JP2023090132A - METHOD FOR ANALYZING γ-CARBOXY GLUTAMIC ACID MODIFICATION LEVEL IN VITAMIN K DEPENDENT PROTEIN - Google Patents

Abstract

To provide a method for analyzing Gla modification levels in vitamin K dependent protein.SOLUTION: An analytical method at least comprises the steps of: digesting vitamin K dependent protein with chymotrypsin, to cleave a Gla domain, to obtain Gla domain peptides; and sorting the Gla domain peptides by liquid chromatography according to the Gla modification levels. Such an analytical method enables the Gla modification levels to be determined quantitatively and qualitatively.SELECTED DRAWING: Figure 2

Description

The present invention relates to a method for analyzing the degree of γ-carboxyglutamic acid (Gla) modification of vitamin K-dependent proteins such as vitamin K-dependent blood coagulation factors.

Vitamin K is a fat-soluble vitamin that acts as a cofactor for γ-carboxylase, an enzyme that converts glutamic acid residues (Glu) to γ-carboxyglutamic acid residues (Gla). Proteins with such Gla are called vitamin K-dependent proteins. For example, vitamin K-dependent blood coagulation factors such as blood coagulation factor X (FX) have a Gla domain containing around 10 Gla at their N-terminus. have. Gla is an amino acid that is not found in general proteins, but in vitamin K-dependent proteins, Gla is introduced by post-translational modification during the intracellular production process (Non-Patent Document 1).

This Gla domain has a role of coordinating Ca ions and plays an important role in the physiological functions of vitamin K-dependent proteins. Therefore, in order to use vitamin K-dependent proteins as pharmaceuticals, when purifying them from human plasma or expressing and purifying them as recombinants, it is necessary to accurately analyze the degree of Gla modification in the Gla domain of vitamin K-dependent proteins. is important, and there is a need for analytical methods that allow it.

As one of such analysis methods, for example, since Gla is decomposed by acid hydrolysis into glutamic acid, the total amount of Gla in protein can be analyzed by alkaline hydrolysis. However, since only the total amount is quantified as the amino acid composition, when the degree of Gla modification in the protein is heterogeneous, only the average value can be obtained. However, no information about the heterogeneity of the molecular species present can be obtained.

Moreover, mass spectrometry is mentioned as a technique for analyzing Gla modification. However, mass spectrometry of peptides and proteins containing Gla affects ionization due to their negative charge and their diversity, making quantitative analysis difficult. In order to overcome this problem, methylation of the Gla carboxyl group has also been attempted (Non-Patent Document 2). However, confirmation of sufficient methylation of each carboxyl group is difficult and uncommon.

Moreover, since Gla is rich in negative charges, it has been reported that proteins with different degrees of Gla modification can be separated by anion exchange chromatography (Non-Patent Document 3). Human blood coagulation factor IX (FIX), a vitamin K-dependent blood coagulation factor, contains 12 Gla residues. Separation into peaks according to degree of modification is shown. Furthermore, based on the fact that each peak remained separated even in the neuraminidase digestion of the recombinant FIX protein and the results of its peptide map analysis, while human FIX has 12 Gla residues, this recombinant FIX It is reported that the protein has 10Gla-modified, 11Gla-modified and 12Gla-modified (Non-Patent Document 3).

However, with regard to blood coagulation factor X (FX), anion exchange chromatography of bovine FX showed that the tyrosine residue (Tyr) in the activating peptide on the NH2 _- terminal side of the FX heavy chain was not the difference in Gla modification. It has been reported that two peaks are separated depending on the presence or absence of sulfate group modification (Non-Patent Document 4). Therefore, these reports indicate that not all vitamin K-dependent blood coagulation factors, including Gla, can be separated by degree of Gla modification based on the negative charge of Gla by anion exchange chromatography.

Furthermore, as another method for analyzing Gla modification, site-specific analysis of Gla modification can be estimated from peptide maps. However, when performing site-specific analysis of Gla modification of human FX, which has 11 Gla modification sites, it is difficult to confirm some modification sites from the peptide map.

Conventionally, it has been reported that a vitamin K-dependent blood coagulation factor is digested with chymotrypsin to cleave the Gla domain to prepare a vitamin K-dependent blood coagulation factor without the Gla domain (Non-Patent Document 5). In this non-patent document 5, Tyr44-Lys45 of bovine FX is cleaved by chymotrypsin digestion of bovine FX, and Gla domain-deleted FX is purified by anion exchange chromatography and prepared.

However, to the present inventors' knowledge, it is not possible to target the Gla domain cleaved by chymotrypsin and use it to determine the degree of Gla modification of vitamin K-dependent proteins such as vitamin K-dependent blood coagulation factors. , has not yet been reported.

K. Hansson, et al., Journal of Thrombosis and Haemostasis 2005, 3: 2633-2648 Hallgren, et al., J Proteome Res 2013, 12: 2365-2374 S. Gillis, et al., Protein Science 1997, 6: 185-196 T. Morita, et al., J Biol Chem 1986, 261:4008-4014 T. Morita, et al., J Biol Chem 1986, 261:4015-4023 T. Zama, et al., Br J Haematol 1999, 106:809-811 L. Thim, et al., Biochemistry 1988, 27:7785-7793

The present inventors have recently discovered that the degree of Gla modification of vitamin K-dependent proteins can be analyzed in detail by allowing chymotrypsin to act on vitamin K-dependent proteins and analyzing the Gla domain obtained by cleavage. and obtained knowledge about its more appropriate analytical conditions. The present invention is based on such findings.

Accordingly, an object of the present invention is to provide a new analytical method for the degree of Gla modification of vitamin K-dependent proteins.

The method for analyzing the degree of Gla modification of vitamin K-dependent proteins according to the present invention is a method for analyzing the degree of γ-carboxyglutamic acid (Gla) modification of vitamin K-dependent proteins, comprising:
digesting the vitamin K dependent protein with chymotrypsin to cleave the Gla domain and obtain Gla domain peptides;
and a step of separating the Gla domain peptides by liquid chromatography according to the degree of Gla modification.

According to the method of the present invention, the degree of Gla modification of vitamin K-dependent proteins can be properly analyzed.

Amino acid sequence of the Gla domain of human vitamin K-dependent blood coagulation factor (Fig. 4(A) of Non-Patent Document 1). In the figure, FVII is blood coagulation factor VII, FIX is blood coagulation factor IX, FX is blood coagulation factor X, PT is blood coagulation factor II (prothrombin), PC is protein C, PZ is protein Z, and PS stands for protein S. Reversed-phase HPLC chromatogram (A) of the chymotryptic digest of FX and the blank at a wavelength of 280 nm obtained in Example 1 and an enlarged view of the chromatogram at wavelengths of 214 nm and 280 nm for a retention time of 35 to 40 minutes ( B). ₄ is an overlaid C4 reverse-phase HPLC chromatogram at a wavelength of 214 nm when calcium salt or trivalent chromium salt was added to FX chymotrypsin-digested Gla domain peptide sample obtained in Example 2. FIG. Fig. 3 is a _C18 reverse phase HPLC chromatogram at a wavelength of 214 nm of the human FX Gla domain peptide and blank obtained in Example 3; The assignment result of each peak is shown in the figure. _C4 reversed-phase HPLC chromatogram (A) at a wavelength of 214 nm of the chymotryptic digest of human FVIIa and human rFVIIa obtained in Example 4 and an enlarged view of the chromatogram (B) with a retention time of 36 to 40 minutes. be. ₂ is an overlaid C18 reversed-phase HPLC chromatogram at a wavelength of 214 nm with and without the addition of trivalent chromium salt to the human FVIIa Gla domain peptide sample obtained in Example 5. FIG.

Vitamin K-Dependent Proteins and Gla-Modification The subject of the analytical method according to the present invention is the degree of Gla-modification of vitamin K-dependent proteins. Here, the vitamin K-dependent protein is a protein whose glutamic acid residue (Glu) is converted into γ-carboxyglutamic acid residue (Gla) depending on vitamin K. Specific examples thereof include vitamin K dependent blood clotting factors such as blood coagulation factor II, blood coagulation factor VII, blood coagulation factor IX and blood coagulation factor X; vitamin K dependent blood clotting factors such as protein C or protein S; blood coagulation regulators; furthermore osteocalcin, protein Z.

The vitamin K-dependent protein that is the subject of the analysis method according to the present invention has a Gla domain containing around 10 γ-carboxyglutamic acid residues (Gla) at its NH ₂ -terminal. Its structure, for example, the amino acid sequence of the Gla domain of human vitamin K-dependent blood coagulation factor, is as shown in FIG. 1 (FIG. 4(A) of Non-Patent Document 1). In FIG. 1, Gla is indicated by γ. Amino acid residues common to each blood coagulation factor are shaded. From the figure, it can be seen that the vicinity of about 40 residues from the N-terminus is rich in aromatic amino acid residues (Phe (F), Trp (W) and Tyr (Y)) that serve as cleavage sites for chymotrypsin.

This Gla domain has a role of coordinating Ca ions and plays an important role in the physiological functions of vitamin K-dependent proteins. Therefore, it is important to accurately analyze the degree of Gla modification in the Gla domain of vitamin K-dependent proteins. According to the analytical method of the present invention, peptides with different degrees of Gla modification derived from vitamin K-dependent proteins can be separated by chromatography, and their quantification is also possible.

When vitamin K is deficient, blood coagulation factors are produced with their Gla modifications immature. can know In addition, it is possible to know the degree of modification not only of vitamin K-dependent proteins derived from living organisms but also of vitamin K-dependent proteins obtained by genetic recombination. Can be qualitative and quantitative.

A specific qualitative determination of the degree of modification will be described below using vitamin K-dependent blood coagulation factor X (FX) as an example. The FX N-terminal Gla domain shown in FIG. 1 consists of 45 amino acid residues and contains 11 Gla residues. And, in general, 11Gla-modified forms in which all 11 residues from the NH2 _- terminus to the 6th Gla6 to the 39th Gla39 from the NH2-terminus of human FX are modified, and similarly 10 residues from the _NH2- terminus to the 32nd Gla32. It is conceivable that there are a 10Gla modification with modified residues, a 9Gla modification with modification of 9 residues from the NH ₂ terminal to the 29th Gla29, and the like. On the other hand, a molecular disorder of the Gla32 site of FX (Gla32Gln) is known as FX Tokyo, and has been reported to significantly prolong prothrombin time (Non-Patent Document 6). Therefore, the 9Gla-modified form, in which Gla32 is not modified and Gla29 is modified, is considered to be an inactive FX. According to the analysis method according to the present invention, the existence and abundance of these substances can be known.

A specific qualitative determination of the degree of modification will be described below using blood coagulation factor VII (FVII) as an example. The N-terminal Gla domain of FVII shown in FIG. 1 consists of 45 amino acid residues and contains 10 residues of Gla. And, in general, 10Gla modifications in which all 10 residues from the NH ₂ terminal to the 6th Gla6 to the 35th Gla35 from the NH 2 terminal of human FVII are modified, and similarly, 9 from the NH ₂ terminal to the 29th Gla29 It is conceivable that there are 9Gla-modified forms with modified residues. It has been reported that recombinant activated human blood coagulation factor VII (rFVIIa) has a 9Gla modification in which Gla35 is not modified (Non-Patent Document 7).

Digestion with Chymotrypsin In the analysis method according to the present invention, first, a step of digesting vitamin K-dependent proteins with chymotrypsin is performed. As described above, vitamin K-dependent proteins have a region rich in aromatic amino acid residues (Phe, Trp, and Tyr) that serves as a cleavage site for chymotrypsin near about 40 residues from the N-terminus. The amino acid residue is used as the cleavage site. and obtain the Gla domain peptide.

Chymotrypsin that can be used in the present invention may be an enzyme commercially available for biochemical use, and α-chymotrypsin may be used. Furthermore, chymotrypsin treated with a trypsin inhibitor such as TLCK (tosyl-L-lysyl chloromethyl ketone) treatment may be used to promote substrate-specific cleavage of chymotrypsin.

In the analysis method according to the present invention, the conditions for digestion of vitamin K-dependent proteins with chymotrypsin are such that cleavage is possible in a region rich in aromatic amino acid residues (Phe, Trp, and Tyr) around about 40 residues from the N-terminus described above. is not limited as long as For example, the weight ratio of vitamin K-dependent protein and chymotrypsin is about 1/5 to 1/10000, and they are reacted at a temperature of 10 to 40°C for about 1 to 900 minutes. For example, in Non-Patent Document 5, FX is added to TLCK-treated chymotrypsin at a weight ratio of 1/700 and reacted in 0.05 mol/L Tris-HCl, 0.1 mol/L NaCl, pH 7.5 at 22°C for 50 minutes. Without being limited to this example, any amount of chymotrypsin can be used to cleave the Gla domain peptide from the vitamin K-dependent protein under conditions where chymotrypsin acts. can.

Liquid Chromatography In the analytical method of the present invention, the Gla domain peptides obtained by digestion with chymotrypsin are then subjected to liquid chromatography to separate them according to the degree of Gla modification.

According to a preferred embodiment of the invention, the separation of Gla domain peptides is performed by reverse phase liquid chromatography (HPLC). The column is functionalized with C ₁ (methyl), C ₄ (butyl), C ₈ (octyl), or C ₁₈ (octadecyl) groups for reversed-phase chromatography and can be used to separate peptides and proteins. It is not limited as long as it is a column. As the mobile phase, a system in which trifluoroacetic acid, formic acid, etc. are added to water and an organic solvent such as acetonitrile, respectively, and mixed in a linear gradient is used, but if peptides and proteins can be separated, but not limited to them. In addition, eluted peptides can be detected and quantified by ultraviolet absorption at a wavelength near 210 nm or at a wavelength of 280 nm due to the presence of aromatic amino acids. When quantifying by detection at a wavelength of 280 nm, it is necessary to consider the extinction coefficient of each component.

In the present invention, in separation by liquid chromatography, some peptides may coordinate metal ions that are believed to originate from stainless steel parts in the high performance liquid chromatography (HPLC) apparatus. Therefore, according to a preferred embodiment of the present invention, for example, a trivalent chromium salt or the like can be added in advance to an analysis sample that has been digested with chymotrypsin. Alternatively, to avoid such metal ion coordination, a chelating agent such as, for example, an ethylenediaminetetraacetic acid (EDTA) salt can be added to the chymotrypsin-digested analytical sample in advance. Although the amount to be added is not particularly limited, according to one aspect of the present invention, the final addition concentration of the trivalent chromium salt is at least 0.25 mol/L, or the final addition concentration of the chelating agent is at least 0.005 mol/L. It is said that More preferably, the final concentration of trivalent chromium salt added is 0.25 mol/L to 0.5 mol/L. In addition, depending on the subject of analysis, the preferred amount to be added is, for example, in the case of chymotrypsin-digested peptide of human blood coagulation factor X (FX), chromium chloride (III) (chromium trichloride) is added to the analysis sample at a final concentration of 0.5 mol. By adding about /L, Gla domain peptides can be separated by reversed-phase chromatography in the state of coordinated chromium ions. In addition, for example, human activated blood coagulation factor VII (FVIIa) is digested with chymotrypsin while suppressing the effect of calcium salts that may be contained in the sample by adding EDTA to a final concentration of about 5 mmol/L, and Gla domain peptides can be separated by reversed-phase chromatography in the absence of coordinating metal ions. Depending on the peptide, it is of course possible to separate the Gla domain peptide by reversed-phase chromatography without adding such a metal salt or chelating agent to the sample for analysis.

EXAMPLES The present invention will be described in detail below with reference to Examples, but the present invention is not limited to these Examples.

Example 1:
(a) Digestion of human blood coagulation factor X (FX) with chymotrypsin 0.3 mg of human blood coagulation factor X (FX) purified from human plasma, in a buffer of 0.1 mol/L Tris-HCl, pH 7.5, It was digested with bovine pancreas-derived TLCK-treated α-chymotrypsin (product number C3142, Sigma-Aldrich). Chymotrypsin was added at a weight ratio of 1/500 to FX and allowed to react at room temperature for 1 hour. The reaction was then stopped by the addition of acid (trifluoroacetic acid (TFA)) and used below as the human FX chymotryptic digest.

(b) Separation by HPLC The human FX chymotryptic digest was subjected to reverse phase HPLC using a _C4 column. As a result, Gla domain-deleted FX eluted at a retention time of 42 minutes. As a result of alkaline hydrolysis of the peak fractions, γ-carboxyglutamic acid was detected between retention times of 37 and 39 minutes indicated by double arrows (Fig. 2A). The peak of this portion was fractionated into 8 fractions A1 to D2 (Fig. 2B). The analysis conditions of reverse phase HPLC at this time are as follows.
・Column: 214TP5410, 4.6 mm×10 cm, Vydac ・Mobile phase A: 0.05% TFA
・Mobile phase B: 0.05% TFA, 80% _CH3CN

(c) Peak identification
Each peak fraction of Gla-containing peptides was analyzed by ESI-MS by flow injection method. Using a single quadrupole ESI-MS instrument LCMS-2020 (manufactured by Shimadzu Corporation), 60% CH ₃ CN as a mobile phase was fed at a flow rate of 0.3 mL/min, and the fractionated peptide sample was injected. Both positive and negative ions were scanned in the m/z range 300-2000. When modified to Gla, m/z 44 higher than Glu residues will be observed per modification site. Each peak was assigned as shown in Table 2 from the observed m/z. Chymotrypsin digestion cleaved the Gla domain peptide of human FX at the COOH terminus of Trp41 or Tyr44. Peak fractions C1, C2, D1 and D2 showed m/z +52 higher than peak fractions A1, A2, B1 and B2, respectively, suggesting the possibility of peptides coordinated with chromium ions. .

Example 2
(a) Analysis of Gla domain peptides of human blood coagulation factor X (FX)
FX sample 0.5 mg was digested with 1/500 volume of TLCK-treated α-chymotrypsin in 0.1 mol/L Tris-HCl, pH 7.5 (room temperature, 1 hour). After digestion, 10% TFA was added to 1/40 of the reaction volume to stop the reaction. Then, according to Example 1(b), separation was performed by HPLC, and the Gla domain peptide fractions of human FX were collected together.

(b) Separation by HPLC Gla domain peptide fractions of human FX were added with calcium chloride or chromium(III) chloride prior to HPLC injection analysis in amounts equivalent to 20 or 10 μg FX and were treated with the It was subjected to HPLC according to

The resulting chromatogram was as shown in FIG. When calcium chloride was added at a final concentration of 100 mmol/L to the human FX Gla domain peptide fraction and subjected to HPLC analysis, the peak shape of the human FX Gla domain peptide fraction did not change much. Although not shown, the peak shape did not change even when nickel (II) sulfate, magnesium chloride, or zinc chloride was added.

Next, when chromium (III) chloride was added to the Gla domain peptide fraction of human FX, the A1, A2, B1 and B2 peaks decreased depending on the added chromium (III) chloride concentration. and the D2 peak increased. Then, adding the same concentration of chromium (III) chloride to a relatively small amount of human FX Gla domain peptide fraction further increased the C1, C2, D1 and D2 peak contents, and the final concentration of 250 mmol/L chromium chloride ( III), the A1, A2, B1 and B2 peaks almost disappeared and were converted to C1, C2, D1 and D2 peaks (Fig. 3). Therefore, peaks C1, C2, D1 and D2 were considered Gla domain peptides coordinated with chromium ions.

When comparing the retention time range of peaks A1 to B2 with the retention time range of peaks C1 to D2, the latter retention time range is larger, so the chromium ion coordination is more effective for each peak. It was thought that the separation could be improved.

Example 3
(a) Analysis of the degree of Gla modification of human FX 15 µg of human blood coagulation factor X (FX) was treated with TLCK-treated α-chymotrypsin at a weight ratio of 1/500 in 0.1 mol/L Tris-HCl, pH 7.5 at room temperature. Digested for 1 hour. After that, 10% TFA was added in an amount of 1/40 of the reaction volume to terminate the reaction. Chromium (III) chloride was then added to the reaction solution at a final concentration of 0.5 mol/L. This sample was subjected to _C18 reverse phase HPLC under the following conditions.
・Column: 5C18AR300, 4.6 mm×25 cm, manufactured by nacalai tesuque ・Mobile phase A: 0.05% TFA
・Mobile phase B: 0.05% TFA, 80% _CH3CN

The resulting chromatogram was as shown in FIG.

(c) Quantitative determination of degree of Gla modification ESI-MS analysis was performed according to Example 1(c), and each peak was assigned as shown in FIG. From the results of the peak area ratio, it was found that human FX mainly consisted of 80% 11Gla-modified and 20% 10Gla-modified. This coincided with the abundance ratio of peptides containing Gla39 and peptides containing Glu39 in the Lys-C peptide map of human FX. It was also found that a 41-residue-long 9Gla-modified product was eluted at the position indicated by the arrow in FIG. 4, but almost no 9Gla-modified product was observed in human plasma-derived FX. The formation of two peptides with different lengths of 44 residues and 41 residues was considered to depend on the amount of chymotrypsin used and the degree of Gla modification. From the above, the degree of Gla modification of human FX could be determined from the peak area ratio of HPLC.

Example 4
(a) Digestion of activated human blood coagulation factor VII (FVIIa) with chymotrypsin Activated human blood coagulation factor VII (FVIIa) was purified from human plasma (manufactured by KM Biologics Co., Ltd.). Recombinant activated human blood coagulation factor VII (rFVIIa) was purchased from Novo Nordisk. 0.27 mg of FVIIa and 0.16 mg of rFVIIa were each added in a buffer of 0.1 mol/L Tris-HCl, 0.005 mol/L EDTA, pH 7.5 with p-amidinophenylmethanesulfonyl fluoride hydrochloride at a final concentration of 10 mmol/L. The autolysis of FVIIa and rFVIIa was inhibited by reaction at room temperature for 30 minutes. Then, 1/500 weight ratio of TLCK-treated α-chymotrypsin was added and allowed to react at room temperature for 1 hour. Thereafter, 10% TFA was added in an amount of 1/40 of the reaction volume to stop the reaction, and the mixture was used as a chymotryptic digest of FVIIa and rFVIIa below.

(b) Separation by HPLC
Chymotryptic digests of FVIIa and rFVIIa were subjected to _C4 reverse-phase HPLC according to Example 1(b). As a result, Gla domain-deleted FVIIa and rFVIIa eluted at a retention time of 48 minutes. As a result of alkaline hydrolysis of the peak fractions, γ-carboxyglutamic acid was detected between 37 and 40 minutes retention time indicated by the double arrow (Fig. 5A). The peak of this portion was fractionated into 4 fractions a1 to b2 (Fig. 5B).

(c) Quantification of Gla modification degree
Each peak fraction of the Gla-containing peptide was analyzed according to Example 1 by ESI-MS using the flow injection method. Each peak was assigned as shown in Table 4 from the observed m/z. Chymotryptic digestion cleaved the Gla domain peptides of FVIIa and rFVIIa at the COOH termini of Leu39 or Phe40. It is well known that chymotrypsin cleaves Leu at the COOH terminus.

From the results of the peak area ratio, it was found that FVIIa consisted of almost 10Gla modifications. And rFVIIa was considered to consist of approximately 30% of the 10Gla-modified and 70% of the 9Gla-modified.
NH2 _- terminal amino acid sequence analysis has reported the presence of a 9Gla-modified form in which Gla35 is not modified in recombinant activated human blood coagulation factor VII (rFVIIa), but its abundance is not quantitative. , about half the amount (Non-Patent Document 7). In addition, in the Lys-C peptide map of FVIIa, peptides containing Gla35 were extremely hydrophilic and could not be separated by reversed-phase HPLC, so the degree of modification of Gla35 could not be evaluated. However, this method could determine the degree of Gla modification of FVIIa and rFVIIa from the peak area ratio of HPLC.

Example 5
Gla domain peptide peaks a1 and b1 of FVIIa were fractionated, and chromium (III) chloride was added to a final concentration of 0.5 mol/L to part of each peak fraction. It was subjected to _C18 reverse phase HPLC according to Example 3 with or without its addition. As a result, the Gla domain peaks a1 and b1 of FVIIa each eluted later on _C18 reverse-phase HPLC by adding chromium(III) chloride (Fig. 6).

Claims (12)

A method for analyzing the degree of γ-carboxyglutamic acid (Gla) modification of a vitamin K-dependent protein, comprising:
digesting the vitamin K dependent protein with chymotrypsin to cleave the Gla domain and obtain Gla domain peptides;
and a step of separating the Gla domain peptides by degree of Gla modification by liquid chromatography. 2. The analysis method according to claim 1, wherein said vitamin K-dependent protein is a vitamin K-dependent blood coagulation factor or a vitamin K-dependent blood coagulation regulator. The analysis method according to claim 2, wherein said vitamin K-dependent blood coagulation factor is selected from the group consisting of blood coagulation factor II, blood coagulation factor VII, blood coagulation factor IX, and blood coagulation factor X. . 3. The analysis method according to claim 2, wherein said vitamin K-dependent blood coagulation regulator is protein C or protein S. 2. The analysis method according to claim 1, wherein said vitamin K-dependent protein is osteocalcin or protein Z. The analysis method according to claim 1, wherein the vitamin K-dependent protein is an activated form of a vitamin K-dependent blood coagulation factor or a vitamin K-dependent blood coagulation regulator. The analytical method according to any one of claims 1 to 6, wherein the separation of the Gla domain peptides is performed by reversed-phase liquid chromatography. The analysis method according to any one of claims 1 to 7, wherein the Gla domain peptides are separated by degree of Gla modification by liquid chromatography in the presence of a trivalent chromium salt or a chelating agent. 9. The analysis method according to claim 8, wherein the final concentration of trivalent chromium salt added is at least 0.25 mol/L or the final concentration of chelating agent added is at least 0.005 mol/L. The analysis method according to claim 9, wherein the final concentration of trivalent chromium salt added is 0.25 mol/L to 0.5 mol/L. When the vitamin K-dependent blood coagulation factor is blood coagulation factor X, 11-Gla-modified, 10-Gla-Gla-modified, and 9-Gla-Gla-modified The analysis method according to any one of claims 1 to 10, which separates. 12. The analysis method according to claim 11, wherein the Gla-modified one is the 44 residues from the N-terminus of blood coagulation factor X, or the 41 residues from the N-terminus.

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