METHODS AND KITS FOR DETERMINING BINDING SITES

Abstract

The invention provides methods, compositions, and kits for the characterisation and analysis of proteins. Methods are provided for determining, on a protein, a binding site for a binding partner, the methods comprising: contacting a protein with a plurality of monomers, and polymerising the monomers to create a protein:polymer complex; digesting the protein in the complex to produce a peptide:polymer complex; isolating the peptide:polymer complex; and sequencing the peptide, wherein the peptide corresponds to a binding site for a binding partner.

Claims

1. A method for determining, on a protein, a binding site for a binding partner, comprising: contacting the protein with a plurality of monomers; polymerising the monomers to create a protein:polymer complex; digesting the protein in the complex to produce a peptide:polymer complex; and isolating the peptide:polymer complex wherein the peptide corresponds to a binding site for a binding partner.

2. The method of claim 1, comprising sequencing the peptide.

3. The method of claim 1, wherein the protein is a folded protein and/or wherein the binding site is a novel binding site, optionally wherein the binding site is an epitope to which an antibody binds.

4. The method of claim 1, wherein a complex between the protein and a molecularly imprinted polymer (MIP) is created by contacting the protein with the plurality of monomers and polymerising the monomers to create the protein:polymer complex.

5. The method of claim 1, wherein the plurality of monomers comprises cross-linking monomers, or magnetic nanoparticles.

6. (canceled)

7. The method of claim 1, wherein the protein contacted with a plurality of monomers is in solution or immobilised on a solid surface, and the solid surface is selected from a surface comprising polysaccharide, silica, organic or inorganic polymer, metal, or a combination thereof, and/or the form of the solid surface is selected from beads, magnetic beads, arrays, the surface of waveguides, fibres, membranes, or capillaries.

8-9. (canceled)

10. The method of claim 1, wherein the polymer product: (a) has a magnetic core; (b) is linear or cross-linked; (c) is covalently linked to the target protein; and/or (d) is in a soluble or colloidal form.

11. The method of claim 1, further comprising the step: isolating the protein:polymer complex, optionally wherein the isolation of the protein:polymer complex comprises the use of ultrafiltration, ultracentrifugation, electrophoresis, sonication, chromatographic separation, washing, adding urea, guanidine, magnetic forces, or any combination thereof, and/or wherein the protein or polymer is linked to a solid support to facilitate the isolation of the protein:polymer complex.

12. (canceled)

13. The method of claim 1, wherein digesting the protein is a partial proteolysis reaction, such that the parts of the protein that are not bound to the polymer are separated from the complex with the polymer, optionally comprising the use of one or more protein cleaving agent.

14. The method of claim 1, wherein isolating the peptide:polymer complex comprises the separation of the peptide:polymer complex from unbound protein and/or unbound peptide, optionally wherein comprising the use of ultrafiltration, ultracentrifugation, electrophoresis, sonication, chromatographic separation, washing, adding urea, guanidine, magnetic forces, or any combination thereof.

15. The method of claim 1, further comprising the step: releasing bound peptide from the peptide:polymer complex.

16. The method of claim 2, wherein sequencing the peptide comprises the use of Edman degradation, mass spectrometry, mass spectrometry using matrix assisted laser desorption ionization (MALDI), electrospray ionization (ESI) sources, atmospheric pressure ionisation techniques or tandem mass spectrometric analysis (MS/MS), or any combination thereof.

17. Use of a molecularly imprinted polymer (MIP) for producing a composition suitable for determining, on a protein, a novel binding site for a binding partner, optionally wherein the binding site is an epitope to which an antibody binds.

18. The use according to claim 17, wherein the use comprises: (i) forming a protein:MIP complex; (ii) digesting the protein in the complex to produce a peptide:MIP complex; and (iii) isolating the peptide:MIP complex wherein the peptide corresponds to a binding site for a binding partner.

19. The use according to claim 17, wherein the complex is formed between a pre-formed MIP and the protein, and/or the protein:MIP complex is formed by the contacting of the protein with a plurality of monomers, and the initiation of polymerisation of the monomers.

20. (canceled)

21. The use according to claim 17, comprising: isolating the protein:MIP complex before digesting the protein in the complex.

22. The use according to claim 17, wherein the digestion of the protein in the protein:MIP complex comprises the partial proteolysis of the protein in the complex, such that the parts of the protein that are not bound to the polymer are separated from the complex with the polymer, optionally wherein the digestion comprises the use of one or more protein cleaving agent, such as a protease.

23. The use according to claim 17, comprising: releasing the bound peptide from the peptide:MIP complex, and/or determining the sequence of the bound peptide.

24. (canceled)

25. A kit for determining, on a protein, a binding site for a binding partner, the kit comprising: (i) a plurality of monomers suitable for contacting a target protein and polymerising the monomers to create a protein:polymer complex; and (ii) one or more protein cleaving agent.

26. The kit according to claim 25, wherein the plurality of monomers are suitable for forming a MIP in complex with a target protein.

27. The kit according to claim 25, further comprising one or more initiators suitable for initiating polymerisation, magnet nanoparticles, and/or materials comprising a solid surface suitable for the immobilisation of a target protein.

28-29. (canceled)

30. The kit according to claim 25, further comprising materials for use in the isolation of a protein:polymer complex or for use in the isolation of a peptide:polymer complex, optionally wherein the materials are for ultrafiltration and/or centrifugation.

31. The kit according to claim 25, wherein the one or more protein cleaving agent comprises an enzyme, optionally a protease, and/or one or more buffers.

32. (canceled)

33. The method of claim 1, wherein the plurality of monomers comprises one or more monomer selected from the group consisting of: vinyl monomers, allyl monomers, acetylenes, acrylates, methacrylates, acrylamides, methacrylamides, chloroacrylates, itaconates, trifluoromethylacrylates, derivatives of amino acids, nucleosides, nucleotides, carbohydrates, ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, divinylbenzene, methylene bisacrylamide, ethylene bisacrylamide and N,N-bisacryloylpiperazine, N-Isopropylacrylamide (NIPAM), tert-butylacrylamide, acrylic acid, 3-aminopropyl methacrylate, methacrylic acid, or any combination thereof.

Description

[0125] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:

[0126] FIG. 1 shows a schematic representation of an embodiment of the methods of the invention. The embodiment shown uses molecular imprinting and mass spectrometry for the identification of peptide sequences exposed on a protein surface.

EXAMPLES

[0127] The present invention will now be further described particularly with references to the following non-limiting examples, and FIG. 1.

Example 1

Epitope Mapping using Molecularly Imprinted Polymers

[0128] Referring to FIG. 1, there is shown an antigen 2, which can be any proteinaceous material, such as a protein or polypeptide. The antigen 2 has two epitopes 4, 6, which are exposed on the surface of an inner region 14, and it is these epitopes 4, 6 which the method seeks to identify as binding sites of a binding partner, such as an antibody, aptamer, affimer, small drug molecule, MIP or other natural or synthetic receptor. Sequence information for these epitopes 4, 6 can be obtained using molecular imprinting and sequencing techniques. Knowledge about these amino acid sequences may be subsequently used for generating binding partners, such as high affinity antibodies, or other natural or synthetic receptors, for research, diagnostic, or therapeutic applications.

[0129] In one embodiment of the method, the antigen 2 is contacted with a plurality of monomers, which are then polymerised to create a molecularly imprinted polymer (MIP) nanoparticle 8, which complexes with epitope 4 such that epitope 6 is exposed (see top of FIG. 1). The resulting complex is shown as MIP nanoparticle 8. In the same reaction vessel, the antigen 2 is also contacted with a plurality of monomers, which are then polymerised to create a molecularly imprinted polymer (MIP) nanoparticle 10, which complexes with epitope 6 such that epitope 4 is exposed (see bottom of FIG. 1). The resulting complex is shown as MIP nanoparticle 10.

[0130] Following creation of the two MIP nanoparticles 8, 10, the antigen 2 is digested with an enzyme, which digests off either the inner region 14 and epitope 6 leaving just epitope 4 complexed with the MIP nanoparticle 8 (see top of FIG. 1), or the inner region 14 and epitope 4 leaving just epitope 6 complexed with MIP nanoparticle 10 (see bottom of FIG. 1). The epitope:MIP complexes 8, 10 can then be analysed using mass spectrometry to determine the sequences of epitope 4 and epitope 6.

Example 2

Epitope Mapping of AChE

[0131] To demonstrate the method described in Example 1, the target for identification of peptide epitope sequences on a protein surface was acetylcholine esterase (AChE) enzyme from electric eel Electrophorus electricus (Sigma, C-3389). 4.4 mg of the AChE was dissolved in 2 mL of PBS. 0.7 mL of sample was mixed with 10 mL of the deoxygenated monomeric mixture (19.5 mg of NIPAM, 3 mg of methylene bisacrylamide (MBAA), 15 mg of tert-butylacrylamide, 50 L, of the solution of 22 L of acrylic acid in 1 mL of H.sub.2O, and 3 mg of 3-aminopropyl methacrylate dissolved in 50 mL of phosphate buffered saline (PBS) and purged with nitrogen for 20 min) and polymerisation initiated by adding 100 L of freshly prepared solution of 12 mg of ammonium persulfate (APS) and 6 L of TEMED dissolved in 400 L of PBS. The polymerisation was carried out for 1 h at a room temperature (20 C.). In order to remove the unreacted functional monomers and low-affinity particles, the polymerised samples were filtered through the 50 kDa centrifuge filter for 30 min at a 3500 rpm. 10 mL of PBS were added, incubated for 10 min and passed through centrifuge filter as described above.

[0132] MIP nanoparticles bound to protein were reconstituted in 5 mL of PBS containing 0.5 mg of trypsin (Bovine pancrease, Sigma, T 9201) and incubated at a room temperature for 36 h. Free fragments of digested AChE and trypsin were removed by centrifugation of the samples using 50 kDa centrifuge cartridge for 15 min at a 3500 rpm followed by 25 mL wash with PBS. The peptides bound to MIPs were separated from polymers using 21 mL of hot water, lyophilised and reconstituted in 0.1% formic acid. The peptides were initially loaded onto a Waters 2G-V/M Symmetry C18 trap column (180 m20 mm, 5 m) to desalt and chromatographically focus the peptides prior to elution onto a Waters Acquity HSS T3 analytical UPLC column (75 m250 mm, 1.8 m). Single pump trapping was used with 99.9% solvent A and 0.1% solvent B at flow rate of 5 L/min for 3 min. Solvent A was LC-MS grade water containing 0.1% formic acid and solvent B was acetonitrile containing 0.1% formic acid.

[0133] For the analytical column, the flow rate was set at 0.3 L/min and the temperature maintained at 40 C. The 50 min run time gradient elution was initiated as the peptides were eluted from the trap column. The following gradient was used: 0 min-3% B, 30 min-40% B, 32 min-85% B, 40 min-85% B and 41 min-3% B. The NanoAcquity UPLC was coupled to a Waters Synapt G2 HDMS mass spectrometer. The instrument was operated in positive electrospray ionisation (ESI) mode. The capillary voltage was set at 2.4 kV and cone voltage at 3 0V. PicoTip emitters (New Objective, US, lovtm internal diameter) were used for the nanostage probe. A helium gas flow of 180 mL/min and ion mobility separator nitrogen gas flow of 90 mL/min with a pressure of 2.5 mbar were used. The IMS wave velocity was set at 650 m/s and the IMS wave height at 40 V.

[0134] During the HDMSE acquisition a low collision induced dissociation (CID) energy of 4V was applied across the transfer ion guide. For the high CID energy acquisition a ramp of 20 to 40 V was applied. Argon was used as the CID gas. Lockspray provided mass accuracy throughout the chromatographic run using [Glu1]-Fibrinopeptide (GFP) with 785.8427 m/z. The data was acquired using MassLynx 4.1. All raw data were processed using ProteinLynx Global SERVER (PLGS) (Waters Corporation, Milford, Mass., USA). PLGS was used to assemble the data for alignment, peak picking, peptide and protein identification and limited upstream statistics. Data was searched against Uniprot Electrophorus electricus database (downloaded December 2016).

[0135] Table 1 shows the peptide sequences that are the most prevalent (40% peak intensity) in the I-TASSER model [16] found using molecular imprinting and mass spectrometry. The sequence identified here matches these found using the Immune Epitope Database and Analysis Resource (IEDB) search tool http://www.iedb.org [17]. The imprinting method has correctly identified two out of three known epitopes. Four other sequences have not been previously identified as epitopes for acetylcholine esterase.

TABLE-US-00001 TABLE1 Peptidesequencesthatarethemostprevalent(40%peakintensity) intheI-TASSERmodelfoundusingmolecularimprintingandmass spectrometryandthesefoundusingtheImmuneEpitopeDatabaseand AnalysisResource(IEDB). Peptide PeptidesequenceinI-TASSERMODEL PeptideSequences sequence foundusingmolecularimprinting foundinepitope location andmassspectrometry database(IEDB) 200-217 LALQWVQDNIHFFGGNPK (SEQIDNO:1) 313-320 FRFSFVPV FRFSFVPV(human) (SEQIDNO:2) (SEQIDNO:7) 218-243 QVTIFGESAGAASVGMHLLSPDSRPK GESAGAA.sup.12(eel) (SEQIDNO:3) (SEQIDNO:8) 375-382 EDFLQGVK (SEQIDNO:4) 526-532 YWANFAR(human) (SEQIDNO:9) 533-547 TGNPNINVDGSIDSR (SEQIDNO:5) 549-559 RWPVFTSTEQK (SEQIDNO:6)

Example 3

Epitope Mapping of AChE using Magnetic Beads

[0136] 19.5 mg of NIPAM, 3 mg of methylene bisacrylamide (MBAA), 15 mg of tert-butylacrylamide, 50 L of the solution of 22 L of acrylic acid in 1 mL of H.sub.2O, 3 mg of 3-aminopropyl methacrylate were dissolved in 50 mL of phosphate buffered saline (PBS) and purged with nitrogen for 20 min. Before the polymerisation 10 mg of the iron oxide nanoparticles (10-30 nm diameter), modified with 3-(trimethoxysilyl) propyl methacrylate, were added to 10 mL of the monomeric mixture and purged with nitrogen for 5 min. 5 mg of AChE (Sigma, C-3389) was solubilised in 10 mL of monomeric mixture and purged for 5 min with nitrogen. The polymerisation was initiated by adding 100 L of freshly prepared solution of 12 mg of ammonium persulfate (APS) and 6 L of TEMED dissolved in 400 L of PBS. The polymerisation was carried out for 1 h at a room temperature (20 C.).

[0137] Magnetic material was collected using magnet and washed 310 mL of PBS. Collected magnetic materials with bound AChE were treated by adding 10 mL of the solution of 1 mg of trypsin (Bovine pancrease, Sigma, T 9201) in PBS and incubating at a room temperature (20 C.) for 3 days. The magnetic particles bound to peptides were collected using magnet and washed 310 mL of the cold water. The specific peptides were eluted from magnetic particles using 31 mL of hot water (90 C.). The eluted peptides were sequenced as described in Example 1.

Example 4

Analysis of Sequences of AChE Epitopes Identified by Molecular Imprinting

[0138] Only four of the Electrophorus electricus (Ee) AChE sequences: 200-217 LALQWVQDNIHFFGGNPK (SEQ ID No:1), 218-243 QVTIFGESAGAASVGMHLLSPDSRPK (SEQ ID No:3), 313-320 FRFSFVPV (SEQ ID No:2) and 526-532 YWANFAR (SEQ ID No:9) have matches with human (h) AChE. Parts of these sequences are known epitopes for anti-AChE antibodies [18-20].

[0139] The following sequences: 209-215 (hAChE) LALQWVQ (SEQ ID No:io), 231-247 (hAChE) FGESAGAASVGMHLLSP (SEQ ID No:11), 326-333 (hAChE) FRFSFVPV (SEQ ID No:7), and 526-532 (hAChE) YWANFAR (SEQ ID No:9) are identical for both EeAChE and hAChE.

TABLE-US-00002 TABLE2 PeptidesequencesidentifiedinMIP,andtheircorrelationwith knownepitopes Sequenceidentifiedin molecularimprintingwork Sequenceofknownepitope[1-3] LALQWVQDNIHFFGGNPK(SEQID LLDQRLALQW(SEQIDNo:12) No:1) QVTIFGESAGAASVGMHLLSPDSRP TLFGESAGAA(SEQIDNo:13); K(SEQIDNo:3) KTVTIFGESAGGASVGMHILSPGSR(SEQID No:14) FRFSFVPV(SEQIDNo:7) VFRFSFVPV(SEQIDNo:15) EDFLQGVK(SEQIDNo:4) YWANFAR(SEQIDNo:9) YWANFAR(SEQIDNo:9) TGNPNINVDGSIDSR(SEQIDNo:5) RWPVFTSTEQK(SEQIDNo:6)

Example 5

Synthesis and Characterisation of MIP Nanoparticles for Epitopes Identified for AChE

[0140] The corresponding peptides, including a linker consisting of two glycine residues and a terminal cysteine were first synthesised for use as templates and immobilized onto the surface of amine derivatized glass beads. A solid-phase approach described by Canfarotta et al. [21] was adapted for MIP nanoparticle synthesis. The affinity of nanoMIPs imprinted against each epitope of AChE was assessed using a surface plasmon resonance (SPR) instrument (MP-SPR Navi 220A NAALI). For these experiments, MIP nanoparticles were covalently immobilized onto the sensor surface and a kinetic titration performed, increasing concentration of protein with each injection. All MIPs exhibited excellent affinity for AChE, with KD values in the low nanomolar range (Table 3). Importantly, MIP nanoparticles synthesised for epitope present mainly in denatured AChE were able to bind to native AChE (Table 3).

TABLE-US-00003 TABLE3 AffinityofMIPnanoparticlessynthesisedusingepitopes identifiedforAChE K.sub.Dofsynthesised MIPnanoparticles EpitopeusedinsynthesisofMIPnanoparticles tonativeAChE YWANFAR(SEQIDNo:9) 4.04nM MSIntensity*-45%inboth,nativeanddenaturedprotein FGESAGAASVGMHLLSP(SEQIDNo:11) 0.85nM MSintensity-86%innativeprotein,10%indenatured FRFSFVPV(SEQIDNo:7) 26.3pM MSintensity-100%indenaturedprotein,10%innative protein LALQWVQ(SEQIDNo:10) 0.84M MSintensity-100%inboth,nativeanddenaturedprotein *relative percentage of peptide intensity in MS spectrum

CONCLUSIONS

[0141] Advantages of the invention reside in the ability to experimentally determine peptide sequences that are binding sites, without prior knowledge of the protein, protein structure, or any candidate binding sites. Thus, the method may be used to identify completely novel binding sites. Furthermore, the invention does not require the use of complex reagents, such as antibodies, which may be either unavailable or challenging to produce due to complexity, expense, and/or the time required.

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