JP4835933B2 - Method for quantification of proteins and peptides with constituent amino acids - Google Patents

Description

  The present invention relates to a method for accurately determining the amount of a protein or peptide from the amount of a specific amino acid constituting the protein or peptide.

As a method for quantifying proteins, there is gel chromatography. As a quantitative method close to the present invention, a method is known in which a protein is decomposed and obtained from amino acids.
As an example of this, prior art No. 1) John W. Baynes, Suzanne R. Thorpe, Martha H .; 3. Muthashaw: Nonenzymatic Glucosylation of Lysine Residues in Album, Methods in Enzymology, 106: 88-98, 198 . There is. However, when this method is applied to a protein or peptide having a modified amino group, a considerable amount of the modified amino acid is also decomposed into various forms of compounds, so that the protein or peptide having the modified group is correctly and accurately detected. There is no method for quantification as long as the authors conducted a literature survey . Moreover, efficiency with decomposition influenced by steric hindrance or the like, not uniformly decided amount of protein from the resulting amino acid content.
Furthermore, there is an enzymatic measurement method using protease and ketoamine oxidase , which is used as a glycoalbumin measurement kit in clinical tests . However, since the enzymatic degradation product of the protein by this method is a mixture of amino acids and peptides, it is impossible to obtain only glycated lysine, which is a glycated amino acid , and the degradation is incomplete, so it is accurate. Quantification is difficult .

The final object of the present invention is to disclose a method for measuring the saccharification rate with high accuracy since the glycation ratio of blood proteins such as hemoglobin and albumin is an index for diagnosis of diabetes.
Moreover, since it became clear from the technique for achieving this objective that protein, a peptide, etc. can be measured with high precision, it is providing the method.

  The sample to be used in the present invention is mainly blood, plasma, serum, urine, etc., and glycohemoglobin, glycoalbumin, albumin, globulin, C-peptide, insulin, etc. therein are mainly measured.

Means to be Solved by the Invention

Those forming a framework of the present invention is to decompose the protein, to obtain the amino acids constituting them, but it is to measure certain modified amino acids amount among them, upon degradation, stable isotopes of modified amino acids of interest In addition, the ratio of the obtained modified amino acid to the remaining stable isotope is measured by mass spectrometry, and the target modified amino acid is quantified from a separately obtained calibration curve.
At this time, the amino acid is modified with glucose, and if the amount of glycated amino acid is similarly determined by mass spectrometry, the glycated protein is obtained.
The present invention can be applied even when the modifying group is other than glucose.

In the present invention, proteins must be decomposed into amino acids, and examples of the decomposition methods include enzyme decomposition, acid decomposition, and alkali decomposition. Its Although both can be applied, enzymatic degradation, specificity is high casting, often for decomposition is insufficient, even amino acid or glycated amino acid and an object is being obtained, et al., Then back-calculated to proteins, peptides It is not required.
Therefore, acid decomposition or alkali decomposition is used in many cases. However, in these methods, since the modifying group of the modified amino acid is decomposed, generally, hydrogenation or cyanation is used to reduce the decomposition rate. However, since decomposition occurs, the sugar content of the target modified amino acid is extremely poor. Thus, the inventors have found that if a stable isotope of a modified amino acid is added at the time of decomposition to compensate for the decomposition, the accuracy is remarkably increased by measuring the ratio . The amount of modified protein is generally determined by the ratio to the total amount of the target protein. The present invention also discloses a method for obtaining this ratio with high accuracy.

An example of quantification of albumin, which is the main component of serum protein, according to the present invention will be described.
After separating albumin from serum using an eluent such as phosphate by gel filtration chromatography or ion exchange chromatography, the eluent components are washed away with water by ultrafiltration to obtain a purified solution of albumin. Add a certain amount of stable isotopes of the main amino acids constituting albumin to this solution, mix well, transfer to a sealed glass tube, add hydrochloric acid, and purge with an inert gas such as nitrogen. From the sealed tube under reduced pressure, it is heated and hydrolyzed.

Hydrolyzed hydrochloric acid and water in a sealed tube that has been hydrolyzed are removed under reduced pressure. The resulting dried product dissolved in a solution suitable for liquid chromatography mass spectrometry (LC / MS) such as formic acid is contained in albumin. The isotope intensity ratio of the ions of mass derived from each of the added amino acid and the added stable isotope amino acid is measured by LC / MS. For a calibration solution with multiple isotope ratio compositions prepared by adding a certain amount of stable isotopes of amino acids to a standard amino acid solution consisting of natural isotope compositions, the isotopic intensity ratios were similarly measured by LC / MS. The molar concentration of amino acid in the albumin purification solution is determined from the obtained calibration curve.
The measured molar concentration of amino acid is divided by the number of amino acids constituting one molecule of albumin to determine the molar concentration of albumin in the purified albumin solution.

  A quantitative example according to the present invention of glycated albumin in which glucose is modified with lysine, which is one of the amino acids constituting albumin, will be described.

  After separating albumin from serum using an eluent such as phosphate by gel filtration chromatography or ion exchange chromatography, the eluent components are washed away with water by ultrafiltration to obtain a purified solution of albumin. To this solution, a certain amount of a stable isotope of glycated lysine is added and mixed thoroughly and homogenized, and then an alkali solution of sodium borohydride is added and hydrogenated at room temperature for 4 hours. This is transferred to a glass tube for sealing, added with hydrochloric acid and purged with an inert gas such as nitrogen, sealed in a reduced pressure state, heated and hydrolyzed.

  Hydrochloric acid and water in the sealed tube after hydrolysis were removed under reduced pressure, and the resulting dried product dissolved in a solution suitable for LC / MS such as formic acid was added with glycated lysine contained in albumin. The isotope intensity ratio of ions of mass derived from each stable isotope glycated lysine is measured by LC / MS. Similarly, LC / MS measures the isotope intensity ratio of a calibration solution with multiple isotope ratio compositions prepared by adding a fixed amount of a stable isotope of glycated lysine to a standard solution of glycated lysine having a natural isotope composition The molar concentration of glycated lysine in the albumin purification solution is determined from the calibration curve obtained in this way.

  The measured glycated lysine molar concentration is divided by the molar concentration of albumin in the albumin purification solution to determine glycated albumin as the number of glycated lysine per molecule of albumin.

Embodiment of the Invention

The target protein (or peptide) is separated and purified from other proteins by affinity chromatography, gel filtration chromatography, ion exchange chromatography, reverse phase chromatography, thin phase chromatography, electrophoresis or the like.
From there, weighed liquid containing the desired protein having mg equivalent amount in a sealed container, a stable isotope of the modified amino acids you attempt to quantify added already modified amino acids corresponding amount included in the target protein were weighed. The amount of modified amino acid can be estimated from the measured values of commercially available daily methods. Later, the modified amino acid to the more stable hydride by adding sodium borohydride.
Thereto, hydrochloric acid is added so as to have a final concentration of 4 to 6 M, sealed under nitrogen gas, and thermally decomposed at 110 to 120 ° C. for 15 to 24 hours.
After decomposition, the container is opened, and water containing hydrochloric acid is evaporated to dryness in a warm bath under reduced pressure.
Dissolve in 5 mM heptafluorobutyric acid (LC / MS sensitivity can be obtained, other solvents may be used if there is no interference), inject into a liquid chromatograph mass spectrometer (LC / MS), and measure.

FIG. 1 Procedure for measuring glycated lysine in albumin Column: Shim-pac HP-5 (5 μm, 0.2 mm id × 150 mm L)
Eluent: (A) 0.05% HFBA (B) Solvent gradient mixed with acetonitrile Flow rate: 0.2 mL / min Sample injection amount: 1 μL Ionization method: ESI
Measurement of the isotope ratio: area ratio in the SIM mode measurement R ⁼ 311M ⁺ / 317M +
R = 147M ⁺ / 151M ⁺
FIG. 2 The degree of agreement G / A ratio between this method and a commercial measurement method (enzyme method) indicates the molar ratio of albumin-linked glycated lysine and albumin.
G and A were determined according to Example 1.

A gel filtration chromatography column was injected with 25 uL of serum and monitored at a wavelength of 280 nm under the condition that an eluent (30 mM phosphate buffer containing 150 mM Na ₂ SO ₄ , pH 6.87) was flowed at 0.8 ml / min. An approximately 8 mL aliquot containing 10,000 to 70,000 albumin was obtained. Next, an eluent component having a molecular weight of 5,000 or less in the fractionated solution was washed and removed with water using a centrifugal ultrafiltration filter to obtain 180 uL of a purified albumin solution.

Weigh the above purified albumin solution (equivalent to 1 mg of estimated albumin amount) into a glass tube for sealed tube, and add 4,4,5,5-D4-lysine solution (lysine amount in 1 mg of albumin) which is a stable isotope of lysine. Saccharified lysine saccharified with ¹³ C ₆ -glucose (estimated amount of saccharified lysine in 1 mg of albumin) and mixed thoroughly and homogenized into an alkaline solution of sodium borohydride And hydrogenated at room temperature over 4 hours. Thereto is added 10M hydrochloric acid so that the final concentration is 5M, and it is sealed and dried under a reduced pressure of N2 gas with a gas burner, and then kept in a heater set at 110 ° C. for 20 hours to hydrolyze the peptide bond of albumin.

  The sealed tube after the hydrolysis was opened and kept in a 45 ° C. water bath, and the dried product obtained by volatilizing and removing hydrochloric acid and water by connecting to a water flow vacuum pump was transferred to 500 μL of 5 mM HFBA (heptafluorobutyric acid). The dissolved one was used as an LC / MS measurement solution.

Each of lysine glycated with lysine and glycated lysine of natural isotopic composition and added stable isotopes 4,4,5,5-D ₄ -lysine and ¹³ C ₆ -glucose contained in albumin is ionized. The isotope intensity ratio of the protonated molecular ions was measured by LC / MS. Lysine glycated with 4,4,5,5-D4-lysine and ¹³ C ₆ -glucose in a mixed standard solution of high-purity lysine (Sigma, purity 98% or more) and glycated lysine (self-synthesized, purified product) For a calibration solution with three isotope ratio compositions (isotope ratios 0.8, 1.0, 1.2) prepared by addition, albumin was obtained from a calibration curve obtained by measuring the isotope intensity ratio in the same manner. The molar concentration of lysine and saccharified lysine in the purified solution was determined. The measured molar concentration of lysine was divided by the number 59 of lysine constituting one molecule of albumin to determine the molar concentration of albumin in the purified albumin solution. These results and measurement conditions are shown in FIGS. 2 and 1, respectively. FIG. 2 shows a comparison between the conventional method for determining the amount of glycated albumin by an enzymatic method and the present method, and there is no fundamental contradiction between the present method and the conventional method .
In this example, the amount of glycated albumin and the total amount of albumin were determined simultaneously, but only the amount of glycated albumin or only the amount of total albumin can also be determined separately. In this case, the amino acid is not limited to lysine, and other amino acids may be measured (for example, stable amino acids such as alanine and leucine may be used).

Claims (6)

Adding a stable isotope of a specific modified amino acid constituting the protein or peptide to a solution containing the protein or peptide, and then performing an acid decomposition or alkali decomposition operation of the protein or peptide; The ratio of the added stable isotope to the remaining amount is measured by mass spectrometry, and the amount of the modified amino acid is obtained from a calibration curve obtained for the ratio of the desired amount of the modified amino acid and the amount of the stable isotope. A protein / peptide quantification method for calculating the target protein or peptide amount by back-calculating from the number of specific modified amino acids constituting one peptide molecule.   The protein / peptide quantification method according to claim 1, wherein the decomposition method is acid decomposition or alkali decomposition, and the decomposition operation is performed after the modifying group is reduced or cyanated with hydride or cyanide.   The method for quantifying proteins and peptides according to claim 2, wherein the modifying group is glucose.   The specific amino acid amount and the modified amino acid amount of the amino acid are determined by the operation of claim 1, and the ratio of both or the ratio of the modified amino acid amount and the sum of the two is calculated, and the ratio of the protein or peptide modified. For quantifying proteins and peptides.   5. A method for quantifying a protein or peptide according to claim 1, wherein when the solution contains a mixed protein or peptide, the target protein or peptide and another coexisting protein or peptide are separated and purified in advance, and then the operation according to any one of claims 1 to 4 is performed.   6. The protein / peptide quantification method according to claim 5, wherein the separation / purification method is any one or a combination of ion exchange separation method, gel filtration method, precipitation method, electrophoresis method, thin layer chromatography and affinity chromatography.

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