Method for labeling isoaspartic acid (lβ-aspartic acid) residues

Abstract

To provide a method for labeling Lβ-aspartic acid residues in a target peptide containing aspartic acid residues.SOLUTION: A method according to the present invention includes the steps of: (1) degrading a peptide containing Lα-aspartic acid residues by processing the target peptide with a protease that specifically recognizes Lα-aspartic acid residues in the peptide and cuts the peptide; (2) removing the protease that specifically recognizes the Lα-aspartic acid residues and cuts peptides from a reaction system; and (3) methyl-esterifying the Lβ-aspartic acid residues using a methyltransferase that specifically methyl-esterifies the Lβ-aspartic acid residues and converts them into the Lα-aspartic acid residues, here, the hydroxy group of the converted Lα-aspartic acid residues is labeled with 18O by performing the step (3) in 18O water.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for labeling isoaspartic acid (Lβ-aspartic acid) residues in a peptide of interest that contains aspartic acid residues.The present invention also relates to a method for analyzing Lβ-aspartic acid residues in a protein of interest. [Background technology]

[0002] Amino acids have two enantiomers, L- and D-. For a long time, it was thought that all amino acids that make up proteins were L-amino acids. However, in recent years, proteins and peptides containing D-amino acids have been discovered in various tissues of the body.

[0003] It has been suggested that isomerization of amino acid residues changes the three-dimensional structure of proteins and affects their functions (Non-Patent Documents 1-3). Therefore, the accumulation of proteins containing amino acid isomers is thought to be related to the onset of various diseases.

[0004] In particular, aspartic acid (Asp) residues are the most susceptible to isomerization among the amino acids that constitute proteins. Non-Patent Document 4 describes the calculation of the isomerization rate to each Asp isomer using synthetic peptides and the simulation of the accumulation of Asp isomers during a life span. It has been reported that without protein metabolism, Lα-Asp is rapidly replaced by Lβ-Asp in a few years, and then gradually replaced by Dα-Asp and Dβ-Asp (Non-Patent Document 2). This result indicates that a technique for large-scale identification of Lβ-Asp is very important in the comprehensive search for Asp isomerizing proteins.

[0005] Asp has a unique isomerization mechanism, and isomerization proceeds under mild conditions in the body, isomerizing from the usual Lα form to three isomers, Lβ, Dα, and Dβ. As shown in the following formula 1, Asp is isomerized without directly abstracting the α-hydrogen of the Asp residue due to a chemical equilibrium reaction mediated by succinimide.

[0006] [ka]

[0007] Since Asp isomerization occurs through a chemical equilibrium reaction, it is believed that the main factor in isomerization in the body is time. Therefore, it is believed that isomerization of Asp residues occurs in proteins in tissues with slow metabolic turnover, such as the lens and bone. In particular, in the lens, proteins formed during fetal development are not metabolized throughout life, so isomerized proteins accumulate. Furthermore, the Asp isomerization rate of lens proteins in cataract patients is higher than that of healthy subjects of the same age, and Asp isomerization is thought to trigger the onset of cataracts (Non-Patent Document 5). Thus, Asp isomerization has been thought to accumulate and be detectable only in limited tissues with long turnover. However, in recent years, the presence of Asp isomers has been confirmed in IgG in serum, etc., suggesting the possibility that Asp isomers exist widely in the body (Non-Patent Document 1).

[0008] In addition, asparagine (Asn) residues in proteins and peptides are deamidated to form L-succinimide, which has the same structure as an aspartic acid residue, and are converted to aspartic acid residues. That is, aspartic acid isomers are formed from Asp and Asn residues.

[0009] Mass spectrometry-based proteomics is useful for large-scale exploration of post-translational modifications that occur in proteins. However, unlike phosphorylation and glycosylation, Asp isomerization is difficult to apply to proteomics because it does not change the molecular weight of the protein.

[0010] The present inventors previously reported a method for identifying Asp isomer peptides based on the characteristic that peptides with Asp isomers elute at different elution times in LC chromatograms (Non-Patent Documents 6, 7). This can be achieved by matching the elution time with a synthetic peptide or by using an enzyme that specifically cleaves or converts Asp isomers. Specifically, Lα-Asp is cleaved by endopeptidase "AspN", Lβ-Asp is treated with protein D-aspartate methyltransferase (PIMT), and Dα-Asp is degraded by D-aspartate endopeptidase (DAEP). However, this method requires the selection of peptides to be analyzed one by one, making it difficult to search for Asp isomer peptides on a large scale.

[0011] Alternatively, it has been reported that peptides with β-Asp have higher y-ion intensity in MALDI-TOF / TOF spectra than peptides with α-Asp. By utilizing this, y-46 fragment ions can be detected (and c+58 and c-57 are detected in ECD / ETD spectra) (Non-Patent Documents 8-10). These methods can be analyzed from the MS / MS spectrum of the target peptide, so Asp isomers can be detected more easily. However, these methods also require individual peptide analysis, so the presence of Asp isomers cannot be confirmed on a large scale.

[0012] On the other hand, protein D-aspartate methyltransferase (PIMT), an enzyme that specifically methylates Lβ-Asp, 18A method for specifically labeling Lβ-Asp by an enzymatic reaction in water has been reported (Non-Patent Document 11). It has also been reported that the methyl ester generated by PIMT can be further converted with hydrazine or hydroxylamine and recovered using an aldehyde resin (Non-Patent Documents 12 and 13). By this method, Lβ-Asp can be 18 O Specific to the enzymatic reaction of PIMT in water 18 Upon O labeling, the mass shifts by 2 Da, making Asp isomerization amenable to proteomics in the same way as other post-translational modifications such as phosphorylation and glycosylation, and these methods are useful for large-scale analysis of Asp isomers.

[0013] However, PIMT 18 O-labeling is problematic in that it is difficult to control. This is because PIMT is an enzyme that catalyzes the conversion of Lβ-Asp to succinimide, which is further hydrolyzed to Lβ and Lα in a ratio of about 7:3. In other words, for each turn of the enzymatic reaction by PIMT, endogenous Lβ-Asp is converted to 70% labeled Lβ-Asp and 30% labeled Lα-Asp. The labeled Lβ-Asp is catalyzed again by PIMT, and finally, most of the endogenous Lβ-Asp is converted to labeled Lα-Asp. Although labeled Lα-Asp can be analyzed by mass spectrometry, it is difficult to detect it in biological samples because it is mixed with the signal of Lα-Asp, which is abundant in biological samples, and sufficient identification results cannot be obtained.

[0014] On the other hand, there is a method in which the enzyme reaction is limited and 70% of the labeled Lβ-Asp is analyzed; however, this method is not suitable for large-scale analysis because the 30% converted to Lα-Asp is lost in the analysis and the catalytic efficiency of PIMT is expected to vary depending on the peptide. [Prior art documents] [Non-patent literature]

[0015] [Non-Patent Document 1] Gossler-Schofberger,R.,et al.,Arch Biochem Biophys 2012,522(2),100-106.DOI:10.1016 / j.abb.2012.04.006 From NLM Medline.

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Summary of the Invention

Problems to be Solved by the Invention

[0016] In order to solve the above problems, the present inventors first treated peptides with endopeptidase AspN to degrade peptides having endogenous Lα-Asp residues, and then removed AspN with magnetic beads to prevent the degradation of PIMT used in the next reaction, and to prevent the degradation of Lα-Asp labeled via PIMT by AspN, thereby finding that Lβ-Asp can be identified with high sensitivity, thus achieving the present invention.

[0017] The present invention provides a method for labeling Lβ-aspartic acid residues, among aspartic acid residues in target peptides and proteins, in a manner that distinguishes them from other Asp isomers. [Means for solving the problem]

[0018] The present invention includes, but is not limited to, the following aspects. [1] A method for labeling an Lβ-aspartic acid residue in a target peptide containing an aspartic acid residue, comprising the steps of: (1) degrading a peptide having an Lα-aspartic acid residue by treating the peptide with a protease that specifically recognizes and cleaves the Lα-aspartic acid residue in the peptide; (2) removing from the reaction system a protease that specifically recognizes the Lα-aspartic acid residue and cleaves the peptide; and (3) methyl-esterifying the Lβ-aspartic acid residues with a methyltransferase that specifically methyl-esterifies the Lβ-aspartic acid residues and converts them to Lα-aspartic acid residues; Here, step (3) 18 By carrying out the conversion in water, the hydroxy group of the Lα-aspartic acid residue is 18 Labeled with O, The method comprising: [2] The method according to [1], wherein the methyltransferase that specifically methylates Lβ-aspartic acid residues in step (3) and converts them to Lα-aspartic acid residues is L-isoaspartic acid methyltransferase. [3] The method according to [1], wherein the target peptide is a peptide having a length of 3-100 amino acid residues. [4] The method according to [1], wherein the target peptide is obtained by treating a protein with a protease that degrades the protein. [5] Before step (1), (0-i) treating a target protein with a protease that degrades proteins to obtain a target peptide; (0-ii) removing the protease that degrades the protein from the reaction system The method according to claim 1, comprising: [6] The method according to [5], wherein the treatment step with a protease that degrades proteins in step (0-i) is carried out under conditions of pH 8 to 9. [7] The method according to [4] or [5], wherein the protease that degrades the protein is selected from the group consisting of trypsin, chymotrypsin, elastase, thermolysin, pepsin, Glu-C, Glu-N, Lys-C, Lys-N, Arg-C, V8 protease, IdeS, IdeZ, and collagenase. [8] A method according to any one of [1] to [5], in which a buffer of the same pH is used from the step of (1) degrading a peptide having an Lα-aspartic acid residue by treating the target peptide with a protease that specifically recognizes Lα-aspartic acid residues in the peptide and cleaves the peptide, or (0-i) treating the target protein with a protease that degrades proteins to obtain the target peptide, to the step of (3) methyl esterifying the Lβ-aspartic acid residue with a methyltransferase that specifically methyl esterifies the Lβ-aspartic acid residue and converts it to an Lα-aspartic acid residue. [9] 1. A method for analyzing a protein of interest, comprising: (0-i) treating a target protein with a protease that degrades proteins to obtain a target peptide; (0-ii) removing a protease that degrades the protein from the reaction system; (1) degrading a peptide having an Lα-aspartic acid residue by treating said peptide with a protease that specifically recognizes and cleaves the Lα-aspartic acid residue in the peptide; (2) removing from the reaction system a protease that specifically recognizes the Lα-aspartic acid residue and cleaves the peptide; (3) methyl-esterifying the Lβ-aspartic acid residues with a methyltransferase that specifically methyl-esterifies the Lβ-aspartic acid residues and converts them to Lα-aspartic acid residues; Here, step (3) 18 By carrying out the conversion in water, the hydroxy group of the Lα-aspartic acid residue is 18 Labeled with O, and, (4) subjecting the aspartic acid residues in the peptide labeled in step (3) to mass spectrometry; The method comprising:

[10] The method according to [9], wherein the mass spectrometry in step (4) is carried out by LC-MS or MALDI-MS. Effect of the Invention

[0019] The labeling / analysis methods of the present invention make it possible to identify Lβ-aspartic acid residues in a peptide, distinguishing them from other isomers.

[0020] In the method of the present invention, step (3) "a step of methyl esterifying Lβ-aspartic acid residues with a methyltransferase that specifically methylates Lβ-aspartic acid residues and converts them to Lα-aspartic acid residues" can be carried out until the Lβ-aspartic acid residues are completely converted to Lα-aspartic acid residues. Therefore, no special control is required for the labeling of Lβ-aspartic acid residues. Furthermore, the method of the present invention includes step (2) "a step of removing a protease that specifically recognizes the Lα-aspartic acid residues and cleaves the peptide from the reaction system". This step improves the sensitivity of isomer detection and makes it possible to identify the isomerization site.

[0021] This allows the method of the present invention to enable non-targeted comprehensive analysis using database searches. [Brief description of the drawings]

[0022] [Figure 1-1] Figure 1 shows the conversion of Lβ-synthetic peptide to Lα-peptide via succinimide and methyl ester by PIMT. (a) Total ion chromatogram. (b) Base peak chromatogram. (c) Extracted ion chromatogram (EIC) of m / z 298.1. [Figure 1-2] Figure 1 shows the results of the conversion of Lβ-synthetic peptide to Lα-peptide via succinimide and methyl ester by PIMT: (d) Extracted ion chromatogram EIC of m / z 588.4, representing the TVLDSGISEVR peptide with Asp replaced by four Asp isomers; (e) EIC of m / z 579.4, representing the TVLDSGISEVR peptide with Asp replaced by succinimide; (f) EIC of m / z 595.4, representing the TVLDSGISEVR peptide with Asp replaced by methyl ester. [Diagram 2] FIG. 2 shows the change in the amount of TVLDSGISEVR synthetic peptide (a) with or (b) without AspN removal. [Diagram 3] FIG. 3 shows EIC results showing the change in the amount of TVLDSGISEVR peptide containing each Asp isomer at each reaction step: (a) untreated, (b) AspN-treated, and (c) AspN+PIMT-treated. [Figure 4] Figure 4 shows the EIC and MS signals of each isomeric peptide after the Lβ peptide detection experiment: (a) EIC of 588.6-589.6; (b) signal of peptide in which Lβ-Asp was converted to 18O-labeled Lα-Asp by PIMT; (c) signal of peptide having Dβ-Asp; (d) signal of peptide having Dα-Asp. [Figure 5-1] Figure 5 shows the results of analyzing the TVLDSGISEVR peptide in bovine αA crystallin by high-resolution mass spectrometry: (a) the amount of peptide present at each step (a1: trypsin digestion, a2: AspN digestion, a3: 18O-labeling with PIMT); (b) the signal of Lα-peptide (b1) before and (b2) after labeling. [Figure 5-2] Figure 5 shows the results of analyzing the TVLDSGISEVR peptide in bovine αA crystallin by a high-resolution mass spectrometer. (c) MS / MS spectrum of the Lα-peptide (c1) before labeling and (c2) after labeling. [Figure 6-1] Figure 6 shows the results of analyzing the HFSPEDLTVK peptide in bovine αA-crystallin by high-resolution mass spectrometry: (a) the amount of peptide present at each step (a1: trypsin digestion, a2: AspN digestion, a3: 18O-labeling with PIMT); (b) the signal of Lα-peptide (b1) before and (b2) after labeling. [Figure 6-2] 6 shows the results of analyzing the HFSPEDLTVK peptide in bovine αA-crystallin by a high-resolution mass spectrometer. (c) MS / MS spectra of the Lα-peptide (c1) before labeling and (c2) after labeling. [Figure 7-1] Figure 7 shows the results of analyzing the VQEDFVEIHGK peptide in bovine αA-crystallin by a high-resolution mass spectrometer: (a) the amount of peptide present at each step (a1: trypsin digestion, a2: AspN digestion, a3: 18O-labeling with PIMT); (b) the signal of the Lα-peptide (b1) before and (b2) after labeling. [Figure 7-2] 7 shows the results of analyzing the VQEDFVEIHGK peptide in bovine αA-crystallin by a high-resolution mass spectrometer. (c) MS / MS spectra of the Lα-peptide (c1) before labeling and (c2) after labeling. [Figure 8-1] Figure 8 shows the results of high-resolution mass spectrometry analysis of IPSGVDAGHSER peptide in bovine αA-crystallin: (a) amount of peptide present at each step (a1: trypsin digestion, a2: AspN digestion, a3: 18O-labeling with PIMT); (b) signal of Lα-peptide (b1) before and (b2) after labeling. [Figure 8-2] 8 shows the results of high-resolution mass spectrometry analysis of the IPSGVDAGHSER peptide in bovine αA-crystallin: (c) MS / MS spectrum of the Lα-peptide (c1) before labeling and (c2) after labeling. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention includes the following embodiments without limitation. Unless otherwise specified in the present specification, technical and scientific terms used in the present specification have the same meaning as those commonly understood by those skilled in the art. The substances, materials and examples disclosed in the present specification are merely illustrative and are not intended to be limiting. When referring to "in one embodiment" in the present specification, it means that the embodiment is not limited, i.e., it is not limited.

[0024] 1. Labeling method In one aspect, the present invention relates to a method for labeling Lβ-aspartic acid residues in a peptide of interest that contains an aspartic acid residue. The method of the present invention also includes a method for labeling Lβ-aspartic acid residues in a protein of interest that contains an aspartic acid residue.

[0025] The method comprises: (1) degrading a peptide having an Lα-aspartic acid residue by treating the peptide with a protease that specifically recognizes and cleaves the Lα-aspartic acid residue in the peptide; (2) removing from the reaction system a protease that specifically recognizes the Lα-aspartic acid residue and cleaves the peptide; and, (3) methyl-esterifying the Lβ-aspartic acid residues with a methyltransferase that specifically methyl-esterifies the Lβ-aspartic acid residues and converts them to Lα-aspartic acid residues; Here, step (3) 18 By carrying out the conversion in water, the hydroxy group of the Lα-aspartic acid residue is 18 Labeled with O, Includes.

[0026] The "protease that specifically recognizes and cleaves an Lα-aspartic acid residue in a peptide" is not particularly limited as long as it specifically recognizes and cleaves an Lα-aspartic acid residue in a peptide or protein. Examples of such proteases include AspN, V8 protease, and Glu-C.

[0027] In one embodiment, the above-mentioned "protease that specifically recognizes Lα-aspartic acid residues in a peptide and cleaves the peptide" is a "protease that cleaves the N-terminus or C-terminus of Lα-aspartic acid residues in a peptide", particularly an "endopeptidase that cleaves the N-terminus of Lα-aspartic acid residues in a peptide". The "endopeptidase that cleaves the N-terminus of Lα-aspartic acid residues in a peptide" is a metalloendopeptidase that cleaves the N-terminus of Lα-aspartic acid residues, also called "AspN". The "endopeptidase that cleaves the N-terminus of Lα-aspartic acid residues in a peptide" is specific to Lα-Asp, and other isomers of aspartic acid residues, such as Lβ-Asp, Dα-Asp, and Dβ-Asp, are not cleaved by the peptidase.

[0028] The above-mentioned "protease that specifically recognizes Lα-aspartic acid residues in a peptide and cleaves the peptide" does not necessarily have to cleave the peptide at the N-terminus or C-terminus of the Lα-aspartic acid residue, but any protease that specifically recognizes Lα-aspartic acid residues and cleaves the peptide at either site can be used in the present invention. Such proteases include, for example, Factor Xa protease.

[0029] The conditions for treating a target peptide with a protease that specifically recognizes Lα-aspartic acid residues in the peptide and cleaves the peptide are not particularly limited. It is preferable that the target peptide is treated under conditions that specifically recognize endogenous Lα-aspartic acid residues in the peptide and cleave the peptide in its entirety.

[0030] In one embodiment, the buffer for the protease treatment in step (1) is a neutral buffer (preferably, pH 8.6) such as Tris-HCl, MOPS, or HEPES, more preferably about 50 mM Tris-HCl (preferably, pH 8.6).

[0031] The temperature of the protease treatment in step (1) is preferably, but not limited to, about 35°C to about 39°C, about 36°C to about 38°C, or about 37°C. The treatment time is preferably, but not limited to, about 15 minutes to about 5 hours, about 30 minutes to about 2 hours, about 45 minutes to about 1.5 hours, or about 1 hour. The target peptide is treated with the protease at about 36°C to about 38°C for about 45 minutes to about 1.5 hours, or at about 37°C for about 1 hour, but not limited to. To facilitate the enzymatic reaction of the protease, components according to the type of protease, such as a catalyst (e.g., metal, etc.), coenzyme, denaturant, surfactant, etc., may be added to the reaction system.

[0032] Preferably, step (1) results in substantially all of the target peptides (or target proteins) containing Lα-aspartic acid residues being degraded, more preferably all of the target peptides containing Lα-aspartic acid residues being degraded.

[0033] In one embodiment, after step (1), (2) a protease that specifically recognizes the Lα-aspartic acid residue and cleaves the peptide is removed from the reaction system, thereby suppressing decomposition of the labeled Lα-aspartic acid residue after conversion in the subsequent step (3).

[0034] The protease that specifically recognizes the Lα-aspartic acid residue and cleaves the peptide can be removed by removing the protease protein itself from the reaction system (physical removal), or by inactivating the enzymatic activity of the protease.

[0035] The method for physically removing the protease that specifically recognizes the Lα-aspartic acid residue and cleaves the peptide is not particularly limited, and any known method for removing a specific protein can be used. For example, a purification tag may be attached to the protease, and the protease may be removed from the reaction system by a substance that binds to the tag. The type of tag is not particularly limited as long as it does not affect the activity of the protease. In the examples of the present specification, recombinant AspN with a His tag was used, and AspN was removed from the reaction system using magnetic beads for His tag purification. Alternatively, for example, biotin-avidin binding, antibodies (immunoprecipitation), etc. can be used.

[0036] The method for inactivating the enzymatic activity of the protease that specifically recognizes Lα-aspartic acid residues to cleave the peptide can be a known method, and is not particularly limited. For example, the enzymatic activity can be inactivated by adding a chelating agent, a denaturant, a protein inhibitor, or the like to the reaction system. Alternatively, high heat treatment, ultrafiltration, peptide purification by various separation modes, such as gel filtration, reverse phase, HILIC, ion exchange, or other HPLC, may be performed. However, it is preferable that these enzyme inactivation treatments do not affect the structure of the target peptide treated with the protease.

[0037] In one embodiment, the peptide of interest may be lyophilized after step (2) and before the next step (3). Since the reaction solution is removed by lyophilization, the lyophilized peptide can be 18 In step (3), the reaction solution is prepared by redissolving the compound in water. 18 O water can be easily used.

[0038] After step (2), "(3) the Lβ-aspartic acid residue is methyl-esterified by a methyltransferase which specifically methyl-esterifies the Lβ-aspartic acid residue and converts it to an Lα-aspartic acid residue." The type of "methyltransferase that specifically methylates Lβ-aspartic acid residues and converts them to Lα-aspartic acid residues" is not particularly limited as long as it is an enzyme having such activity. In one embodiment, the enzyme is "L-isoaspartic acid methyltransferase" (PIMT). There are two types of "PIMT", one that acts specifically only on Lβ-aspartic acid residues and one that acts on both L-isoaspartic acid (Lβ-aspartic acid residues) and D-aspartic acid (Dα-aspartic acid). The latter is sometimes called "protein D-aspartic acid / L-isoaspartic acid methyltransferase".

[0039] Since its first discovery in human brain tissue, PIMT has been isolated and identified in blood cells and brain tissues of various animals. It has also been revealed that it is widely distributed in the biological kingdom, both prokaryotes and eukaryotes, including Escherichia coli, nematodes, and wheat. It is known that the substrate specificity of PIMT differs depending on the organism from which it is derived.

[0040] The PIMT used in the labeling method is preferably a PIMT specific to Lβ-aspartic acid residue. However, even if a PIMT classified as protein D-aspartic acid / L-isoaspartic acid methyltransferase has a high affinity to Lβ-aspartic acid residue and a very low affinity to Dα-aspartic acid, it hardly acts on Dα-aspartic acid. Such a PIMT can be used as the PIMT used in the labeling method.

[0041] For example, it has been reported that PIMT from the hyperthermophilic archaeon Thermotoga maritima recognizes only Lβ-aspartic acid residues. However, this PMIT is not commercially available and is difficult to obtain. Human-derived PIMT is classified as protein D-aspartate / L-isoaspartate methyltransferase. In the examples of this specification, the present inventors used recombinant human protein D-aspartate-L-isoaspartate-O-methyltransferase (PIMT) (NKMAX (Sungnam, Korea)) for four types of asparagine residue isomers, Lα-Asp, Lβ-Asp, Dα-Asp, and Dβ-Asp, and found that it acted only on Lβ-Asp and not on Dα-Asp, making it possible to use the labeling method without any problems. Therefore, even PIMT classified as protein D-aspartate / L-isoaspartate methyltransferase can be used in step (3) of the labeling method as long as it acts substantially only on Lβ-aspartate residues and does not act detectably on other isomers of aspartate residues.

[0042] Examples of PIMT that can be used in the labeling method include PIMTs derived from various organisms registered as Enzyme entry 2.1.1.77.

[0043] PIMT is an enzyme that specifically recognizes the free α-carboxyl group of Lβ-aspartic acid residues and catalyzes the methyl group transfer reaction from the coenzyme S-adenosyl-L-methionine (SAM). The Lβ-aspartic acid residue that has been methyl esterified by PIMT is rapidly converted to a cyclic imide intermediate accompanied by elimination of methanol, and the generated cyclic imide intermediate is converted to an Lα-aspartic acid residue. In the method of the present invention, preferably, SAM is present in the reaction system together with PIMT in step (3).

[0044] In addition to PIMT, any enzyme having the activity of "esterifying Lβ-aspartic acid residues by an ester transferase that specifically esterifies Lβ-aspartic acid residues and converts them to Lα-aspartic acid residues," and in particular, "methyl-esterifying Lβ-aspartic acid residues by a methyl transferase that specifically methyl-esterifies Lβ-aspartic acid residues and converts them to Lα-aspartic acid residues," can be used in the labeling method of the present invention.

[0045] Step (3) 18 By carrying out the conversion in water, the hydroxy group of the Lα-aspartic acid residue is 18 For example, in one embodiment, the target peptide lyophilized after step (2) is labeled with O before the next step (3). 18 In step (3), the cellulose acetate solution may be redissolved in water and used. 16 It is important that the water content is free.

[0046] The conditions for the treatment of "(3) methyl-esterifying Lβ-aspartic acid residues with a methyltransferase that specifically methyl-esterifies Lβ-aspartic acid residues and converts them to Lα-aspartic acid residues" are not particularly limited. The treatment is preferably carried out under conditions that allow complete conversion to Lα-aspartic acid residues. The temperature in step (3) is preferably about 35°C to about 39°C, about 36°C to about 38°C, or about 37°C. The treatment time is preferably about 1 hour to about 24 hours, about 3 hours to about 20 hours, about 5 hours to about 18 hours, about 7 hours to about 12 hours, or about 8 hours or more. The treatment with the methyltransferase is preferably carried out at about 36°C to about 38°C for about 7 hours to 10 hours, or at about 37°C for about 8 hours.

[0047] In one embodiment, the buffer for the methyl esterification treatment in step (3) is a neutral buffer (preferably, pH 8.6) such as Tris-HCl, MOPS, or HEPES. More preferably, it is about 50 mM Tris-HCl (preferably, pH 8.6). For example, if lyophilization is performed before step (3), the solvent 16Only the water evaporates and disappears, while Tris, MOPS, HEPES, etc. and peptides remain as solids. 18 When water is added, the Tris concentration and pH remain the same. 16 O Water 18 The water can be exchanged for O water and then the treatment in step (3) can be carried out.

[0048] The length of the target peptide to be labeled by the labeling method is not particularly limited. In one embodiment, the target peptide is preferably 3 or more amino acid residues, 4 or more amino acid residues, 5 or more amino acid residues, 6 or more amino acid residues, 7 or more amino acid residues, or 8 or more amino acid residues. In one embodiment, the target peptide is 100 or less amino acid residues, 50 or less amino acid residues, 30 or less amino acid residues, 25 or less amino acid residues, 20 or less amino acid residues, 18 or less amino acid residues, 16 or less amino acid residues, 15 or less amino acid residues, or 14 or less amino acid residues. In one embodiment, the target peptide is a peptide having a length of 3-100 amino acid residues, 3-30 amino acid residues, 4-25 amino acid residues, 5-20 amino acid residues, 6-16 amino acid residues, or 8-14 amino acid residues.

[0049] The peptide of interest may be a peptide derived from a natural or non-natural protein. In one embodiment, the peptide of interest is obtained by treating a protein with a protease that degrades the protein. Alternatively, the peptide of interest may be a synthetic peptide.

[0050] In one embodiment, the peptide of interest may be derived from a biological sample, such as a natural protein, food, or pharmaceutical sample derived from tissue or cell of an animal, plant, bacteria, etc. Non-limiting examples of animal biological samples include body fluids such as blood, urine, and sweat, feces, skin, hair, excised organs (lens, uterus, stomach, intestine, breast, lung, etc.), and cells of an animal. Non-limiting examples of plant biological samples include leaves (including blades and petioles), stems, roots, seeds, flowers, pollen, anthers, ovules, pedicels, meristems, cotyledons, hypocotyls, pericycles, embryos, endosperms, explants, calluses, tissue cultures, buds, cells, protoplasts, and the like.

[0051] In a non-limiting example, the biological sample is dissolved in a dissolution buffer to extract the target protein. The buffer for protein extraction is not particularly limited. Preferably, the buffer for protein extraction is under conditions of pH 7.5 to 9.5, pH 8 to 9, more preferably under conditions of about pH 8.6. In one embodiment, the buffer for protein extraction is Tris-HCl (preferably pH 8.6). Other buffers such as Good's buffers such as HEPES and MOPS can also be used.

[0052] In one embodiment, the labeling method further comprises the steps of: (0-i) treating a target protein with a protease that degrades proteins to obtain a target peptide; (0-ii) removing a protease that degrades the protein from the reaction system; Includes.

[0053] The type of protease used in step (0-i) for decomposing a protein is not particularly limited, as long as a peptide of an appropriate length for labeling by the labeling method can be obtained by treating a target protein with the protease for decomposing a protein. The protease used in step (0-i) may be an endopeptidase that cleaves an internal peptide bond of a protein, or an exopeptidase that cleaves a terminal amino acid residue from a protein chain. Preferably, the protease is an endopeptidase.

[0054] In one embodiment, the protein-degrading protease is a serine protease, including trypsin, chymotrypsin, and the like.

[0055] The protease that degrades the protein is selected from the group consisting of trypsin, chymotrypsin, elastase, thermolysin, pepsin, Glu-C, Glu-N, Lys-C, Lys-N, Arg-C, V8 protease, IdeS, IdeZ, and collagenase, but is not limited thereto. Trypsin is preferred. Trypsin is a type of endopeptidase, classified as a serine protease, and hydrolyzes peptide bonds on the carboxyl side of basic amino acids (lysine, arginine).

[0056] The conditions for treating the target protein with a protease that degrades the protein in step (0-i) are not particularly limited, and appropriate conditions can be used depending on the type of protease. In one embodiment, the treatment step with a protease that degrades the protein in step (0-i) is preferably performed under conditions of pH 7.5 to 9.5, pH 8 to 9, and more preferably under conditions of about pH 8.6. In one embodiment, the protein extraction buffer is Tris-HCl (preferably, pH 8.6), more preferably about 50 mM Tris-HCl (preferably, pH 8.6). Other buffers such as Good's buffers such as HEPES and MOPS can also be used.

[0057] The temperature of the treatment with the protease that degrades the protein is preferably, but not limited to, about 0° C. to about 45° C., about 10° C. to about 32° C., about 15° C. to about 30° C., about 18° C. to about 26° C., about 20° C. to about 25° C., or room temperature. The treatment time is preferably, but not limited to, about 10 minutes to about 3 hours, about 15 minutes to about 2 hours, about 20 minutes to about 1 hour, about 25 minutes to about 45 minutes, or about 30 minutes. The target protein is treated with the protease, but not limited to, preferably, at room temperature (e.g., about 18° C. to about 26° C.) for about 25 minutes to about 45 minutes, or at about 20° C. to about 25° C. for about 30 minutes.

[0058] Without limitation, the target protein may be subjected to a reduction treatment before the treatment with a protease that degrades the protein in step (0-i). The reduction treatment cleaves disulfide bonds in the target protein, making it easier for the protease to act on the protein. The reduction treatment of the target protein can be performed using a known method. For example, it can be performed by adding a reducing agent to an extract of the target protein. The reducing agent is not particularly limited, and for example, dithiothreitol (DTT), 2-mercaptoethanol, tris(2-carboxyethyl)phosphine (TCEP), etc. can be used.

[0059] The conditions for the reduction treatment are not particularly limited. In one embodiment, the reduction treatment is preferably carried out under a condition of pH 7.5 to 9.5, a condition of pH 8 to 9, and more preferably under a condition of about pH 8.6. In one embodiment, the reduction treatment is carried out in Tris-HCl (preferably, pH 8.6), and more preferably, in about 50 mM Tris-HCl (preferably, pH 8.6).

[0060] Although not limited thereto, the reduction treatment is preferably carried out at about 35° C. to about 39° C., about 36° C. to about 38° C., or about 37° C. Although not limited thereto, the treatment time is preferably about 15 minutes to about 5 hours, about 30 minutes to about 2 hours, about 45 minutes to about 1.5 hours, or about 1 hour. Although not limited thereto, the reduction treatment is preferably carried out at about 36° C. to about 38° C. for about 45 minutes to about 1.5 hours, or at about 37° C. for about 1 hour.

[0061] The reduced cysteine ​​residue may be alkylated non-initially by alkylating the reduced target protein. The method of alkylation is not particularly limited, and any known method for alkylating reduced cysteine ​​residues may be used. Examples of alkylating agents that can be used include iodoacetamide (IAA), iodoacetic acid, chloroacetamide (CAA), and methyl methanethiosulfonate (MMTS). For example, IAA may be used to carbamidomethylate the cysteine ​​residue.

[0062] The conditions for the alkylation treatment are not particularly limited. In one embodiment, the alkylation treatment is preferably carried out under a condition of pH 7.5 to 9.5, a condition of pH 8 to 9, and more preferably under a condition of about pH 8.6. In one embodiment, the alkylation treatment is carried out in Tris-HCl (preferably, pH 8.6), and more preferably, in about 50 mM Tris-HCl (preferably, pH 8.6).

[0063] Although not limited thereto, the alkylation treatment is preferably performed at about 35° C. to about 39° C., about 36° C. to about 38° C., or about 37° C. Although not limited thereto, the treatment time is preferably about 15 minutes to about 5 hours, about 30 minutes to about 2 hours, about 45 minutes to about 1.5 hours, or about 1 hour. Although not limited thereto, the alkylation treatment is preferably performed at about 36° C. to about 38° C. for about 45 minutes to about 1.5 hours, or at about 37° C. for about 1 hour.

[0064] Non-limiting examples of the method include a step (0-ii) of removing the protease from the reaction system after the treatment of the target protein with the protease in step (0-i) and before step (1). This treatment preferably prevents the protease from acting in the reaction system in steps after step (1).

[0065] The removal of the protease that degrades the protein in step (0-ii) may be accomplished by removing the protease protein itself from the reaction system (physical removal), or by inactivating the enzymatic activity of the protease.

[0066] The method for physically removing the protease that degrades the protein is not particularly limited, and a known method for removing a specific protein can be used. For example, a solution containing the protein is added to a spin column loaded with beads to which the protease is immobilized, and the treatment with the protease that degrades the protein in step (0-i) is performed. After the treatment with the protease, the spin column is transferred to a new tube, and a solution containing only the fragmented peptides can be collected by spinning down. Alternatively, for example, as in step (2), a tag for purification may be attached to the protease, and the protease may be removed from the reaction system by a substance that binds to the tag. The type of tag is not particularly limited as long as it does not affect the activity of the protease.

[0067] The method for inactivating the enzymatic activity of proteases that degrade proteins can be a known method, and is not particularly limited. For example, the enzymatic activity can be inactivated by adding an inhibitor, such as a trypsin inhibitor, a denaturant, or a surfactant corresponding to each protease to the reaction system. Alternatively, high heat treatment or the like may be performed. However, it is preferable that the inactivation treatment of these enzymes does not affect the structure of the peptide treated with the protease.

[0068] In one embodiment, A buffer of the same pH is used from the step of (1) degrading a peptide having an Lα-aspartic acid residue by treating the target peptide with a protease that specifically recognizes Lα-aspartic acid residues in the peptide and cleaves the peptide, or (0-i) treating the target protein with a protease that degrades proteins to obtain the target peptide, to the step of (3) methyl-esterifying the Lβ-aspartic acid residue with a methyltransferase that specifically methyl-esterifies the Lβ-aspartic acid residue and converts it to an Lα-aspartic acid residue.

[0069] That is, the labeling method (0-i) treating a target protein with a protease that degrades proteins to obtain a target peptide; (0-ii) removing a protease that degrades the protein from the reaction system; (1) degrading a peptide having an Lα-aspartic acid residue by treating said peptide with a protease that specifically recognizes and cleaves the Lα-aspartic acid residue in the peptide; (2) removing from the reaction system a protease that specifically recognizes the Lα-aspartic acid residue and cleaves the peptide; (3) methyl-esterifying the Lβ-aspartic acid residues with a methyltransferase that specifically methyl-esterifies the Lβ-aspartic acid residues and converts them to Lα-aspartic acid residues; Here, step (3) 18 By carrying out the conversion in water, the hydroxy group of the Lα-aspartic acid residue is 18 Labeled with O, The same pH buffer may be used in all steps.

[0070] The buffer with the same pH is, but is not limited to, Tris-HCl (preferably, pH 8.6), more preferably about 50 mM Tris-HCl (preferably, pH 8.6). Other buffers such as Good's buffers such as HEPES and MOPS can also be used.

[0071] The buffer for extracting proteins from tissues or cells may be the same as the buffer used in steps (0-i) to (3).

[0072] In the step (0-i) of reducing the target protein prior to the treatment with a protease that degrades the protein, a buffer common to steps (0-i) to (3) may also be used.

[0073] In the step of alkylating the reduced cysteine ​​residue by alkylating the reduced target protein, a buffer common to steps (0-i) to (3) may be used.

[0074] In the step (0-ii) of removing the protease that degrades the protein from the reaction system, a buffer common to steps (0-i) to (3) may also be used.

[0075] By using a common buffer, buffer exchange through dialysis, column operation, etc. is not required in the series of steps in the described method.

[0076] By the labeling method, the hydroxy group of the Lα-aspartic acid residue after conversion is 18 The peptide is labeled with O. As a result, of the four isomers of aspartic acid residues in the target peptide, Lα-Asp, Lβ-Asp, Dα-Asp, and Dβ-Asp, only the isomer originally contained in the target peptide as Lβ-Asp will have a molecular weight that is 2 Da larger. Therefore, by performing mass spectrometry on the labeled peptide, it is possible to identify only Lβ-Asp, distinguishing it from the other isomers, Lα-Asp, Dα-Asp, and Dβ-Asp. In addition, when Asp generated by deamidation of Asn is converted to Lβ-Asp, the molecular weight will be 3 Da larger than that of asparagine. This technology can also be used to analyze the case where Asn is converted to Lβ-Asp.

[0077] 2. Protein Analysis Methods In one aspect, the present invention relates to a method for analyzing a protein of interest.

[0078] The analysis method includes: (0-i) treating a target protein with a protease that degrades proteins to obtain a target peptide; (0-ii) removing a protease that degrades the protein from the reaction system; (1) degrading a peptide having an Lα-aspartic acid residue by treating said peptide with a protease that specifically recognizes and cleaves the Lα-aspartic acid residue in the peptide; (2) removing from the reaction system a protease that specifically recognizes the Lα-aspartic acid residue and cleaves the peptide; (3) methyl-esterifying the Lβ-aspartic acid residues with a methyltransferase that specifically methyl-esterifies the Lβ-aspartic acid residues and converts them to Lα-aspartic acid residues; Here, step (3) 18 By carrying out the conversion in water, the hydroxy group of the Lα-aspartic acid residue is 18 Labeled with O, and, (4) subjecting the aspartic acid residues in the peptide labeled in step (3) to mass spectrometry; Includes.

[0079] Each step of step (0-i), step (0-ii), step (1), step (2), and step (3) is as described in "1. Labeling method." All the contents described in "1. Labeling method" also apply to "2. Analytical method" unless otherwise contradicted.

[0080] In step (4), the aspartic acid residues in the labeled peptide are subjected to mass spectrometry. The method for mass spectrometry of the aspartic acid residues in the peptide is not particularly limited, and any known method can be used.

[0081] Mass spectrometry (MS) When molecules are ionized and separated in an electric or magnetic field, the force that the molecules experience is proportional to the ratio of their mass m to their charge z, and so mass spectrometry is an analytical method that accurately measures the mass of ions or molecules by measuring m / z. Instruments used for mass spectrometry are called "mass spectrometers."

[0082] A mass spectrometer is usually composed of a "mass introduction section," an "ion source," an "analysis section," an "ion detection section," and a "data processing section." The "sample introduction section" is the section where the sample is introduced into the device. The introduction method differs depending on whether the sample is a gas or a volatile substance, or a liquid, solid, or a non-volatile substance. The "ion source" is the section where the target sample is charged. Various methods have been developed, such as the EI method, the CI method, the FD method, the FAB method, the MALDI method, and the ESI method. The "analysis section" separates the ionized sample, and several methods have been developed with different resolutions (mass resolutions) to distinguish m / z values ​​and different measurable ranges. There are magnetic sector types, quadrupole types, ion trap types, time-of-flight types, etc., and they are selected according to the purpose. The "ion detection section" detects the ions selected in the analysis section by sensitizing them with an electron multiplier or a microchannel plate. The "data processing section" creates a mass spectrum from the obtained data. Moreover, it is possible to directly connect a high performance liquid chromatography (HPLC), gas chromatography (GC), or capillary electrophoresis (CE) to the front stage of the mass spectrometer for analysis. These are called LC-MS, GC-MS, and CE-MS, respectively. In one embodiment, the analysis method is performed by LC-MS using a nanoflow liquid chromatograph (nanoLC) with a flow rate of 300 nL / min.

[0083] ESI (ElectroSpray Ionization) method This is an ionization method mainly used in LC / MS, and is a type of atmospheric pressure ionization (API) method. The sample is dissolved in a solvent and introduced and sprayed into a capillary with a high voltage applied, forming charged droplets, from which the solvent molecules are evaporated, causing the charge on the droplet surface to overcome the surface tension and splitting the droplets. This process is repeated, ultimately producing ions. Like MALDI, which will be described later, this method shows particularly excellent properties for ionizing high molecular weight compounds (it is easy to produce multivalent ions). It is one of the softest ionization methods. The applicable molecular weight range is about 200 to 1 million.

[0084] MALDI (Matrix Assisted Laser Desorption Ionization) method This method involves mixing the sample into a matrix (such as an aromatic organic compound) to create crystals, which are then ionized by irradiating them with a laser. Even high molecular weight compounds such as proteins can be ionized stably. The applicable molecular weight range is about 1 to 1,000,000.

[0085] In the case of ESI, separation by LC is required, resulting in LC-MS, whereas in the case of MALDI, no connection with LC is required (MALDI-MS). In one embodiment, the analysis method is carried out by MALDI-MS.

[0086] FAB (Fast Atom Bombardment) method A method in which a sample is mixed into a matrix (such as glycerin) and ionized by colliding neutral atoms (such as Ar and Xe) at high speed. Since there is no need to vaporize the sample, it can be used for a wide range of substances. The applicable molecular weight range is about 500 to 5000.

[0087] APCI (Atmospheric Pressure Chemical Ionization) method This is an ionization method mainly used in LC / MS. After the sample solution is forcibly vaporized by heating at high temperatures of 400-500°C, ions are generated using the discharge of a corona needle. As the name suggests, this is CI under atmospheric pressure, and the vaporized solvent acts as the reactant gas. It is a harder ionization method than ESI, and a small amount of fragment ions are generated. It is suitable for ionizing compounds with low to medium polarity, i.e. compounds that can be separated by normal phase chromatography.

[0088] In one embodiment, the mass spectrometry in step (4) is carried out by LC-MS or MALDI-MS.

[0089] In step (3) of the analysis method, the hydroxy group of the Lα-aspartic acid residue after conversion is 18The peptide is labeled with O. Thus, of the four isomers of aspartic acid residues in the peptide, Lα-Asp, Lβ-Asp, Dα-Asp, and Dβ-Asp, only the isomer originally contained in the peptide as Lβ-Asp has a molecular weight that is 2 Da larger. In addition, the isomer in which asparagine is deamidated and converted to Lβ-Asp has a molecular weight that is 3 Da larger. Therefore, by performing mass spectrometry on the peptide labeled in step (4), it becomes possible to identify only Lβ-Asp, distinguishing it from the other isomers, Lα-Asp, Dα-Asp, and Dβ-Asp.

[0090] Table 1 shows a comparison of the labeling method / analysis method of the present invention (AspN Digestion before 18O label by PIMT (this method)) with the Asp isomer analysis methods reported prior to the present invention.

[0091] [Table 1]

[0092] The labeling method / analysis method of the present invention can identify only Lβ-Asp, distinguishing it from other isomers, Lα-Asp, Dα-Asp, and Dβ-Asp. The method of the present invention can also identify other aspartic acid isomers, preferably in combination with other methods. For example, in order to improve the reliability of the identification of the method of the present invention, it can be combined with MS / MS to confirm β-Asp-specific fragments from the MS / MS spectrum.

[0093] Asp has a unique isomerization mechanism, and it has been reported that isomerization proceeds under mild conditions in the living body, and the normal Lα form is isomerizes into three isomers, Lβ, Dα, and Dβ. Since Asp isomerization occurs through a chemical equilibrium reaction mediated by succinimide, it is believed that the main factor in isomerization in the living body is time. The method of the present invention enables the identification of proteins that have Lβ-Asp in proteins, and further allows for a deeper analysis of the presence of other Asp isomers. The method of the present invention allows the identification of which tissues, which proteins, and which Asp residues are isomers at once.

[0094] 3. Application of the present invention The labeling method / analysis method of the present invention is a technique that enables comprehensive analysis of which amino acid site in a protein Lβ-Asp occurs in a single analysis. Therefore, the present invention also includes the identification of Lβ-Asp as a biomarker (for disease or aging) or as an indicator for monitoring the quality of protein preparations such as antibodies. The present invention can be widely applied to the discovery of biomarkers and the quality control of biopharmaceuticals.

[0095] Regarding quality control, it is known that Lβ-Asp occurs in antibodies due to deamidation of asparagine, and that aspartic acid is directly isomerized. This may occur during the manufacturing process, or the antibody may gradually change to Lβ-Asp when stored. In any product using a protein, deamidation and isomerization are unavoidable spontaneous modifications, and isomerization has been incorporated into the quality evaluation (MAM) of protein preparations (e.g., antibody drugs). For this purpose, it is important to identify which aspartic acid residues in a protein preparation are prone to isomerization and to grasp at once which should be monitored. There is no method other than the present invention that can comprehensively search for Lβ-Asp in a protein for the quality control of such protein preparations. EXAMPLES

[0096] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Those skilled in the art can easily modify and alter the present invention based on the description in this specification, and such modifications and alterations are within the technical scope of the present invention.

[0097] material In the examples herein, the following materials were used unless otherwise specified.

[0098] All chemicals were of reagent grade or higher. Recombinant human protein D-aspartate-L-isoaspartate-O-methyltransferase (PIMT) was purchased from NKMAX (Sungnam, Korea). S-Adenosyl-methionine hydrochloride (SAM) was purchased from Cayman Chemicals (Sedaratna Laboratories, Canada). 18 O water was purchased from Taiyo Nippon Sanso Corporation (Tokyo).

[0099] α-Crystallin purified from bovine eye lenses was obtained from Sigma (St. Louis, MO, USA). Immobilized trypsin and recombinant AspN were purchased from Promega (Madison, WI, USA).

[0100] Example 1 Analysis of synthetic peptides (1) Preparation of synthetic peptides As a verification peptide, an α-crystallin peptide containing four different Asp isomers at Asp58 was used. α-crystallin is a complex of αA-crystallin (SEQ ID NO: 1) and αB-crystallin (SEQ ID NO: 2). Of the amino acid sequence of SEQ ID NO: 2, a sequence (TVLDSGISEVR (SEQ ID NO: 3)) corresponding to 55 to 65 was synthesized.

[0101] The peptides were synthesized on a 0.03 mmol scale by Fmoc solid-phase synthesis on 0.03 mmol of Fmoc-Arg(Pbf) Wang resin (0.8 mmol / g substitution) using a microwave peptide synthesizer (Liberty Blue; CEM). Fmoc deprotection was performed with 20% piperidine in N,N'-dimethylformamide (DMF). Coupling reactions of amino acids were performed using 0.2 M Fmoc-amino acids dissolved in DMF, with the addition of a solution (containing 0.5 M N,N'-diisopropylcarbodiimide (DIC) and 0.5 M Oxyma Pure) in a volume 5 times the volume of the resin. Fmoc-amino acids and other coupling reagents were purchased from Watanabe Chemical Industry (Hiroshima).

[0102] During synthesis, in order to replace the fourth aspartic acid residue with the four isomers Lα, Lβ, Dα, and Dβ, Fmoc-aspartic acid was synthesized using Fmoc-L-Asp(OtBu)-OH for Lα, Fmoc-L-Asp-OtBu-OH for Lβ, Fmoc-D-Asp(OtBu)-OH for Dα, and Fmoc-D-Asp-OtBu-OH for Dβ.

[0103] The peptide synthesized on the resin was treated in 92.5% trifluoroacetic acid (TFA), 2.5% water, 2.5% triisopropylsilane, and 2.5% 3,6-dioxa-1,8-octanedithiol for 2 hours to simultaneously cleave the peptide from the resin and remove the protecting groups. Ice-cold diethyl ether was added to the peptide solution after cleavage to precipitate the peptide. The precipitate was air-dried and then dissolved in 0.1% TFA / water. The peptide solution was subjected to reverse-phase chromatography using a C18 Shiseido column (Capcell Pak C18 ACR, 10 × 250 mm; Shiseido, Tokyo) to purify the target peptide. The chromatography conditions were: Solution A: 0.1% TFA / water, Solution B: 0.1% TFA / acetonitrile, gradient: initial condition was 100% Solution A. The analysis was performed with a linear gradient reaching 50% B in 60 minutes, a flow rate of 3 mL / mL, and a detection wavelength of 230 nm. The purified peptide was lyophilized to a powder, dissolved in water to a concentration of 10 mg / mL, and stored at -30°C until use.

[0104] (2) AspN digestion of synthetic peptides and removal of AspN The synthetic peptide synthesized in (1) above was dissolved in 50 mM Tris-HCl (pH 8.6) to 1 ng / μL. AspN was added to the peptide solution at a ratio of peptide:AspN = 1:5, and the mixture was treated at 37 °C for 1 hour. AspN was a recombinant AspN with a His tag attached, which is sold by Promega. During the enzymatic reaction with AspN, magnetic beads for His tag purification were prepared. Specifically, 10 μL of magnetic beads for His tag purification (Dynabeads His-Tag Isolation & Pulldown) were washed three times with 50 μL of 50 mM Tris-HCl (pH 8.6). The solution after the AspN enzymatic reaction of the peptide was added to the washed magnetic beads, and the mixture was stirred at 1200 rpm at 4 °C for 1 hour to adsorb AspN to the magnetic beads. The tube was placed on a magnet, the magnetic beads were adsorbed to the wall of the tube, and AspN was removed, and only the solution was transferred to another tube.

[0105] (3) PIMT reaction and 18O labeling Using the above synthetic peptide, the conversion reaction from Lβ-Asp to Lα-Asp was analyzed over time.

[0106] To the peptide solution from which AspN was removed in (2) above, 1 μg of PIMT (10 times the amount) and 10 μg of SAM (10 times the amount of PIMT) were added to 50 ng of synthetic peptide, and incubated at 37 ° C. After 1, 3, 5, 7, 9 and 15 hours, the peptides were collected over time and analyzed by LC-MS (G6125B LC / MSD (Agilent technology, USA). The quantitative changes were analyzed by tracing the doubly charged ions m / z 588.3 of the synthetic peptide TVLDSGISEVR, m / z 579.4 (-18 Da) where Asp was converted to a succinimide form, and m / z 595.3 (+14 Da) where Asp was converted to a methyl ester form.

[0107] 18 For O labeling, freeze-dry the peptide solution (excluding AspN) and the PIMT / SAM to dryness, then lyophilize the peptide. 18 The cells were resuspended in 0. This solution was transferred to the lyophilized PIMT / SAM and incubated at 37° C. for 1 hour.

[0108] (4) Mass spectrometry of synthetic peptides The reaction solution (3) above was subjected to mass spectrometry using an LC-MS system consisting of a G7137A multisampler, a G7131A quaternary pump, a G7132A high-speed pump, a G7116B multicolumn thermostat, a G717A diode array detector, a G5664B fraction collector, a G1330B thermostat, a G7110B isocratic pump, and a G6125B LC / MSD (Agilent technology, USA).

[0109] The sample solution was injected onto a Poloshell C18 column (2.1 μm, 100 mm × 2.1 mm; Agilent technology, USA) with a linear gradient of 10–20% acetonitrile for 7 min at a flow rate of 0.6 mL / min in the presence of 0.1% formic acid. The column temperature was set at 40 °C. MS scan collection was performed over an m / z range of 200–2000. The capillary voltage was 3 kV and the drying gas temperature was 350 °C. Extracted ion chromatography (EIC) was used to quantify the relative amounts of Lβ-peptide and its Asp isomer. EICs were drawn using m / z 588.317 for the unlabeled peptide and m / z 589.317 for the labeled peptide.

[0110] (5) Results (A) Methyl esterification of Lβ-Asp catalyzed by PIMT The conversion reaction of Lβ-Asp to Lα-Asp catalyzed by PIMT was confirmed using a synthetic peptide TVLDSGISEVR in which the Asp residue was replaced with Lβ-Asp. The Lβ-Asp peptide peak disappeared at the early stage of the reaction and was replaced by succinimide and methyl ester forms. The succinimide and methyl ester were gradually replaced by Lβ-Asp and Dβ-Asp. The succinimide and methyl ester forms decreased over time, almost disappearing after 8 hours and completely disappearing after overnight treatment. On the other hand, the m / z 298.1 peak was observed to increase with the extension of the reaction time at an elution time of about 2 minutes (Figure 1). This is considered to be a by-product derived from SAM or PIMT, since its mass does not match the peptide cleavage.

[0111] (B) Effect of AspN removal To confirm the effect of AspN removal, four peptide mixture samples, Lα-, Lβ-, Dα-, and Dβ-peptides, were subjected to AspN digestion and then removed with magnetic beads. The peptide mixture was divided into two test tubes. In one test tube, magnetic beads were added immediately after the addition of AspN, and the solution was stirred at 1,400 rpm at 4 °C for 1 h. In the other tube, after the addition of AspN, the solution was left stationary at 4 °C for 1 h without magnetic beads. Each peptide solution was incubated at 37 °C for 1 h and then analyzed by LC-MS. Figure 2 shows the EIC results of the synthetic peptides with and without AspN removal.

[0112] Removal of AspN inhibited the cleavage of Lα-peptide. In contrast, without AspN removal, only Lα-peptide was completely lost. This result indicates that AspN is inactivated by collection with magnetic beads and that AspN acts specifically on Lα-Asp and does not degrade other Asp isomer peptides.

[0113] (C) Sequential processing of synthetic peptides The sequential treatment from AspN digestion to Lβ-Asp conversion by PIMT was performed on a mixture of peptides with Lα-, Lβ-, Dα- and Dβ-peptides. Figure 3 shows the EIC of the synthetic peptides at each reaction step. Lα-peptide completely disappeared at the AspN digestion step. In the next step of PIMT treatment, Lβ-peptide disappeared and Lα-peptide was reconstituted. This result also indicates that AspN is completely removed by magnetic beads and does not affect the subsequent PIMT treatment. AspN removal is a very important step because the residual AspN will degrade not only PIMT itself but also the Lα-peptide reconstituted by PIMT, significantly reducing the detection sensitivity.

[0114] (D) 18 PIMT reaction in water A synthetic peptide mixture containing four Asp isomer peptides was processed up to the AspN removal step. The peptide solution was lyophilized, and the PIMT reaction was carried out by removing the solvent. 18The experiment was carried out under conditions where the ion exchange ratio was completely replaced with O and water. Figure 4 shows the EIC of the synthetic peptides and the mass spectra of the Lα-, Dα-, and Dβ-peptides. The mass spectra of the Dα- and Dβ-peptides showed a signal at m / z 588.3 as a monoisotopic doubly charged mass. On the other hand, a mass shift of 2 Da was observed for the Lα-peptide, with the signal at m / z 589.3. These results indicate that the PIMT reaction is responsible for the 18 This shows that O labeling proceeds specifically with the Lβ-peptide.

[0115] Example 2 Processing and Analysis of Bovine Alpha-Crystallin (1) Trypsin digestion of bovine lens α-crystallin A commercially available powder of bovine crystalline lens-derived α-crystallin (10 μg) was dissolved in 50 mM Tris-HCl (pH 8.6) to prepare a solution of 0.1 mg / mL. Of this, 1 μL (10 μg, weight ratio of protein:DTT=1:1) of dithiothreitol (DTT) prepared at 10 μg / μL was added to 10 μL (10 μg) of the protein solution, and the solution was reduced at 37°C for 1 hour to cleave the disulfide bonds.

[0116] Next, 1 μL (25 μg, weight ratio of protein:IAA=1:2.5) of iodoacetamide (IAA) adjusted to 25 μg / μL was added to the reduced protein, and the cysteine ​​residues were alkylated (carbamidomethylated) at 37° C. for 1 hour.

[0117] 100 μL of trypsin-immobilized beads (Promega, V9012) were transferred onto a spin column, spun down (2000 rpm, 5 seconds), and the solution was removed. 100 μL of 50 mM Tris-HCl (pH 8.6) was added to the trypsin-immobilized beads, and the beads were washed by spinning down again (2000 rpm, 5 seconds). Washing was performed three times. The alkylated protein was added to the washed trypsin-immobilized beads, and the beads were left to stand at room temperature for 30 minutes to fragment the peptides on the spin column. The spin column was transferred to a new tube, and the solution containing the fragmented peptides was collected by spinning down.

[0118] (2) AspN digestion of bovine lens α-crystallin and removal of AspN 500 ng of AspN was added to the trypsin-digested peptide fragment (10 μg) in (1) above, and the mixture was treated at 37°C for 1 hour. AspN was a recombinant AspN with a His tag attached, available from Promega. During the enzymatic reaction with AspN, magnetic beads for His tag purification were prepared. Specifically, 10 μL of magnetic beads for His tag purification (Dynabeads His-Tag Isolation & PμLldown) were washed three times with 50 μL of 50 mM Tris-HCl (pH 8.6) during the enzymatic reaction.

[0119] The solution after AspN enzymatic reaction was added to the washed magnetic beads and stirred at 1200 rpm for 1 hour at 4°C to allow AspN to be adsorbed onto the magnetic beads. The tube was placed on a magnet to allow the magnetic beads to be adsorbed onto the tube wall, and the AspN was removed and only the solution was transferred to another tube.

[0120] (3) PIMT reaction and 18 O labeling Using the trypsin-digested peptide fragments described above in (2), the conversion reaction from Lβ-Asp to Lα-Asp was analyzed over time.

[0121] The solution of trypsin-digested peptide fragments (10 μg) from which AspN was removed as described above in (2) and PIMT / SAM (PIMT: 1 μg; 1 / 10 the amount of peptide, SAM: 10 μg; 10 times the amount of PI) were lyophilized to complete drying. The peptides were then immersed in an equal amount of H2 18 The mixture was resuspended in 0.05% ethanol and transferred to the lyophilized PIMT / SAM and incubated at 37° C. The reaction solution was time-dependently collected after 1, 3, 5, 7, 9, and 15 hours and injected into an LC-MS / MS system to identify the isomerization site of the Asp residue using a database search.

[0122] The samples were analyzed by LC-MS / MS using a Q-Exactive mass spectrometer (Thermo Fisher Scientific, USA) equipped with a nanoflow LC system (Easy nLC 1200, Thermo Fisher Scientific, USA).

[0123] Peptides were collected on a C18 trap column, desalted, and then separated and analyzed on a C18 nanocolumn (C18 column, 0.15 mm id × 100 mm; 3 μm particles; Nikko Technos, Japan). Peptides were separated using a linear gradient of acetonitrile concentration increasing from 5 to 60% in 90 min at a flow rate of 200 nl / min with a mobile phase of water-acetonitrile containing 0.1% formic acid. The spray voltage was 1.5 kV and the capillary temperature was 250 °C. All MS scans were obtained on an Orbitrap analyzer with a resolution of 70,000 at m / z 400 (m / z 150–2000), and HCD MS / MS scans of the top 10 most abundant ions were obtained on an Orbitrap analyzer with a resolution of 17,500, a normalized collision energy (NCE) of 27, and 15 s of exclusion. EIC was used to quantify the relative amounts of Lβ-Asp peptide and its Asp isomer. The eluate was ionized via an electrospray interface and directly introduced into a mass spectrometer. Mass spectrometry was performed in positive ion mode with data-dependent acquisition.

[0124] The obtained MS / MS spectra were converted to mgf by Mascot distiller, and the mgf files were applied to a database search using the Mascot search engine (ver. 2.6.2; Matrix Science) and the UniProtKB database (downloaded on August 3, 2018; 7330 entries for bovine).

[0125] The search parameters were the fragmentation method used (trypsin digestion), the number of miscleavages allowed: 2, the fixed modification: carbamidomethylation of cysteine, the variable modifications: pyroglutamylation of N-terminal glutamine (terminal glutamine), deamination of the N-terminal amino group (terminal cysteine), oxidation (methionine),18 Deamidation with incorporation of O (asparagine), 18 Aspartic acid incorporating O, MS spectrum tolerance: 7 ppm, MS / MS spectrum tolerance: 0.02 Da were used.

[0126] A search was performed under the above conditions, and from the results obtained, an identification was deemed to have been made if the identification certainty score (probability of a coincidence: Expect) was less than the smaller of 0.05 or the Expect value that results in a False Discover Rate of 1%.

[0127] (4) Identification of the isomerization site of α-crystallin Alpha-crystallin (SEQ ID NO: 1) from bovine eye lens was digested with immobilized trypsin after reduction and alkylation. Lβ-Asp was then 18 The peptides were labeled with O. The resulting peptides were analyzed by high-resolution LC-MS / MS, and database searches were performed using Mascot to identify peptides with a mass increase of +2 Da at the Asp site.

[0128] As a result, four isomerization sites were found in four peptides derived from α-crystallin.

[0129] TVLD 58 SGISEVR (SEQ ID NO: 3), HFSPED 84 LTVK (SEQ ID NO: 4), VQED 92 FVEIHGK (SEQ ID NO:5), IPSGVD 151 AGHSER (SEQ ID NO:6), Figure 5 shows the TVLD 58The results of SGISEVR are shown. Figure 5a is the extracted ion chromatogram of the peptide sequence at m / z 588.3170 ± 5 ppm or 589.3191 ± 5 ppm, which is TVLDSGISEVR. The EICs of three types of trypsin treatment, trypsin-AspN treatment, and trypsin-AspN-PIMT treatment were compared from top to bottom. The intensity of the main peak at 61.7 min was decreased by AspN digestion compared to trypsin digestion, therefore, the peak at 61.7 min is assigned to Lα-peptide. Furthermore, the peak at 58 min was decreased by PIMT treatment, so the peak at 58 min was assigned to Lβ-peptide. The other peaks detected at 62 min and 63.7 min were considered to be other isomeric peptides with the same mass, such as Dα- or Dβ-peptides.

[0130] Figure 5b compares the mass spectra of the Lα-peptide from the tryptic digest and the Lα-peptide after PIMT treatment. A monoisotopic mass of m / z 588.317 was detected in the Lα-peptide from the tryptic digest. Meanwhile, the intensity of m / z 588.317 was decreased, and the intensity of m / z 589.139, which is shifted by 2 Da, was enhanced in the Lα-peptide after PIMT treatment. The spectrum detected at m / z 588.317 corresponds to the endogenous Lα-peptide that is not completely digested by AspN and 18 O- 5% of the water 16 O - It is believed that water may be the cause.

[0131] Comparing the MS / MS spectra of Lα-peptide treated with trypsin and treated with PIMT, the same fragment ions were detected from y1 to y7 ions, but a 2 Da mass shift was observed in y8 and y9 ions. 18 It was shown that the O label was introduced into the fourth amino acid residue in the peptide, i.e., an Asp residue.

[0132] HFSPED 84 LTVK, VQED 92 FVEIHGK and IPSGVD 151The spectra of each of the three peptides of AGHSER are shown in Figures 6-8. [Industrial Applicability]

[0133] The labeling method / analysis method of the present invention is a technique that enables comprehensive analysis of which amino acid site in a protein Lβ-Asp occurs in a single analysis. Therefore, the present invention also includes the use of Lβ-Asp as a biomarker (for disease or aging) or as an indicator for monitoring the quality of protein preparations such as antibodies. The present invention can be widely applied, such as in the discovery of biomarkers and the quality control of biopharmaceuticals.

Claims

1. 1. A method for labeling an L β-aspartic acid residue in a subject peptide that contains an aspartic acid residue, comprising: (1) degrading a peptide having an L α-aspartic acid residue by treating the peptide of interest with a protease that specifically recognizes and cleaves the L α-aspartic acid residue in the peptide; (2) removing from the reaction system a protease that specifically recognizes the Lα-aspartic acid residue and cleaves the peptide; and (3) methyl-esterifying the Lβ-aspartic acid residue with a methyltransferase that specifically methyl-esterifies the Lβ-aspartic acid residue and converts it to an Lα-aspartic acid residue; Here, step (3) １８ By carrying out the reaction in water, the hydroxy group of the L α-aspartic acid residue after the conversion is １８ Labelled with O, The method comprising:

2. The method according to claim 1, wherein the methyltransferase in step (3) which specifically methylates Lβ-aspartic acid residues and converts them to Lα-aspartic acid residues is L-isoaspartic acid methyltransferase.

3. The method of claim 1, wherein the peptide of interest is a peptide of 3-100 amino acid residues in length.

4. The method of claim 1 , wherein the target peptide is obtained by treating a protein with a protease that degrades the protein.

5. Before step (1), (0-i) treating a target protein with a protease that degrades the protein to obtain a target peptide; (0-ii) removing the protease that degrades the protein from the reaction system The method of claim 1 , comprising:

6. The method according to claim 5, wherein the treatment step with a protease that degrades proteins in step (0-i) is carried out under conditions of pH 8 to 9.

7. 6. The method of claim 4 or 5, wherein the protein-degrading protease is selected from the group consisting of trypsin, chymotrypsin, elastase, thermolysin, pepsin, Glu-C, Glu-N, Lys-C, Lys-N, Arg-C, V8 protease, IdeS, IdeZ, and collagenase.

8. The method according to any one of claims 1 to 5, wherein the same pH buffer is used for the steps from (1) treating a target peptide with a protease that specifically recognizes Lα-aspartic acid residues in the peptide to degrade the peptide having Lα-aspartic acid residues, or (0-i) treating a target protein with a protease that degrades proteins to obtain a target peptide, to (3) methyl-esterifying the Lβ-aspartic acid residues with a methyltransferase that specifically methyl-esterifies the Lβ-aspartic acid residues and converts them to Lα-aspartic acid residues.

9. 1. A method for analyzing a protein of interest, comprising: (0-i) treating a target protein with a protease that degrades the protein to obtain a target peptide; (0-ii) removing a protease that degrades the protein from the reaction system; (1) degrading a peptide having an L α-aspartic acid residue by treating said peptide with a protease that specifically recognizes and cleaves the L α-aspartic acid residue in the peptide; (2) removing from the reaction system a protease that specifically recognizes the Lα-aspartic acid residue and cleaves the peptide; (3) methyl-esterifying the Lβ-aspartic acid residue with a methyltransferase that specifically methyl-esterifies the Lβ-aspartic acid residue and converts it to an Lα-aspartic acid residue; Here, step (3) １８ By carrying out the reaction in water, the hydroxy group of the L α-aspartic acid residue after the conversion is １８ Labelled with O, and, (4) subjecting the aspartic acid residues in the peptide labeled in step (3) to mass spectrometry; The method comprising:

10. The method according to claim 9, wherein the mass spectrometry in step (4) is carried out by LC-MS or MALDI-MS.

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