WO2016056094A1

WO2016056094A1 - Device for amino acid sequence analysis and method for amino acid sequence analysis

Info

Publication number: WO2016056094A1
Application number: PCT/JP2014/077018
Authority: WO
Inventors: 伸幸秋永; 松本　博幸; 竜此下; 徹江連
Original assignee: 株式会社島津製作所
Priority date: 2014-10-08
Filing date: 2014-10-08
Publication date: 2016-04-14

Abstract

The method for amino acid sequence analysis according to the present invention, said method involving a process for subjecting a sample disposed on a reaction tank to Edman degradation reaction, comprises: a cleavage step for introducing a reagent to the reaction tank and thus releasing the N-terminal amino acid of the sample; a first extraction step for introducing a reagent to the reaction tank and thus extracting the amino acid released in the cleavage step from the reaction tank to an extraction container; and a second extraction step for further introducing a reagent to the reaction tank and thus extracting the released amino acid that remains in the reaction tank from the reaction tank to the extraction container.

Description

Amino acid sequence analyzer and amino acid sequence analysis method

The present invention relates to an amino acid sequence analyzer and an amino acid sequence analysis method.

Edman degradation is known as a method for sequentially analyzing the primary structures of proteins and peptides, that is, amino acid sequences from the N-terminus. An amino acid sequence analyzer that analyzes the amino acid sequence of a sample using this Edman degradation reaction is known. (For example, refer to Patent Document 1).

The amino acid sequence analyzer is equipped with a reaction tank for reacting the sample. The reaction vessel is provided with a sample support that holds a sample. The sample support is formed of a porous material such as a glass fiber filter or a polyvinylidene fluoride (PVDF) film.

During the analysis, a predetermined reagent is supplied to the reaction tank in which the sample support is installed. Then, amino acids are sequentially released from the N-terminal side of the sample by Edman degradation reaction. The released amino acid is introduced into, for example, a high performance liquid chromatograph, and the amino acid sequence is analyzed.

JP-A-9-068534

The object of the present invention is to improve the recovery rate of amino acids released by the Edman degradation reaction.

An amino acid sequence analyzer according to an embodiment of the present invention includes an Edman decomposition reaction unit that performs an Edman decomposition reaction of a sample disposed in a reaction vessel, a reagent supply unit that supplies a reagent to the Edman decomposition reaction unit, and the Edman decomposition reaction An amino acid analysis unit for analyzing amino acids released from the sample by Edman degradation reaction in the unit, and a control unit for controlling operations of the Edman degradation reaction unit, the reagent supply unit, and the amino acid analysis unit, After introducing the reagent from the reagent supply unit to release the amino acid at the N-terminal part of the sample, the released amino acid is introduced into the reaction vessel and extracted from the reaction vessel to the extraction container, and A control system is provided that includes an extraction control unit that introduces a reagent into the reaction tank and extracts the liberated amino acid remaining in the reaction tank into the extraction container. And parts, in which with a.

An amino acid sequence analysis method according to an embodiment of the present invention is an amino acid sequence analysis method in which a step of performing an Edman degradation reaction of a sample placed in a reaction vessel is sequentially performed a plurality of times, and each step includes a reagent in the reaction vessel. A cutting step for introducing and releasing an amino acid at the N-terminal portion of the sample; a first extraction step for introducing a reagent into the reaction vessel and extracting the amino acid liberated in the cutting step from the reaction vessel; and the reaction vessel And a second extraction step of extracting the liberated amino acid remaining in the reaction vessel from the reaction vessel into an extraction container.

The amino acid sequence analyzer and amino acid sequence analysis method of the embodiment of the present invention can improve the recovery rate of amino acids released by the Edman degradation reaction.

It is a schematic flow-path block diagram for demonstrating one Embodiment of an amino acid sequence analyzer. It is a schematic block diagram of the same embodiment. It is a schematic sectional drawing for explaining an example of a reaction vessel. 6 is a schematic cross-sectional view for explaining another example of the reaction vessel 6. FIG. FIG. 5 is a schematic cross-sectional view and a plan view of blocks constituting the reaction tank of FIG. It is the schematic sectional drawing and top view of the block which comprise the reaction tank of FIG. It is a flowchart for demonstrating one Embodiment of the amino acid sequence analysis method. It is a figure for comparing the amount of amino acids recovered in the first extraction and the amount of amino acids recovered in the second extraction. It is a figure which expands and shows the 2nd extraction of FIG. It is a figure for demonstrating the recovery amount of an amino acid when performing extraction operation of the 3rd time. It is a figure for demonstrating the collection amount of the amino acid in the 2nd extraction operation when the 1st extraction operation conditions are changed.

In the amino acid sequence analyzer and the amino acid sequence analysis method of the embodiment of the present invention, the reaction vessel has a reaction space and a main plane for holding a sample, and the reaction space is divided into upper and lower spaces on the main plane. A sample support disposed in the reaction space, a reagent inlet that is provided on an inner wall surface of the space above the reaction space, into which a reagent from the reagent supply unit flows, and an inner wall surface of the space below the reaction space And a reagent outlet for recovering the reagent in the reaction space.

An embodiment of the present invention will be described with reference to the drawings.
FIG. 1 is a schematic flow path configuration diagram for explaining an embodiment of an amino acid sequence analyzer. FIG. 2 is a schematic block diagram of the embodiment.

The amino acid sequence analyzer 1 includes a first reagent supply unit 2, a second reagent supply unit 3, an Edman degradation reaction unit 4, an amino acid analysis unit 10, and a control unit 35. The first reagent supply unit 2 supplies gas and reagents to the reaction tank 6 of the Edman decomposition reaction unit 4. The second reagent supply unit 3 supplies gas and a reagent to the conversion flask 8 (extraction container) of the Edman decomposition reaction unit 4. The first reagent supply unit 2 and the second reagent supply unit 3 constitute a reagent supply unit.

The first reagent supply unit 2 is connected to the reaction tank 6 of the Edman decomposition reaction unit 4 via the first reagent supply channel 11. The first reagent supply unit 2 is provided with reagent containers 5a to 5e. Reagents are stored in the reagent containers 5a to 5e, respectively. Ethyl acetate is accommodated in the reagent container 5a. The reagent container 5b contains n-butyl chloride. Trimethylamine is accommodated in the reagent container 5c. The reagent container 5d contains an n-heptane solution of PITC (phenyl isothiocyanate). The reagent container 5e contains trifluoroacetic acid.

The reagent containers 5a to 5e are connected to the first reagent supply channel 11 via three-way solenoid valves 12a to 12e, respectively. The reagent containers 5a to 5e are connected to drains via electromagnetic valves 13a to 13e, respectively. Gas supply channels 7a to 7e are connected to the reagent containers 5a to 5e, respectively. The gas supply channels 7a to 7e are provided with solenoid valves 9a to 9e for controlling the supply of gas for pressurizing the reagent containers 5a to 5e. This gas is, for example, N ₂ gas.

One of the reagent containers 5a to 5e is selectively connected to the first reagent supply channel 11 by switching the three-way solenoid valves 12a to 12e, and N2 gas is supplied to the reagent container and pressurized to thereby form the reagent. Is supplied to the reaction vessel 6. The first reagent supply channel 11 is also provided with an electromagnetic valve 9h that controls the supply of gas.

The second reagent supply unit 3 is connected to the conversion flask 8 of the Edman decomposition reaction unit 4 via the second reagent supply channel 20. The second reagent supply unit 3 is provided with

reagent containers

5f and 5g.

Reagent containers

5f and 5g are accommodated as reagents. The reagent container 5f contains a mixture of acetonitrile and water. The reagent container 5g contains a trifluoroacetic acid solution.

The

reagent containers

5f and 5g are connected to the second reagent supply channel 20 via three-

way solenoid valves

12f and 12g, respectively. A gas supply channel 7f is connected to the reagent container 5f. A gas supply channel 7g is connected to the reagent container 5g. The

gas supply passages

7f and 7g are provided with

electromagnetic valves

9f and 9g for controlling the supply of gas for pressurizing the

reagent containers

7f and 7g.

One of the

reagent containers

5f and 5g is selectively connected to the second reagent supply flow path 20 by switching the three-

way solenoid valves

12f and 12g, and a gas is supplied to the reagent container and pressurized to thereby supply the reagent. It is supplied to the conversion flask 8. The second reagent supply channel 20 is also provided with an electromagnetic valve 9i that controls the supply of gas.

In the Edman decomposition reaction section 4, a flow path 14 is connected to the outlet side of the reaction tank 6. The flow path 14 is connected to a flow path 18 that leads to the drain and the conversion flask 8 via a three-way solenoid valve 16. The outlet side of the reaction vessel 6 is connected to either the conversion flask 8 or the drain by switching the three-way electromagnetic valve 16.

In addition to the flow path 18 from the reaction tank 6 side, a second reagent supply flow path 20, an introduction flow path 22 and a drain are connected to the conversion flask 8. The drain of the conversion flask 8 is opened and closed by a solenoid valve 19. The introduction flow path 22 communicates with one port of the 6-way valve 24 of the amino acid analyzer 10.

The sample is held in a reaction space provided inside the reaction vessel 6. The reaction tank 6 has a structure in which the reagent passes through the sample support holding the sample. By passing the reagent through the sample support, amino acids are cut out from the N-terminal side of the sample, and the cut-out amino acids are guided to the conversion flask 8. The conversion flask 8 is connected to the amino acid analysis unit 10 via the introduction flow path 22, and the amino acid cut out from the sample in the reaction vessel 6 is converted and dissolved in the conversion flask 8, and then the amino acid analysis unit. 10 is introduced.

FIG. 3 is a schematic cross-sectional view for explaining an example of the reaction vessel 6.
The reaction tank 6 includes a reaction space 42 inside. The reaction tank 6 includes an inlet channel 44 (reagent inlet) and an outlet channel 46 (reagent outlet) that communicate with the reaction space 42.

The reaction tank 6 is configured by two

blocks

36 and 38 being stacked one above the other. The

blocks

36 and 38 are, for example, cylindrical glass blocks. The reaction space 42 is a space composed of a recess 42 a provided on the lower surface of the upper block 36 and a recess 42 b provided on the upper surface of the lower block 38. For example, the concave portion 42a and the concave portion 42b have a conical shape symmetrical to each other.

A sample support 48 is disposed in the reaction space 42. The sample support 48 has a main plane, and can hold the sample in the main plane and allow the reagent to pass therethrough. The shape of the main plane of the sample support 48 is, for example, the same circle as the plane shape of the reaction space 42. As the sample support 48, a glass fiber filter or a PVDF membrane can be used. The sample support 48 is disposed at the lower boundary portion of the block 36 so that its main plane is horizontal.

The block 36 is detachably attached to the block 38. By removing the block 36 from the block 38, the sample support 48 can be installed in or removed from the reaction space 42.

A PTFE film 40 is sandwiched between the

blocks

36 and 38. This is because when the

blocks

36 and 38 are removed, dust and foreign substances may enter between the

blocks

36 and 38, and those contaminants enter the downstream solenoid valve through the outlet channel 46 and break down. This is to prevent this from occurring.

The inlet channel 44 is provided in the block 36. The outlet channel 46 is provided in the block 38. The reaction space 42 is partitioned into two upper and lower spaces by a sample support 48. The opening at the end of the inlet channel 44 on the reaction space 42 side is provided on the inner wall surface of the space above the reaction space 42 partitioned by the sample support 48. The opening at the end of the outlet channel 46 on the reaction space 42 side is provided on the inner wall surface of the space below the reaction space 42 partitioned by the sample support 48. Both the opening of the inlet channel 44 and the opening of the outlet channel 46 are provided at positions opposite to each other across the center of the sample support 48 near the end of the main plane of the sample support 48. . As a result, the reagent that has flowed into the reaction space 42 through the inlet channel 44 reaches the entire reaction space 42, and the entire main plane of the sample support 48 is passed by the reagent.

The end of the inlet channel 44 on the upper surface side of the block 36 is provided on the central axis of the block 36, for example. The end of the outlet channel 46 on the lower surface side of the block 38 is provided on the central axis of the block 38, for example. Thereby, the positioning of the piping connected to the inlet channel 44 and the outlet channel 46 can be made with reference to the central axis of the reaction tank 6, and the connection of the pipe to the reaction tank 6 is facilitated.

In addition, the structure of the reaction tank 6 is not restricted to what was shown by FIG.
FIG. 4 is a schematic cross-sectional view for explaining another example of the reaction vessel 6. FIG. 5 is a schematic cross-sectional view and a plan view of blocks constituting the reaction tank of FIG.

4 and 5, the reaction space 42 is formed in a cylindrical shape whose central axis is inclined from the vertical direction. The

recesses

42a and 42b forming the reaction space 42 are both formed by, for example, inclining a flat bottom drill with respect to the recess forming surfaces of the

blocks

36 and 38 to a certain depth.

The opening at the end of the inlet channel 44 on the reaction space 42 side is provided at the deepest portion of the recess 42 a, that is, at the uppermost portion of the reaction space 42. This makes it easier for the reagent to reach the entire reaction space 42. Further, the opening at the end of the outlet channel 46 on the reaction space 42 side is provided at the deepest part of the recess 42 b, that is, at the lowest part of the reaction space 42. This makes it difficult for the reagent to accumulate in the reaction space 42.

It should be noted that the structure of the reaction vessel 6 is not limited to that shown in FIG. 3 or FIG. For example, the position where the inlet channel 44 and the outlet channel 46 are connected to the reaction space 42 is not particularly limited, and may be, for example, the central portion of the reaction space 42.

Returning to FIG. 1, the description will be continued.
The amino acid analyzer 10 is a high performance liquid chromatograph. The amino acid analysis unit 10 includes a six-way valve 24, a sample retention channel 26, a mobile phase feeding channel 27, and an analysis channel 28. The sample retention channel 26, the mobile phase feeding channel 27, and the analysis channel 28 are connected to respective ports of the six-way valve 24. The six-way valve 24 is connected between the introduction flow path 22 and the sample retention flow path 26 and the drain (state shown in FIG. 1), and between the mobile phase liquid supply flow path 27, the sample retention flow path 26, and the analysis flow path 28. Switch to one of the connected states.

The sample retention channel 26 includes a sample loop 26a that temporarily retains the amino acid introduced from the introduction channel 22. The mobile phase liquid supply flow path 27 includes a liquid supply pump 30 for supplying the mobile phase solvent, and the analysis flow path 28 includes an analysis column 32 and a detector 34 downstream thereof.

As shown in FIG. 2, the operations of the first reagent supply unit 2, the second reagent supply unit 3, the Edman decomposition reaction unit 4 and the amino acid analysis unit 10 are controlled by the control unit 35. The control unit 35 introduces the reagent from the first reagent supply unit 2 into the reaction vessel 6 to release the amino acid at the N-terminal portion of the sample, and then introduces the released amino acid into the reaction vessel 6 to remove the amino acid. An extraction control unit 35a is provided for extracting from the reaction tank 6 into the conversion flask 8 and introducing the reagent into the reaction tank 6 and extracting the liberated amino acid remaining in the reaction tank 6 into the conversion flask 8. Further, detection data obtained by the detector 34 of the amino acid analysis unit 10 is input to the control unit 35, and amino acid identification, quantification, yield calculation, and the like are performed in the control unit 35. The control unit 35 is realized by, for example, a personal computer or a dedicated computer.

FIG. 6 is a flowchart for explaining an embodiment of the amino acid sequence analysis method. An embodiment of an amino acid sequence analysis method will be described with reference to FIGS.

[Sample Installation (Step S1)]
The sample support 48 (for example, PVDF film) holding the sample is sandwiched between the

blocks

36 and 38 together with the PTFE film 40, so that the sample support 48 is installed in the reaction space 42 of the reaction tank 6.

[Coupling reaction (step S2)]
Under the control of the control unit 35, the electromagnetic valve 9 c is switched, N ₂ gas is supplied to the trimethylamine reagent container 5 c, and the pressure in the reagent container 5 c is increased, whereby the gas is supplied to the reaction tank 6 through the three-way electromagnetic valve 12 c. Trimethylamine is supplied to fill the reaction vessel 6. An n-heptane solution of PITC is supplied from the first reagent supply unit 2 to the reaction vessel 6 and reacted with the N-terminal amino group of the sample protein to produce PTC (phenylthiocarbamyl) -protein. Ethyl acetate is supplied from the first reagent supply unit 2 to the reaction tank 6, and excess reagents and by-products in the reaction tank 6 are washed and discharged to the drain. This coupling reaction is carried out with the flow path 14 on the outlet side of the reaction vessel 6 connected to the drain.

[Cleavage reaction (step S3)]
Trifluoroacetic acid is supplied from the first reagent supply unit 2 to the reaction tank 6, and the N-terminal peptide bond of the PTC-protein is cleaved to form an ATZ (anilinothiazolinone) -amino acid. This cutting reaction is also performed with the flow path 14 on the outlet side of the reaction tank 6 connected to the drain.

[First extraction (step S4)]
The flow path 14 on the outlet side of the reaction tank 6 is connected to the conversion flask 8 side, and n-butyl chloride is supplied from the first reagent supply unit 2 to the reaction tank 6 so that the ATZ-amino acid released from the sample is converted into the conversion flask. 8 is extracted. For example, about 400 μL (microliter) of n-butyl chloride is introduced into the reaction vessel 6. A predetermined amount of n-butyl chloride is sent by being pushed by N ₂ gas. For example, after feeding n-butyl chloride at a flow rate of 1 mL / min (milliliter / minute) for 20 seconds, the liquid supply is stopped for 30 seconds, and again at a flow rate of 1 mL / min for 30 seconds. Thereafter, N ₂ gas is supplied to the reaction vessel 6. The inside of the reaction vessel 6 after the passage of n-butyl chloride is filled with N ₂ gas.

A specific example of the extraction operation will be described.
(1) The electromagnetic valve 9b is opened, the inside of the reagent container 5b is pressurized with N ₂ gas, and n-butyl chloride contained in the reagent container 5b is transferred to the first reagent supply channel 11 via the electromagnetic valve 12b. For example, 400 μL is introduced. For example, a liquid sensor is provided in the middle of the first reagent supply channel 11, and when the liquid sensor detects a liquid, the liquid feeding is stopped, whereby the chloride introduced into the first reagent supply channel 11. n-Butyl is weighed. Measurement can also be performed by controlling the opening time of the

electromagnetic valves

9b and 12b.

(2) The electromagnetic valve 9h is opened and N ₂ gas is introduced into the first reagent supply channel 11 at a flow rate of 1 mL / min for 20 seconds, for example. Thereafter, the electromagnetic valve 9h is closed. As a result, n-butyl chloride in the first reagent supply channel 11 is fed and introduced into the reaction vessel 6. At this time, the reaction vessel 6 is filled with n-butyl chloride. In this state, liquid feeding is stopped for 30 seconds.

(3) The electromagnetic valve 9h is opened again and N ₂ gas is introduced into the first reagent supply channel 11 at a flow rate of 1 mL / min for 30 seconds, for example. Thereafter, the electromagnetic valve 9h is closed. Thereby, the released ATZ-amino acid in the reaction vessel 6 is extracted into the conversion flask 8 with n-butyl chloride. The reaction tank 6 is filled with N ₂ gas.

[Second Extraction (Step S5)]
In the state where the flow path 14 on the outlet side of the reaction tank 6 is connected to the conversion flask 8 side, n-butyl chloride is supplied again from the first reagent supply unit 2 to the reaction tank 6, thereby remaining in the reaction tank 6. ATZ-amino acids are extracted into the conversion flask 8. For example, as in the first extraction, about 400 μL of n-butyl chloride is introduced into the reaction vessel 6. For example, after feeding n-butyl chloride at a flow rate of 1 mL / min for 20 seconds, the liquid supply is stopped for about 30 seconds and again at a flow rate of 1 mL / min for 30 seconds. Thereafter, N ₂ gas is supplied to the reaction vessel 6. A specific example of the extraction operation is the same as the first extraction.

[Conversion reaction (step S6)]
A trifluoroacetic acid solution is supplied from the second reagent supply unit 3 to the conversion flask 8 to convert the ATZ-amino acid into a stable PTH (phenylthiohydantoin) -amino acid.

[Dissolution (Step S7)]
A mixture of acetonitrile and water is supplied from the second reagent supply unit 3 to the conversion flask 8, and the PTH-amino acid is dissolved in the mixture of acetonitrile and water. The reaction from the coupling reaction (step S2) to the dissolution (step S7) is an Edman decomposition reaction.

[Introduction to liquid chromatograph (step S8)]
The PTH-amino acid obtained by the above Edman decomposition reaction is introduced into the high performance liquid chromatograph 10. For this purpose, the introduction flow path 22-sample retention flow path 26-drain are connected by a 6-way valve 24, and N ₂ gas is supplied to the conversion flask 8 via the

electromagnetic valves

9i, 12f and 12g. , The mixed solution (sample solution) of acetonitrile and water containing PTH-amino acid in the conversion flask 8 is led out to the introduction flow path 22 side. The sample solution led out to the introduction channel 22 side stays in the sample loop 26 a of the sample retention channel 26. The 6-way valve 24 is switched so as to connect between the mobile phase liquid flow path 27, the sample retention flow path 26, and the analysis flow path 28, so that the sample solution depends on the mobile phase solvent from the mobile phase liquid flow path 27. It is transferred to the analytical column 32. The sample solution is separated for each component in the analysis column 32, and the composition component is detected in the detector 34. The detection signal obtained by the detector 34 is taken into the control unit 35, and amino acid identification, quantification, yield calculation, and the like are performed.

The operations in steps S2 to S8 are repeatedly executed, amino acids are sequentially released from the N-terminal of the sample, and amino acid separation analysis is performed with the high performance liquid chromatograph 10.

Thus, in the above embodiment, the operation of extracting the amino acid at the N-terminal part released by the Edman degradation reaction is performed twice. This improves the recovery rate of the free N-terminal amino acid. The amino acid extraction operation in the Edman degradation method is usually one time.

Explain that the recovery rate is improved by recovering the free N-terminal amino acid by two extraction operations.

FIG. 7 is a diagram for comparing the recovery amount of amino acid in the first extraction with the recovery amount of amino acid in the second extraction. FIG. 8 is an enlarged view showing the second extraction of FIG. 7 and 8, the vertical axis represents the peak area (arbitrary unit), and the horizontal axis represents the detected amino acid symbol and detection cycle number.

Only the amino acid extracted by the first extraction operation and only the amino acid extracted by the second extraction operation were detected by high performance liquid chromatography. The conditions for the first extraction and the second extraction are the same as in the above embodiment. The sample is a horse-derived myoglobin sample (50 pmol). The first extraction operation was measured twice (first collection 1, first collection 2). The second extraction operation was measured four times (second recovery 1, second recovery 2, second recovery 3, second recovery 4).

FIG. 7 shows that amino acids are also recovered by the second extraction operation. The amount is about 0 to 25% with respect to the amino acid recovery amount in the first extraction operation. It was confirmed that not all amino acids could be recovered by the first extraction operation alone. This shows that the recovery rate of amino acids is improved by performing the extraction operation twice.

Also, regarding the second extraction operation, there is a peak area value that is low depending on the analysis (see second recovery 3). This is considered to be due to variations in amino acid recovery by the first extraction operation. The fact that the extraction operation is performed twice can reduce variation in the recovery rate of amino acids as compared to the case where the extraction operation is performed only once.

Since recovery of amino acids was confirmed by the second extraction operation, it was verified whether or not amino acids were recovered by further extraction operation (third time).
FIG. 9 is a diagram for explaining the recovery amount of amino acids when the third extraction operation is performed. In FIG. 9, the vertical axis represents the peak area (arbitrary unit), and the horizontal axis represents the detected amino acid symbol and detection cycle number.

In the third extraction operation, the second extraction operation was performed under the same conditions. In the third extraction operation, n-butyl chloride (third recovery S3 in the figure) and ethyl acetate (third recovery S2 in the figure) were used as extraction solvents. Other operations are the same as in the above embodiment.

In the third extraction operation, almost no peak was detected. From this, it is considered that amino acids were almost recovered by the second extraction operation.
Similarly, no peak was detected even when the extraction solvent was changed from n-butyl chloride to ethyl acetate. In the experiment using ethyl acetate as the extraction solvent in the third extraction operation, no peak was detected from the first cycle, so it was judged that there was no effect and the analysis was stopped in 9 cycles (3 in FIG. 9). (See second recovery S2).

Next, since the recovery of amino acids was confirmed by the second extraction operation, studies were carried out to increase the first extraction efficiency.
FIG. 10 is a diagram for explaining the amino acid recovery amount in the second extraction operation when the first extraction operation condition is changed. In FIG. 10, the vertical axis represents the peak area (arbitrary unit), and the horizontal axis represents the detected amino acid symbol and detection cycle number.

In the above embodiment, in the first extraction operation, the extraction solvent (n-butyl chloride) was fed at a rate of 1 mL / min, stopped for 30 seconds, and again at a rate of 1 mL / min. The inventor of this application assumed that a single stoppage of liquid feeding is insufficient to extract all free amino acids from the sample support (PVDF membrane). Therefore, the inventor of the present application changed the extraction solvent supply condition in the first extraction operation, and increased the stoppage of the supply to three times (each stopped for 30 seconds) (the first three-stage extraction in the figure, the second recovery). ). In addition, about 10 seconds at 1 mL / min about the liquid feeding between the 1st liquid feeding stop and the 2nd liquid feeding stop, and the liquid feeding between the 2nd liquid feeding stop and the 3rd liquid feeding stop It was broken. The total amount of extraction solvent at this time is about 400 μL.

In addition, the inventors of the present application assumed that the extraction solvent mainly passes through the central part of the reaction tank and the amino acid remains in the peripheral part of the space in the reaction tank at the liquid feeding speed in the above embodiment. Therefore, the inventor of the present application changed the liquid feeding speed from 1 mL / min to 0.4 mL / min and performed the first extraction operation (the first low-flow extraction in the figure, the second collection, the first low-flow extraction). Second recovery 2). The total amount of extraction solvent at this time is about 400 μL.

FIG. 10 also shows the data of FIG. 8 (second recovery 1, second recovery 2, second recovery 3, second recovery 4) for comparison.

As shown in FIG. 10, in any of the data of the first three-stage extraction, the second collection, the first low flow rate extraction, the second collection 1, the first low flow rate extraction, the second collection 2, the amino acid in the second extraction operation Has been detected. And compared with the said embodiment demonstrated with reference to FIG. 6, the big improvement was not seen.
It should be noted that slowing the extraction solvent feed rate in the first extraction operation seems to have a slightly higher extraction efficiency because the amount of amino acid recovered in the second extraction operation is reduced.

As mentioned above, although embodiment of this invention was described, the structure, arrangement | positioning, numerical value, etc. in the said embodiment are examples, This invention is not limited to this, The present invention described in the claim Various modifications within the range are possible.

1 Amino Acid Sequence Analyzer 2 First Reagent Supply Unit (Reagent Supply Unit)
3 Second reagent supply unit (reagent supply unit)
4 Edman decomposition reaction part 6 Reaction tank 8 Conversion flask (container for extraction)
10 High performance liquid chromatograph (Amino acid analysis part)
35 Control unit 35a

Extraction control unit

36, 38 Block 42 Reaction space 44 Inlet channel (reagent inlet)
46 Outlet channel (reagent outlet)
48 Sample support

Claims

An Edman decomposition reaction part for performing an Edman decomposition reaction of the sample placed in the reaction vessel;
A reagent supply unit for supplying a reagent to the Edman decomposition reaction unit;
An amino acid analysis unit for analyzing amino acids released from the sample by Edman degradation reaction in the Edman degradation reaction unit;
A control unit for controlling operations of the Edman degradation reaction unit, the reagent supply unit, and the amino acid analysis unit, wherein a reagent is introduced from the reagent supply unit into the reaction tank to release an amino acid at the N-terminal portion of the sample. After that, the released amino acid is introduced into the reaction vessel and extracted from the reaction vessel to the extraction vessel, and further introduced into the reaction vessel and the free amino acid remaining in the reaction vessel. An amino acid sequence analyzer comprising: a control unit including an extraction control unit for extracting the product into the extraction container.
The reaction vessel has a reaction space, a main plane for holding the sample, a sample support disposed in the reaction space so as to divide the reaction space into upper and lower spaces on the main plane, and an upper side of the reaction space A reagent inlet for allowing a reagent from the reagent supply section to flow in, and a reagent outlet for recovering the reagent in the reaction space. The amino acid sequence analyzer according to claim 1.
An amino acid sequence analysis method for sequentially performing a step of performing an Edman degradation reaction of a sample placed in a reaction vessel a plurality of times,
Each process is
A cleavage step of introducing a reagent into the reaction vessel to liberate an amino acid at the N-terminal portion of the sample;
A first extraction step of introducing a reagent into the reaction vessel and extracting the amino acid liberated in the cutting step from the reaction vessel into an extraction container;
And a second extraction step of introducing a reagent into the reaction tank and extracting the liberated amino acid remaining in the reaction tank from the reaction tank to an extraction container.
The reaction vessel has a reaction space, a main plane for holding the sample, a sample support disposed in the reaction space so as to divide the reaction space into upper and lower spaces on the main plane, and an upper side of the reaction space A reagent inlet for allowing a reagent from the reagent supply section to flow in, and a reagent outlet for recovering the reagent in the reaction space. The amino acid sequence analysis method according to claim 3.