JP2004222568A - Proteome column and analytical method using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a proteome column enabling amino acid sequence analysis quickly and easily through solving the problem that, for analyzing the amino acid sequence of a protein or peptide, amino acids need to be cleaved one by one, however, the analysis is highly difficult because of random cleavage of the bonds in the case of a physical technique, and 30 min or longer is needed for analyzing one amino acid in the case of a chemical technique. <P>SOLUTION: The proteome column is provided by the following way: An enzyme having the ability to hydrolyze peptide linkages is immobilized on a carrier or a resin, and a carrier or resin immobilized with another enzyme or that immobilized with no enzyme is then added to the above carrier so that the 1st enzyme becomes heterogeneous in the final column. By passing a sample protein or peptide through the column, amino acids are cleaved one by one at the ends, thus effecting a simple and quick analysis compared to conventional physical and chemical techniques, furthermore, enabling analyzing an amino acid sequence longer than that in the case using an enzyme-immobilized carrier alone. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a column for obtaining a fragment of a protein or peptide by cleaving from the end of the protein or peptide, and an analytical method using the column.
[0002]
2. Description of the Related Art Determining the amino acid residue sequence of peptides and proteins is also important for elucidating the various functions of proteins, which are the main components of living organisms. In order to analyze the amino acid sequence, it is necessary to extract and analyze the amino acids from the peptide or protein. As a method of cleaving a peptide bond between amino acids, there is a method of physically cleaving (for example, see Patent Documents 1 and 2) and a method of cleaving using a chemical reaction (for example, see Patent Document 3).
[0003]
As a biological method, a protein or peptide is decomposed with an enzyme such as aminopeptidase or carboxypeptidase, and a portion is collected over time, and the reaction solution is analyzed with an amino acid analyzer to quantify the released amino acid. A method has been used (for example, see Non-Patent Document 1). In addition, a method has been reported in which the reaction solution is subjected to a mass spectrometer to measure the molecular weight of a protein or peptide fragment, and the type of amino acid is determined from the difference from the molecular weight of the original protein or peptide (for example, non-patented). Reference 2).
[0004]
[Patent Document 1]
JP-A-2001-235377
[Patent Document 2]
JP-A-2002-131284
[Patent Document 3]
JP-A-10-293130
[Non-patent document 1]
Edited by The Biochemical Society of Japan, Biochemistry Laboratory Course, Volume 1, Protein Chemistry II, p203-211, published in 1976 [Non-Patent Document 2]
A. Tsugita, R .; van den Broek, M.E. Pyzybylski, FEBS Lett. 137, 19 (1982) (A. Tsugita, R. van den Brook, M. Pigibirsky, Febs Letter 137, 19 (1982))
[0005]
However, it is difficult to analyze a sequence by determining the sequence because it is randomly cleaved by the physical method, and the chemical method requires more than 30 minutes to analyze one amino acid. And it takes an enormous amount of time.
[0006]
In addition, the biochemical method described above is complicated in operation, the enzyme cannot be reused, a new enzyme is required each time, the enzyme has autolytic properties, and the purification of the enzyme is insufficient. However, there is a problem that amino acids or peptides are mixed due to the above. Furthermore, when introduced into a mass spectrometer for analysis, electrospray ionization (hereinafter abbreviated as ESI) is a simple and useful method as an ionization method. However, a disadvantage of this method is that multivalent ions are easily generated. There is a problem that it is difficult to estimate an accurate molecular weight.
[0007]
One of the methods used to solve the problem of enzyme recycling and enzyme autolysis is immobilization of the enzyme. An immobilized enzyme is an enzyme that is confined in a certain space, and is capable of performing an enzymatic reaction continuously, collecting the enzyme after the reaction, and reusing the enzyme in a state that can be reused. That is. Due to the limited movement of the enzymes, the autolysis of the enzymes resulting from the reaction between the enzymes is also less likely to occur. By washing well before use, impurities such as amino acids or peptides can be removed.
[0008]
By packing this immobilized enzyme into a column, a fragment from which a terminal amino acid has been deleted from a protein or peptide can be obtained simply by passing a sample through the column.
[0009]
Although this method can reuse the enzyme, the immobilized enzyme is evenly packed in the column, so passing it through the column causes only a fixed number of enzyme reactions, which is limited. Only fragments having a length in the above range could be obtained, and it was difficult to determine the amino acid sequence from the obtained fragments.
[0010]
Means for Solving the Problems As a result of intensive studies to solve the above problems, the following column was invented.
[0011]
(1) A column in which an immobilized enzyme that hydrolyzes a peptide bond of a protein or peptide and a carrier or resin for holding the immobilized enzyme are added with a carrier or resin on which the enzyme is not immobilized, A) a column comprising two or more types of immobilized enzymes containing the enzyme according to the first invention, and one or more carriers or resins for holding the respective immobilized enzymes; (3) the enzyme according to the first invention; (4) a column in which two or more types of immobilized enzymes including the above, and one or more types of carriers or resins for holding the respective immobilized enzymes are filled with a carrier or resin to which the enzymes are not immobilized; A column in which two or more types of immobilized enzymes including the enzyme according to the first invention are immobilized on the same carrier or resin; (5) the enzyme is immobilized on the carrier or resin according to the fourth invention; A column packed with a carrier or resin not added, (6) the first invention or the second invention characterized in that the packed carrier or resin serves as a means for separating a substance obtained by an enzymatic reaction; The column according to the third or fourth or fifth invention, (7) an ion-exchange resin as a carrier or resin to be packed, a packing material for reversed-phase and normal-phase chromatography, a packing material for size-exclusion chromatography, The first invention, the second invention, the third invention, the fourth invention, or the fifth invention, characterized in that the packing material for hydrophobic chromatography and the packing material for adsorption chromatography are used alone or as a mixture of two or more kinds. (8) a protein or peptide sample according to the first invention, the second invention, the third invention, the fourth invention, the fifth invention or Through sixth invention or the seventh column of the invention described in, by introducing into the mass spectrometer, the analysis method [0012] to determine the sequence of amino acids from the mass of each fragment
The column described in the first to seventh inventions is called a proteome column.
[0013]
By passing a protein or peptide sample through a proteome column and introducing it into a mass spectrometer, the amino acid sequence can be determined from the difference in the mass of each fragment. It became an economically superior column.
[0014]
EXAMPLES Examples of the present invention will be described below, but the present invention is not limited to these examples. In addition, the amino acids of peptides and proteins described below are all represented by one letter.
[0015]
Example 1 First step, 528 mg of bromocyan-activated Sepharose 4B (Amsham Pharmacia) was immersed in a 1 mmol / l hydrochloric acid solution at room temperature for 30 minutes to activate the resin, and then the solution was adjusted to pH 8.0 and 100 mmol / l. The mixture was replaced with a sodium phosphate buffer, and 50 μl of a 10 μg / μl carboxypeptidase Y (hereinafter abbreviated as CP-Y) solution was added thereto, followed by shaking at 30 ° C. for 4 hours. After completion of the shaking, the sodium phosphate buffer was removed, the resin was washed, 2 ml of a 10 mg / ml bovine serum albumin solution was added, and the mixture was shaken at 30 ° C. for 4 hours, and the remaining active groups were washed with bovine serum albumin. Blocked. Thereafter, the resin was washed with a phosphate buffer to obtain a resin in which the enzyme was immobilized on the resin.
[0016]
The resin obtained in the second step and the first step was mixed well with Toyopearl SP-650M as an anion exchange resin at a volume ratio of 1: 3, and packed into a stainless steel column having an inner diameter of 2 mm and a length of 100 mm. Separately, the resin obtained in the first step was mixed well with a cation exchange resin, Toyopearl DEAE-650M, at a volume ratio of 1: 3, and packed in a stainless steel column having an inner diameter of 2 mm and a length of 100 mm. .
[0017]
The proteome column obtained in the third step and the second step was incorporated in a liquid chromatography mass spectrometer, and the amino acid sequence of the bradykinin solution was analyzed by mass spectrometry. A degasser DGV-12AM (manufactured by Shimadzu) and a pump LC10-ADVP (manufactured by Shimadzu) were used as liquid chromatography devices, and LCMS-2010α (manufactured by Shimadzu) was used as a mass spectrometer. As an experimental condition, a 100 mmol / l ammonium acetate buffer was passed through the mobile phase at a flow rate of 0.2 ml / min, and the ESI method was used as an ionization method. As a control experiment, a column to which no ion exchange resin was added was prepared and used in the experiment.
[0018]
The results of mass spectrometry are shown in FIGS. In a proteome column to which an anion exchange resin was added, a fragment cut from the carboxy terminal (hereinafter abbreviated as C-terminal) side of bradykinin (amino acid sequence RPPGFSPFR) had a molecular weight of 904.25, 757.35, 660.30, 573.25, A peak was obtained at 426.10. By taking these differences and analyzing the values, it was revealed that the amino acid sequence of FSPF exists on the C-terminal side (FIG. 1).
[0019]
Next, in the proteome column to which the cation exchange resin was added, peaks were obtained at the molecular weights of 904.25, 757.35, 660.25, 573.20, and 426.10. Was found to have the amino acid sequence of FSPF (FIG. 2).
[0020]
On the other hand, in the control experiment according to the conventional method, only peaks having molecular weights of 757.35, 573.25, and 426.10 were obtained from FIG. Only the sequence of the F and SP portions of the above was obtained.
[0021]
The results showed that the proteome column to which the carrier was added was able to analyze a longer amino acid sequence than the conventional column.
[0022]
Example 2 First step, 564 mg of bromocyan-activated Sepharose 4B (Amsham Pharmacia) was immersed in a 1 mmol / l hydrochloric acid solution at room temperature for 30 minutes to activate the resin, and then the solution was adjusted to pH 8.0 and 100 mmol / l. The solution was replaced with a sodium phosphate buffer, and 200 μl of a 3.4 mg / ml aminopeptidase M (hereinafter abbreviated as AP-M) solution was added thereto, followed by shaking at 30 ° C. for 4 hours. After completion of the shaking, the sodium phosphate buffer was removed, the resin was washed, 2 ml of a 10 mg / ml bovine serum albumin solution was added, and the mixture was shaken at 30 ° C. for 4 hours, and the remaining active groups were washed with bovine serum albumin. Blocked. Thereafter, the resin was washed with a phosphate buffer to obtain a resin in which the enzyme was immobilized on the resin.
[0023]
The resin obtained in the second step and the first step was mixed well with Toyopearl SP-650M as an anion exchange resin or Toyopearl DEAE-650M as a cation exchange resin in the same manner as in Example 1, and the inner diameter was 2 mm. And a 100 mm long stainless steel column.
[0024]
The columns obtained in the third step and the second step were incorporated into a liquid chromatography mass spectrometer, and the amino acid sequence of the angiotensin III solution was analyzed by mass spectrometry. As an experimental condition, a 100 mmol / l ammonium acetate buffer was passed through the mobile phase at a flow rate of 0.2 ml / min, and the ESI method was used as an ionization method. As a control experiment, a column to which no ion exchange resin was added was prepared and used in the experiment.
[0025]
The results of mass spectrometry are shown in FIGS. In the proteome column to which an anion exchange resin was added, fragments cut from the amino terminus (hereinafter abbreviated as N-terminus) side of angiotensin III (amino acid sequence RVYIHPF) had a molecular weight of 931.05, 775.25, 676.30, 513. Peaks were obtained at .25, 400.05 and 262.85. By taking these differences and analyzing the values, it was possible to analyze that the amino acids were arranged in the order of RVYIH from the N-terminal side (FIG. 4).
[0026]
In the case of the proteome column to which the cation exchange resin was added, peaks were obtained at molecular weights of 775.40, 676.35, 513.05, 400.15, and 263.05. Similar analysis revealed that the amino acid sequence of VYIH was present on the N-terminal side (FIG. 5).
[0027]
On the other hand, in the control experiment, peaks with molecular weights of 775.40, 676.40, 513.30, and 400.15 were obtained. As a result of analysis in the same manner, the sequence of the N-terminal amino acid sequence VYI could be analyzed (FIG. 6).
[0028]
From these results, the amino acid sequence can be longer by one residue in the proteome column to which the cation exchange resin is added and by two residues in the proteome column to which the anion exchange resin is added, as compared with the column by the conventional method. Was.
[0029]
Example 3 The anion exchange resin-added proteome column obtained in Example 1 was incorporated into a liquid chromatography mass spectrometer, and the amino acid sequence of a synthetic peptide (amino acid sequence DRVYIHPFHLVIH) solution was analyzed by mass spectrometry. As an experimental condition, a 100 mmol / l ammonium acetate buffer was passed through the mobile phase at a flow rate of 0.2 ml / min, and the ESI method was used as an ionization method. After the injection of the sample, molecular ions having a mass / charge ratio of 50 to 2,000 were measured, and a change in the amount of each molecular ion with time was measured. FIG. 7 shows the results of total ion chromatography and the chromatography of the fragment formed by cleavage of the amino acid at the C-terminal of the peptide.
[0030]
From among the peaks derived from the fragment obtained by cleavage from the C-terminal side of the synthetic peptide from FIG. 7, the fragment having the amino acid sequence of DRVD and the fragment of RVY have a retention time of 0.9 minutes, the fragment having the amino acid sequence of DRVYIHP and DRVYIHPF fragments had retention times of 2.4 minutes and 2.8 minutes, respectively, indicating a correlation between fragment length and retention time.
[0031]
When ionized by the ESI method, the DRVD fragment and the RVY fragment have a charge number of 1 under these conditions, whereas the DRVYIHP fragment and the DRVYIHPF fragment have a charge number of 2, whereas in FIG. The fragment and the RVY fragment were observed at a retention time of 1 minute, while the DRVYIHP fragment and DRVYIHPF fragment with a charge number of 2 appeared at a retention time of the order of 2 minutes, indicating a correlation between the charge number and the retention time. Therefore, by using this, analysis can be easily performed.
[0032]
As described above, according to the first invention, it is possible to analyze a longer amino acid sequence than the conventional method in which only the immobilized enzyme is filled, and at the same time, the amount of immobilized enzyme used Is reduced to 25%.
[0033]
In the second invention, the same effect as in the first invention can be obtained only by adding a carrier or a resin on which an enzyme different from the enzyme described in the first invention is immobilized, instead of the carrier or the resin to be added. In addition, it is possible to remove the modified or modified portion of the protein or peptide of the sample, which has the effect of making the amino acid sequence more readable.
[0034]
In the third invention, the effect of the second invention can be realized with a smaller amount of enzyme.
[0035]
In the fourth invention, it is possible to obtain the effect obtained in the second invention with one carrier or resin, and it is possible to omit the step of mixing the carrier or resin when producing a proteome column. Become.
[0036]
In the fifth invention, while having the effect of the second invention, by further localizing the enzyme, there is an effect that the analysis of the amino acid sequence in a longer range can be performed.
[0037]
In the sixth aspect, by adding a carrier or a resin having a separating ability, impurities can be separated and measurement can be performed with higher sensitivity. In addition, since the reaction and the separation can be performed simultaneously in one column by the carrier or resin to be added, there is an effect that analysis of expensive ions generated when the ESI method is used can be easily performed.
[0038]
By using the carrier or resin described in the seventh invention, there is an effect that information on amino acid sequence analysis can be obtained while performing separation according to the target protein or peptide with one proteome column. .
[0039]
In the eighth invention, the use of the proteome column according to the first to seventh invention for amino acid sequence analysis has an effect that analysis can be performed more easily and more quickly than in the conventional method.
[Brief description of the drawings]
FIG. 1: Mass spectrometry of a digested fragment of bradykinin by a column using only a resin on which CP-Y is immobilized. FIG. 2: Bradykinin by a column containing a cation exchange resin added to a resin on which CP-Y is immobilized. Mass spectrometry of digested fragment [Fig. 3] Mass spectrometry of digested fragment of bradykinin using a column in which anion exchange resin is added to resin on which CP-Y is immobilized [Fig. 4] Only resin on which AM-P is immobilized Mass spectrometry of the digested fragment of angiotensin III using the column used. [FIG. 5] Mass spectrometry of the digested fragment of angiotensin III using a column in which a cation exchange resin was added to a resin on which AM-P was immobilized. Angiotensin using a column in which anion exchange resin is added to a resin on which P is immobilized Mass spectrometry of digested fragment of II. [FIG. 7] Total ion chromatography of a synthetic peptide (amino acid sequence DRVYIHPFHLVIH) using a column in which anion exchange resin is added to a resin on which CP-Y is immobilized, and C-terminal amino acids are cleaved. Chromatography of the isolated fragments (amino acid sequences: DRV, DRVY, DRVYIHP and DRVYIHPF)

Claims (8)

A column in which an immobilized enzyme that hydrolyzes the peptide bond of a protein or peptide, and a carrier or resin that holds the immobilized enzyme, and a carrier or resin on which the enzyme is not immobilized are added and packed. A column comprising two or more types of immobilized enzymes containing the enzyme according to claim 1, and one or more types of carriers or resins for holding the respective immobilized enzymes. A carrier or resin to which no enzyme is immobilized is added to two or more kinds of immobilized enzymes containing the enzyme according to claim 1 and one or more kinds of carriers or resins for holding the respective immobilized enzymes, and filled. Column A column in which two or more immobilized enzymes including the enzyme according to claim 1 are immobilized on the same carrier or resin. A column filled with a carrier or resin to which the enzyme is not immobilized, added to the carrier or resin according to claim 4. 6. The column according to claim 1, wherein the carrier or resin to be packed serves as a means for separating a substance obtained by an enzyme reaction. Ion-exchange resin, packing material for reversed phase and normal phase chromatography, packing material for size exclusion chromatography, packing material for hydrophobic chromatography, packing material for adsorption chromatography, alone or as a mixture of two or more kinds The column according to claim 1, claim 2, claim 3, claim 4, or claim 5, wherein the column is used as Each fragment is obtained by passing a protein or peptide sample through a column according to claim 1 or claim 2 or claim 3 or claim 4 or claim 5 or claim 6 or claim 7 and introducing the sample into a mass spectrometer. Method to determine amino acid sequence from mass

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