Method for analyzing extracellular vesicle

Abstract

To provide a method that enables efficient and precise omics analysis of extracellular vesicles.SOLUTION: The problem to be solved is solved by subjecting extracellular vesicles to omics analysis through a method that includes the steps of: causing an adsorbent to adsorb extracellular vesicles contained in a sample, where the adsorbent contains an insoluble carrier and a sphingomyelin-recognizing molecule containing a sphingomyelin-binding protein, immobilized to the carrier; and analyzing molecules present in the extracellular vesicles adsorbed onto the adsorbent.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for analyzing extracellular vesicles contained in a sample, and in particular to a method capable of performing omics analysis of the extracellular vesicles with high accuracy. [Background technology]

[0002] Extracellular vesicles contain substances such as proteins and nucleic acids such as microRNA and messenger RNA, and are thought to be involved in the transmission of information between cells. As such, they are not only attracting attention as biomarkers for various diseases such as cancer, but are also expected to be used as novel therapeutic agents and drug delivery systems (Non-Patent Document 1).

[0003] When using extracellular vesicles for the above-mentioned purposes, it is necessary to isolate the extracellular vesicles contained in a sample and analyze the proteins, nucleic acids, etc. contained in the isolated extracellular vesicles (so-called omics analysis).

[0004] As a method for isolating extracellular vesicles for use in omics analysis, a method using annexin V or Tim4 that has binding affinity to phosphatidylserine present on the surface of extracellular vesicles is known (Patent Document 1 and Non-Patent Document 2), and this method can isolate extracellular vesicles with a simple operation. However, the degree of purification of extracellular vesicles isolated by this method is low, and is insufficient for omics analysis applications. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2016 / 088689 issue [Non-patent literature]

[0006] [Non-Patent Document 1] Proteomics, 2013, vol. 13, pp. 1637-1653 [Non-Patent Document 2] Japanese Pharmacological Journal, 2017, Vol. 149, pp. 119-122 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a method for easily and accurately performing omics analysis of extracellular vesicles. [Means for solving the problem]

[0008] As a result of intensive research aimed at solving the above-mentioned problems, the inventors have discovered that by isolating extracellular vesicles contained in a sample using an adsorbent comprising an insoluble carrier and a sphingomyelin recognition molecule immobilized on the carrier, the extracellular vesicles can be easily isolated with a degree of purity sufficient for use in omics analysis, and have thus completed the present invention.

[0009] That is, the present invention includes the following aspects [1] to [5].

[0010] [1] A method for omics analysis of extracellular vesicles, comprising the steps of: adsorbing extracellular vesicles contained in a sample to an adsorbent capable of adsorbing extracellular vesicles; and analyzing molecules present in the extracellular vesicles adsorbed to the adsorbent, wherein the adsorbent comprises an insoluble carrier and a sphingomyelin recognition molecule comprising a sphingomyelin-binding protein immobilized on the insoluble carrier.

[0011] [2] The analytical method according to [1] above, wherein the sphingomyelin recognition molecule further comprises a sphingomyelin-binding protein and an immobilization assisting protein capable of binding to an insoluble carrier.

[0012] [3] The analysis method according to [1] or [2] above, wherein the molecule present in extracellular vesicles is a protein present in extracellular vesicles.

[0013] [4] The method of any one of [1] to [3] above, wherein the sphingomyelin-binding protein is equinatoxin.

[0014] [5] The method for analyzing according to the above [4], wherein the equinatoxin is a protein selected from the following (a) to (c): (a) a protein comprising at least amino acid residues consisting of the amino acid sequence set forth in SEQ ID NO:1; (b) a protein comprising at least the amino acid residues of the amino acid sequence set forth in SEQ ID NO:1, further comprising one or more of substitutions, deletions, insertions, and additions of one or several amino acid residues at one or several positions in the amino acid residues, and having sphingomyelin-binding ability; (c) A protein comprising at least amino acid residues consisting of an amino acid sequence having 70% or more homology to the entire amino acid sequence set forth in SEQ ID NO:1 and having sphingomyelin-binding ability.

[0015] The present invention will now be described in further detail.

[0016] In the present invention, extracellular vesicles refer to particles of 20 nm to several μm composed of lipid bilayers secreted from eukaryotic cells, and are classified into exosomes, microvesicles, and apoptotic vesicles depending on their size and origin. Among them, exosomes and microvesicles with a particle size of 50 nm to 500 nm are preferred because they are effective in disease diagnosis and treatment (for example, Biology (Basel), 2021, Vol. 10, p. 359).

[0017] The present invention is characterized in that, when performing omics analysis of extracellular vesicles contained in a sample, the vesicles are isolated by adsorbing them to an adsorbent (hereinafter also referred to as "extracellular vesicle adsorbent") comprising an insoluble carrier and a sphingomyelin recognition molecule including a sphingomyelin-binding protein immobilized on the carrier, and then the vesicles are analyzed.

[0018] The insoluble carrier is not particularly limited as long as it is insoluble in an aqueous solution. Examples include carriers made from polysaccharides such as agarose, alginate (alginic acid salt), carrageenan, chitin, cellulose, dextrin, dextran, and starch; carriers made from synthetic polymers such as polyvinyl alcohol, polymethacrylate, poly(2-hydroxyethyl methacrylate), and polyurethane; carriers made from ceramics such as silica; and magnetic microparticles made from paramagnetic iron oxide. Among them, carriers made from polysaccharides and carriers made from synthetic polymers are preferred as insoluble carriers, and specific examples include polymethacrylate gels with hydroxyl groups introduced such as Toyopearl (manufactured by Tosoh), agarose gels such as Sepharose (manufactured by Cytiva), and cellulose gels such as Cellufine (manufactured by JNC). The shape of the insoluble carrier is not particularly limited, and it may be granular or non-granular, porous or non-porous. Among these, granular materials are preferred because they have a large surface area and can immobilize a large amount of sphingomyelin recognition molecules. In addition, the particle diameter of the granular materials is preferably 1 μm or more and 10 μm or less because the granular materials allow the extracellular vesicles to pass through the gaps of the insoluble carrier without clogging and are easily available.

[0019] The sphingomyelin recognition molecule may contain at least a protein having a specific binding ability to sphingomyelin (hereinafter, also referred to as "sphingomyelin-binding protein"), and a polypeptide or molecule having no sphingomyelin-binding ability may be added to the N-terminus or C-terminus of the protein. Examples of the polypeptide or molecule that may be added include protein tags such as SpyTag or SpyCatcher, the Z domain of Protein A (SpA) derived from Staphylococcus aureus, which is a highly soluble protein, linker sequences such as GS linker (a linker consisting of repeated sequences of 4 glycine residues and 1 serine residue), protease recognition sequences such as the HRV 3C protease recognition sequence, tags for separation and purification such as polyhistidine, c-myc tag, and FLAG tag, and proteins for immobilization on insoluble carriers such as glutathione S-transferase (GST), maltose-binding protein (MBP), and cellulose-binding domain (CBD). Among these, the Z domain of SpA is preferred in that it can improve the productivity of recombinant sphingomyelin-binding protein in E. coli, and SpyCatcher is preferred in that it can control the molecular density and orientation of the sphingomyelin-binding protein.

[0020] Examples of sphingomyelin-binding proteins include Equinatoxin II (hereinafter also referred to as "EqtII") derived from the sea anemone Actinia equina, Lysenin derived from the earthworm Eisenia foetida, and Pleurotolysin A2 and Ostreolysin A derived from the oyster mushroom Pleurotus ostreatus. Among them, EqtII is a preferred embodiment of the sphingomyelin-binding protein because of the ease of recombinant protein production by Escherichia coli.

[0021] Furthermore, the sphingomyelin-binding protein is not limited to naturally occurring sphingomyelin-binding proteins (including partial regions thereof, the same applies below), and may have one or more of the following substitutions, deletions, insertions, and additions of amino acid residues in the protein, so long as the protein has sphingomyelin-binding ability.

[0022] In a preferred embodiment when the sphingomyelin-binding protein is EqtII, the protein is selected from any one of the following (a) to (c): SEQ ID NO: 1 is a partial sequence of native (non-mutation-introduced) EqtII, and specifically corresponds to the amino acid sequence from the 36th to the 214th amino acids of UniProt Accession No. P61914. (a) a protein comprising at least the amino acid residues of the amino acid sequence set forth in SEQ ID NO:1; (b) a protein that contains at least the amino acid residues of the amino acid sequence set forth in SEQ ID NO:1, and further has one or more of the following substitutions, deletions, insertions, and additions of one or several amino acid residues at one or several positions in the amino acid residues, and has sphingomyelin-binding ability. (c) A protein comprising at least amino acid residues consisting of an amino acid sequence having 70% or more homology to the entire amino acid sequence set forth in SEQ ID NO:1 and having sphingomyelin-binding ability.

[0023] In the above (b), "one or several" varies depending on the position of the amino acid residue in the three-dimensional structure of the protein and the type of amino acid residue, but means, for example, any of 1 to 20, 1 to 10, 1 to 5, or 1 to 3. Furthermore, "any one or more of substitution, deletion, insertion, and addition" in the above (b) also includes naturally occurring mutations (mutants or variants) based on individual differences, species differences, and the like of the organism from which the gene is derived.

[0024] An example of the "substitution" described in (b) is the substitution of valine at position 8 and lysine at position 69 of SEQ ID NO: 1 with cysteine ​​(SEQ ID NO: 2). This substitution detoxifies the native EqtII (SEQ ID NO: 1), which is a pore-forming toxin, and increases safety (J. Biol. Chem., 2004, vol. 279, pp. 46509-46517).

[0025] The homology of the amino acid sequences in (c) may be 70% or more, and may be higher (e.g., 80% or more, 85% or more, 90% or more, or 95% or more). In the present invention, homology may mean similarity or identity, and may particularly mean identity. The identity between amino acid sequences means the ratio of amino acid residues of the same type in those amino acid sequences (Experimental Medicine, February 2013, Vol. 31, No. 3, Yodosha). The similarity between amino acid sequences means the sum of the ratio of amino acid residues of the same type in those amino acid sequences and the ratio of amino acid residues with similar side chain properties (Experimental Medicine, February 2013, Vol. 31, No. 3, Yodosha). The homology of amino acid sequences can be determined using an alignment program such as BLAST (Basic Local Alignment Search Tool) or FASTA.

[0026] There is no limit to the number of sphingomyelin-binding proteins contained in one sphingomyelin recognition molecule, but in terms of high affinity to sphingomyelin and ease of preparation using recombinant proteins in E. coli, the number is preferably 3 to 9. In addition, there is no limit to the type of sphingomyelin recognition molecule contained in the extracellular vesicle adsorbent, and two or more types of sphingomyelin recognition molecules may be immobilized.

[0027] A specific example of a sphingomyelin-binding protein is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3. In SEQ ID NO: 3, the amino acid sequence from serine at position 5 to alanine at position 183 is the amino acid sequence of NT-EqtII (SEQ ID NO: 2), the amino acid sequence from glycine at position 189 to glycine at position 202 is a GS linker sequence, and the amino acid sequence from glycine at position 207 to isoleucine at position 322 is the amino acid sequence of the protein tag SpyCatcher (Chain A and B [116 residues] of Protein Data Bank No. 4MLI, hereinafter also referred to as "SpyC").

[0028] In addition, as long as the sphingomyelin-binding protein has a specific binding ability to extracellular vesicles, a substance that does not recognize sphingomyelin (hereinafter, also referred to as a "sphingomyelin non-binding substance") may be bound (immobilized) on the insoluble carrier. Examples of the sphingomyelin non-binding substance include immobilization auxiliary proteins, proteins such as bovine serum albumin that prevent non-specific adsorption on the surface of the insoluble carrier, and non-protein components such as dextran that improve the hydrophilicity of the carrier surface. Among these, the immobilization auxiliary proteins are proteins that have a binding ability to the sphingomyelin-binding protein and have a function of controlling the density and orientation of the protein when the sphingomyelin-binding protein is immobilized on the insoluble carrier. There is no limit to the number or type of sphingomyelin non-binding substances bound to one sphingomyelin recognition molecule-immobilized carrier, and two or more types of sphingomyelin non-binding substances may be immobilized (bound).

[0029] As the non-sphingomyelin-binding substance to be immobilized on the insoluble carrier, an immobilization auxiliary protein is preferred in terms of controlling the density and orientation of the sphingomyelin-binding protein and optimizing the binding ability of the sphingomyelin-binding protein to extracellular vesicles, and an immobilization auxiliary protein having a trimer structure is even more preferred in terms of improving affinity due to the multivalent effect of the sphingomyelin-binding protein and being easy to prepare.

[0030] A specific example of an immobilization assistant protein when using a sphingomyelin-binding protein having the above-mentioned SpyC attached to the C-terminus and an insoluble carrier having a biotin-binding protein such as avidin or streptavidin immobilized thereon is a protein consisting of the amino acid sequence set forth in SEQ ID NO:4.

[0031] In addition, the fifth residue in SEQ ID NO: 4 is a cysteine ​​residue used for biotin labeling when immobilizing to the insoluble carrier, the 15th aspartic acid to the 72nd valine are the amino acid sequence of the Z domain of SpA (the 4th to 61st amino acids of GenBank Accession No. AL052730), the 78th glycine to the 92nd serine, the 112th glycine to the 126th serine, and the 162nd glycine to the 176th serine are GS linker sequences, the 129th alanine to the 142nd alanine, and the 179th alanine to the 192nd alanine are α-helix linker sequences, and the 95th alanine to the 107th lysine, the 145th alanine to the 157th lysine, and the 200th alanine to the 212th lysine are the amino acid sequence of the protein tag SpyTag (hereinafter also referred to as "SpyT").

[0032] There are no particular limitations on the method for immobilizing the sphingomyelin recognition molecule on an insoluble carrier, so long as it is a common method commonly used by those skilled in the art. For example, After introducing a reactive functional group such as a maleimide group or a carboxy group into the insoluble carrier, the sphingomyelin recognition molecule may be directly immobilized to the functional group. A sphingomyelin-binding protein and an immobilization assistant protein may be bound via a multimer-forming protein containing the amino acid sequence of SpyT / SpyC to prepare a sphingomyelin-recognizing molecule, and then the molecule may be immobilized via the biotin contained in the recognition molecule on an insoluble carrier to which a biotin-binding protein such as avidin or streptavidin has been introduced. The sphingomyelin-binding protein may be immobilized on an insoluble carrier by immobilizing a non-sphingomyelin-binding substance, such as an immobilization assistant protein, on the insoluble carrier via a reactive functional group, and then binding the sphingomyelin-binding protein to the non-sphingomyelin-binding substance, thereby immobilizing the protein on the insoluble carrier. The sphingomyelin recognition molecule may be brought into contact with an insoluble carrier, and the recognition molecule may be immobilized on the insoluble carrier by physical adsorption.

[0033] Among these, immobilizing the sphingomyelin-binding protein on an insoluble carrier via an immobilization assistant protein, thereby immobilizing the sphingomyelin recognition molecule on an insoluble carrier, is preferred because the reaction during the immobilization process is milder, resulting in less denaturation of the sphingomyelin-binding protein, and because the orientation and density of the sphingomyelin-binding protein on the carrier can be controlled.

[0034] In the present invention, the step of adsorbing extracellular vesicles contained in a sample to an extracellular vesicle adsorbent is not particularly limited as long as it is a general method commonly used by those skilled in the art. The sample containing extracellular vesicles is not particularly limited, and may be derived from a living organism such as a biological tissue or a cultured cell, or may be a solution in which extracellular vesicles are suspended in a medium or a buffer solution. The solvent for preparing the sample containing the extracellular vesicles is not particularly limited as long as it can hold the extracellular vesicles in a stable state and does not inhibit the adsorption of the extracellular vesicles to the extracellular vesicle adsorbent, but a buffer solution adjusted to pH 6.0 to 8.0 is preferable in terms of preventing the denaturation of sphingomyelin recognition molecules and extracellular vesicles contained in the solution, and specific examples thereof include TBS (Tris [Tris(hydroxymethyl)aminomethane] Buffered Saline), PBS (Phosphate Buffered Saline), and HBS (HEPES [4-(2-HydroxyEthyl)-1-PiperazineEthaneSulfonic acid] Buffered Saline). Furthermore, the solvent may further contain a surfactant to prevent non-specific adsorption of the sphingomyelin recognition molecule to components in the solution, specifically, a non-ionic surfactant in an amount of 0.00001% (w / v) to 0.5% (w / v) inclusive. A preferred example of the non-ionic surfactant is Tween 20 (trade name).

[0035] The temperature at which the extracellular vesicle adsorbent is brought into contact with the sample containing extracellular vesicles is preferably 2° C. or more and 40° C. or less in order to prevent denaturation of the extracellular vesicles and the sphingomyelin recognition molecules. The contact time between the extracellular vesicle adsorbent and the sample containing extracellular vesicles is preferably 30 minutes or more and 24 hours or less, more preferably 1 hour or more and 12 hours or less, in order to ensure the time for contact between the sphingomyelin recognition molecules and the extracellular vesicles and to prevent denaturation of the extracellular vesicles.

[0036] The extracellular vesicle adsorbent nonspecifically adsorbs impurities as well as extracellular vesicles contained in the sample. Therefore, it is advisable to add a washing step to remove the impurities. The washing step may be appropriately selected from methods commonly used by those skilled in the art. The buffer solution and pH for washing may be, for example, the same as those used in the solvent for preparing the solution containing extracellular vesicles. The temperature may also be the same as that of the step of adsorbing extracellular vesicles to the extracellular vesicle adsorbent.

[0037] When performing omics analysis of extracellular vesicles adsorbed to an extracellular vesicle adsorbent, the extracellular vesicles may be dissociated from the adsorbent and analyzed, or the molecules contained therein may be extracted and analyzed while the extracellular vesicles are still adsorbed. The dissociation may be performed by a method commonly used by those skilled in the art. For example, a method of denaturing and dissociating sphingomyelin recognition molecules by high-temperature treatment at 50°C or higher, or a method of adding a high concentration of salt, amino acid, surfactant, etc., may be used to dissociate the sphingomyelin recognition molecules. The extraction may also be performed by a method commonly used by those skilled in the art. For example, a method of treating the membrane of the extracellular vesicles with a surfactant at a concentration capable of solubilizing the membrane of the extracellular vesicles and extracting the membrane, or a method of disrupting the membrane of the extracellular vesicles with ultrasound may be used. The surfactant for solubilizing the membrane is not particularly limited, and may be appropriately selected from Tween 20, Tween 80, Triton X-100, NP-40 (all trade names), SDS (Sodium Dodecyl Sulfate), etc.

[0038] In the present invention, the omics analysis is not particularly limited as long as it is an analysis of molecules present in the extracellular vesicles to be analyzed. Examples include proteomics analysis for analyzing proteins present in the vesicles, genomics analysis and transcriptomics analysis for analyzing nucleic acids present in the vesicles, and metabolomics analysis for analyzing metabolic substances present in the vesicles. Among them, the present invention is a particularly useful method for performing proteomics analysis.

[0039] The analysis method is not particularly limited, and as an example, when the omics analysis is proteomics analysis, a method using a mass spectrometer can be mentioned. Specifically, using a commercially available pretreatment kit such as S-Trap micro kit (manufactured by ProtiFi) or Detergent-free Exosomal Protein Extraction Kit (manufactured by 101 Bio), proteins present in extracellular vesicles can be extracted, reduced, alkylated, and treated with trypsin, and then subjected to a mass spectrometer for analysis. Effect of the Invention

[0040] The present invention is characterized in that, when performing omics analysis of extracellular vesicles contained in a sample, the vesicles are isolated by adsorption to an adsorbent containing an insoluble carrier and a sphingomyelin recognition molecule, including a sphingomyelin-binding protein, immobilized on the carrier, and then analyzed, thereby enabling the analysis to be performed simply and accurately. [Brief description of the drawings]

[0041] [Figure 1] Schematic diagrams of the sphingomyelin-binding protein, immobilization assisting protein, sphingomyelin recognition molecule, and extracellular vesicle adsorbent prepared in the examples. EXAMPLES

[0042] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to these examples.

[0043] Example 1: Preparation of sphingomyelin-binding protein (1) A fusion protein ((NT-EqtII)-SpyC, SEQ ID NO: 3) of detoxified equinatoxin (NT-EqtII, SEQ ID NO: 2) and SpyCatcher (Chain A and B [116 residues] of Protein Data Bank No. 4MLI, hereinafter also referred to as "SpyC") was inserted at the N-terminus of the Z domain of Protein A derived from Staphylococcus aureus (SpA-Z, GenBank Accession No. A polynucleotide (SEQ ID NO: 6) encoding the polypeptide (SpA-Z)-(NT-EqtII)-SpyC (SEQ ID NO: 5) to which a polypeptide consisting of the 4th to 61st amino acid residues of No. AL052730 has been added was inserted into the multicloning site of pET28a(+) (Merck) to prepare a vector pET_(SpA-Z)-(NT-EqtII)-SpyC capable of expressing (SpA-Z)-(NT-EqtII)-SpyC (SEQ ID NO: 5) in E. coli.

[0044] (2) The expression vector pET_(SpA-Z)-(NT-EqtII)-SpyC prepared in (1) was transformed into E. coli BL21(DE3) strain to obtain recombinant E. coli (transformant).

[0045] (3) The obtained transformants were inoculated into Luria-Bertani (LB) medium (10 g / L tryptone, 5 g / L yeast extract, and 5 g / L NaCl) supplemented with 30 μg / mL kanamycin, and pre-cultured overnight at 37°C with shaking.

[0046] (4) The preculture solution from (3) was inoculated into TB (Terrific broth) medium (24 g / L yeast extract, 12 g / L tryptone, 9.4 g / L K2HPO4, 2.2 g / L KH2PO4, and 4 mL / L glycerol) supplemented with 30 μg / mL kanamycin, and cultured with shaking at 37°C.

[0047] (5) Turbidity of the culture medium (OD 600nmWhen the ΔEqtII ratio reached approximately 0.6, the culture temperature was switched to 20°C, 0.1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added, and the mixture was further cultured overnight to express (SpA-Z)-(NT-EqtII)-SpyC.

[0048] (6) After collecting the bacteria from the culture medium, a soluble protein extract was recovered from the bacteria using a BugBuster Protein extraction kit (Merck) according to the manufacturer's protocol.

[0049] (7) (SpA-Z)-(NT-EqtII)-SpyC was purified from the soluble protein extract by nickel chelate affinity chromatography and the storage buffer (50 mM Tris-HCl (pH 7.5) containing 150 mM sodium chloride) was replaced using an ultrafiltration filter.

[0050] (8) To remove the histidine tag and SpA-Z from (SpA-Z)-(NT-EqtII)-SpyC, (SpA-Z)-(NT-EqtII)-SpyC was digested with HRV 3C protease fused with a histidine tag peptide (Merck) according to the manufacturer's protocol.

[0051] (9) After adding 5 mM imidazole to the digestion solution, the digest was purified by nickel chelate affinity chromatography to obtain a (NT-EqtII)-SpyC (SEQ ID NO: 3) solution.

[0052] Example 2 Preparation of immobilization assistant protein (1) A polynucleotide encoding a fusion protein (Cys-(SpA-Z)-3SpyT, SEQ ID NO: 4) of SpA-Z with a cysteine ​​residue (Cys) for biotin labeling added to the N-terminus and three copies of SpyTag (Chain B [13 residues] of Protein Data Bank No. 4MLI, hereinafter also referred to as "SpyT") was inserted into the multicloning site of pGEX (manufactured by Cytiva) to prepare a vector pGEX_GST-Cys-(SpA-Z)-3SpyT capable of expressing the fusion protein GST-Cys-(SpA-Z)-3SpyT (SEQ ID NO: 7) of glutathione S-transferase (GST) and Cys-(SpA-Z)-3SpyT in E. coli (the sequence of the polynucleotide encoding GST-Cys-(SpA-Z)-3SpyT is shown in SEQ ID NO: 8).

[0053] (2) The expression vector pGEX_GST-Cys-(SpA-Z)-3SpyT prepared in (1) was transformed into E. coli BL21 strain to obtain recombinant E. coli (transformant).

[0054] (3) The obtained transformant was inoculated into LB medium supplemented with 60 μg / mL carbenicillin and pre-cultured overnight at 37°C with shaking, and then the pre-culture solution was inoculated into LB medium supplemented with 60 μg / mL carbenicillin and cultured with shaking at 37°C.

[0055] (4) Turbidity of the culture medium (OD 600nm When the β-amyloid ratio (α,β-amyloid) reached approximately 0.6, the culture temperature was changed to 30°C, 0.5 mM IPTG was added, and the culture was continued for an additional 5 hours to express GST-Cys-(SpA-Z)-3SpyT.

[0056] (5) After collecting the bacteria from the culture medium, a soluble protein extract was recovered from the bacteria using a BugBuster Protein extraction kit (Merck) according to the manufacturer's protocol.

[0057] (6) GST-Cys-(SpA-Z)-3SpyT was purified from the soluble protein extract by affinity chromatography using glutathione-immobilized support.

[0058] (7) The GST-Cys-(SpA-Z)-3SpyT bound to the immobilized carrier was digested with GST-fused HRV 3C protease (Merck) according to the manufacturer's protocol, and the supernatant was collected to obtain a Cys-(SpA-Z)-3SpyT (sequence number 4) solution.

[0059] Example 3: Preparation of sphingomyelin recognition molecule (1) The solution of the immobilized auxiliary protein Cys-(SpA-Z)-3SpyT (sequence number 4) obtained in Example 2 was replaced with D-PBS (Dulbecco's Phosphate Buffered Saline) using an ultrafiltration filter, and the sulfhydryl group of the cysteine ​​residue was labeled with biotin using EZ-Link Maleimide-PEG2-Biotin (manufactured by Thermo Fisher Scientific) according to the manufacturer's protocol.

[0060] (2) The immobilized auxiliary protein Cys-(SpA-Z)-3SpyT (sequence number 4) labeled with biotin in (1) and the sphingomyelin-binding protein (NT-EqtII)-SpyC (sequence number 3) obtained in Example 1 were each suspended in D-PBS(+) or TBS-T (25 mM Tris-HCl (pH 7.2) containing 0.15 M NaCl and 0.05% (w / v) Tween 20 (trade name)) at a concentration of 0.3 mg / mL or more.

[0061] (3) After mixing the two suspensions so that the molar ratio of biotin-labeled Cys-(SpA-Z)-3SpyT to (NT-EqtII)-SpyC was 4 or more, the two suspensions were left at 4°C for at least one day to allow SpyT and SpyC to bind to each other, producing ((NT-EqtII)-SpyC)3 / Cys-(SpA-Z)-3SpyT, a sphingomyelin-recognizing molecule in which three sphingomyelin-binding proteins are bound to an immobilization auxiliary protein.

[0062] Example 4 Preparation of extracellular vesicle adsorbent containing sphingomyelin recognition molecule (1) Magnoshere MS300 / Streptavidin (particle size 3 μm) (manufactured by JSR Life Sciences), which is a streptavidin-immobilized magnetic microparticle, was used as the insoluble carrier. 250 μL of a slurry solution of the magnetic microparticles (microparticle content 1% (w / v)) was collected, and after removing the supernatant, the microparticles were washed with PBS-T (D-PBS containing 0.05% (w / v) Tween 20 (trade name)).

[0063] (2) The sphingomyelin recognition molecule prepared in Example 3 was adjusted to a concentration of 0.1 mg / mL or more in terms of the concentration of the immobilization auxiliary protein Cys-(SpA-Z)-3SpyT (SEQ ID NO: 4).

[0064] (3) To the magnetic particles washed in (1), 25 μg of the sphingomyelin recognition molecule solution prepared in (2), calculated as the amount of immobilization auxiliary protein, was added, and the mixture was mixed at room temperature for 15 minutes to allow the reaction.

[0065] (4) After the reaction, the supernatant was removed and the cells were washed three times with PBS-T to prepare an extracellular vesicle adsorbent containing sphingomyelin recognition molecules.

[0066] Example 5 Omics analysis of extracellular vesicles contained in a sample using an extracellular vesicle adsorbent containing a sphingomyelin recognition molecule (1) Preparation of samples containing extracellular vesicles (1-1) Prostate cancer cell line (PC3 cells) was cultured at 37°C in Ham's F-12K medium (Fujifilm Wako Pure Chemical Industries, Ltd.) containing 15% (v / v) fetal bovine serum (FBS).

[0067] (1-2) The cultured PC3 cells were further cultured for 2 days in Ham's F-12K medium containing 15% (v / v) FBS (extracellular vesicles had been removed in advance by ultrafiltration), and 150 mL of the culture supernatant was then collected.

[0068] (1-3) The collected culture supernatant was centrifuged at 300×G for 10 minutes at room temperature to remove floating cells, and the supernatant was collected. The filtrate was then passed through a 0.22 μm filter to remove cell debris, and used as the cell culture supernatant.

[0069] (1-4) The cell culture supernatant obtained in (1-3) was concentrated to 500 μL using an ultrafiltration filter, and this was used as a sample containing extracellular vesicles.

[0070] (2) Adsorption of extracellular vesicles contained in a sample by an extracellular vesicle adsorbent (2-1) 500 μL each of the sample prepared in (1) and PBS-T was added to a container containing the extracellular vesicle adsorbent containing the sphingomyelin recognition molecule prepared in Example 4, and the container was then shaken at 4° C. for 3 hours.

[0071] (2-2) After removing the supernatant, the sample was washed twice with 500 μL of PBS-T to obtain an adsorbent that adsorbed extracellular vesicles contained in the sample.

[0072] (3) Omics analysis of extracellular vesicles adsorbed to adsorbents (3-1)(2) was obtained. The adsorbed extracellular vesicles were solubilized by adding 50 μL of RIPA buffer (Nacalai Tesque) containing 0.1% (w / v) SDS (sodium dodecyl sulfate) to the container containing the adsorbent with the adsorbed extracellular vesicles.

[0073] (3-2) After removing a portion of the solvent using a centrifugal concentrator, protein extraction, reduction and alkylation of the extracted protein, and enzyme (trypsin / lysyl endopeptidase) treatment were performed using an S-Trap micro kit (ProtiFi) according to the manufacturer's protocol.

[0074] (3-3) The peptide fragments recovered in the process of (3-2) were dissolved in 15 μL of 0.1% (v / v) formic acid-5% (v / v) acetonitrile aqueous solution to prepare a measurement sample. The amount of protein contained in the sample was quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific).

[0075] (3-4) 10 μg of the measurement sample obtained in (3-3) was subjected to liquid chromatography analysis using Easy-nLC 1200 (Thermo Fisher Scientific). The column used was a 3 μm C18 NANO HPLC CAPILLARY COLUMN (Nikkyo Technos), and the eluents used were 0.1% (v / v) formic acid-distilled water and 0.1% (v / v) formic acid-80% (v / v) acetonitrile, respectively.

[0076] (3-5) After the liquid chromatography analysis, the samples were subsequently subjected to mass spectrometry using Orbitrap Fusion (Thermo Fisher Scientific) in electrospray ionization positive mode.

[0077] (3-6) Proteins were identified using Proteome Discoverer 2.2 (Thermo Fisher Scientific) from the mass spectrometry results. The database used was Homo sapiens (SwissProt). Proteins were identified when the total number of peptide amino acid sequences that belonged to the protein was two or more.

[0078] Comparative Example 1 Preparation of an extracellular vesicle adsorbent containing a phosphatidylserine recognition molecule (1) As an insoluble carrier, streptavidin-immobilized magnetic microparticles, Magnoshere MS300 / Streptavidin (particle size 3 μm) (manufactured by JSR Life Sciences) were used. 250 μL of the slurry solution of the magnetic microparticles (microparticle content 1% (w / v)) was collected, the supernatant was removed, and the microparticles were washed with Exosome Capture Immobilizing / Washing Buffer (1×) included in the MagCapture Exosome Isolation Kit PS Ver.2 (manufactured by Fujifilm Wako Pure Chemical Industries, hereafter also referred to as the "FW kit").

[0079] (2) 41.65 μL of a phosphatidylserine recognition molecule solution (Biotin-labeled Exosome Capture, included in the FW kit) was added to the magnetic particles washed in (1), and the mixture was mixed at room temperature for 15 minutes to allow the reaction to occur.

[0080] (3) After the reaction, the supernatant was removed and the mixture was washed three times with Exosome Capture Immobilizing / Washing Buffer (1x) provided with the FW kit to prepare an extracellular vesicle adsorbent containing phosphatidylserine recognition molecules.

[0081] Comparative Example 2 Omics analysis of extracellular vesicles contained in a sample using an extracellular vesicle adsorbent containing a phosphatidylserine recognition molecule (1) 500 μL of the sample prepared in Example 5(1), 500 μL of TBS-T, and 2 μL of Exosome Binding Enhancer (500×) included in the FW kit were added to a container containing the extracellular vesicle adsorbent containing the phosphatidylserine recognition molecule prepared in Comparative Example 1, and the container was then shaken at 4° C. for 3 hours.

[0082] (2) After removing the supernatant, the sample was washed twice with 500 μL of Exosome Capture Immobilizing / Washing Buffer (1×) provided with the FW kit to obtain an adsorbent that had adsorbed extracellular vesicles contained in the sample.

[0083] (3) Omics analysis of extracellular vesicles adsorbed to the adsorbent was performed using the method described in Example 5(3).

[0084] Table 1 shows the proteins that could be identified by the analysis of Example 5 and Comparative Example 2. From the analysis result (Example 5) of the extracellular vesicles adsorbed with an adsorbent containing a sphingomyelin recognition molecule, only proteins related to the extracellular vesicles (specifically, four types: CD9, Syntenin-1, Alix, and HSP60) could be identified. On the other hand, from the analysis result (Comparative Example 2) of the extracellular vesicles adsorbed with an adsorbent containing a phosphatidylserine recognition molecule, in addition to the four types of proteins related to the extracellular vesicles described above, proteins related to the endoplasmic reticulum (specifically, Calnexin, Calreticulin, DNAJA1, GANAB, HSPH1, P4HB, PDIA6, PRKCSH, Rpn1, Ubiquilin-2, and VCP) were also identified. From the above results, it can be seen that when performing omics analysis of extracellular vesicles, by adsorbing the extracellular vesicles contained in the sample with an adsorbent containing a sphingomyelin recognition molecule, it is possible to perform analysis with higher accuracy than by adsorbing with an adsorbent containing a phosphatidylserine recognition molecule.

[0085] [Table 1]

Claims

1. A method for omics analysis of extracellular vesicles, comprising the steps of: adsorbing extracellular vesicles contained in a sample to an adsorbent capable of adsorbing the extracellular vesicles; and analyzing molecules present in the extracellular vesicles adsorbed to the adsorbent, The aforementioned analytical method, wherein the adsorbent comprises an insoluble carrier and a sphingomyelin recognition molecule, which comprises a sphingomyelin-binding protein, immobilized on the insoluble carrier.

2. The analytical method according to claim 1 , wherein the sphingomyelin recognition molecule further comprises a sphingomyelin-binding protein and an immobilization assisting protein capable of binding to an insoluble carrier.

3. The analysis method according to claim 1 , wherein the molecule present in the extracellular vesicle is a protein present in the extracellular vesicle.

4. The method of any one of claims 1 to 3, wherein the sphingomyelin-binding protein is equinatoxin.

5. The method of claim 4, wherein the equinatoxin is a protein selected from the following (a) to (c): (a) a protein comprising at least the amino acid residues consisting of the amino acid sequence set forth in SEQ ID NO:1; (b) a protein comprising at least the amino acid residues of the amino acid sequence set forth in SEQ ID NO:1, and further comprising any one or more of substitutions, deletions, insertions, and additions of one or several amino acid residues at one or several positions in the amino acid residues, and having sphingomyelin-binding ability; (c) A protein comprising at least amino acid residues consisting of an amino acid sequence having 70% or more homology to the entire amino acid sequence set forth in SEQ ID NO:1 and having sphingomyelin-binding ability.

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