WO2015126077A1

WO2015126077A1 - Method for production, purification and analysis of stable isotope-labeled protein

Info

Publication number: WO2015126077A1
Application number: PCT/KR2015/001063
Authority: WO
Inventors: 최준혁; 김숙경
Original assignee: 한국표준과학연구원
Priority date: 2014-02-19
Filing date: 2015-02-02
Publication date: 2015-08-27
Also published as: KR20150098040A

Abstract

The present invention relates to a method for intracellular production, extraction and purification of a stable isotope-labeled protein and, more specifically, to a method for purification of a protein and analysis of the protein, the method allowing high-yield production of proteins having a biological activity while maximizing solubility in an extraction process of a target protein in the case of expressing the proteins on a large scale using an Escherichia coli system. A large quantity of high-purity hGH was purified according to the present invention, a stable isotope-labeled Sl-His-hGH was synthesized, and whether the protein is labeled or not was successfully confirmed.

Description

Specification

Name of invention: stable isotope labeling protein production, purification and analysis method

[1] The present invention relates to intracellular production, extraction, and purification of stable isotopically labeled proteins. More specifically, stable isotopically labeled proteins that can be used as internal standards for the quantitative analysis of proteins in E. coli systems. It is about the purification method and the analysis method of purified protein which can maximize the solubility and obtain high yield in the process of mass expression and extraction.

Background

[2] Human growth hormone (hGH) is a single-chain polypeptide synthesized in the pituitary gland with 191 amino acid residues.Human growth hormone is known to have a variety of biological functions, including cell proliferation and metabolism. It is one of the most important hormones in the human body (Annu. Rev. Physiol., 47, 1985, 483-499) .Human growth hormone is a synthetic process agent of recombinant protein and is known as somatropin.

Use names that are distinct from somatotropin.

[3] Recent studies on protein quantification have focused on the development of techniques for the absolute determination of target proteins in biologically complex samples, for which the stable isotope is an internal standard. Labeled

Many experimental principles have been used to deduce the amount of target protein cleaved at the peptide level using peptides.However, this method requires expensive peptide synthesis, and the cleavage efficiency of the target protein is used for quantitative analysis. It is a disadvantage that has a big impact and a better alternative is needed.

Detailed description of the invention

Technical task

[4] recombinant DNA technology

isotopically labeled proteins were synthesized using stable isotopically labeled amino acids and developed as internal standards for the quantitative analysis of target proteins. The greatest advantage of this approach is that the internal standard is very similar structurally and chemically to the target protein. The present inventors have used an Escherichia coli expression system to optimize the production process for isotopically labeled human growth hormone. The present invention has been completed by confirming the process. ᅳ

Task solution

[5] One aspect of the present invention is

[1] (1) transforming Escherichia coli into an expression vector encoding a target protein;

[7] (2) culturing the transformed Escherichia coli in a culture medium; (3) inoculating the cultured Escherichia coli with a glucose minimum medium;

[9] (4) culturing Escherichia coli vaccinated to the minimum medium;

[1] (5) containing amino acids labeled with stable isotopes in the cultured Escherichia coli

Injecting an amino acid mixture solution and inducing the expression of the isotopically labeled protein;

(6) incubating the E. coli culture solution containing the amino acid solution at a temperature of 15 to 25 ° C. for 8 to 18 hours;

[12] relates to the production of stable isotopically labeled proteins, including

[13] In the present invention, the target protein of step (1) is not limited,

Escherichia coli in step (1) above can be used, but not limited to, E. coli BL21 strain, and can be cultured at 37 ° C until the OD ₆₀₀ value is between 0.4 and 8. Culture media also include, but are not limited to, 5 to 15 g / L Bacto Tryptone, 3 to 8 g / L yeast extract and 5 to 15 containing 40 to 60 mg / mL kanamycin. Fresh LB (Luria-Breta i) medium with g / L sodium chloride can be used. Also in the present invention, the stable isotope of step (5) above may be, but not limited to, "C ₆ ^ N ₄ -arginine (Argine).

[14] In another aspect of the present invention,

(1) transforming Escherichia coli into an expression vector encoding a target protein;

[2] (2) culturing the transformed Escherichia coli in a culture medium;

[3] (3) inoculating the cultured Escherichia coli into a glucose minimum medium;

[4] (4) culturing Escherichia coli vaccinated to the minimum medium;

(5) injecting an amino acid solution labeled with a stable isotope to the cultured Escherichia coli and inducing the expression of the isotopically labeled protein;

(7) dissolving E. coli as a complete solution of step (6);

[22] (8) Ultrasonic treatment of the E. coli and centrifugation to obtain isotopically labeled proteins.

Recovering; and

[9] (9) purifying the stable isotopic labeling protein;

[24] on methods of purification of stable isotopically labeled proteins, including

[25] In the present invention, the target protein of step (1) is not limited,

It may be a human growth hormone, preferably a histidine-labeled human growth hormone. The stable isotopically labeled amino acid is not limited, but ⁱ³ C ₆ ¹⁵ N ₄

It may be arginine.

[26] In the present invention, the above-mentioned tablets of stable isotope marker proteins are not limited.

One or more of affinity chromatography and anion-exchange chromatography can be used, preferably the column of the affinity chromatography is Ni-NTA column, and the column of the anion-exchange chromatography is Mono Q column. Available have.

[27]

In the present invention, the lysis buffer solution of step (7) is not limited but 0.3 to 0.8 mM EDTA, 0.5 to L5 mg / mL lysozyme, lx protease inhibitor cocktail and It may be Tris-HCl, pH 7.5 to 8.5, including a detergent. The nonionic denaturant is preferably 0.01 to 2% (v / v) Triton X-100 or Tween. May be 20).

In addition, in the present invention, the dissolved complete solution of step (7) may include, but is not limited to, salts, and preferably, the salt may be sodium chloride or potassium chloride.

[30] In the present invention, the use of the above-mentioned buffer buffer solution is not limited,

It may be 0.25 to 2 mL for 30 mg of E. coli cells (pellets).

[31]

[32] Another aspect of the present invention is

(2) culturing the transformed Escherichia coli in a culture medium;

(4) culturing Escherichia coli inoculated on the minimum medium;

(6) culturing the E. coli culture solution containing the amino acid solution at a temperature of 15 to 25 ° C. for 8 to 18 hours;

(7) dissolving E. coli as a buffer solution in step (6);

(8) ultrasonically treating the E. coli and centrifuging to recover isotopically labeled proteins;

(9) purifying the stable isotopic labeling protein; and

[10] (10) Purified stable isotopic marker protein by MALDI-TOF MS or HPLC

Analyzing;

The MALDI-TOF MS in the present invention is not limited, but may range from 600 m to 3,500 m / z, and the column temperature of the HPLC is not limited to 20 to 50. ° C, flow rate can be α 1-1.0 mL / min.

Effects of the Invention

[44] The present invention relates to a stable isotope labeled in the process of transforming Escherichia coli using an expression vector that encodes a protein of interest to produce a stable isotope marker hGH in the E. coli system. Specific amino acids To induce the mass expression of isotopically labeled hGH, to maximize the solubility of hGH in the extraction process, to refine it to very high purity, and to analyze the isotopically labeled hGH. Highly efficient expression of stable isotopically labeled proteins, which can be used as internal standards for analysis, maximizes solubility during protein extraction, resulting in high yields.Affinity chromatography and ion-exchange chromatography can then be used to obtain high yields. It was possible to purify high purity hGH and based on this, a large amount of high purity

Stable isotopic labeling SI-His-hGH was produced, and MALDI-TOF MS was used to confirm the isotopic labeling.

Brief description of the drawings

1 shows the expression of recombinant hGH and His-hGH in E. coli.

[46] M: protein marker, U: transformed and unexpressed cells, I: transformed and expressed cells, Empty: transformed cells, hGH: non-tagged -hGH expressing cells, His- hGH: histidine marker -hGH expressing cells.

FIG. 2 shows the purification of His-hGH in non-optimal native conditions.

[48] L: cell lysate, S: supernatant (soluble protein), P: pellet (insoluble protein), El: elution

1, E2: elution 2, E3: elution 3, FT: flow through.

FIG. 3 shows the purification of His-hGH under optimized native conditions.

[50] 1: unexpressed cells, 2: expressed cells, 3: cell lysate, 4: pellets (insoluble protein), 5: supernatant (soluble protein), 6: elution 1, 7: elution 2 ， 8: flow through, 9: secondary elution 1, 10: secondary elution 2, 11: secondary flow through.

4 shows the expression of His-hGH and SI (stable isotope) -His-hGH in Escherichia coli.

FIG. 5 shows the primary purification of SI-His-hGH under optimized native conditions.

[53] 1: uninduced cells, 2: induced cells, 3: cell lysates, 4: pallets (insoluble protein),

5: supernatant (soluble protein), 6: elution 1, 7: elution 2, 8: elution 3, 9: flow through, 10: control LG-hGH (2 g), 11: control LG-hGH (10 g) ).

FIG. 6 shows a peptide predicted after trypsinizing His-hGH.

FIG. 7 is a diagram illustrating mass measurement values of four representative peptides generated after trypsin treatment of the positive control LG-hGH and SI-His-hGH using a MALDI-TOF mass spectrometer.

FIG. 8 is a graph comparing the Lift TOF TOF (MS / MS) spectrum of the LG-hGH trypsin peptide of m / z 1205.574 with the spectrum of SI-His-hGH peptide of m / z 1215.429.

9 shows ion-exchange chromatography of His-hGH using a monocube column. 10 shows the results of silver staining analysis of the monocube fraction.

11 shows the results of HPLC analysis of His-hGH purified with LG-hGH. Best Mode for Carrying Out the Invention

[60] The present invention will be described in detail below with reference to Examples and accompanying drawings. However, these are intended to explain the present invention in more detail, and the scope of the present invention is not limited to the following Examples.

[61]

(Example 1) Preparation of Vector for Expression of Human Growth Hormone

[63] Human Growth Hormone Gene for Expression of Recombinant Human Growth Hormone (NCBI Reference:

NM_000515.3: 141-719) requested the synthesis of BIONIA (BIONEER, Daejeon, Korea), Nhel restriction enzyme cleavage site (GCTAGC) at the 5 'end and Xhol restriction enzyme cleavage site (CTCGAG) at the 3' end. The synthesized cDNA was ligation to pET-28a (Novagen, Madison, WI, USA) using restriction enzymes Nhel and Xh to cut 6-histidine label and truncated bin at the N-terminus. A His-hGH expression vector expressing a recombinant human growth hormone with site was completed.

[64] Also, for untagged hGH expressing vectors expressing human growth hormone without histidine, forward primer 5 '-££ ggcgatgttcccaaccatt-3' (SEQ ID NO: 2) and reverse primer 5'- £ i gagctagaagccacagct-3 ' CDNA comprising Ncol restriction enzyme cleavage site (CCATGG) at 5 'end and Xhol restriction enzyme cleavage site (CTCGAG) at 3' end using polymerase chain reaction using the synthesized cDNA as a template using (SEQ ID NO: 3). The result of the dimerase chain reaction was cut into restriction sites of Nc and Xh and followed by Nc and Xhol restriction sites of the pET28a expression vector to express untagged hGH.

Nucleotide sequences of the gene to be expressed were confirmed through automatic sequencing.

[65]

Example 2 Expression and Extraction of Human Growth Hormone Using Escherichia Coli Expression System

Three His-hGH presenting combination DNAs were transformed into Escherichia coli BL21 (DE3). The E. coli was inoculated into a 250 mL flask containing 50 mg / mL kanamycin and incubated overnight at 37 ° C. 5 mL of the culture medium was centrifuged at 10,000 xg for 5 minutes, followed by M9 medium containing 0.2 wt% glucose (Cat No. The treated culture was inoculated into a 4L flask containing 500 mL of M9 medium containing kanamycin and incubated at 37 ° C until a value of OD _{600 of} about 0.6 was obtained. To make an amino acid solution, a 25 X amino acid complex was prepared by adding 19 amino acids lg, except arginine (Arg), to 200 mL distilled water, followed by "C ¹⁵ N-arginine (Arg) labeled with radioisotopes in 1 mL distilled water. A solution containing 250 g was prepared: 20 mL of 25 X amino acid complex and ¹³ C ^{15 at a} concentration of 250 mg / mL. N-arginine (Arg) 400 was added and incubated for 30 minutes. The solution ().

Separately stored for SDS-PAGE and from the above E. coli culture

0.5 M to induce the expression of human growth hormone

1 mL of beta-di-1-galactopyranoside (β-D-l-thiogalactopyranoside, IPTG) was added to express the protein. The protein was named SI-His-hGH. After 16 hours of incubation at 16 ° C, 0.5 mL was stored separately for SDS-PAGE, and the remaining solution was centrifuged at 10,000 xg for 20 minutes at 4 ° C to obtain cells and frozen for storage. E. coli cells were disrupted with 25 mL of lysate buffer solution (1 mg / mL lysozyme, 1 X protease inhibitor cocktail (Rosh, Spain) and 50 mM Tris-HCl containing 0.5 mM EDTA) and then ultrasonically at 10,000 xg. Centrifuged for 20 minutes to obtain insoluble and soluble fractions

SDS-PAGE was performed and stained with Coomassie Blue Staining Reagent. Also, ImageQuant ™ TL 5.2 analysis for analysis of human growth hormone.

Software (ImageQuant ™ TL 5.2 analysis software)

Densitometry analysis of soluble (S) and insoluble (P) protein levels

Analyzed.

In Fig. 1, it can be seen that His-hGH is expressed by IPTG, which induces protein synthesis. From the above results, His-hGH expressing DNA was selected as a target of the following examples.

[69]

[Example 2 (Example 3) Optimal Conditions of a Dissolved Complete Solution for Solubilization of Insoluble Ingredients

[71]

[72] solubilizing the insoluble ingredient fraction so that it can be included in the availability fraction

The optimum composition of the dissolved complete solution was confirmed. Insoluble component obtained by performing the same method as Example 2 was confirmed using the dissolved buffer solution of Table 1 below to determine the optimum composition.

[73]

[74] Table 1

[Table 1]

Preparation of Dissolved Complete Solution for Acquisition of Solubilized Protein from Insoluble Component

Example 4 Purification of Recombinant Protein and Human Growth Hormone

[0109] The optimal human growth hormone expression and extraction conditions established in Examples 1 to 3 were used to extract and purify the human growth hormone.

E. coli BL21 (DE3), which expresses human growth hormone (His-hGH and SI-ffis-hGH), was cultured in 250 mL culture medium and harvested for 16 hours at 16 ° C. The harvested cells were dissolved in 25 mL lysate layer solution (pH 8.0 50 mM Tris-HCl) containing 0.5 mM EDTA, 0.1% Triton X-100, 1 mg / mL lysozyme and 1 X Protease Inhibitor Cocktail (Rosh, Spain). After sonication, centrifugation was performed at 10,000 xg for 20 minutes to obtain and purify.

[78] (1) Purification of His-hGH and SI-His-hGH

[79] First purification by Ni-NTA column ^^ affmitv chromatographv

[80] After the centrifugation, the optimally dissolved His-hGH and SI-His-hGH supernatants were transferred to Ni-NTA.

Each column was infused with agarose beads hnL (Qiagen, Valencia, CA, USA). The His-hGH and SI-His-hGH proteins injected into the column were washed with the wash buffer solution of Table 2, which was three times the volume of the column, eluted with 10 mL elution buffer of Table 2, and then His-hGH and SI. -His-hGH was first purified. His-hGH and

Fractions containing SI-His-hGH were dialyzed with anion exchange column complete solution (pH 8.0 50 mM Tris-HCl and 10% glycerol), respectively. TABLE 2

[Table 2]

Composition of complete solution for affinity chromatography

[83] a pH of 7.4 containing the above composition with a wash and elution layer solution.

Phosphate buffered saline (PBS) was used.

[84]

[85] Secondary purification by ion-exchange chromatography

[86] Dialysis fractions are 5/50 monocuum columns, anion-exchange chromatography (GE)

Healthcare, USA) was eluted by elution with a linear gradient of 0 to 500 mM sodium chloride concentration.

[0087] The lane S (supematant, soluble protein) and lane P (pellet, insoluble protein) of Fig. 2 showed that most of His-hGH was insoluble. Thus, Ni-NTA column chromatography showed Only a small amount of His-hGH could be recovered, such as E1-E3. The results of protein expression and Ni-NTA affinity chromatography under optimized conditions in this example are shown in Figure 3. Lane 4 and lane 5 We confirmed that most of His-hGH is in the soluble fraction, and 30 mg, which is more than 15 times higher than the total protein yield of 2 mg / 250 mL of the previous test results in lanes 6-7 and 9-10. It was confirmed that a / 250 mL culture solution was obtained.

4 shows whether SI-His-hGH is expressed. Comparing lanes 2 and 4, the difference in gel mobility between ^I3 C ¹⁵ N-Arg labeled protein and unlabeled protein can be confirmed. Based on the above results, Ni-NTA affinity column chromatography was performed as shown in Fig. 5. The solubility of the protein was confirmed in lanes 4 and 5, and the protein was separated at a high concentration in lanes 6-8. Could.

[89]

[90] (Example 5) Analysis of purified protein

[91] (1) Analysis of purified protein using MALDI-TOF mass spectrometer

[92] The above for the analysis of purified proteins using MALDI-TOF mass spectrometry

The purified protein obtained in the Examples was stained with Coomassie stained gel. Pretreatment for MALDI-TOF MS was performed using an in-gel digest method, in which the target protein was separated from the gel to treat trypsin. After cutting off each of the gel fragments containing His-hGH and SI-His-hGH, induction of reduction and alkylation is incubated for 10 minutes at 66 ° C. Trypsin is 25 mM ammonium bicarbonate (ammonium) at a concentration of 22.5 ng ^ L. Dissolved in bicarbonate, NH ₄ HC0 ₃ ), gel fragments and reaction at 37 ° C overnight to induce peptide formation. The matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) was induced. 1 mg / mL of the obtained protein was analyzed using MALDI matrix.

Mix with alpha-cyano-4-hydroxycinnamic acid at a ratio of 1: 1 (v / v) and spot on MALDI mass spectrometry plates Analysis was performed on an Autoflex III Smartbeam (Brukerdaltonix, USA) apparatus. Externally deficient peptides and protein correction kits (Sigma-Aldrich, USA) were used, and the mass spectrometry spectra ranged from 600 to 3,500 m / z. Performed in mode to confirm the mass of His-hGH and SI-His-hGH.

[93]

[94] (2) Purification of purified protein using HPLC

[95] Prominence the His-hGH fraction identified by the negative-exchange chromatography

UFLC system (Prominence UFLC system, Shimadzu) and Kinetex CI 8 column (2.6 μπι, 50 χ 2.10 mm; Phenomenex, Torrance, CA, USA) were analyzed using LG-hGH (LG Life Science, Korea) as a positive control. The complete solution was A (0.1%).

Trifluoroacatic acid (TFA) in water) and B (0.1% Trifluoroacatic acid (TFA) in acetonitrile (ACN)) were used, and the linear gradient of elution layer B was changed from 35% to 100% for 7 min. ^o eluted from C. the flow rate was 0.4 mL / min, to measure the UV absorption at 220 nm wavelength.

[96]

6 shows the predicted peptides that are formed when His-hGH is treated with trypsin.

Figure 7 shows the mass control peptides generated after trypsin treatment with LG-hGH and SI-His-hGH in the control group using a MALDI-TOF mass spectrometer.

As a result of the mass measurement, it was confirmed that the four peptides analyzed by SI-His-hGH caused a difference of about 10 m / z due to C ₆ ¹⁵ N ₄ -arginine, which is SI-His-hGH. The parent synthesis detected at m / z 1205.5 of the dual comparison control and the parent ion detected at m / z 1215.4 of SI-His-hGH.

MALDI-TOF / TOF (MS / MS) using a LIFT mode and FlexControl (Bruker, Germany) for further analysis results are shown in Fig. Was confirmed the formation of a number of daughter ions in the spectrum of Figure 8, yijeung ¹³ C ₆ ¹⁵ N ₄ ᅵ All 10 ions containing arginine are found to have a difference of about 10 m / z. Also, Mascot software (Matrix Science Inc, UK) analyzes the specifics of hGH as expected. ^• Identified peptides.

[98]

In FIG. 9, after the ion-exchange chromatography was performed for the secondary purification of SI-His-hGH, the result was shown. The fraction of SI-His-hGH detected at 15 minutes of elution time was separated. In the present invention, SI-His-hGH purified purely from the fractions was identified by silver staining, and it was confirmed that it was well separated from other impurities. The fraction of SI-His-hGH identified in FIG. 10 was analyzed by HPLC. 11 is shown. As a result of analyzing the positive control group LG-hGH and SI-His-hGH, it was confirmed that the purified SI-His-hGH had a high level of purity.

Sequence List Free Text

[100] The sequence listing was attached as a sequence listing file.

Claims

Claim

(1) Escherichia coli as an expression vector that encodes a target protein.

Transforming;

(2) culturing the transformed Escherichia coli in a culture medium;

(3) inoculating the cultured Escherichia coli on glucose minimum medium;

(4) culturing E. coli inoculated to the minimum medium;

(5) injecting an amino acid solution labeled with a stable isotope to the cultured E. coli, and inducing the expression of isotopically labeled proteins;

(6) incubating the E. coli culture solution into which the amino acid solution is introduced at a temperature of 15 to 25 ° C. for 8 to 18 hours;

Method of producing stable isotopically labeled protein comprising a.

In paragraph 1,

The target protein of step (1) is a method for producing a stable isotope labeled protein of human growth hormone.

In paragraph 1,

The stable isotope of step (5) above is a ¹³ C N-arginine phosphorus isotopic labeled protein production method.

(1) Escherichia coli as an expression vector that encodes a target protein.

Transforming;

(2) culturing the transformed Escherichia coli in a culture medium;

(3) inoculating the cultured Escherichia coli on glucose minimum medium;

(4) culturing E. coli inoculated to the minimum medium;

(6) culturing the E. coli culture solution to which the amino acid solution is added at a temperature of 15 to 25 ⁰ C for 8 to 18 hours;

(7) dissolving E. coli as a buffer solution in step (6);

(8) ultrasonically treating the E. coli and centrifuging to recover the isotopically labeled protein; and

(9) purifying the stable isotopic labeled protein;

Methods for Purification of Stable Isotope Labeling Proteins

In paragraph 4,

The target protein of step (1) is a method for purifying a stable isotope labeled protein of human growth hormone.

In paragraph 5,

The above-mentioned protein is a histidine-labeled human growth hormone stable stable isotopic protein. [Claim 7] In paragraph 4,

The stable isotopically labeled amino acid is ¹³ C ¹⁵ N-arginine.

[Claim 8] In paragraph 4,

The stable isotopically labeled protein is a method for the purification of stable isotopically labeled protein using at least one of affinity chromatography and anion-exchange chromatography.

[Claim 9] In paragraph 8,

The column of the affinity chromatography is Ni-NTA column

Methods for Purifying Stable Isotope Labeling Proteins

[Claim 10] In paragraph 8,

The method of purifying stable isotopically labeled proteins wherein the column of the anion-exchange chromatography is a monocube column.

[Claim 11] In paragraph 4,

Purification method of stable isotopically labeled protein of step (9) wherein biological stability is not inhibited.

[Claim 12] In paragraph 4,

Dissolving buffer solution of step (7) is a method for the purification of stable isotopically labeled protein comprising a nonionic denaturing agent.

13. The method of claim 12,

Said nonionic denaturant is 0.01 to 2% (v / v) Triton X-100 or twen 20 phosphorus isotopically labeled protein.

[Claim 14] In paragraph 4,

Dissolution buffer solution of step (7) does not contain salt

Methods for Purifying Stable Isotope Labeling Proteins.

[Claim 15] In paragraph M,

Said salt is sodium chloride or potassium chloride.

[Claim 16] In paragraph 4,

A method for the purification of stable isotopically labeled proteins, wherein the amount of the soluble complete layer solution is 0.25-2 mL per 30 mg of E. coli cells (pellets).

[Claim Π] (1) An E. coli is expressed as an expression vector encoding a protein

Transforming;

(2) culturing the transformed Escherichia coli in a culture medium;

(3) inoculating the cultured Escherichia coli into a glucose minimal medium;

(4) culturing E. coli inoculated to the minimum medium;

(5) injecting an amino acid solution labeled with a stable isotope to the cultured E. coli, and inducing the expression of isotopically labeled proteins; (6) culturing the E. coli culture solution to which the amino acid solution is added at a temperature of 15 to 25 ⁰ C for 8 to 18 hours;

(7) dissolving E. coli as a complete solution of step (6);

(8) ultrasonically treating the E. coli and centrifuging to recover the isotopically labeled protein;

(9) purifying the stable isotopic labeled protein; and

(10) analyzing the purified stable isotope marker protein by MALDI-TOF MS or HPLC;

Method for the analysis of stable isotopic labeled proteins, including.

[Claim 18] In paragraph Π,

The MALDI-TOF MS is an analysis method of the stable isotope marker protein to identify the mass spectrum in the range of 600 to 3,500 m / z.

[Claim I ⁹ ] In paragraph Π,

The HPLC column temperature is 20 to 50 ° C, the flow rate is set to 0.1 to 1.0 mL Anin, the detection method of the stable isotopic labeling protein.