Protein analysis methods

Abstract

To solve the problem in which: since a long time is required for pretreatment prior to protein analysis, a quicker pretreating method is required.SOLUTION: Provided is a pretreatment method for structural analysis of a protein, including the steps for chemical modifying and fragmenting a protein to be analyzed. In the fragmenting step, the protein is treated at a temperature of 60 to 99°C in the presence of a heat-resistant protease that has four or more cleavage sites and is soluble or immobilized on a carrier.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a pretreatment method in protein analysis and a protein analysis method using the method.

Background Art

[0002] Macromolecules such as proteins have complex higher-order structures and thereby exhibit interactions with other biomolecules. Understanding such interactions and complex formation between biomolecules is important for elucidating the mechanisms of complex phenomena in vivo and drug development.

[0003] As methods for analyzing interactions between biomolecules, methods such as single crystal X-ray diffraction, nuclear magnetic resonance spectroscopy (NMR), surface plasmon resonance (SPR), quartz crystal microbalance (QCM), and imaging mass cytometry (IMC) have been developed, but there are limitations in throughput and the quality and quantity of information obtained. On the other hand, a method for interaction analysis using liquid chromatography-mass spectrometry (LC-MS) has been developed.

[0004] For a protein having a higher-order structure, by reacting a drug capable of chemically modifying it for a short time, functional groups present inside the protein molecule can be not modified, and only functional groups exposed on the surface can be selectively modified. It is known that by utilizing this, information on the higher-order structure can be obtained by peptide mapping using LC-MS for the modified protein (Patent Document 1). It has also been reported that by applying such a method to a protein complex, the presence or absence of formation of the protein complex can be determined (Non-Patent Document 1).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Non-Patent Documents

[0006] [Non-Patent Document 1] Chem. Rev. 2007, 107, 3514-3543 [Overview of the project] [Problems that the invention aims to solve]

[0007] However, a challenge with the above analytical methods was that they required a long time for pre-processing before analysis. In other words, there was a need for a method to rapidly pre-process proteins for analysis. [Means for solving the problem]

[0008] In light of the above-mentioned problems, the inventors of this invention have conducted various studies and found that by using a proteolytic enzyme with a wide variety of cleavage sites in protein analysis, pretreatment can be performed in a significantly shorter time compared to conventional methods. Furthermore, it is preferable to use a heat-resistant enzyme as the proteolytic enzyme, in which case pretreatment can be carried out at high temperatures. [Effects of the Invention]

[0009] The present invention enables the completion of protein pretreatment for analysis in a short time. By rapidly determining the higher-order structure of a protein using the method of the present invention, it is possible to confirm whether or not the protein has denatured and to identify factors that may contribute to the stability of the protein. Furthermore, the method of the present invention makes it possible to identify substances that can interact with proteins, for example, to find substances that are useful as pharmaceuticals. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 shows the results of HPLC analysis of peptides obtained when BSA aggregates in the present invention were treated with thermolysin at 80°C (solid line) and when they were not treated (dotted line). The vertical axis shows the absorbance at 220 nm. [Figure 2]Figure 2 shows the results of HPLC analysis of peptides pretreated by the method of the present invention, with BSA aggregates and non-aggregates respectively. The arrows indicate peaks that showed a significant difference between aggregates and non-aggregates. The vertical axis shows the absorbance at 220 nm. [Modes for carrying out the invention]

[0011] The embodiments of the present invention will be described in detail below, but the present invention is not limited to these descriptions, and those skilled in the art can make various modifications within the scope of the technical ideas disclosed herein.

[0012] The present invention relates to a pretreatment method for protein analysis and a method for analyzing proteins using the said method. Specifically, the present invention provides a pretreatment method for protein structural analysis, comprising a chemical modification step and a fragmentation step of the protein to be analyzed, characterized in that the fragmentation step uses a protease having four or more cleavage sites. The method of the present invention is intended to carry out the chemical modification step and the fragmentation step in a liquid on a sample containing protein.

[0013] In this specification, "protein" may include low and medium molecular weight molecules, sometimes referred to as peptides and polypeptides. Furthermore, "protein" may also include, but is not limited to, glycoproteins and nucleoproteins formed by binding with sugars or nucleic acids. Proteins may be naturally occurring or synthetically produced, and may further encompass those forming the following complexes.

[0014] The method of the present invention is not limited to, but aims to analyze the higher-order structure of a protein whose amino acid sequence as its primary structure is known. Here, higher-order structure includes the three-dimensional structure of the protein itself, as well as complex structures obtained by interactions between multiple identical or different proteins.

[0015] Specifically, the method of the present invention can be used for analysis to identify amino acids present on the surface of a protein to be analyzed and to obtain information about its three-dimensional structure. For example, since proteins undergo structural changes when they are denatured, it is possible to determine whether or not denaturation has occurred.

[0016] Furthermore, the higher-order structure of a protein may change depending on whether it exists alone or in a complex. Therefore, the method of the present invention can be used for analysis to determine whether a protein molecule to be analyzed is in a complex (protein complex) with another substance. A protein complex is a state in which a protein (including peptides and polypeptides) forms an aggregate with another substance. Examples include, but are not limited to, protein-ligand complexes, enzyme protein-substrate complexes, nucleic acid-protein complexes, protein-peptide complexes, antibody protein-antigen complexes, aggregates of the same protein, and subunit aggregates. Complexes can be formed by covalent or non-covalent bonds, such as hydrogen bonds, hydrophobic bonds, and electrostatic interactions.

[0017] While the purpose of determining the presence or absence of protein complex formation is not limited, it can be used for screening candidate substances in drug development. False positives are often a problem when screening peptides that can recognize specific proteins. For example, phage display may include substances that do not actually interact as intended. In contrast, if information about the state of complex formation between the protein and the candidate substance can be obtained quickly, false positives can be eliminated, and screening can proceed more efficiently. It can also be used for screening candidate structures of medium-molecule drugs.

[0018] The protein analysis method provided in the present invention includes a pretreatment step and a fragmentation step.

[0019] <Chemical modification process> In the chemical modification process, a reagent highly reactive with the protein to be analyzed is reacted for a short time to progress chemical modification that reflects the actual complex formation state in the solution.

[0020] In the chemical modification process, among the functional groups in the protein, modification preferentially proceeds at highly reactive sites. Since highly reactive sites depend on the higher-order structure, when the higher-order structure changes due to denaturation or complex formation, the modification state changes accordingly. Therefore, denaturation or complex formation can be determined by analyzing the modification state. In particular, as described above, quickly determining complex formation is important.

[0021] For example, whether a protein to be analyzed and a candidate peptide with unknown binding affinity to this protein form a complex can be determined as follows.

[0022] When no complex is formed, the modification state obtained by the chemical modification process is almost the same as when the protein to be analyzed and the candidate peptide are chemically modified separately. Therefore, by comparing with the results of each alone, if the difference in the analysis results is within the range of measurement error, it can be determined that no complex is formed.

[0023] On the other hand, when the protein to be analyzed and an enzyme peptide form a complex, the modification state can change because the local environment changes compared to the single state around the site involved in complex formation and its vicinity. Also, when an allosteric effect occurs due to complex formation, the modification state can change because the structure changes at a site far from the site involved in complex formation in the protein to be analyzed. Therefore, if the difference in the analysis results is clear (outside the range of measurement error) compared to the results when chemically modified alone, it can be determined that a complex is formed. In this case, it can be determined that the vicinity of the site where the difference in the analysis results is large is involved in complex formation (the binding site with the candidate peptide or the site affected by the allosteric effect).

[0024] The analysis results can be confirmed, for example, by calculating the modification rate of a specific amino acid residue (the proportion of a particular amino acid residue that is modified in a specific protein in the sample). For example, the presence or absence of complex formation can be observed as a difference in the modification rate of a specific amino acid residue. The difference in modification rates can be compared by comparing the modification rates of individual amino acid residues, or by comparing the modification rates of multiple amino acid residues and, in some cases, calculating the average value of the difference in modification rates.

[0025] In the method of the present invention, the reactions used for chemical modification are not limited. For example, these include modification of nucleophilic amino acid residues (histidine, lysine, serine, threonine, tyrosine, etc.) using electrophiles such as diethyl pyrocarbonate (DEPC) or succinimide ester, oxidation of amino acid residues using photolysis of hydrogen peroxide, carbene insertion into amino acid residues using photolysis of dialidine, selective modification of tryptophan using [(2-hydroxy-5-nitrophenyl)methyl]dimethylsulfonium bromide (HNSB), hydrogen-deuterium exchange, and the like. It is not necessarily required to use irreversible chemical modifications, but from the viewpoint of the amount of information obtained and reproducibility, it is preferable that the modifications have sufficient stability to be detectable in a series of analyses. Furthermore, it is desirable that the reaction conditions be simple.

[0026] Modification of nucleophilic amino acid residues using electrophiles is a method that offers a certain degree of stability in chemical modification and simple reaction conditions. Examples of electrophiles include, but are not limited to, carbonyl compounds with good leaving groups and electron-deficient multiple bond compounds. Examples of carbonyl compounds with good leaving groups include pyrocarbonate compounds such as DEPC, active ester compounds such as succinimide esters, carbonylimidazoles, carboxylic acid anhydrides, and carboxylic acid halides. Examples of electron-deficient multiple bond compounds include carbodiimides, maleimides, isocyanates, and isothiocyanates. Chemically modified residues can be detected by measuring the characteristic absorbance of the reagents used for chemical modification.

[0027] <Fragmentation process> In the fragmentation step, chemically modified proteins are fragmented to make them analyzable. The fragmentation method is preferably hydrolysis using proteolytic enzymes (also referred to as "enzyme digestion" in this specification), but is not limited to this method.

[0028] Conventional peptide mapping techniques utilize highly specific proteolytic enzymes such as trypsin, chymotrypsin, and Lys-C. Proteolytic enzymes are generally known to cleave specific amino acid residues at either the C-terminus or N-terminus, and "highly specific proteolytic enzymes" here refer to those that can cleave a limited number of sites. By adjusting the enzyme-to-substrate ratio, reaction temperature, reaction time, and other factors to set higher reaction conditions, the number of cleavable sites can increase. Therefore, in this specification, for each enzyme, the sites that are generally considered cleavable in this field are referred to as "cleavage sites" or "preferred cleavage sites."

[0029] For example, trypsin preferentially cleaves the C-terminal sides of lysine and arginine (two preferred cleavage sites). Chymotrypsin preferentially cleaves the C-terminal sides of phenylalanine, tyrosine, and tryptophan (three preferred cleavage sites). Lys-C preferentially cleaves the C-terminal side of lysine (one preferred cleavage site). When proteins are degraded using these enzymes, the resulting peptides have longer amino acid lengths, which may be suitable for identifying the proteins from which these peptides originate. Therefore, it is advantageous to use these enzymes, for example, when identifying proteins whose primary structure is unknown.

[0030] On the other hand, because these enzymes have few preferred cleavage sites, it is necessary to denature or reduce the protein for digestion to increase the probability of contact between the enzyme and the cleavage site. For example, when denatured for digestion, it is necessary to apply a denaturing agent such as urea, arginine, or a surfactant, and then to remove these denaturing agents, which contributes to the long pretreatment time. Similarly, when reducing, it is necessary to apply a reducing agent such as DTT or TCEP, perform an alkylation step to cap the reduced site, and inactivate or remove excess reagents, which also contributes to the long pretreatment time.

[0031] As described above, the method of the present invention is intended for the analysis of the higher-order structure of proteins. In this case, it is not necessary to use highly specific proteolytic enzymes as described above. On the contrary, as a result of our investigations, we have found that by using enzymes with lower specificity, i.e., enzymes with a relatively large number of preferential cleavage sites, the steps of denaturation and reduction using denaturing agents can be omitted, and pretreatment can be performed in a short time.

[0032] Examples of proteolytic enzymes that can be suitably used in the method of the present invention include thermolysin (preferentially cleaving the N-terminus of hydrophobic amino acid residues such as leucine, phenylalanine, valine, isoleucine, alanine, and methionine; preferred cleavage sites: 6 types), elastase (preferentially cleaving the C-terminus of alanine, valine, serine, glycine, leucine, and isoleucine; preferred cleavage sites: 6 types), and pepsin (preferentially cleaving the C-terminus of phenylalanine, leucine, tyrosine, and tryptophan; preferred cleavage sites: 4 types).

[0033] When these enzymes are used, proteins can be digested without requiring lengthy denaturation or reduction processes. The enzymes that can be suitably used in the method of the present invention are not particularly limited, but those with four or more preferential cleavage sites are considered preferable. Even if natural enzymes have three or fewer preferential cleavage sites, the number of preferential cleavage sites may increase due to genetic modification or chemical modification, so such enzymes can also be used as enzymes with a relatively large number of preferential cleavage sites.

[0034] There is no upper limit to the number of preferential cleavage sites. While too many preferential cleavage sites may result in fragments that are too short and difficult to analyze, any number of enzymes can be used as long as analysis is possible. While shorter fragments may reduce amino acid sequence coverage, detecting changes in chemical modification is sufficient to determine the presence or absence of protein complex formation; therefore, high coverage is not necessarily required. However, from the standpoint of ease of analysis and reproducibility, a number of preferential cleavage sites of around 10 or less is considered practical. Note that chemical modification of amino acid residues may affect reactivity with proteases; in such cases, the number of preferential cleavage sites can be considered as that for an unmodified protein. An example of how chemical modification affects reactivity is when lysine is chemically modified, making it less susceptible to cleavage by trypsin.

[0035] The reaction temperature in the fragmentation process can be increased as long as it does not adversely affect enzymatic digestion. This is because, as long as the enzymatic activity is sufficient, a higher temperature will complete the digestion in a shorter time.

[0036] General enzymatic reactions are often carried out at temperatures of around 30-50°C because enzyme activity decreases at high temperatures. For example, existing peptide mapping technologies typically use trypsin at around 37°C. However, enzymatic digestion under these conditions generally takes 2-18 hours, depending on the amount of trypsin used. In contrast, the fragmentation process can be completed in a shorter time by using a heat-resistant enzyme and performing enzymatic digestion at high temperatures. As a heat-resistant enzyme, one can use an enzyme that is naturally heat-resistant, or one that has acquired heat resistance through genetic modification or chemical modification.

[0037] Examples of heat-resistant proteolytic enzymes include thermolysin, genetically modified thermolysin, modified thermolysin, genetically modified elastase, modified elastase, genetically modified pepsin, and modified pepsin. When using such heat-resistant enzymes, the enzyme digestion temperature is preferably 60-99°C, more preferably 65-95°C, and even more preferably 70-90°C. Furthermore, when enzyme digestion is performed at high temperatures using heat-resistant enzymes, an effect of improved reactivity can be expected due to the thermal denaturation of the target protein to be fragmented.

[0038] The enzyme used in the fragmentation process may be used in solution or immobilized on a support. Immobilizing the enzyme on a support prevents autolysis of the enzyme, thus preventing a decrease in activity due to autolysis even when used at high concentrations. For example, immobilized thermolysin, such as the thermolysin mentioned above, can be used. Immobilized enzymes also have the advantage of being easier to separate from the sample. The support is not limited, but for example, magnetic beads (Absolute Mag TM Amine Magnetic Nanoparticles, Suitable materials include CD Bioparticles, silica beads (silica gel, Sigma), metal beads (steel balls SUS304, mm size), resin beads (Melamine resin beads Supelco), and polysaccharide beads (Alginate and cellulose beads).

[0039] The solvent and pressure used in the fragmentation process are not limited. Organic solvents, salts, and surfactants can be used as long as sufficient enzyme activity is obtained. Furthermore, pressurization / depressurization is permitted as long as sufficient enzyme activity is obtained. Pressurization can improve enzyme activity in some cases, making it a viable option for shortening digestion time, although atmospheric pressure is also acceptable.

[0040] <Analysis process> The protein analysis method provided in the present invention includes an analysis step in addition to the steps described above. The analytical process allows for the quantitative evaluation of the modification status of chemically modified and fragmented proteins. Since samples containing chemically modified and fragmented proteins are complex mixtures, separation and analysis are preferable.

[0041] For example, chemically modified and fragmented proteins in a sample can be separated using liquid chromatography (LC). The LC separation mode is not limited. Reverse-phase, normal-phase, pi-electron density, hydrophobic and ionic combined modes, size exclusion, anion exchange, cation exchange, affinity, and other separation modes can be selected as needed, and multiple columns can be used. Solvent conditions and pressure in LC are also not limited. HPLC, UPLC, or capillary column LC systems can be used. Another example is that separation can also be performed using electrophoresis.

[0042] There are no limitations on the methods for quantitatively evaluating the modification status of proteins. For example, using mass spectrometry as a quantitative evaluation method facilitates the identification of fragmented chemical species and the quantification of the modification rate. Mass spectrometry can be combined with separation by LC, for example (LC-MS). Furthermore, if affinity labels are assigned through chemical modification, separation and analysis can be performed simultaneously by detecting the components obtained through affinity separation. On the other hand, it is also possible to quantitatively evaluate only the relative changes in the modification status without identifying the components in the chemically modified and fragmented sample. For example, by using sites that exhibit characteristic light absorption or fluorescence properties during chemical modification, it is possible to quantitatively evaluate the relative changes in the modification status using only LC by absorbance detection or fluorescence detection. Also, if the components in the chemically modified and fragmented sample are sufficiently separated, quantitative changes in the modification status can be detected from changes in the shape of the LC chart even without characteristic light absorption or fluorescence. It is also possible to perform the analysis without separation if the modification rate can be evaluated with sufficient quantitative accuracy without separation. NMR is one possible method for evaluating the modification status without separation. By using the method of the present invention, it becomes possible to identify the structure of a target protein by detecting chemically modified residues and fragmented proteins.

[0043] The method provided in the present invention may further include the following steps in addition to the steps described above.

[0044] <Quenching process> For example, a quenching step may be included after the chemical modification step. By inactivating unreacted reagents used in the chemical modification, it is possible to prevent them from reacting with the digestive enzymes used in the fragmentation step. The means of inactivation can be appropriately selected by those skilled in the art depending on the chemical modification used, and are not limited to those methods.

[0045] For example, when using electrophilic reagents for chemical modification, nucleophiles with a nucleophilic heterocyclic structure, such as imidazole, can be added as quenchers.

[0046] Furthermore, when using photodegradation of hydrogen peroxide for chemical modification, sacrificial agents such as hydrogen peroxide-degrading enzymes or methionine, which react with secondary oxides, can be added as quenchers.

[0047] While the quencher is not limited, from the perspective of speeding up the pretreatment process, it is preferable to use one that is easy to remove in subsequent processes or has little impact on subsequent processes. One example of a condition that makes it easy to remove in subsequent processes is that the quencher is heterogeneous or can be easily made heterogeneous.

[0048] Heterogeneous quenchers include those supported on a carrier and poorly soluble quenchers. Examples include particles with imidazole moieties on their surface and particles with catalase immobilized on their surface. Quenchers that are easily heterogeneous include those that are easily adsorbed onto adsorbents and those that easily reduce solubility. Examples of quenchers that are easily adsorbed onto adsorbents include polymers such as polyhistidine and polyvinylimidazole, which have imidazole substructures. Molecules with multiple imidazole substructures are known to be easily adsorbed onto adsorbents, for example, those supporting chelated Ni ions. Quenchers that easily reduce solubility include those with temperature-responsive moieties that change solubility, those with chelate moieties that easily precipitate with precipitating agents, and those that become hydrophobic in a pH-dependent manner, resulting in decreased solubility.

[0049] <Purification process> Another example is the inclusion of a purification step between the chemical modification and fragmentation steps. Since residual by-products of the reaction reagents used in the chemical modification step, or chemical species resulting from the deactivation of the reaction reagents, could interfere with the activity of the digestive enzymes used in the fragmentation step, a purification step can be included to remove them. While the purification method is not limited, from the perspective of speeding up the sample preparation, the heterogeneity described above can be utilized. For example, the sample solution obtained by chemical modification and the quencher can be separated by filtration or centrifugation. Furthermore, if magnetic materials are used as the carrier for the quencher or adsorbent, magnetic separation can also be performed. For example, in the above example, magnetic particles with imidazole moieties on their surface or magnetic particles with catalase immobilized on their surface could be used.

[0050] <Buffer replacement process> Another example is the inclusion of a buffer exchange step after the fragmentation step. Since the solvent conditions used for chemical modification and fragmentation are not necessarily suitable for analysis, buffer exchange can be appropriately performed using methods such as dialysis, desalting column, or solid-phase extraction.

[0051] <Enzyme removal process> Another example is the inclusion of an enzyme removal step after the fragmentation step. In particular, when immobilized enzymes are used, the enzymes can be easily removed by filtration, centrifugation, or magnetic separation, depending on the properties of the support material.

[0052] <Preservation process> Another example is that a preservation step may be included after the fragmentation step. The fragmented proteins can be prevented from changing state before analysis by, for example, cooling, freezing, or freeze-drying.

[0053] <Judgment process> When the protein analysis method provided in the present invention is used to determine whether or not a protein complex has formed, the invention includes a determination step.

[0054] For example, in the determination process, the analytical results of a sample containing a mixture of protein and candidate substances can be compared with those of a sample containing protein alone. Alternatively, the analytical results of a sample containing protein and candidate substances can be compared with those of a sample containing protein and a reference substance (a substance whose complex formation with protein is known).

[0055] Comparison can involve determining whether there is a significant difference between the results of each analysis. The method for determining significance is not limited; for example, it could be determined by focusing on the modification rate of a specific functional group, or by focusing on the modification rates of multiple functional groups. Alternatively, it could be determined based on the standard deviation obtained from multiple trials, or by using statistical tests. Examples of statistical tests, though not limited, include the t-test. In statistical tests, the significance level can be arbitrarily set as long as the results are reliable. [Examples]

[0056] [Comparative Example 1] We investigated conventional pretreatment methods using BSA aggregates prepared by heat-treating globulin-free BSA (Wako) at 80°C for 30 minutes as a biomolecular complex, DEPC as an electrophile, and trypsin (TPCK-treated trypsin, Sigma-Aldrich) as a digestive enzyme. Note that BSA is a protein consisting of 583 amino acids, listed as 3V03 in databases such as the PDB (protein data bank), and information such as the amino acid sequence can be obtained by those skilled in the art (see, for example, https: / / www.rcsb.org / structure / 3v03).

[0057] BSA aggregates (10 mg / mL, 500 μL) were mixed with a DEPC acetonitrile solution (11 mg / mL, 10 μL) and heated at 37°C for 5 minutes. Then, an imidazole aqueous solution (95 mg / mL, 25 μL) was added and heated at 37°C for 30 minutes. The resulting DEPC-modified BSA solution was denatured with urea, the urea was removed by ultrafiltration, and then reduced and alkylated using a DTT / IAA system. The reagent residue was removed again by ultrafiltration.

[0058] The obtained solution was adjusted in concentration with Tris buffer, then trypsin solution was added to BSA in a ratio of 25:1 (BSA:enzyme, the same applies below), and the mixture was heated at 37°C for 12 hours. After that, the resulting solution was buffer-changed and analyzed by LC-MS under the following conditions. LC: ODS column, 40°C, water / acetonitrile + 0.1% formic acid MS:TOF, ESI positive

[0059] When BSA aggregates without enzyme treatment were analyzed under the above conditions, a single peak was observed around 24 minutes of retention. This peak intensity was used as the initial state, and the percentage decrease in peak intensity was calculated as the digestion rate. As a result, the BSA digestibility rate was over 90%, and the total time required for pretreatment was 18 hours.

[0060] [Example 1] We investigated a pretreatment method that does not involve reduction and alkylation, using BSA aggregates as the biomolecular complex, DEPC as the electrophile, and thermolysin (Promega) as the digestive enzyme. BSA aggregates (10 mg / mL, 500 μL) were mixed with a DEPC acetonitrile solution (11 mg / mL, 10 μL) and heated at 37°C for 5 minutes. Then, an imidazole aqueous solution (95 mg / mL, 25 μL) was added and heated at 37°C for 30 minutes.

[0061] The obtained solution was adjusted in concentration with Tris-CaCl2 buffer, then thermolysin solution was added to BSA in a ratio of 25:1 and heated at 80°C for 30 minutes. After that, the obtained solution was buffer-changed and analyzed in the same manner as in Comparative Example 1. As a result, the BSA digestion rate was over 80% as calculated from HPLC, and the total time required for pretreatment was 1.5 hours, which was shorter than conventional methods.

[0062] The obtained solution was analyzed by HPLC (220 nm absorbance) using a C18 column (Cosmosil Protein-R) under gradient elution conditions of water:acetonitrile = 10:90 to 90:10, in the same manner as in Comparative Example 1.

[0063] As shown in Figure 1, when the fragmentation process was carried out at high temperature using thermolysin, the peak observed at a retention time of 24 minutes almost disappeared, and peaks of fragmented peptides were observed at shorter retention times.

[0064] [Comparative Example 2] We investigated pretreatment at typical temperatures using thermolysin. The conditions were the same as in Example 1, except that the reaction was carried out at 37°C. BSA aggregates (10 mg / mL, 500 μL) were mixed with a DEPC acetonitrile solution (11 mg / mL, 10 μL) and heated at 37°C for 5 minutes. Then, an imidazole aqueous solution (95 mg / mL, 25 μL) was added and heated at 37°C for 30 minutes.

[0065] The obtained solution was adjusted in concentration with Tris-CaCl2 buffer, then thermolysin solution was added to BSA in a 25:1 ratio and heated at 37°C for 30 minutes. After that, the resulting solution was buffer-changed and analyzed by LC-MS. As a result, the digestibility of BSA was 10%, indicating that fragmentation at 37°C using thermolysin was not sufficient as a pretreatment.

[0066] [Comparative Example 3] We investigated high-temperature pretreatment using a non-heat-resistant digestive enzyme. The conditions were the same as in Example 1, except that trypsin was used. BSA aggregates (10 mg / mL, 500 μL) were mixed with a DEPC acetonitrile solution (11 mg / mL, 10 μL) and heated at 37°C for 5 minutes. Then, an imidazole aqueous solution (95 mg / mL, 25 μL) was added and heated at 37°C for 30 minutes.

[0067] The obtained solution was adjusted in concentration with Tris buffer, then trypsin solution was added to BSA in a 25:1 ratio and heated at 80°C for 30 minutes. After that, the resulting solution was buffer-changed and analyzed by LC-MS. As a result, the digestibility of BSA was less than 5%, indicating that it was not sufficient pretreatment.

[0068] [Comparative Example 4] We investigated pretreatment without reductive alkylation using an enzyme with a small number of preferential cleavage sites. BSA aggregates (10 mg / mL, 500 μL) were mixed with a DEPC acetonitrile solution (11 mg / mL, 10 μL) and heated at 37°C for 5 minutes. Then, an imidazole aqueous solution (95 mg / mL, 25 μL) was added and heated at 37°C for 30 minutes.

[0069] The obtained solution was adjusted in concentration with Tris buffer, then trypsin solution was added to BSA in a 25:1 ratio and heated at 37°C for 1 hour. After that, the resulting solution was buffer-changed and analyzed by LC-MS. As a result, the digestibility of BSA was less than 10%, indicating that it was not sufficient for proper pretreatment.

[0070] [Example 2] In addition to the conditions of Example 1, a pretreatment method was investigated that included a step to easily remove the quencher during the chemical modification process. Polyvinylimidazole was used as the quencher.

[0071] BSA aggregates (10 mg / mL, 500 μL) were mixed with DEPC acetonitrile solution (11 mg / mL, 10 μL) and heated at 37°C for 5 minutes. Then, polyvinylimidazole ethanol solution (10 mg / mL, 25 μL) was added as the first adsorbent and heated at 37°C for 30 minutes. Ni-supported polysaccharide beads (HisTrap) were added to the resulting solution as the second adsorbent and stirred for 10 minutes. The supernatant was taken, the concentration was adjusted with Tris-CaCl2 buffer, and then thermolysin solution was added to the BSA in a ratio of 25:1 and heated at 80°C for 30 minutes. After that, the resulting solution was buffer-changed and analyzed by LC-MS. As a result, the BSA digestion rate was over 90%, and the total time required for pretreatment was 1.7 hours, which was shorter than conventional methods.

[0072] [Example 3] In addition to the conditions of Example 1, a pretreatment method was investigated that included a step to easily remove the quencher during the chemical modification process. Magnetic beads (sicaster-M) having amino groups on their surface were used as the quencher.

[0073] BSA aggregates (10 mg / mL, 500 μL) were mixed with DEPC acetonitrile solution (11 mg / mL, 10 μL) and heated at 37°C for 5 minutes. Then, sicaster-M Tris dispersion (500 μL) was added as an adsorbent and stirred for 30 minutes. After the magnetic beads were precipitated using a magnet, the supernatant was taken, the concentration was adjusted with Tris-CaCl2 buffer, and then thermolysin solution was added to the BSA in a ratio of 25:1. The mixture was heated at 80°C for 30 minutes, and the resulting solution was then buffer-changed and analyzed by LC-MS. As a result, the BSA digestion rate was over 90%, and the total time required for pretreatment was 1.7 hours, which was shorter than conventional methods.

[0074] [Example 4] The same pretreatment and analysis as in Example 1 were performed on non-aggregated BSA (globulin-free BSA, Wako), and the results were compared to determine the formation of aggregates as a complex.

[0075] Analysis of Example 1 and this example was performed with N=3 for each, and a t-test was conducted on representative peaks in HPLC. As a result, more than six peaks with a significance probability of 99% or higher were found (e.g., ADEKK (SEQ ID NO: 1), LPK, IQK, AKD, FDK, and LKT), and it was determined that there was a significant difference in the modification rate.

[0076] Next, LC-MS was used to identify which residues produced the significantly different peaks. Lys131, Lys180, Lys204, Lys322, Lys375, and Lys544 were identified as residues with significantly different modification rates.

Claims

1. A pretreatment method for analyzing the higher-order structure of a protein, comprising a step of chemical modification of the protein to be analyzed with an electrophile and a fragmentation step, wherein in the fragmentation step, the protein is treated at a temperature of 60 to 99°C in the presence of a heat-resistant protease having four or more cleavage sites and being soluble or immobilized on a carrier.

2. The method according to claim 1, wherein the heat-resistant proteolytic enzyme comprises one or more selected from thermolysin, recombinant thermolysin, modified thermolysin, elastase, recombinant elastase, modified elastase, pepsin, recombinant pepsin, and modified pepsin.

3. The method according to claim 1, further comprising the step of removing one or more selected from the electrophile, by-products of the electrophile, a quencher for the electrophile, and by-products of the quencher for the electrophile using an adsorbent.

4. The method according to claim 3, wherein the electrophile is a carbonyl compound containing a leaving group or an electron-deficient multiple bond compound.

5. The method according to claim 3, wherein the adsorbent is a polymeric or insoluble adsorbent.

6. A method for analyzing the higher-order structure of a protein, The chemical modification process of the protein to be analyzed using an electrophile, The fragmentation process of chemically modified proteins, Analysis process of fragmented proteins and The method comprising, wherein the fragmentation step is characterized by treating the protein at a temperature of 60 to 99°C in the presence of a heat-resistant proteolytic enzyme having four or more cleavage sites and being soluble or immobilized on a carrier, and the analysis step is carried out by one or more analytical methods selected from liquid chromatography, mass spectrometry, liquid chromatography-mass spectrometry, and NMR.

7. The method according to claim 6 for determining whether or not a complex is formed between a protein and a candidate substance.

8. The method according to claim 7, comprising comparing the analytical results of a sample containing a protein and a candidate substance with the analytical results of a sample containing a protein but not a candidate substance.

9. The method according to claim 7, comprising comparing the analytical results of a sample containing a protein and a candidate substance with the analytical results of a sample containing a protein and a reference substance.

10. The method according to claim 7, further comprising the step of determining whether or not complex formation has occurred based on the chemical modification rate of the fragmented protein using a statistical test.

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