JPH1123558A - Separation method and apparatus using the same - Google Patents

Abstract

(57) [Problem] To separate a sample containing a peptide or a protein, a peptide or a protein can be separated and, after separation, can be purified without a complicated operation such as desalting, and a method using the method. Provide the equipment that was used. When a sample containing a peptide and / or protein is separated by normal phase liquid chromatography, the sample is injected into a separation column, and then a mixture of an organic solvent and water and / or an alcohol is added to the mixture. A method and apparatus for separating peptides and / or proteins by developing a mobile phase to which an amine is added is used.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a method for separating peptides and peptides using a separation column used in normal phase chromatography and a mobile phase obtained by adding an acid and / or an amine to a mixture of an organic solvent and water or an alcohol. The present invention relates to a method for separating a sample containing a protein and an apparatus using the same.

[0002]

2. Description of the Related Art Techniques for separating peptides and proteins using liquid chromatography include reverse phase chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, and affinity chromatography. However, there have been no reports or reports on the separation and purification of native peptides and proteins that are not chemically modified using normal phase chromatography.

[0003] Among them, reversed-phase chromatography is a very useful method for separating and purifying peptides and proteins since a highly volatile organic solvent or acid is used as a mobile phase. Is performed by utilizing the difference in hydrophobic interaction between each peptide or protein in the sample and the stationary phase of the separation column.Therefore, each peptide or protein in the sample has no hydrophobicity or the difference in hydrophobic interaction is small. Without it, it was difficult to separate.

On the other hand, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography,
In the method using affinity chromatography,
Although it is possible to separate peptides and proteins in a sample that has no hydrophobicity or a small difference in hydrophobic interaction that cannot be separated by the method using reversed-phase chromatography, the mobile phase can be used in any method. However, there is a problem that complicated operations such as desalting are involved in order to purify the peptide or protein after separation, since a buffer solution containing a salt is used.

[0005] On the other hand, a classical and general method using normal phase chromatography using a column such as a silica column or an alumina column performs separation using the polarity of a target substance in a sample. It has the opposite property to the method using reverse phase chromatography for separation, but because it is performed using a non-aqueous solvent such as hexane as the mobile phase, the solubility of the target substance in the sample in the mobile phase is poor, It was not applied to the separation of biological materials such as peptides and proteins.

In recent years, chemically bonded stationary phases in which polar groups such as amino groups, hydroxyl groups (OH) and amide groups are bonded to a gel base material such as silica or resin have been developed for normal phase chromatography. Since a mixed solution of a solvent and water or an alcohol can be used as a mobile phase, it becomes possible to apply the method to a biological sample that could not be used due to the problem of solubility in the conventional method using normal phase chromatography.

[0007]

SUMMARY OF THE INVENTION Under the above-mentioned background, an object of the present invention is to separate peptides and proteins when separating samples containing peptides and proteins.
It is still another object of the present invention to provide a method that can be purified without performing a complicated operation such as desalting after separation, and an apparatus using the method.

[0008]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above problems, and as a result, a polar group such as a hydroxyl group (OH) or an amide group has been added to a gel base such as silica or resin. Reversed-phase chromatography, rather than normal-phase chromatography using a mobile phase in which an acid or amine is added to a mixture of a highly volatile organic solvent and water or alcohol using a bonded chemically bonded stationary phase. The ability to separate samples containing peptides and proteins that are difficult to separate by the method, and the selection of highly volatile organic solvents, acids, and amines used in the reverse phase chromatography method as the mobile phase As a result, the present inventors have found that after separation of peptides and proteins, purification can be performed without complicated operations such as desalting, and the present invention has been completed.

[0009]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the drawings.

[0010] In the present specification, the term "peptide" is used in a sense that includes the amino acids constituting the peptide.

FIG. 1 shows an apparatus for separating peptides and / or proteins using the normal phase chromatography method of the present invention.

In FIG. 1, a first mobile phase bottle 1 and a second mobile phase bottle 2 are bottles for storing a mobile phase (also referred to as an eluent in this specification). 1. Eluent 2 is stored. These stored eluents 1 and 2 differ in the concentration of the organic solvent therein. The size, shape, and material of the bottle are not particularly limited. The size may be any size as long as it can be stored in an amount exceeding a mobile phase required for separation and purification. Therefore, any solution can be used as long as the stored solution can be sent. The material may be any material as long as the solution can be stored stably when the mobile phase is stored, and examples thereof include glass and a polymer resin.

The liquid sending pump 3 is for sending the first mobile phase 1 and the second mobile phase 2 in a gradient manner. The conditions for the liquid sending are not particularly limited, and when it is carried out by a small-scale system for analyzing peptides and / or proteins in a sample, the conditions vary depending on the size of the separation column.
It is usually carried out at a flow rate of 1.5 ml / min or less. More specifically, when the inner diameter of the column is about 4.6 mm, the flow rate in the range of 0.7 to 1.0 ml / min is used. ~ 0.2ml
A flow rate in the range of / min is preferably used, and any liquid feed pump that can achieve such a flow rate may be used. When a specific peptide or protein in a sample is purified by a large-scale system, the required performance is appropriately determined according to the size of the column or the separation pattern of the specific peptide or protein in the sample. A liquid pump may be used.

When it is not necessary to send the first mobile phase 1 and the second mobile phase 2 at a gradient, only one of the first mobile phase 1 and the second mobile phase 2 may be used. .

Reference numeral 4 denotes a sample injecting device, and the volume of the sample to be used can be changed to an arbitrary amount, and the injection can be performed manually or automatically. The sample injection volume differs between the case where the sample is used for small-scale analysis and the case where the sample is used for large-scale purification, and a sample injection device that can appropriately inject the required sample volume may be used.

Reference numeral 5 denotes a separation column, and 6 denotes a constant temperature bath. The separation column 5 is maintained at a constant temperature in the constant temperature bath 6. Alternatively, when temperature adjustment is not required, the temperature of the separation column 5 does not need to be adjusted in a constant temperature bath.

The thermostat 6 is used to keep the temperature of the separation column 5 constant, and the temperature is kept constant by a heating and / or cooling mechanism using a gas such as air or a liquid such as water as a medium. It has a function. Here, the temperature that can be maintained in the thermostatic oven depends on the object to be separated and the purpose, and therefore cannot be unconditionally determined, but is 10 to 70 ° C.
Is preferable, and a temperature range of 30 to 50 ° C. is more preferable. This is because the temperature must be higher than the freezing point of the mobile phase, and the temperature must not exceed the boiling point. Furthermore, if the conditions deviate from this range, peptides and proteins in the sample to be separated may be degraded.

Reference numeral 7 denotes a detector, which is not particularly limited as long as it can detect peptides and / or proteins in a sample to be separated and is not disturbed by a mobile phase. Any known means such as an ultraviolet absorption detector, a visible absorption detector, a fluorescence detector, and a luminescence detector can be used, and the detection wavelength may be determined under known conditions. Further, the signal obtained by connecting this detector to a recorder can be output to paper or the like or recorded on an electric recording medium and used for analysis and purification.

Further, as for the liquid feed pipe connecting between each of the above-mentioned parts 1 to 7, if it has pressure resistance, its material,
The inner diameter, outer diameter, length, and the like are not particularly limited, and usually, metals such as stainless steel, polymer resins, and the like are preferably used.

In the chromatographic apparatus thus constructed, in the embodiment described below, the first mobile phase bottle 1 is mixed with a solution of acetonitrile: water = 97: 3 (by volume) in a ratio of 0.1: 0.1. % Trifluoroacetic acid was used, but it is essential that the organic solvent does not significantly contribute to the donation and acceptance of protons and is miscible with water or alcohol. Such organic solvents include
Cyanated hydrocarbons and the like are preferably used, and acetonitrile is more preferably used. The concentration is preferably 60% by volume or more, more preferably 70% by volume or more of the total amount of the mobile phase from the viewpoint of the resolving power.

The water is not particularly limited as long as it does not affect the detector described above, and either cold water or hot water can be used. Furthermore, the alcohol that may be used in place of water is not particularly limited as long as it is miscible with an organic solvent, but is completely miscible with the organic solvent at the time of separation, and may be used in a sample to be separated. It is sufficient that the peptide or protein does not deteriorate. For example, methyl alcohol, ethyl alcohol and the like can be exemplified. Further, water and alcohol can be used in combination.

The mixing ratio of the organic solvent to water or alcohol may be (water or alcohol) /
(Organic solvent) is preferably in the range of 0 to 0.3 (by volume). Here, the mixing ratio refers to a case where the mixture is substantially used for separating a sample containing a peptide and / or a protein.

Further, an acid is added to the mobile phase as a measure against ionic adsorption to the gel base material in the mobile phase, that is, as a means for improving the recovery of a sample containing peptides and the like. As the type of acid, organic acids such as trifluoroacetic acid, formic acid, and acetic acid, and inorganic acids are preferably used.Furthermore, organic acids are used from the viewpoint of volatility which does not require a desalting operation which is effective when used for purification. Acids, especially trifluoroacetic acid, are preferably used. As the content ratio, based on the total amount of the mobile phase,
It is preferable to use it in the range of 0.01 to 20% on a volume basis.

Also, amines can be used together with or as an acid. As the kind of the amine used, a tertiary amine is preferable, and triethylamine and trimethylamine are more preferably used.
Regarding the content ratio, it can be added in the range of 0.01 to 20% by volume with respect to all mobile phases.

In the embodiment, the mobile phase stored in the second mobile phase bottle 2 is acetonitrile: water = 55: 4.
A solution obtained by adding 0.1% of trifluoroacetic acid to a solution mixed at a ratio of 5 was used. In this case, as in the case of the first mobile phase bottle 1, the type and the ratio of the organic solvent, water, alcohol, acid or amine can be changed.

As an example of a separation column, an amide group (carbamoyl group) is covalently bonded to a gel base material.
TSKgel Amide-80 (Tosoh Corporation)
Was used. Such a gel base material is not particularly limited as long as it can perform normal phase chromatography with a chemical bond type, but inorganic silica, alumina, an organic polymer resin and the like are preferably used, and in particular, Inorganic silica is preferably used in terms of separation ability, durability and the like.

The functional group bonded to the gel base material is not particularly limited as long as it can perform normal phase chromatography. Examples of the functional group include an amino group, an amide group (carbamoyl group), and a hydroxyl group. , A carboxyl group and a sulfonic acid group are preferably used. Further, an amide group (carbamoyl group) and a hydroxyl group are preferably used in view of separation ability and durability.
Are preferably used. Further, two or more of these functional groups may be present.

More specifically, the above-described TSKge
l Amide-80 or TSKgel OH-120 (manufactured by Tosoh Corporation) in which a hydroxyl group is covalently bonded to a gel base material, TSKgel commonly used in a normal phase method.
Silica-60 (manufactured by Tosoh Corporation), TSKgel SP-2SW, TSKgel SP, which are ion exchange columns involved in donating and accepting protons in the separation mechanism.
-2PW, TSKgelSP-5PW, TSKgel
CM-2SW, TSKgel CM-2PW (all manufactured by Tosoh Corporation) and the like.

In the method of the present invention, the sample may be difficult to dissolve in the mobile phase due to the use of a high ratio of the organic solvent as the mobile phase. It may be diluted in the order of 2, mobile phase 1. The final formic acid concentration in the sample can be used up to 70% by volume based on the total volume of the injected sample.

The sample containing the adsorbed peptide and / or protein is eluted shortly after injection of the sample by gradiently increasing the concentration of water or alcohol in the mobile phase by the liquid sending pump 3. In the examples, the gradient for elution was performed by a gradient curve as shown in Table 1 as an example.

[0031]

[Table 1]

Here, the gradient conditions for elution may be not only a linear gradient but also a curve gradient, and may be appropriately changed depending on the substance to be separated in the sample.

Thus, the peptides and proteins eluted from the separation column are detected. In FIG. 2, chromatograms obtained by detecting with an ultraviolet absorption detector having an absorption wavelength of 215 nm and separating various peptides were obtained. Here, as a detection method, the eluted substance to be separated may be directly detected, but a liquid feed pump and a solution for detection are added before the detector shown in FIG. Alternatively, a method of merging with the solution may be used. In this case, the liquid sending pump may have the same configuration as that described above, and the detection solution may react with the substance to be separated and become a substance having ultraviolet or visible absorption. This can be achieved by using a solution containing a substance which becomes a fluorescent substance, for example, a compound such as ninhydrin or orthophthaldialdehyde.

FIG. 2 is a chromatogram shown as an example. In FIG. 2, (A) shows a conventional method using reverse phase chromatography, in which TSKge is used as a column.
IODS-80Ts is used, (B) is a method by normal phase chromatography of the present invention, and T
SKgel Amide-80 was used. As the conditions for the chromatography, in (A), 0.1% by volume of trifluoroacetic acid and 5% by volume as the mobile phase 1 were used.
Aqueous solution containing acetonitrile, 0.1 as mobile phase 2
An aqueous solution containing trifluoroacetic acid (volume%) and acetonitrile (volume 55%) was eluted from mobile phase 1 to mobile phase 2 by a linear concentration gradient in 83.3 minutes.
In (B), 55% by volume containing 0.1% by volume of trifluoroacetic acid and 97% by volume of acetonitrile as the mobile phase 1 and 0.1% by weight of trifluoroacetic acid as the mobile phase 2
Was eluted from mobile phase 1 to mobile phase 2 with a linear concentration gradient over 70 minutes. Sample volume 20 μl, separation temperature 40
° C. As is apparent from FIG. 2, according to the method of the present invention, each peptide is eluted in an order depending on the hydrophobic interaction with its separation column, and the separation ability is the same as that of the conventional reverse phase chromatography. It turns out that it is superior in comparison.

The target sample that can be separated by the method or apparatus of the present invention may be any sample containing at least one peptide and / or protein, such as a body fluid component such as serum, plasma, and urine; Any sample such as a solution and a waste liquid used for production of a protein or the like can be used.

Further, according to the method of the present invention, the peptide and / or protein in the sample is eluted depending on the hydrophobic interaction with the column. Quantification is possible, and qualitative determination is also possible by determining the peak retention time.

[0037]

According to the present invention, peptides and / or peptides in a sample which cannot be separated due to lack of hydrophobicity which is not retained by the method of reverse phase chromatography or which has little difference in hydrophobicity with the separation column substrate. Alternatively, it is useful because the protein can be separated and further purified after a separation without a complicated operation such as desalting.

[Brief description of the drawings]

FIG. 1 is a chromatography apparatus showing one embodiment of the present invention.

FIG. 2 is a chromatogram shown as an example.
In the figure, the X axis (horizontal axis) indicates the retention time (minutes),
The axis (vertical axis) indicates the absorbance at 215 nm.

[Explanation of symbols]

1: In FIG. 1, a bottle for the first mobile phase of the chromatography apparatus, in which the mobile phase 1 is contained. 2: In FIG. 1, a bottle for the second mobile phase of the chromatography apparatus, in which mobile phase 2 is contained. 3: In FIG. 1, a liquid sending pump of the chromatography apparatus. 4: In FIG. 1, it is a sample injection device of the chromatography device. 5: In FIG. 1, it is a separation column of a chromatography device. 6: In FIG. 1, it is a thermostat of the chromatography apparatus. 7: In FIG. 1, it is an ultraviolet absorption detector of the chromatography apparatus. 8: Pro Gly, the peptide shown in FIG. 9: Leu Gly, the peptide shown in FIG. 10: Phe Gly, peptide shown in FIG.
It is. 11: Glu His, peptide shown in FIG.
Xaa. Here, Xaa are those Pro carboxyl group (-COOH) has become -CONH _2. 12: Val Gly, peptide shown in FIG.
Ser Gly. 13: Gly Gly, a peptide shown in FIG.
Tyr Arg. 14: Trp Ala, peptide shown in FIG.
Gly Gly Asp Ala Ser Gly
Glu. 15: Arg Ser, peptide shown in FIG.
Arg Phe Arg.

Claims (6)

[Claims] When a sample containing a peptide and / or protein is separated by normal phase liquid chromatography, the sample is injected into a separation column, and then a mixture of an organic solvent and water and / or an alcohol is added to the mixture. And / or separating a peptide and / or protein by developing a mobile phase to which an amine is added. 2. The method according to claim 1, wherein the acid is trifluoroacetic acid. 3. The separation method according to claim 1, wherein the mobile phase according to claim 1 or 2 contains 0.01 to 20% by volume of the total amount of the mobile phase. 4. The method according to claim 1, wherein the organic solvent is acetonitrile, and its concentration is 60% by volume or more of the total amount of the mobile phase. 5. A separation column comprising a substrate having at least one functional group selected from the group consisting of an amino group, a hydroxyl group, an amide group, a carboxyl group and a sulfonic acid group. 5. The separation method according to any one of items 1 to 4. 6. An apparatus using the separation method according to claim 1.