe. The second probe may comprise a chromophore, configured to generate (acceptor) emission radiation upon irradiation with excitation radiation. In embodiments, the second probe may be configured to transiently bind the protein at the second binding site with a second frequency, wherein the second binding site may be comprised by a second amino acid type. In embodiments, the second frequency may be dependent on one or more of the concentration of the second probe and the concentration of the second binding site (on the protein, and the concentration of the protein). Hence, in embodiments, measuring the second frequency may facilitate determining a concentration of the second binding sites (on the protein), and thus a concentration of a second amino acid type (in the protein). Further, in embodiments, the method may comprise sequentially exposing the protein to x probes, wherein each of the x probes may be configured to transiently bind the protein at a different binding site with a respective (binding) frequency, wherein said different binding sites may each correspond to a different amino acid type. In embodiments, the method may thus comprise (obtaining reference frequencies, especially standard curves, and) measuring binding frequencies for x different labels to facilitate determining a concentration of x different (types of) binding sites (on the protein), and thus a concentration of x different amino acid types (in the protein). In specific embodiments, x may be selected from the range of 2-20, such as from the range of 2-15, especially from the range of 3-10. In embodiments, different proteins may comprise different concentrations of amino acid types. Hence, in embodiments, determining the concentration of x different amino acid types (in the protein) may facilitate characterizing, such as identifying, the protein. Hence, in specific embodiments, the method may comprise (a) a labelling stage comprising exposing the protein to a first proximity labelling enzyme, wherein the first proximity labelling enzyme may be configured to label amino acids of a first amino acid type with a first label; and (b) an exposure stage comprising: (i) exposing the protein to a first probe, (ii) providing excitation radiation to the protein, and (iii) measuring emission radiation to provide an emission signal; wherein: (A) the first probe may be configured to transiently bind the protein at a first binding site, wherein the first label may provide at least part of the first binding site, and wherein the first probe may comprise a first chromophore; (B) the first chromophore may have an excitation radiation range and an emission radiation range, wherein the radiation may comprise a wavelength selected from the excitation radiation range, and wherein the emission may be measured in the emission radiation range. Further, in specific embodiments, the method may comprise determining a concentration of the first binding site based on a first frequency, wherein the method may optionally further comprise determining a number of amino acids (of the first amino acid type) in the protein based on the first frequency.

[0041] Such a method may facilitate characterizing, such as identifying, a protein based on the total number of amino acids (of the first and / or further amino acid types). Hence, such a method may provide a relatively fast method to characterize a protein. Further, proximity labelling enzymes may have a high labelling efficiency, i.e., most (such as > 80%) of the targeted amino acids may be labelled. As such, the calculated number of amino acids may have a relatively small error margin (such as < 20%) compared to the actual number of amino acids. Hence, the method may be relatively accurate in determining the number of amino acids of a specific amino acid type.

[0042] In embodiments, the method may comprise exposing the protein to a first probe and a second probe. Exposing the protein to two (different) probes, and determining a FRET efficiency between said probes, may provide additional information regarding the protein (e.g. distances between amino acid residues). Hence, such a method may facilitate characterizing, such as identifying, a protein with a higher degree of confidence. Further, such a method (using FRET efficiencies) may be less sensitive to incomplete labelling. In general, a probe (such as e.g. the first probe and the second probe) may comprise a (transient) affinity-based probe. Especially, in embodiments, the probe may be a (transient) affinity-based probe. Further, in embodiments, the probe may comprise a single molecule, i.e., a single molecule may be configured to (transiently) bind the protein (at a binding site) and may comprise a chromophore. In such embodiments, the probe may comprise one or more of an aptamer, an antibody, a nanobody, a (small) peptide (such as proteins, homomeric protein complexes and heteromeric protein complexes), and an organic ligand. Hence, in specific embodiments, the first probe and the second probe may comprise affinity-based probes selected from the group comprising an aptamer, an antibody, a nanobody, a peptide, and an organic ligand. In embodiments wherein the probe comprises one or more of a protein, a homomeric protein complex, and a heteromeric protein complex, the probe may especially have a height h between the binding site and the chromophore. In embodiments, the height h may be selected from the range of 0.05-34 nm, such as from the range of 0.1-29 nm, especially from the range of 0.1-20 nm. In further embodiments, the height h may be selected from the range of 2-20 nm, such as from the range of 3-15 nm, especially from the range of 3-10 nm. Further, in specific embodiments, the first probe and the second probe may comprise aptamers.

[0043] Aptamers are single stranded DNA, RNA, PNA or peptide nanostructures, typically of tens of monomers (such as nucleotides, or such as amino acids) in length. Aptamers may, in embodiments, be selected from the group comprising DNA aptamers (including synthetic DNA aptamers, such as L-DNA aptamers), RNA aptamers, short peptide aptamers, peptide nucleic acid (PNA) aptamers, locked nucleic acid (LNA) aptamers, and slow off-rate modified aptamers (SOMAmers). Aptamers may be partially self-complementary and may fold into secondary structures and may then be able to transiently bind a specific region (or epitope) in a target molecule. Currently, antibodies or nanobodies may be the standard probes for protein staining, but aptamers may have several advantages. For example, aptamers may be chemically synthesized, which may make production faster, cheaper and purer than antibodies. Furthermore, the increased stability of aptamers may translate into a longer shelf-life and resistance against degrading conditions (such as e.g. high temperatures, high or low pH, etc.) than antibodies would. Also, unlike the amino acids that are the building blocks of antibodies, the nucleotides that may comprise the DNA or RNA aptamer backbone can easily be sequenced for identification, enabling highly parallel assays. Different aptamers may have different binding kinetics.

[0044] Hence, in embodiments, the first probe (and / or the second probe) may comprise an aptamer. The aptamer may comprise 2-80 aptamer monomers such as nucleotides, or such as amino acids. Further, the aptamer may comprise 5-80 aptamer monomers, such as 5-70 aptamer monomers. The aptamer may in embodiments comprise 10-70 aptamer monomers, especially 10-60 aptamer monomers, Moreover, the aptamer may comprise 20-60 aptamer monomers, such as 20-50 aptamer monomers.

[0045] In embodiments, the aptamer may especially comprise a DNA aptamer. The DNA aptamer may comprise 10-80 aptamer monomers, i.e., 10-80 nucleotides. Further, the DNA aptamer may comprise 12-80 aptamer monomers, such as 12-70 aptamer monomers. The DNA aptamer may in embodiments comprise 15-70 aptamer monomers, especially 15-60 aptamer monomers, Moreover, the DNA aptamer may comprise 20-60 aptamer monomers, such as 20-50 aptamer monomers.

[0046] In certain embodiments, the first probe (and / or the second probe) may comprise an antibody (or: “immunoglobulin”). Antibodies are glycoproteins that may be naturally produced by the immune system of vertebrate animals and may be configured to selectively bind specific molecules or molecule domains, such as protein domains. The target molecule or molecule domain of an antibody is called an epitope. Through the recombination of so-called “variable domains” comprised by antibodies, the immune system of vertebrate animals may be able to produce many varieties of antibodies with different epitopes, thereby facilitating the ability of the vertebrate animal immune system to recognize and neutralize foreign compounds. Antibodies with specific epitopes may be generated for use in biotechnological applications. Hence, antibodies may be generated for use as a first probe (and / or a second probe) in the current invention. In specific embodiments, the invention may provide a method to investigate the epitope of an antibody on a protein.

[0047] In specific embodiments, the first probe (and / or the second probe) may comprise a nanobody. Nanobodies are the recombinant variable domains isolated from antibodies, retaining the specific binding abilities for an epitope but losing other parts of the antibody glycoprotein. Nanobodies may hence have a smaller molecular weight and better solubility than antibodies. Hence, nanobodies may be generated for use as a first probe (and / or as a second probe) in the current invention.

[0048] In other embodiments, the first probe (and / or the second probe) may comprise an organic ligand. An organic ligand may comprise an organic compound with one or more of (i) a molecular weight of < 2000 Da, such as < 1000 Da, especially < 500 Da, and (ii) a molecular size from the range of 0.01 - 50 nm, such as from the range of 0.02 - 20 nm, especially from the range of 0.05 - 10 nm. Such organic ligands may be efficiently synthesized and may transiently bind a localized protein substructure. The organic ligand may be selected from the group comprising thiols and reducing agents. Especially, the organic ligand may be selected from the group comprising tris(2-carboxyethyl)phosphine (TCEP), 2- Mercaptoethanol (P-mercaptoethanol], and iodoacetamide.

[0049] The unit Dalton (Da) herein refers to the atomic mass constant, which may typically be used to indicate the mass of proteins, as well as of small molecules. 1 Dalton may correspond to one twelfth of the mass of a free carbon-atom at rest. As will be known to the person skilled in the art, 1 Dalton corresponds to (approximately) 1.660* KT27kg.

[0050] In embodiments, the probe may comprise two or more different molecules (or “entities”). For instance, in embodiments, the probe may comprise a protein binding moiety and a chromophore molecule. In such embodiments, the protein binding moiety may bind the protein at the binding site, especially transiently bind the protein at the binding site, and the chromophore molecule may associate with or be covalently bound to the protein binding moiety. In such embodiments, the protein binding moiety may be one or more of an aptamer, an antibody, a nanobody, a (small) peptide, and an organic ligand.

[0051] Further, in embodiments, the chromophore may be selected from the group comprising chromophores selected from the Cyanine family, the Alexa family, the Atto family, the Dy family, and the Rhodamine family. In embodiments, the chromophore may be covalently bound to the protein binding moiety. Additionally or alternatively, the chromophore may be associated with the protein binding moiety. For example, the chromophore may be configured to (transiently) bind the protein binding moiety. Further, in embodiments, the chromophore may be associated with the protein binding moiety through one or more non- covalent interactions (e.g. hydrogen bonding and / or ionic interactions), such that, under the conditions present in the exposure stage, the chromophore may (essentially) not dissociate from the protein binding moiety.

[0052] In embodiments, the first probe may comprise a first protein binding moiety and the first chromophore. Further, in embodiments, the second probe may comprise (the same) first protein binding moiety, yet another chromophore, such as the second chromophore. In embodiments where the second probe comprises the first protein binding moiety, the second probe may especially be configured to transiently bind the protein at the first binding site, e.g., the first label may provide a binding site for both the first probe and the second probe. Alternatively, in embodiments, the second probe may comprise a second protein binding moiety, different from the first protein binding moiety, and the second chromophore. In such embodiments, the second probe may be configured to transiently bind the protein at the second binding site, wherein the second binding site differs from the first binding site.

[0053] Hence, in embodiments, the protein may comprise the first binding site and the second binding site. In general, a binding site may be the localized protein substructure, such as especially the (labelled) amino acid type (or the (labelled) PTM type), at which a probe is targeted. Thus, in embodiments, the first probe may be configured to transiently bind the protein at the first binding site. Conversely, depending on the protein binding moiety used, the second probe may be configured to transiently bind the protein at either the first binding site or the second binding site. Especially, the first probe and second probe may comprise affinitybased probes, and hence may reversibly bind to the first binding site and (optionally) the second binding site during the method, especially during the exposure stage. The first (affinity -based) probe and the second (affinity-based) probe may especially be selected from the group comprising an aptamer, an antibody, a nanobody, and a small-molecule moiety. Probes selected from this group of organic compounds may typically be able to target localized protein substructures, such as labelled amino acids or labelled PTMs, with high affinity. This may provide an advantage as the characterization method may not require covalent conjugation of chromophore-comprising probes to the protein of interest. This is an advantage as chromophores, especially fluorophores, may be susceptible to photobleaching. Using transient binding, probes comprising chromophores (and / or fluorophores) may be continuously replaced at binding sites on the protein, thereby reducing photobleaching of said probes. Hence, reducing, especially avoiding, covalent conjugation of chromophores (and / or fluorophores) to the protein may extend the measuring time during the exposure stage, allow for higher intensity excitation radiation, and facilitate reducing sample size.

[0054] The first binding site (and / or the second binding site) may be a localized protein substructure with a known localization within the protein structure, especially in embodiments wherein the method is employed with the goal of identifying the presence or absence of a known protein in a (biological) sample. The first binding site (and / or the second binding site) may also be a localized protein substructure with an unknown localization within the protein structure, especially in embodiments wherein the method is employed with the goal of investigating the structure of a known or unknown protein in a (biological) sample.

[0055] As indicated, the first binding site (and / or the second binding site) may be a localized protein substructure, such as especially a labelled amino acid or a labelled PTM. Further, in embodiments, the first binding site (and / or the second binding site) may be at least partially provided by the first label (and / or by the second label). Hence, in embodiments, the first binding site (and / or the second binding site) may comprise at least part of the first label (and / or of the second label). In specific embodiments, the first binding site (and / or the second binding site) may comprise at least part of a chromophore (or fluorophore), a DNA segment, a peptide, an RNA, a metal ion, and an organic ligand such as selected from the group comprising biotin (conjugates), carbohydrates, and lipids. Further, in embodiments, the first binding site may be different from the second binding site. This may provide the benefit that the first probe may have a high affinity for the first binding site, and a low affinity for the second binding site. The first probe may thus be configured to transiently bind the protein at the first binding site. Such a transient binding may be described using binding kinetics that comprise (i) an on-rate, being the first-order rate constant for the binding of the first probe to the first binding site, and (ii) an off-rate, being the first-order rate constant for the disassociation of the first probe from the first binding site. In embodiments, the on-rate may thus indicate how much of a probe (in moles) binds to the protein over a specific time to form a protein-probe complex. Additionally or alternatively, in embodiments, the off-rate may indicate the amount of probe that dissociates from the protein-probe complex over a specific time. It will be clear to the skilled person that different probes may have different on-rates and off-rates for the protein, especially for binding sites in the protein. In particular, probes may be designed, for instance via computational modeling and / or via binding assays, with specific binding affinities for one or more specific binding sites. In embodiments, the on-rate and off-rate of a probe may be determined using a fluorescence microscope operated in total internal reflection, whereby a sharp increase in emission intensity is observed upon binding of the probe to an immobilized protein (in the focal area of the microscope). Such methods are e.g. described in Jungmann, R. et al. (2010); Single-molecule kinetics and super-resolution microscopy by fluorescence imaging of transient binding on DNA origami, Nano lett. 10, 4756-4761, which is hereby herein incorporated by reference.

[0056] In embodiments, the first probe may be configured to transiently bind the protein at the first binding site with an on-rate selected from the range of 0.02* 106- 5*106M’ 1, such as from the range of 0.05* 106- 5*106M’1, especially from the range of 0.05* 106- 2*106M’V.

[0057] Additionally or alternatively, in embodiments, the first probe may be configured to transiently bind the protein at the first binding site with an off-rate selected from the range of 0.01-10 s’1, such as from the range of 0.02-10 s’1, especially from the range of 0.02-5 s’1. Further, in embodiments, the first probe may be configured to transiently bind the protein at the first binding site with an off-rate selected from the range of 0.05-5 s’1, such as from the range of 0.05-2 s’1, especially from the range of 0.1-2 s’1. Further yet, in embodiments, the first probe may be configured to transiently bind the protein at the first binding site with an off-rate selected from the range of 0.1-10 s’1, such as from the range of 0.2-10 s’1, especially from the range of 0.2-5 s’1. In embodiments, the term “to transiently bind” and conjugations thereof may especially refer to a binding event characterized by an off-rate selected from the range of > 0.01 s’1, such as from the range of 0.01-10 s’1. In embodiments, the first probe may, on average, remain transiently bound to the protein (at the first binding site) for a binding duration correlated to the inverse of the off- rate. i.e. the first probe may have a dwell time on the protein, especially on the binding site. In embodiments, the first probe may have a dwell time (on the first binding site) selected from the range of 0.1-100 s, such as from the range of 0.5-20 s, especially from the range of 5-20 s. Further, in embodiments, the first probe may have a dwell time (on the first binding site) selected from the range of 0.1-10 s, such as from the range of 0.5-10 s. In embodiments, the binding kinetics of the first probe may be controlled. This may be done by e.g. altering the ion strength of the solution which may e.g. affect aptamer folding. Through adjusting specific ion concentrations, the binding kinetics of the probes may be sped up (which may facilitate a faster read-out) or slowed down (which may result in a higher signal-to-noise ratio). Further, the binding kinetics of the first probe may be controlled by altering the labeling (or “binding”) of the first chromophore to a different position on the first probe. Especially, in embodiments where the first probe comprises an aptamer, the first chromophore may be labeled to a different position in the aptamer sequence, which may affect the binding kinetics of the first probe. Hence, the method, especially the exposure stage, may comprise controlling the off-rate (or the on-rate) of the first probe at the first binding site in the range of 0.05-0.2 s'1(or in the range of 0.01 *106- 10* 106M^s’1), and especially in the other ranges mentioned hereabove.

[0058] Similar to the first probe, the second probe may in embodiments be configured to transiently bind the protein at the first binding site. Alternatively, in embodiments, the second probe may be configured to transiently bind the protein at the second binding site. Both the binding of the second probe to the first binding site and the binding of the second probe to the second binding site may be described using binding kinetics. In embodiments, the second probe may be configured to transiently bind the protein at the first binding site (or the second binding site) with an on-rate selected from the range of 0.02* 106- 5*106M^s’1(or 0.02E6 - 5E6 M^s’1), such as from the range of 0.05* 106- 5*106M’1, especially from the range of 0.05*106- 2*106M’V.

[0059] Additionally or alternatively, in embodiments, the second probe may be configured to transiently bind the protein at the first binding site (or the second binding site) with an off-rate selected from the range of 0.01-10 s’1, such as from the range of 0.02-10 s’1, especially from the range of 0.02-5 s’1. Further, in embodiments, the second probe may be configured to transiently bind the protein at the first binding site (or the second binding site) with an off-rate selected from the range of 0.05-5 s’1, such as from the range of 0.05-2 s’1, especially from the range of 0.1-2 s’1. Further yet, in embodiments, the second probe may be configured to transiently bind the protein at the first binding site (or the second binding site) with an off-rate selected from the range of 0.1-10 s’1, such as from the range of 0.2-10 s’1, especially from the range of 0.2-5 s’1.

[0060] In embodiments, the second probe may, on average, remain transiently bound to the protein (at the first binding site or the second binding site) for a binding duration correlated to the inverse of the off-rate. i.e. the second probe may have a dwell time on the protein, especially on the first binding site, or especially on the second binding site. In embodiments, the second probe may have a dwell time (on the first binding site and / or on the second binding site) selected from the range of 0.1-100 s, such as from the range of 0.5-20 s, especially from the range of 5-20 s. Further, in embodiments, the second probe may have a dwell time (on the first binding site and / or on the second binding site) selected from the range of 0.1-10 s, such as from the range of 0.5-10 s. In embodiments, the binding kinetics of the second probe may be controlled. Hence, the method, especially the exposure stage, may comprise controlling the off-rate of the second probe at the first binding site (or the second binding site) in the range of 0.05-0.2 s’1, and especially in the other ranges mentioned hereabove. Hence, in specific embodiments, the first probe may be configured to transiently bind the protein at the first binding site with an off-rate selected from the range of 0.1-10 s’1, and the second probe may be configured to transiently bind the protein at one of (i) the first binding site with an off-rate selected from the range of 0.1-10 s’1, and (ii) the second binding site with an off-rate selected from the range of 0.1-10 s’1. In embodiments, an off-rate of 0.1- 10 s’1(with a related dwell time of 0.1-10 s) may provide long enough binding to detect the fluorescence of the bound probe and measure FRET efficiency between two probes, yet may provide short enough binding to prevent photobleaching of the probe.

[0061] In embodiments, the labelling of one or a few subsets of amino acid types may be sufficient to provide a protein-specific fingerprint. The protein fingerprint may enable characterization, especially identification, of a protein, with more labelled subsets of amino acid types providing a stronger, less error-prone fingerprint. For instance, 90% of human proteins can be correctly identified with reference to a human proteome database by the order in which labelled cysteine (C) and lysine (K) residues appear, while this number increases to 99% when C, K and methionine (M) residues are labelled. The identification of a protein based on the protein fingerprint may, for instance, be performed such as described in Yao et al., “Single-Molecule Protein Sequencing through Fingerprinting: Computational Assessment”, Physical Biology, 2015, or such as described in Ohayon et al., “Simulation of single-protein nanopore sensing shows feasibility for whole-proteome identification”, PLoS Computational Biology, 2019, which are hereby herein incorporated by reference.

[0062] Hence, in embodiments, the method may comprise labelling amino acids of a first amino acid type with a first label (by exposing the protein to a proximity labelling enzyme). In embodiments, the first amino acid type may especially be an amino acid selected from the group comprising cysteine, lysine, tyrosine, histidine, serine, glycine, and tryptophan. Further, in embodiments, the second probe may, in embodiments wherein the second probe comprises the second protein binding moiety, have an (inherent) affinity for (a PTM and / or a binding site on) another amino acid selected from the group comprising cysteine, lysine, tyrosine, histidine, serine, glycine, and tryptophan. Such (inherent) affinity may provide a selective enough (transient) binding for characterization of the protein. Yet, in embodiments, the confidence of the characterization may be improved by labelling amino acids of a second type with a second label, wherein the second protein binding moiety selectively (transiently) binds to the second label. In such embodiments, the second label may especially be different from the first label. Further, as indicated, the second amino acid type may be labelled by a different proximity labelling enzyme. Especially, in embodiments, the (short-lived reactive species generated by the) first proximity labelling enzyme may have a high selectivity for the first amino acid type, and a low selectivity for the second amino acid type and / or a different amino acid type (than the first amino acid type). Conversely, the (short-lived reactive species generated by the) second proximity labelling enzyme may have a high selectivity for the second amino acid type, and a low selectivity for the first amino acid type and / or a different amino acid type (than the second amino acid type). In embodiments, the selectivity of the (short-lived reactive species generated by the) proximity labelling enzyme towards a specific amino acid type may be selected from the range of > 60%, such as from the range of > 70%, especially from the range of > 80%. Further, in embodiments, the selectivity of the (short-lived reactive species generated by the) proximity labelling enzyme towards a specific amino acid type may be selected from the range of > 85%, such as from the range of > 90%, especially from the range of > 95%, like from the range of > 98%. For example, the proximity labelling enzyme APEX may have a labelling specificity of -98% towards tyrosine (residues). Herein, the selectivity of a (proximity labelling) enzyme may refer to the preference of said (proximity labelling) enzyme for one substrate over another substrate, such as over any other substrate. Hence, a labelling selectivity of > 90% may indicate that at least 90% of the amino acids labelled by said (short-lived reactive species generated by said) proximity labelling enzyme may be of the desired type (e.g. the first amino acid type), while at most 10% may be of another (secondary) amino acid type. In embodiments, the secondary (amino acid type) targets of a proximity labelling enzyme may be known. Hence, in embodiments, unselective labelling (i.e. labelling of an amino acid not of the correct amino acid type) may be predictable. In embodiments, the method may therefore comprise labelling a secondary (amino acid type) target of the proximity labelling enzyme on a second (identical) protein using a different proximity labelling enzyme having selectivity for said secondary target, and comparing the emission radiation of the (first) protein and second protein to determine the positions of the unselectively labelled amino acids (of the (first) protein). In specific embodiments, the first proximity labelling enzyme may have a selectivity towards the first amino acid type selected from the range of > 80%, such as from the range of > 85%, especially from the range of > 90%, like from the range of > 95%, such as from the range of > 98%. Additionally or alternatively, in embodiments, the second proximity labelling enzyme may have a selectivity towards the second amino acid type selected from the range of > 80%, such as from the range of > 85%, especially from the range of > 90%, like from the range of > 95%, such as from the range of > 98%. Further, in specific embodiments, each of the n proximity labelling enzymes (see below) may have a selectivity towards a respective amino acid type selected from the range of > 80%, such as from the range of > 85%, especially from the range of > 90%, like from the range of > 95%, such as from the range of > 98%.

[0063] Hence, in embodiments, the labelling stage may comprise exposing the protein to a second (different) proximity labelling enzyme. In embodiments, the second proximity labelling enzyme may be configured to label amino acids of a second amino acid type with a second label (by catalyzing the generation of a (second) short-lived reactive species in the vicinity of the protein). Especially, the second label may provide at least part of the second binding site. Providing (at least part of) the second binding site by a second label may improve the selective (transient) binding of the second probe to the second amino acid type. Hence, a second label may, in embodiments, improve the accuracy of the characterization. Therefore, in specific embodiments, the labelling stage may comprise exposing the protein to a second proximity labelling enzyme, wherein the second proximity labelling enzyme may be configured to label amino acids of a second amino acid type with a second label, and wherein the second label may provide at least part of the second binding site.

[0064] Further, as indicated, the accuracy of the characterization, especially the identification, of the protein may be improved by labelling further amino acid types (or further PTMs) (with further labels). In embodiments, each (further) amino acid type (or PTM) may be labelled by a different (unique) proximity labelling enzyme comprising a higher selectivity for said amino acid type (or PTM) than for other amino acid types (or PTMs). Hence, in embodiments, the labelling stage may comprise exposing the protein to n proximity labelling enzymes. In embodiments, n > 2, such as n > 3, especially n > 5. Yet, in embodiments, n < 20, such as n < 18, especially n < 15. In further embodiments n < 10, such as < 8, especially < 5. Hence, the labelling stage may comprise exposing the protein to n proximity labelling enzymes, wherein n may be selected from the range of 2-20, such as from the range of 3-18, especially from the range of 5-15. In embodiments, the n proximity labelling enzymes may be individually, but uniquely, selected from the group comprising HRP, APEX, APEX2, APEX2C32S, BioID, BIOID2, BASU, TurboID, miniTurbo, AirlD, microID, ultralD, miniSOG, Tyrosinase, mgSrtA, Lipoic Acid Ligase, and Laccase, though other proximity labelling enzymes are herein not excluded. In embodiments, the n different proximity labelling enzymes may be configured to label amino acids of n different amino acid types with n different labels. Hence, in embodiments, the n different proximity labelling enzymes may be configured to provide n different (types of) binding sites on the protein. In embodiments where n amino acid types are labelled, the method may further comprise exposing the protein to n different probes. In embodiments, some of the n different probes may comprise the same (type of) chromophore. Alternatively, in embodiments, the n (different) probes may comprise n different (types of) chromophores. Specifically, in embodiments, the n probes may comprise m different chromophores, wherein m < n. Further, in embodiments, m may be selected from the range of 2-15, such as from the range of 2-10, especially from the range of 2-6. In specific embodiments, m may be selected from the range of 2-4. In embodiments, the n (different) probes may not be introduced to the protein simultaneously. Instead, the exposure stage may, in embodiments, comprise successively exposing the protein to different sets of probes, wherein a first probe of each set may especially comprise a FRET donor, and a second probe of each set may especially comprise a (corresponding) FRET acceptor, i.e., each set of probes may comprise chromophores forming a FRET pair. In embodiments, at least part of the sets may comprise at least two different probes, such as two different probes, configured to transiently bind the protein at (at least) two different labels. In such embodiments, the at least two different probes may comprise (at least) two different chromophores forming a respective FRET pair. In further embodiments, at least part of the sets may comprise at least two different probes configured to transiently bind the protein at the same (type of) label. In such embodiments, the at least two different probes may comprise the same protein binding moiety, yet different chromophores (forming a respective FRET pair). Hence, in specific embodiments, the labelling stage may comprise exposing the protein to n different proximity labelling enzymes, wherein n > 3, wherein the n different proximity labelling enzymes may be configured to label amino acids of n different amino acid types with different labels, and wherein the exposure stage may comprise successively exposing the protein to different sets of probes, wherein each set may comprise at least two different probes configured to transiently bind the protein at at least two different labels, and wherein the at least two different probes may comprise at least two different chromophores forming a respective FRET pair. In further embodiments, each set may comprise two different probes configured to transiently bind the protein at two different labels, and wherein the two different probes comprise two different chromophores forming a FRET pair.

[0065] In embodiments, each set of probes may thus characterize (by FRET) the position of two amino acids types (or PTM types) relative to each other. Further, in embodiments, each set of probes may characterize (by FRET) the position of two amino acids (or PTMs) of the same amino acid type (or PTM type) relative to each other. By combining information from different sets, a map of the protein may be formed. Further, by successively exposing the protein to the different sets, the emission from the chromophores comprised by a first set may not interfere with the emission from the chromophores comprised by a second set. Especially, in embodiments, as the probes are transiently bound (to the protein), the first set of probes may be removed (e.g. by washing) relatively easy before introducing the next set of probes. Hence, in embodiments, sets of probes may be (successively) introduced to the same protein, thus eliminating the need for a large sample size (comprising a plurality of protein each bound to a different set of probes). Hence, the method may facilitate the characterization of single proteins.

[0066] In embodiments, the first probe (from a set of probes) and the second probe (from a set of probes) may thus form a FRET donor-acceptor pair. Especially, in embodiments, the (first) chromophore comprised by the first probe (from a set of probes) may be the FRET donor, and the (second) chromophore comprised by the second probe (from a set of probes) may be the FRET acceptor. Alternatively, in embodiments, the (first) chromophore comprised by the first probe (from a set of probes) may be the FRET acceptor, and the (second) chromophore comprised by the second probe (from a set of probes) may be the FRET donor. The term “chromophore” may herein also be referred to as an “optical emitter”. Further, in embodiments, the optical emitters (chromophores) of the first probe (from a set of probes) and the second probe (from a set of probes) may be referred to as FRET donor-acceptor pair optical emitters. In embodiments, the FRET donor-acceptor pair optical emitters (also: “donoracceptor pair optical emitters”) may comprise fluorescent structures and / or phosphorescent structures. The term “FRET donor-acceptor pair optical emitters” may herein especially refer to two optical emitters (chromophores) capable of FRET energy transfer, i.e., energy transfer in a non-radiative distance-dependent fashion, especially through dipole-dipole coupling of the donor optical emitter and the acceptor optical emitter. Hence, in embodiments, energy transfer takes place through dipole-dipole interactions between an excited donor and an (non-excited) acceptor in a non-radiative process provided that: 1) the emission spectrum of the donor partially overlaps with the excitation spectrum of the acceptor, 2) the proximity of the donor and acceptor is less than a critical distance (typically in the range of <10 nm for fluorescent dye molecules), and 3) the transition dipoles of the donor and acceptor are approximately parallel.

[0067] Hence, the FRET donor-acceptor pair chromophores may comprise a first chromophore and a second chromophore, wherein the FRET donor-acceptor pair chromophores have a donor excitation radiation range, an acceptor excitation radiation range, a donor emission radiation range and an acceptor emission radiation range, wherein one of the FRET donor-acceptor pair chromophores is excitable by donor excitation radiation in the donor excitation radiation range, wherein the other of the FRET donor-acceptor pair chromophores is configured to provide acceptor emission radiation in the FRET acceptor emission radiation range upon excitation with donor excitation radiation in the donor excitation radiation range of the one of the FRET donor-acceptor pair chromophores when the first chromophore and the second chromophore are configured within a FRET distance. Hence, if the donor chromophore and the acceptor chromophore are arranged within a FRET distance, which may vary for different FRET donor-acceptor pairs, the donor chromophore may upon excitation with donor excitation radiation transfer energy to the acceptor chromophore, whereupon the acceptor chromophore may emit acceptor emission radiation. The energy transfer between a FRET donor and FRET acceptor may occur with a specific FRET efficiency depending on the (exact) distance between the donor optical emitter and the acceptor optical emitter. Hence, by measuring the FRET (transfer) efficiency, information regarding the distance between the donor optical emitter and the acceptor optical emitter may be obtained. In particular, the FRET transfer efficiency may be sensitive to sub-nanometer distance changes, which may make FRET an outstanding spectroscopic ruler for probing, for example, biological systems.

[0068] The FRET efficiency (E) may be defined as: wherein Iais the intensity of the acceptor emission, wherein la is the intensity of the donor emission, and wherein IDA / ID is the ratio between the intensity of the donor emission in the presence (IDA) and absence (ID) of an acceptor. The distance between the donor chromophore and the acceptor chromophore may then be calculated by comparing the measured value of E (equation above) to a calculated value of the FRET efficiency Eeas a function of the distance r: wherein R is the Forster radius, which may be specific for the donor-acceptor pair.

[0069] The distance between a first of a plurality of first binding sites and a second of a plurality of first binding sites, or between the first binding site and the second binding site may then be calculated based on the calculated distance between the donor chromophore and the acceptor chromophore, as well as, for example, based on the (length of the) probes. Hence, in embodiments, the method may comprise a distance calculation stage. In embodiments, the distance calculation stage may comprise calculating a distance di between the first of the plurality of first binding sites and the second of the plurality of first binding sites. Additionally or alternatively, the distance calculation stage may comprise calculating a distance d2 between the first binding site and the second binding site. Further, in embodiments, the distance calculation stage may comprise calculating a distance dn2 between (binding sites from) any two types of binding sites comprised by the n different types of binding sites. Further yet, in embodiments, the distance calculation stage may comprise calculating a distance dnibetween any two binding sites comprised by the same type of binding site (comprised by the n different types of binding sites). Especially, the distance calculation stage may comprise calculating one or more of the distance di and the distance d2 (and a distance dn2 and a distance dni) based on the emission signal, especially based on a FRET efficiency. Herein, the emission signal may comprise the intensity of the donor emission radiation over time, especially in the presence and absence of the acceptor. Further, the emission signal may comprise the ratio between the emission from the donor emission radiation range and the emission from the acceptor emission radiation range. This ratio comprises information on the transfer efficiency of the FRET that has occurred between the acceptor and the donor. Hence, the emission signal may allow for a sub-nanometer distance di (or d2) calculation between the first of the plurality of first binding sites and the second of the plurality of first binding sites (or between the first binding site and the second binding site). Hence, in specific embodiments, the protein may comprise a plurality of first binding sites, and the method may comprise a distance calculation stage, wherein one or more applies of: (i) the distance calculation stage may comprise calculating a distance di between a first of the plurality of first binding sites and a second of the plurality of first binding sites based on the emission signal; and (ii) the distance calculation stage may comprise calculating a distance d2 between the first binding site and the second binding site based on the emission signal. Thus, in specific embodiments, the distance calculation stage may comprise calculating a distance di between a first of the plurality of first binding sites and a second of the plurality of first binding sites based on the emission signal. Further, in specific embodiments, the distance calculation stage may comprise calculating a distance d2 between the first binding site and the second binding site based on the emission signal.

[0070] In embodiments, the FRET donor-acceptor pair chromophores may have an excitation radiation range and an emission radiation range for both the donor and the acceptor. Further, in embodiments, the FRET donor-acceptor pair chromophores may be configured to provide an emission signal comprising (i) donor emission radiation in the donor emission radiation range, and (ii) acceptor emission radiation in the acceptor emission radiation range, upon excitation with donor excitation radiation of the donor.

[0071] The FRET excitation and emission ranges may, for example, comprise wavelengths in the ultraviolet (UV) range, the visible light range, and / or the (near)-infrared ((N)IR) range. Hence, the FRET excitation and emission ranges may, for example, comprise a (sub)range selected from within the range of 200-1500 nm, especially from within the range of 400-800 nm. In embodiments, the donor excitation radiation range may comprise a (sub)range selected from the range of 200-1500 nm, especially from the range of 400-800 nm. In embodiments, the donor emission radiation range may comprise a (sub)range selected from the range of 200-1500 nm, especially from the range of 400-800 nm. In embodiments, the acceptor excitation radiation range may comprise a (sub)range selected from the range of 200-1500 nm, especially from the range of 400-800 nm. In embodiments, the acceptor emission radiation range may comprise a (sub)range selected from the range of 200-1500 nm, especially from the range of 400-800 nm. The FRET excitation and emission ranges will in general depend on the used FRET pairs.

[0072] For example, in embodiments, the FRET donor-acceptor chromophore pair may comprise Atto488 and Cy3, wherein Atto488 (the donor chromophore) and Cy3 (the acceptor chromophore) may be excited maximally at about 488 nm and 552 nm respectively, and wherein Atto488 and Cy3 may provide emission radiation at about 521 nm and 568 nm respectively. In further embodiments, the donor-acceptor chromophore pair may comprise Atto488 and Cy5, which may respectively be maximally excited at 488 nm and 650 nm, and may provide emission radiation at about 521 nm and 666 nm. In further embodiments, the donor-acceptor chromophore pair may comprise Cy3 and Cy5, which may respectively be maximally excited at 552 nm and 650 nm, and may provide emission radiation at about 568 nm and 666 nm. In further embodiments, the donor-acceptor chromophore pair may comprise Cy3 and Cy7, which may respectively be maximally excited at 488 nm and 750 nm, and may provide emission radiation at about 568 nm and 788 nm. In further embodiments, the donoracceptor chromophore pair may comprise Cy5 and Cy7, which may respectively be maximally excited at 650 nm and 750 nm, and may provide emission radiation at about 666 nm and 788 nm. In embodiments, the FRET donor-acceptor pair chromophores may comprise a chromophore pair selected from the group comprising Atto488 / Cy3, Atto488 / Cy3b, Atto488 / Cy5, Atto488 / Atto647n, Cy3 / Cy5, Cy3b / Cy5, Cy3 / Cy7, Cy3b / Cy7, and Cy5 / Cy7.

[0073] The term “chromophore” may herein especially refer to a fluorescent chemical molecule that upon excitation with light (e.g. radiation from a laser), emits light of a different wavelength. The FRET donor-acceptor pair chromophores (also: “donor-acceptor pair chromophores”) may especially comprise fluorescent molecules and / or phosphorescent molecules. The term “FRET donor-acceptor pair chromophores” may herein especially refer to two chromophores capable of FRET energy transfer.

[0074] In practice, the selection criteria for donors and acceptors may include sensitivity, stability, biocompatibility, technical availability, ease of conjugation and cost.

[0075] In embodiments, the FRET donor-acceptor pair chromophores may comprise one or more chromophores selected from the group comprising chromophores selected from the Cyanine family, the Alexa family, the Atto family, the Dy family, and the Rhodamine family. Different chromophore pairs may be sensitive at different distances, i.e., may provide a high effect regarding FRET efficiency for subnanometer distance changes (a high resolution). For example, the most commonly used (Cyanine family) FRET pair, Cy3-Cy5, may be most sensitive at a distance of ~5 nm, and may hence be used for probing a second binding site 3-7 nm away from the first binding site. For probing a second binding site less than ~4 nm away from the first binding site, a dye pair such as Cy3-Cy7 may be used that may be most sensitive at a distance of ~3 nm. For a second binding site more than ~6 nm away from the first binding site, a dye pair such as Cy2-Cy3 may be used that may be most sensitive at a distance of ~7- nm. The end-to-end distance of a FRET pair may also be altered by placing a chromophore at a different position in the probe, such as at a different part of the sequence of an aptamer. Hence the FRET donor-acceptor pair may be optimized by altering the chromophore pair. The FRET donor-acceptor pairing may be (essentially) unaffected by being bound to the first probe or second probe and may remain valid if switched, i.e. Cy3 may be bound to the first probe and Cy5 may be bound to the second probe and vice versa.

[0076] In embodiments, movement of the protein during the exposure stage may reduce the resolution of the characterization method. For example, if the protein diffuses and rotates in solution, the information from different sets of probes may not be combined. Hence, it may be beneficial to immobilize the protein, such that the diffusion and / or the rotation of the protein is reduced and / or (essentially) stopped. Thus, in embodiments, the method may comprise an immobilization stage. Especially, the immobilization stage may be executed prior to the exposure stage. Further, in specific embodiments, the immobilization stage may be executed prior to the labelling stage. In embodiments, the immobilization stage may comprise immobilizing the protein on a surface. Specifically, in embodiments, the protein may be immobilized on a surface, wherein the surface may be selected from the group comprising a glass surface, a quartz surface, a semiconductor surface (e.g. a complementary metal-oxide- semiconductor (CMOS) surface), and a (transparent) polymeric surface. In embodiments, the protein may be immobilized at the N terminus. Additionally or alternatively, the protein may be immobilized at the C terminus. Yet, in embodiments, the protein may be immobilized at a immobilization site configured between the N terminus and the C terminus. In embodiments, the immobilization site may comprise a localized protein substructure, such as a (labelled) amino acid. Hence, in specific embodiments, the method may comprise an immobilization stage prior to the exposure stage, wherein the immobilization stage comprises immobilizing the protein on a surface.

[0077] In embodiments, immobilization of the protein may be achieved using covalent chemistry. Further, in embodiments, the protein may be immobilized on a surface (such as a glass surface or quartz surface) by using biotin-Streptavidin binding or by using a covalent chemical approach (such as amine-NHS, 2PCA or thiol chemistry). In embodiments, a biotin or biotinylated polyethylene glycol (PEG) linker may be attached to the protein to facilitate immobilization of the protein on the surface. In embodiments where the protein may be immobilized at the N terminus, especially NHS chemistry, 2PCA chemistry or alkyne-ketene reaction may be used (to attach the PEG linker). Conversely, in embodiments where the protein may be immobilized at the C terminus, a decarboxylative alkylation reaction may be used (to attach the PEG linker). Further, in embodiments, the protein may be immobilized via a different approach, such as via binding to a small-molecule moiety (e.g. inhibitors or other ligands), binding to an antibody, binding to a nanobody, or binding to a capture molecule, such as an aptamer. Further, in embodiments, the protein may be denatured prior to the immobilization stage. Alternatively, in embodiments, the (native) structure of the protein may be (substantially) unaffected by the immobilization stage.

[0078] Further, in embodiments, the exposure stage may not comprise processes or methods that may (substantially) affect the structure, especially the primary structure, of the protein. Such processes or methods may comprise linearization and fragmentation of the protein. The exposure stage may further not comprise the use of enzymes for translocation, linearization, and fragmentation of the protein. Thus, the protein may comprise a “folded” protein, i.e. a protein in its three-dimensional structural conformation. Further, the protein may comprise at least 90% of the amino acid sequence of the protein as obtained from a biological sample, such as at least 95% of the amino acid sequence of the protein as obtained from the biological sample, such as at least 99% of the amino acid sequence of the protein as obtained from the biological sample. Hence, the folded conformation and structure of the (entire) protein may be preserved and characterized with minimal, such as essentially no, enzymatic alteration during the exposure stage.

[0079] This may be especially relevant for the characterization and identification of different protein variants caused by alternative splicing. Alternative splicing may relate to molecular mechanisms resulting in the transcription of many different messenger RNA’s (mRNA’s) from a single gene, with different mRNAs translating into different proteins. One such molecular mechanism is a process called RNA splicing, which facilitates removal of gene segments (termed “introns”) from a primary gene transcript. Alternative splicing is a process in which a single primary transcript results in a variety of mature RNAs leading to the production of a large number of proteoforms that may have different functions. Especially, alternative splicing may be one of the major sources of the diversity of the proteome. However, detection of alternatively spliced proteins may be of high relevance in disease detection and / or monitoring as several diseases, such as cystic fibrosis, cancer and Parkinson disease have been associated with mutations in their spliceoforms that lead to alternative splicing and abnormal protein production. However, current methods that employ protein degradation to characterize and identify proteins may not be able to differentiate between different proteoforms with largely overlapping amino acid sequences. Through protein fingerprinting of the whole protein, the method of the invention may be able to differentiate between such proteoforms with high accuracy.

[0080] In embodiments, the method may, prior to the exposure stage, comprise providing the protein from a protein source comprising the protein. Especially, the protein source may be a biological sample, such as from a bacterial sample, or such as from an archaeal sample, or such as from a protozoan sample, or such as from a fungal sample, or such as from a mammalian sample, or such as from a plant sample. The protein source may be a clinical sample obtained from a patient, or a research sample obtained from a cell culture, or a research sample obtained in situ. In embodiments, the labelling stage may occur within the protein source, e.g. the (native) protein may be labelled inside a (native) cell, such as inside a (native) plant cell. Hence, in embodiments, the protein may be labelled in its native environment, such as inside a cell. However, in embodiments, the protein may need to be immobilized prior to the exposure stage. Thus, in embodiments, the method may comprise an isolation stage. The isolation stage may comprise isolating the (labelled) protein from other cell components. Hence, in embodiments, the isolation stage may be executed between the labelling stage and the exposure stage. Further, the isolation stage may be executed between the labelling stage and the immobilization stage. Hence, in specific embodiments, the labelling stage may be performed inside a cell, and the method may comprise an isolation stage comprising isolating the protein.

[0081] In embodiments, the isolation stage may comprise one or more isolation techniques and purification techniques for proteins. Suitable isolation methods for proteins are known to the person skilled in the art. For instance, isolation methods may include cell lysis, extraction and / or differential centrifugation, though other isolation methods are herein not excluded. Further, purification techniques for proteins are known in the art, and may include one or more of e.g. precipitation and differential solubilization, size exclusion chromatography, ion-exchange chromatography, free-flow-electrophoresis, hydrophobic interaction chromatography, (immune)affinity chromatography, and high pressure liquid chromatography (HPLC). Further, the protein may be isolated using an affinity tag configured to attach to e.g. the first label.

[0082] In embodiments, the method may further be used to study protein-protein interaction (PPI). To this end, in embodiments, the proximity labelling enzyme may be fused (e.g. covalently bound) to at least part of a second protein (of interest). Specifically, the proximity labelling enzyme may be part of a fused protein, wherein the fused protein may further comprise at least part of a second protein. In embodiments, the second protein may be native to the cell, such as native to one (or more) of a bacterial cell, an archaeal cell, a protozoan cell, a fungal cell, a vertebrate cell (e.g. a mammalian cell), and a plant cell. In embodiments, the fused protein may be introduced into the cell. Additionally or alternatively, the fused protein may be expressed by the cell. In embodiments, the fused protein, especially the proximity labelling enzyme, may be configured to label amino acids on proteins interacting with the fused protein, especially with the second protein. Specifically, in embodiments, the fused protein may be configured to label amino acids (on proteins interacting with the fused protein) with a first label. In embodiments, after the labelling stage, the labelled proteins may be isolated (in the isolation stage) using an affinity tag configured to attach to the first label. Further, in embodiments, the isolated (labelled) proteins may be immobilized, and the (characterization) method may be executed on each (type of) labelled protein. Hence, the method may comprise characterizing, such as identifying, proteins interacting with the (fused) protein. Hence, in specific embodiments, the first proximity labelling enzyme may be part of a fused protein, wherein the fused protein may further comprise at least part of a second protein, and wherein the isolation stage may comprise isolating one or more proteins using an affinity tag configured to attach to the first label.

[0083] In embodiments, isolating the protein (or proteins interacting with the fused protein) using an affinity tag may facilitate the selective isolation of labelled proteins. Hence, in embodiments, the method of the invention may be particularly suitable for characterizing proteins available in relatively low abundance, such as with a low concentration in a small sample. In particular, the method of the invention may be suitable to characterize a single protein. Hence, in embodiments, the protein source may comprise less than 10 pM of the protein, further less than 5 pM of the protein, moreover less than 1 pM of the protein, especially less than 0.5 pM of the protein, or even less than 0.1 pM of the protein. In further embodiments, the protein source may comprise less than 100 pL, further less than 50 pL, moreover less than 10 pL, especially less than 5 pL, or even less than 1 pL.

[0084] In embodiments, the method may further comprise a structure prediction stage. In embodiments, the structure prediction stage may comprise predicting the structure of the protein based on the distance di. Further, in embodiments, the structure prediction stage may comprise predicting the structure of the protein based on the distance d2. Further yet, in embodiments, the structure prediction stage may comprise predicting the structure of the protein based on one or more of the distance dniand dn2. The structure may in embodiments be a localized protein substructure and may further be selected from the group comprising a primary protein structure, a secondary protein structure, a tertiary protein structure, and a quaternary protein structure. Especially, the structure may be selected from the group comprising a secondary protein structure, a tertiary protein structure, and a quaternary protein structure, such as particularly a tertiary protein structure. Hence, in specific embodiments, the method may comprise a structure prediction stage comprising predicting a structure of the protein based on one or more of the distance di and the distance d2; wherein the structure is selected from the group comprising a secondary protein structure, a tertiary protein structure, and a quaternary protein structure. In embodiments, by predicting the structure of the protein the FRET method facilitates the characterization and identification of the protein.

[0085] In embodiments, for each type of binding site, more probes may be added than there are binding sites available. Further, due to the nature of the transient binding, each binding site may (transiently) bind a plurality of probes during the exposure stage. Hence, the protein may be probed multiple times using the transiently binding probes. The transient interactions may be monitored in real time using a fluorescence microscope, such as especially a singlemolecule fluorescence microscope. The repetitive probing may allow for the FRET efficiency to be determined with less than 0.1% error, which may provide a smaller than 0.1 nm resolution. Furthermore, as FRET may require the binding of two (different) probes, and thus the colocalization of 2 chromophores, the number of false positives may be reduced compared to assays that use either a single chromophore, or a FRET pair of which one member is covalently bound to the protein. The emission signals from the FRET pairings may be repetitively recorded for the protein until a FRET “fingerprint” sufficient for profiling of the structure of the protein is obtained. Thus, from recording the emission signal per second probe, a FRET histogram may be provided. The FRET histogram may comprise mean FRET efficiency for each observed FRET event. The FRET histogram may be fit using a Gaussian function. The center of the peak may be determined with an accuracy (standard error) of up to a maximum of 2%, such as up to a maximum of 1%, like up to a maximum of 0.5%, especially up to a maximum of 0.1%, moreover up to a maximum of 0.05%, when at least 100 binding events are recorded. The distribution of the center of the peaks from all measurements may be called a ‘FRET fingerprint’ that is unique for the structure of the protein. The term “protein fingerprint” may herein refer to a protein-specific (unique) signal, especially wherein the protein fingerprint is suitable for identification of the protein. Herein, the protein fingerprint may especially refer to one or more of an array of FRET efficiency values; an array of estimated distances; and / or raw data, especially one or more emission signals, obtained according to the method of the invention.

[0086] Thus, in embodiments, the method may further comprise a fingerprint provision stage. Herein, the fingerprint provision stage may comprise providing a protein fingerprint based on the emission signal. The protein fingerprint may in embodiments be provided (in realtime) during the exposure stage. In other embodiments, the protein fingerprint may be provided following the exposure stage. The fingerprint provision stage may in embodiments be followed by a protein identification stage. Herein, the protein identification stage may comprise identifying the protein by comparing the protein fingerprint to protein-related information in reference data. Such reference data may comprise previously acquired protein fingerprints that may allow for the identification of the presence of the same protein in a (biological) sample. Hence, in specific embodiments, the method may further comprise: (i) a fingerprint provision stage comprising providing a protein fingerprint based on the emission signal; and (ii) a protein identification stage comprising identifying the protein by comparing the protein fingerprint to protein-related information in reference data.

[0087] In another aspect, the invention may provide a system for characterization of a structure of a protein. In embodiments, the system may comprise an analytical space, an enzyme supply, a probe supply, a radiation source, a fluorescence microscope, especially a single-molecule fluorescence microscope, and a control system. In embodiments, the analytical space may comprise an analytical surface and may be configured to host the protein. Further, in embodiments, the enzyme supply may be configured to provide proximity labelling enzymes to the analytical space. In embodiments, the probe supply may be configured to provide probes to the analytical space. Additionally or alternatively, the radiation source may be configured to provide radiation, especially radiation in a donor excitation radiation range, to the analytical space. Further, the fluorescence microscope may especially comprise a single-molecule fluorescence microscope, and may be configured to measure emission radiation having a wavelength in a donor emission radiation range and in an acceptor emission radiation range in the analytical space. In embodiments, the (single-molecule) fluorescence microscope may be configured to (then) provide a related emission signal to the control system. The control system may in some embodiments comprise an analysis system, and in other embodiments, the control system may be functionally coupled to an analysis system. In an operational mode (of the system), the system, especially the control system, may be configured to execute the method of the invention. Hence, a system may be provided that performs the method as described above for characterization of a structure of a protein. Especially, the control system may be configured to have the system execute the various stages of the method as described above.

[0088] Hence, in specific embodiments, the invention may provide a system for characterization of a structure of a protein, wherein the system comprises an analytical space, an enzyme supply, a probe supply, a radiation source, a single-molecule fluorescence microscope, and a control system, wherein the analytical space is configured to host the protein, wherein the enzyme supply is configured to provide proximity labelling enzymes to the analytical space, wherein the probe supply is configured to provide probes to the analytical space, wherein the radiation source is configured to provide excitation radiation to the analytical space, wherein the single-molecule fluorescence microscope is configured to measure emission radiation in the analytical space and to provide a related emission signal to the control system, and wherein in an operational mode the control system is configured to execute the method of the invention.

[0089] The term “controlling” and similar terms especially refer at least to determining the behavior or supervising the running of an element. Hence, herein “controlling” and similar terms may e.g. refer to imposing behavior to the element (determining the behavior or supervising the running of an element), etc., such as e.g. measuring, displaying, actuating, opening, shifting, changing temperature, etc.. Beyond that, the term “controlling” and similar terms may additionally include monitoring. Hence, the term “controlling” and similar terms may include imposing behavior on an element and monitoring the element. The controlling of the element can be done with a control system, which may also be indicated as “controller”. The control system and the element may thus at least temporarily, or permanently, functionally be coupled. The element may comprise the control system. In embodiments, the control system and element may not be physically coupled. Control can be done via wired and / or wireless control. The term “control system” may also refer to a plurality of different control systems, which especially are functionally coupled, and of which e.g. one control system may be a master control system and one or more others may be slave control systems. A control system may comprise or may be functionally coupled to a user interface.

[0090] The system, or apparatus, or device may execute an action in a “mode” or “operational mode”. The term “operational mode” may also be indicated as “controlling mode”. Likewise, in a method, an action or stage may be executed in a “mode” or “operational mode.” This does not exclude that the system, or apparatus, or device may also be adapted for providing another controlling mode, or a plurality of other controlling modes. Likewise, this may not exclude that before executing the mode and / or after executing the mode one or more other modes may be executed.

[0091] The control system, or especially the analysis system, may be configured to determine a protein fingerprint based on the emission signal. The control system, or especially the analysis system, may further be configured to identify the protein by comparing the protein fingerprint to protein-related information in reference data. Hence, in specific embodiments, the control system may be configured to estimate a protein fingerprint based on the emission signal, wherein the control system may further be configured to identify the protein by comparing the protein fingerprint to protein-related information in reference data. Such embodiments may be especially suitable for the identification of a protein that has been characterized previously and may provide information about the (biological) sample, e.g. identifying protein biomarkers of disease in clinical samples.

[0092] The control system, especially the analysis system, may be configured to calculate a distance di (or a distance d2) between a first of the plurality of first binding sites and a second of the plurality of first binding sites (or between the first binding site and the second binding site) based on the emission signal. Further, in embodiments, the control system, especially the analysis system, may be configured to calculate one or more of a distance dniand a distance dn2 based on the emission signal. The control system, especially the analysis system, may further be configured to predict the structure of the protein based on one or more of the calculated distance di and the calculated distance d2. Further, in embodiments, the control system, especially the analysis system, may be configured to predict the structure of the protein based on one or more of a distance dniand a distance dn2. Hence, in specific embodiments, the control system may be configured to calculate one or more of a distance di and a distance d2 based on the emission signal, wherein the control system may further be configured to predict the structure of the protein based on one or more of the calculated distance di and the calculated distance d2. Such embodiments may be especially suitable for the characterization of a protein that has not been characterized previously, e.g. the investigation of a protein being developed as a novel drug candidate.

[0093] In embodiments, the control system may be configured to predict the (protein) structure of the protein (using a computational process), especially based on the calculated distance, or especially based on the FRET efficiency. The computational process may especially comprise a computational algorithm. In further embodiments, the computational process may comprise a refinement of a model protein structure based on the calculated distance. In further embodiments, the computational process may comprise a de novo protein structure prediction. In such embodiments, the calculated distance may especially comprise a plurality of calculated distances. In embodiments, the control system may be configured to execute in a controlling mode the method according to the invention. The control system may especially receive program instructions from a data carrier such that the control system (causes the system to) executes the method according to the invention.

[0094] Hence, in a further aspect, the invention may provide a data carrier having stored thereon program instructions. Such program instructions when executed by the system described above may cause the system to execute the method described above. Hence, in specific embodiments, the invention may provide a data carrier having stored thereon program instructions, which when executed by the system described herein may cause the system to execute the method as described herein.

[0095] In embodiments, the data carrier may facilitate the execution of preprogrammed operational modes of the system. This may increase user convenience and adherence to standard use of the system.

[0096] Further, the data carrier may comprise the reference data that may be used in the protein identification stage.

[0097] The term “stage” and similar terms used herein may refer to a (time) period (also “phase”) of a method and / or an operational mode. The different stages may (partially) overlap (in time). For example, the exposure stage may, in general, be initiated prior to the fingerprint provision stage, but may partially overlap in time therewith. However, for example, the isolation stage may typically be completed prior to the exposure stage. It will be clear to the person skilled in the art how the stages may be beneficially arranged in time.

[0098] The method and / or system may be applied in or may be part of analysis methods / sy stems of biological samples, such as protein samples, particularly in relation to protein sequencing, protein structure elucidation, and / or protein interactomics. In further embodiments, the method may be a non-medical method. In further embodiments, the method may be a non-diagnostic method.

[0099] BRIEF DESCRIPTION OF THE DRAWINGS

[0100] 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. 1A-C schematically depict embodiments of the method for characterization of a protein. Fig. 2 schematically depicts embodiments of the method for characterization of protein-protein interactions. Fig. 3 schematically depicts embodiments of the system for characterization of a structure of a protein. The schematic drawings are not necessarily to scale.

[0101] DETAILED DESCRIPTION OF THE EMBODIMENTS

[0102] Fig. 1 A schematically depicts an embodiment of the method for characterization of a protein 10. The method may comprise a labelling stage comprising exposing the protein 10 to a first proximity labelling enzyme 71. Said first proximity labelling enzyme 71 may be configured to label amino acids of a first amino acid type with a first label 81. Further, in embodiments, said first proximity labelling enzyme 71 may be configured to label post- translational modifications (PTMs) of a first post-translational modification type with a first label 81. Especially, as depicted in Fig. 1A, the first proximity labelling enzyme 71 may catalyze the generation of a first short-lived reactive species 810 (comprising the first label 81) in the vicinity of the protein 10. Hence, in embodiments, the first proximity labelling enzyme 71 may activate the first label 81 for reaction with an amino acid (or PTM) of the first amino acid type (or PTM type).

[0103] Following the labelling stage, the method may comprise an exposure stage, as schematically depicted in Fig. IB. The exposure stage may comprise exposing the protein 10 to a first probe 31 and a second probe 32. Further, the exposure stage may comprise providing excitation radiation 50 to the protein 10. Additionally, the exposure stage may comprise measuring emission radiation 60 to provide an emission signal 61.

[0104] In embodiments, the first probe 31 may be configured to transiently bind the protein 10 at a first binding site 11. Especially, the first label 81 may provide at least part of the first binding site 11, such as (essentially) the whole first binding site 11. Further, the first probe 31 may comprise a first chromophore 21. Conversely, the second probe may 32 comprise a second chromophore 22. Further, the second probe 32 may be configured to transiently bind the protein 10 at the first binding site 11. In the embodiment depicted in Fig. IB, the protein 10 may especially comprise two first binding sites 11, wherein a first of the two first binding sites 11 is indicated by reference I la, and the second of the two first binding sites 11 is indicated by reference 1 lb. Further, in embodiments, the second probe 32 may be configured to transiently bind the protein 10 at a second binding site 12. Note that here, for explanatory purposes, both binding of the second probe 32 to the first binding site 11 and to the second binding site 12 is depicted, though generally the second probe 32 may be configured to selectively bind to only one of the first binding site 11 and the second binding site 12, and may therefore not bind to the other of the first binding site 11 and the second binding site 12. In embodiments, the first chromophore 21 and the second chromophore 22 may form a FRET pair having a donor excitation range, a donor emission radiation range and an acceptor emission radiation range. Especially, in embodiments, the excitation radiation 50 may comprise a wavelength selected from the donor excitation radiation range. Further, the emission radiation 60 may be measured in the donor emission radiation range and in the acceptor emission radiation range.

[0105] Returning to Fig. 1 A, the labelling stage may comprise exposing the protein 10 to a second proximity labelling enzyme 72. The second proximity labelling enzyme 72 may in embodiments be configured to label amino acids (or PTMs) of a second amino acid type (or PTM type) with a second label 82. Especially, the second proximity labelling enzyme 72 may catalyze the generation of a second short-lived reactive species 820 (comprising the second label 82) in the vicinity of the protein 10. In embodiments, the second label 82 may provide at least part of a second binding site 12 (on the protein 10). In embodiments, a second probe 32 may be configured to transiently bind the protein 10 at the second binding site 12.

[0106] In embodiments, the first proximity labelling enzyme 71 and the second proximity labelling enzyme 72 may comprise different enzymes selected from the group comprising HRP, APEX, APEX2, APEX2C32S, BioID, BIOID2, BASU, TurboID, miniTurbo, AirlD, microID, ultralD, miniSOG, Tyrosinase, mgSrtA, Lipoic Acid Ligase, and Laccase. Further, in embodiments, the method may employ a plurality of proximity labelling enzymes 70. Hence, in embodiments, the labelling stage may comprise exposing the protein 10 to n different proximity labelling enzymes 70. In embodiments, n > 3, and the n different proximity labelling enzymes 70 may be configured to label amino acids of n different amino acid types with different labels 80. Especially, the n different proximity labelling enzymes 70 may catalyze the generation of (n) different short-lived reactive species 800 (comprising the (n) different labels 80) in the vicinity of the protein 10. In such embodiments, the exposure stage may comprise successively exposing the protein 10 to different sets of probes 33, wherein each set may comprise at least two different probes 33. Said at least two different probes 33 may be configured to transiently bind the protein 10 at at least two different labels 80. Further, said at least two different probes 33 may comprise at least two different chromophores 20 forming a respective FRET pair. A (non-exhaustive) overview of proximity labelling enzymes 70 with their respective substrates, labelling activation stimuli, and reaction times is provided in table 1.

[0107] Table 1: overview of proximity labelling enzymes.

[0108] As can be seen from Table 1, the proximity labelling enzymes 70 may be grouped by the type of amino acid they label. For instance, the proximity labelling enzyme 70 may be selected from the group of tyrosine-selective proximity labelling enzymes, wherein the group of tyrosine-selective proximity labelling enzymes comprises HRP, APEX, APEX2, APEX2C32S, and Laccase. Further, the proximity labelling enzyme 70 may be selected from the group of lysine-selective proximity labelling enzymes, wherein the group of lysineselective proximity labelling enzymes comprises BioID, BioID2, BASU, TurboID, miniTurbo, AirlD, microID, ultralD, and LipoID. In embodiments, the first proximity labelling enzyme 71 and the second proximity labelling enzyme 72 may be selected from different groups, i.e. the first proximity labelling enzyme 71 and the second proximity labelling enzyme 72 may be selective for different amino acid types. For instance, the first proximity labelling enzyme 71 may be selected from the group of tyrosine-selective proximity labelling enzymes, and the second proximity labelling enzyme 72 may be selected from the group of lysine-selective proximity labelling enzymes. Further, in embodiments, the n proximity labelling enzymes 70 may (each) be selected from a different group.

[0109] In (other) embodiments, the method may comprise a labelling stage comprising exposing the protein 10 to a first proximity labelling enzyme 71, wherein the first proximity labelling enzyme 71 may be configured to label amino acids of a first amino acid type with a first label 81. Further, the method may comprise an exposure stage comprising exposing the protein 10 to a first probe 31, providing excitation radiation 50 to the protein 10, and measuring emission radiation 60 to provide an emission signal 61. In embodiments, the first probe 31 may be configured to transiently bind the protein 10 at a first binding site 11, wherein the first label 81 may provide at least part of the first binding site 11. In embodiments, the first probe 31 may comprise a first chromophore 21. Further, in embodiments, the first chromophore 21 may have an excitation radiation range and an emission radiation range. Especially, in embodiments, the radiation 50 may comprise a wavelength selected from the excitation radiation range. Further, in embodiments, the emission 60 may be measured in the emission radiation range. Further, in specific embodiments, the method may comprise determining a concentration of the first binding site 11 based on a first frequency. Additionally or alternatively, the method may comprise determining a number of amino acids (of the first amino acid type) in the protein 10 based on the first frequency.

[0110] Fig. IB schematically shows an embodiment of the method for characterization of a protein 10. In embodiments, the first probe 31 and the second probe 32 may comprise affinity -based probes 35. In embodiments, said affinity -based probes 35 may be selected from the group comprising an aptamer, an antibody, a nanobody, a peptide, and an organic ligand. In the specific embodiment depicted here, the first probe 31 and the second probe 32 may especially comprise aptamers.

[0111] In embodiments, the first probe 31 and the second probe 32 may thus be configured to transiently bind the protein 10 (at the first binding site 11 and optionally the second binding site 12). In embodiments, said transient binding may be described using binding kinetics that at least comprise an off-rate. In embodiments, the off-rate may indicate the amount of probe that dissociates from a protein-probe complex over a specific time. In embodiments, the first probe 31 may be configured to transiently bind the protein 10 at the first binding site 11 with an off-rate selected from the range of 0.1-10 s'1. Further, in embodiments, the second probe 32 may be configured to transiently bind the protein 10 at the first binding site 11 with an off-rate selected from the range of 0.1-10 s'1. Alternatively, in embodiments, the second probe 32 may be configured to transiently bind the protein 10 at the second binding site 12 with an off-rate selected from the range of 0.1-10 s'1.

[0112] Fig. 1C schematically depicts a further embodiment of the method for characterization of a protein 10. In embodiments, the method may comprise an immobilization stage. The immobilization stage may especially be executed prior to the exposure stage. In embodiments, the immobilization stage may comprise immobilizing the protein 10 on a surface 1000. Such immobilization of the protein 10 may be achieved by using biotin-streptavidin binding or by using a covalent chemical approach (such as amine-NHS, 2PCA or thiol chemistry). Fig. 1C schematically depicts an embodiment of a biotin-streptavidin binding, wherein the biotin is attached to the protein 10 via a PEG linker, and wherein the streptavidin is attached to the surface 1000 via a second linker.

[0113] In embodiments, as depicted in e.g. Fig. 1A and Fig. IB, the protein 10 may comprise a plurality of first binding sites 11. Further, in embodiments, the method may comprise a distance calculation stage. The distance calculation stage may, in embodiments, comprise calculating a distance di between a first of the plurality of first binding sites 11 (such as first binding site I la) and a second of the plurality of first binding sites (11) (such as first binding site 11b) based on the emission signal 61. Additionally or alternatively, the distance calculation stage may comprise calculating a distance d2 between the first binding site 11 and the second binding site 12 based on the emission signal 61, as depicted in Fig. 1C. In embodiments, the distance di and / or the distance d2 may be calculated based on the efficiency of the energy transfer between a FRET donor and a FRET acceptor. Schematically depicted in Fig. 1C is irradiation of a FRET donor (comprised by the first chromophore 21) by (donor) excitation radiation 50. Upon absorption of the excitation radiation 50, the first chromophore 21 may especially emit donor emission radiation 63. Said donor emission radiation 63 may in embodiments comprise one or more wavelengths selected from the acceptor excitation radiation range. Hence, in embodiments, the FRET acceptor (comprised by the second chromophore 22) may absorb at least part of the donor emission radiation 63 to generate acceptor emission radiation 64. Hence, the emission radiation 60 may in embodiments comprise donor emission radiation 63 and acceptor emission radiation 64. Further, in embodiments, the first (donor) chromophore 21 may upon excitation with donor excitation radiation transfer energy non-radiatively to the second (acceptor) chromophore 22, whereupon the second (acceptor) chromophore 22 may emit acceptor emission radiation 64. Said energy transfer may occur with a specific FRET efficiency depending on the (exact) distance between the first (donor) chromophore 21 and the second (acceptor) chromophore 22. Hence, by measuring the FRET (transfer) efficiency, information regarding the distance di and / or the distance d2 may be obtained. Note that in the embodiment depicted here and in the description above, the first chromophore 21 comprises the FRET donor and the second chromophore 22 comprises the FRET acceptor, yet this not need to be the case. In embodiments, the method may comprise a structure prediction stage comprising predicting a structure 13 of the protein 10 based on one or more of the distance di and the distance d2. In embodiments, the structure 13 may be selected from the group comprising a secondary protein structure, a tertiary protein structure, and a quaternary protein structure. Further, in embodiments, the method may comprise a fingerprint provision stage comprising providing a protein fingerprint based on the emission signal 61. Additionally, the method may comprise a protein identification stage comprising identifying the protein 10 by comparing the protein fingerprint to protein-related information in reference data.

[0114] In embodiments, the labelling stage may be performed inside a cell. In such embodiments, the method may comprise an isolation stage comprising isolating the protein 10. The isolation stage may especially be executed between the labelling stage and the exposure stage. Alternatively, the isolation stage may be executed between the labelling stage and the immobilization stage.

[0115] Fig. 2 schematically depicts an embodiment of the method for the characterization of protein-protein interactions. In embodiments, the first proximity labelling enzyme 71 may be part of a fused protein 100. The fused protein 100 may (besides the first proximity labelling enzyme 71) further comprise at least part of a second protein 110. The second protein 110 may especially be (any protein or protein substructure) native to the cell. Further, in embodiments, the fused protein 100 may be expressed by the cell. In embodiments, the fused protein 100, especially the first proximity labelling enzyme 71, may be configured to label one or more proteins 1 with the first label 81. Especially, the one or more proteins 1 may comprise proteins 1 configured to interact and / or form a complex with the fused protein 100. Hence, in embodiments, the fused protein 100, especially the first proximity labelling enzyme 71, may be configured to only label one or more interacting proteins 1 (wherein the one or more interacting proteins 1 interact with the fused protein 100) with the first label 81. In such embodiments, the isolation stage may comprise isolating (said) one or more proteins 1 using an affinity tag configured to attach to the first label 81. Hence, one or more proteins 1 interacting with the fused protein 100 may be isolated and characterized, thereby facilitating the studying of protein-protein interactions.

[0116] Fig. 3 schematically depicts an embodiment of the system 200 according to the invention. In particular, it depicts a system 200 for characterization of a structure 13 of a protein 10. The system 200 may comprise an analytical space 210, an enzyme supply 220, a probe supply 230, a radiation source 250, a single-molecule fluorescence microscope 240, and a control system 300. In embodiments, the analytical space 210 may be configured to host the protein 10. Additionally, the enzyme supply 220 may be configured to provide proximity labelling enzymes 70,71,72 to the analytical space 210. Further, the probe supply 230 may be configured to provide probes 31,32,33 to the analytical space 210. Further yet, the radiation source 250 may be configured to provide excitation radiation 50 (especially in a donor excitation radiation range) to the analytical space 210. In further embodiments, the singlemolecule fluorescence microscope 240 may be configured to measure emission radiation 60 (in a donor emission radiation range and in an acceptor emission radiation range) in the analytical space 210, and to provide a related emission signal 61 to the control system 300. In an operational mode, the control system 300 may be configured to execute the method as described above.

[0117] In the depicted embodiment, the radiation source 250 comprises a plurality of radiation sources 250, especially configured to provide different wavelengths of radiation. In particular, the radiation source 250 may be suitable to provide radiation in the donor excitation radiation range of different FRET donors (comprised by different chromophores).

[0118] In the depicted embodiment, the single-molecule fluorescence microscope 240 comprises or is functionally coupled to a plurality of optical elements configured to separate the emission radiation 60 emitted from the analytical space 210 (by the donor chromophore and / or the acceptor chromophore) into the donor emission radiation 63 and acceptor emission radiation 64. The single-molecule fluorescence microscope 240 may comprise an EMCCD camera 241 to measure the donor emission radiation 63 and the acceptor emission radiation 64. It will be clear to the person skilled in the art, that many variations of the single-molecule fluorescence microscope 240 and / or the optical elements may be possible without deviating from the scope of the invention as described herein.

[0119] In embodiments, the control system 300 may be configured to estimate a protein fingerprint based on the emission signal 61. In further embodiments, the control system 300 may be configured to identify the protein 10 by comparing the protein fingerprint to protein- related information in reference data. Further, in embodiments, the control system 300 may be configured to calculate one or more of a distance di and a distance d2 based on the emission signal 61. In such embodiments, the control system 300 may further be configured to predict the structure 13 of the protein 10 based on one or more of the calculated distance di and the calculated distance d2.

[0120] Fig. 3 further schematically depicts a data carrier 400 having stored thereon program instructions, which when executed by the system 200 (especially by the control system 300) according to the invention, causes the system 200 to execute the method according to the invention. In further embodiments, the control system 300 may comprise the data carrier 400.

[0121] Experiments

[0122] Embodiments of the method of the invention were experimentally evaluated using different proximity labelling enzymes 70.

[0123] Experiment 1 - labelling of tyrosine residues with APEX

[0124] In a first experiment, tyrosine residues in the protein 10 alpha-synuclein are labeled using APEX as the proximity labelling enzyme 70. The label 80 used is a phenol- tetramethylrhodamine conjugate, and hydrogen peroxide (H2O2) is added as an activation stimulus. The concentration of the label 80 is varied from 0.5-5.5 mM. The concentration of the other reaction components are provided in Table 1.

[0125] Table 1. Concentrations of reagents in Experiment 1.

[0126] The reagents are mixed in a lx PBS buffer, and incubated at room temperature for 15 minutes. After incubation, the reactions are quenched using 40 mM sodium azide, 10 mM Trolox, and 40 mM sodium ascorbate. The extent of the tyrosine labelling is determined using protein gel electrophoresis. The result of the protein gel electrophoresis is depicted in Fig. 4A, wherein the different lanes indicate different concentrations (Cone.) of the label 80, and wherein a darker color corresponds to a higher concentration of the label 80, specifically of a phenol- tetramethylrhodamine conjugate label. In Fig. 4A, the line indicated by a triangle indicates the labelled protein 10, and the line indicated by the circle indicates the APEX enzyme. As can be seen from Fig. 4A, an increase in the concentration of the label 80 is seen upon increasing the concentration of the label 80 from 0.5 mM to 4.0 mM. A further increase in the concentration of the label 80 does not appear to have a substantial impact on the observed concentration of the label 80.

[0127] The effect of the incubation time is further tested, wherein at least some labelling is observed after an incubation time of 1 min, and no substantial increase in the concentration of the label 80 is observed for incubation times of > 10 minutes (relative to an incubation time of 10 minutes).

[0128] Further, the effect of the concentration of the labelling activation stimulus H2O2 is tested. At an H2O2 concentration of 0.5 mM a higher concentration of the label 80 was observed than at H2O2 concentrations of 0.25 mM and of 1 mM, although labelling is observed for H2O2 concentrations up to 8.0 mM (the highest concentration tested).

[0129] Hence, in embodiments, the labelling stage may comprise providing a stimulus concentration of a labeling activation stimulus to the proximity labelling enzyme. Especially, in embodiments, the proximity labelling enzyme may be H2O2, and the stimulus concentration may be selected from the range of > 0.01 mM, such as from the range of > 0.1 mM, especially from the range of > 0.25 mM. In further embodiments, the proximity labelling enzyme may be H2O2 and the stimulus concentration may be selected from the range of < 2 mM, such as from the range of < 1 mM, especially from the range of < 0.8. In further embodiments, the proximity labelling enzyme may be H2O2 and the stimulus concentration may be selected from the range of 0.1 - 1 mM, such as from the range of 0.25 - 0.8 mM.

[0130] Increasing the concentration of the APEX enzyme 70 from 5 pm to 25 pm appears to improve labelling efficiency. However, using less enzyme may be preferable in terms of costs.

[0131] Experiment 2 - labelling of cysteine residues with Tyrosinase

[0132] In a second experiment, cysteine residues in the protein 10 alpha-synuclein are labeled using megaTYR (Tyrosinase) as the proximity labelling enzyme 70. The label 80 used is a phenol-tetramethylrhodamine conjugate, and no activation stimulus was provided. The concentration of the label 80 is varied from 0.0-5.0 mM. The concentration of the other reaction components are provided in Table 2.

[0133] Table 2. Concentrations of reagents in Experiment 2.

[0134] The reagents are mixed in a 20 mM sodium phosphate buffer (pH 6.5), and incubated at room temperature for 30 minutes. After incubation, the reactions are quenched using 4 mM tropolone. The extent of the cysteine labelling is determined using protein gel electrophoresis. The result of the protein gel electrophoresis is depicted in Fig. 4B, wherein the different lanes indicate different concentrations (Cone.) of the label 80, and wherein a darker color corresponds to a higher concentration of the label 80, specifically the phenol- tetramethylrhodamine conjugate label. In Fig. 4B, the line indicated by a triangle indicates the labelled protein 10, and the line indicated by the circle indicates the megaTYR enzyme. As can be seen from Fig. 4B, an increase in the concentration of the label 80 is seen upon increasing the concentration of the label 80 from 0.5 mM to 3.0 mM. A further increase in the concentration of the label 80 does not appear to have a substantial impact on the concentration of the label 80.

[0135] The effect of the concentration of the megaTYR enzyme is also tested, and labelling of the protein 10 is observed with concentrations as low as 0.1 pM (the lowest concentration tested). Increasing the concentration of megaTYR beyond 0.5 pm did not appear to have a large effect on the labelling efficiency.

[0136] Experiment 3 - labelling of lysine residues with TurboID

[0137] In a third experiment, lysine residues in the protein 10 alpha-synuclein are labeled using TurboID as the proximity labelling enzyme 70. The label 80 used is biotin and ATP. The concentration of the reaction components are provided in Table 3.

[0138] Table 3. Concentrations of reagents in Experiment 3.

[0139] The reagents are mixed in a 40 mM tris-HCl buffer (pH 8.0), and incubated for 0.5-16 h. The extent of the tyrosine labelling is determined using protein gel electrophoresis. The result of the protein gel electrophoresis is depicted in Fig. 4C, wherein the different lanes indicate different incubation times (in hours), and wherein a darker color corresponds to a higher concentration of the label 80, particularly of the biotin label. In Fig. 4C, the line indicated by a triangle indicates the labelled protein 10, and the line indicated by the circle indicates the TurboID enzyme. As can be seen from Fig. 4C, an increase in the concentration of the label 80 is seen upon increasing the incubation time from 0.5 h to 8 h. A further increase in incubation time from 8 h to ~16 h (indicated by reference ON) does not appear to have a substantial impact on the concentration of the label 80. Fig. 4C appears to depict an increase in signal in the line corresponding to the TurboID enzyme upon longer incubation times. This may be the result of self-labelling of the TurboID enzyme, wherein biotin moieties attach to lysine residues in the TurboID.

[0140] The experiments described hereinabove demonstrate successful labelling of proteins 10 using proximity labelling enzymes 70, with different concentrations of the proximity labelling enzymes 70, labels 80, and activation stimuli, and with different labelling durations (or “incubation times”). It will be clear to the person skilled in the art that suitable incubation times and suitable concentrations for the proximity labelling enzyme 70, the label 80, and an activation stimulus may vary, e.g., based on which proximity labelling enzyme 70 is used or which protein 10 is being labelled. The person skilled in the art will be capable of determining suitable incubation times and suitable concentrations.

[0141] The term “plurality” refers to two or more. 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%. 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".

[0142] 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.

[0143] 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.

[0144] 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”, 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.

[0145] 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. In yet a further aspect, the invention (thus) provides a software product, which, when running on a computer is capable of bringing about (one or more embodiments of) the method as described herein.

[0146] 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 is functionally coupled to or comprised by the device, apparatus, or system, controls one or more controllable elements of such device, apparatus, or system.

[0147] 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.

[0148] 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

CLAIMS:

1. A method for characterization of a protein (10), wherein the method comprises: a labelling stage comprising exposing the protein (10) to a first proximity labelling enzyme (71), wherein the first proximity labelling enzyme (71) is configured to label amino acids of a first amino acid type with a first label (81); and an exposure stage comprising: (i) exposing the protein (10) to a first probe (31), (ii) providing excitation radiation (50) to the protein (10), and (iii) measuring emission radiation (60) to provide an emission signal (61); wherein: the first probe (31) comprises an affinity-based probe (35) selected from the group comprising an aptamer, an antibody, a nanobody, a peptide, and an organic ligand; the first probe (31) is configured to transiently bind the protein (10) at a first binding site (11), wherein the first label (81) provides at least part of the first binding site (11), and wherein the first probe (31) comprises a first chromophore (21); and the first chromophore (21) has an excitation radiation range and an emission radiation range, wherein the radiation (50) comprises a wavelength selected from the excitation radiation range, and wherein the emission (60) is measured in the emission radiation range.

2. The method according to claim 1, wherein the first probe (31) is configured to transiently bind the protein (10) at the first binding site (11) with a first frequency; wherein the first frequency is dependent on one or more of the concentration of the first probe (31) and the concentration of the first binding site (11); wherein the method comprises determining (i) a concentration of the first binding site (11) based on the first frequency, and / or (ii) a number of amino acids of the first amino acid type in the protein (10) based on the first frequency.

3. The method according to claim 1, wherein: the exposure stage comprises exposing the protein (10) to a second probe (32); the second probe (32) comprises a second chromophore (22), wherein the second probe (32) is configured to transiently bind the protein (10) at (i) the first binding site (11) or (ii) a second binding site (12); the second probe (32) comprises an affinity -based probe (35) selected from the group comprising an aptamer, an antibody, a nanobody, a peptide, and an organic ligand; andthe first chromophore (21) and the second chromophore (22) form a FRET pair having a donor excitation range, a donor emission radiation range and an acceptor emission radiation range, wherein the excitation radiation (50) comprises a wavelength selected from the donor excitation radiation range, and wherein the emission radiation (60) is measured in the donor emission radiation range and in the acceptor emission radiation range.

4. The method according to claim 3, wherein the labelling stage comprises exposing the protein (10) to a second proximity labelling enzyme (72), wherein the second proximity labelling enzyme (72) is configured to label amino acids of a second amino acid type with a second label (82), wherein the second label (82) provides at least part of the second binding site (12), and wherein the second probe (32) is configured to transiently bind the protein (10) at the second binding site (12).

5. The method according to claim 4, wherein the first proximity labelling enzyme (71) and the second proximity labelling enzyme (72) comprise different enzymes selected from the group comprising HRP, APEX, APEX2, APEX2C32S, BioID, BIOID2, BASU, TurboID, miniTurbo, AirlD, microID, ultralD, miniSOG, Tyrosinase, mgSrtA, Lipoic Acid Ligase, and Laccase.

6. The method according to any one of the preceding claims 3-5, wherein the labelling stage comprises exposing the protein (10) to n different proximity labelling enzymes (70), wherein n > 3, wherein the n different proximity labelling enzymes (70) are configured to label amino acids of n different amino acid types with different labels (80), and wherein the exposure stage comprises successively exposing the protein (10) to different sets of probes (33), wherein each set comprises at least two different probes (33) configured to transiently bind the protein (10) at at least two different labels (80), and wherein the at least two different probes (33) comprise at least two different chromophores (20) forming a respective FRET pair.

7. The method according to any one of the preceding claims 3-6, wherein the first probe (31) is configured to transiently bind the protein (10) at the first binding site (11) with an off-rate selected from the range of 0.1-10 s’1, and wherein the second probe (32) is configured to transiently bind the protein (10) at one of (i) the first binding site (11) with an off-rate selected from the range of 0.1-10 s’1, and (ii) the second binding site (12) with an off- rate selected from the range of 0.1-10 s’1.

8. The method according to any one of the preceding claims, wherein the method comprises: an immobilization stage prior to the exposure stage, wherein the immobilization stage comprises immobilizing the protein (10) on a surface (1000).

9. The method according to any one of the preceding claims 3-8, wherein the protein (10) comprises a plurality of first binding sites (11), wherein the method comprises a distance calculation stage, wherein one or more applies of: the distance calculation stage comprises calculating a distance di between a first of the plurality of first binding sites (11) and a second of the plurality of first binding sites (11) based on the emission signal (61); and the distance calculation stage comprises calculating a distance d2 between the first binding site (11) and the second binding site (12) based on the emission signal (61).

10. The method according to claim 9, wherein the distance calculation stage comprises calculating a distance di between a first of the plurality of first binding sites (11) and a second of the plurality of first binding sites (11) based on the emission signal (61).

11. The method according to claim 9, wherein the distance calculation stage comprises calculating a distance d2 between the first binding site (11) and the second binding site (12) based on the emission signal (61).

12. The method according to any one of the preceding claims 9-11, wherein the method comprises: a structure prediction stage comprising predicting a structure (13) of the protein (10) based on one or more of the distance di and the distance d2; wherein the structure (13) is selected from the group comprising a secondary protein structure, a tertiary protein structure, and a quaternary protein structure.

13. The method according to any one of the preceding claims 3-12, wherein the method further comprises: a fingerprint provision stage comprising providing a protein fingerprint based on the emission signal (61);a protein identification stage comprising identifying the protein (10) by comparing the protein fingerprint to protein-related information in reference data.

14. The method according to any one of the preceding claims, wherein the labelling stage is performed inside a cell, and wherein the method comprises an isolation stage comprising isolating the protein (10).

15. The method according to claim 14, wherein the first proximity labelling enzyme (71) is part of a fused protein (100), wherein the fused protein (100) further comprises at least part of a second protein (110), and wherein the isolation stage comprises isolating one or more proteins (1) using an affinity tag configured to attach to the first label (81).

16. A system (200) for characterization of a structure (13) of a protein (10), wherein the system (200) comprises an analytical space (210), an enzyme supply (220), a probe supply (230), a radiation source (250), a single-molecule fluorescence microscope (240), and a control system (300), wherein the analytical space (210) is configured to host the protein (10), wherein the enzyme supply (220) is configured to provide proximity labelling enzymes (70,71,72) to the analytical space (210), wherein the probe supply (230) is configured to provide probes (31,32,33) to the analytical space (210), wherein the radiation source (250) is configured to provide excitation radiation (50) to the analytical space (210), wherein the single-molecule fluorescence microscope (240) is configured to measure emission radiation (60) in the analytical space (210) and to provide a related emission signal (61) to the control system (300), and wherein in an operational mode the control system (300) is configured to execute the method according to any one of the preceding claims 1-15.

17. The system (200) according to claim 16, wherein the control system (300) is configured to execute the method according to any one of the preceding claims 3-15; wherein the control system (300) is configured to estimate a protein fingerprint based on the emission signal (61), and wherein the control system (300) is configured to identify the protein (10) by comparing the protein fingerprint to protein-related information in reference data.

18. The system (200) according to any one of the preceding claims 16-17, wherein the control system (300) is configured to execute the method according to any one of the preceding claims 3-15; wherein the control system (300) is configured to calculate one or moreof (i) the distance di as defined in claim 9 and (ii) the distance d2 as defined in claim 9 based on the emission signal (61), wherein the control system (300) is configured to predict the structure (13) of the protein (10) based on one or more of the calculated distance di and the calculated distance d2.

19. A data carrier (400) having stored thereon program instructions, which, when executed by the system (200) according to any one of preceding claims 16-18, cause the system (200) to execute the method according to any one of preceding claims 1-15.

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