JP4735289B2 - Screening method and apparatus for identifying reactive compound that binds to target compound - Google Patents

Description

  The present invention relates to a screening method and a screening apparatus for specifying a reactive compound that binds to a target compound.

For example, the following experimental work has been performed off-line for screening for measuring the binding activity of a substance that binds to a protein.
(1) In order to screen for a compound that binds to a protein, the protein and the reaction compound are mixed.
(2) Only the protein is recovered from the mixture.
(3) The reaction compound bound to the recovered protein is dissociated from the protein.
(4) Next, the protein and the reaction compound are separated through a column, and only the reaction compound is recovered.
(5) The recovered reaction compound is measured with a mass spectrometer.

  In addition to the above, there may be mentioned a method of confirming the binding between the reaction compound and the protein by labeling the reaction compound with a radioisotope and binding the protein to the protein and then measuring the radiation dose measured from the protein.

  Conventionally, since all the operations (1) to (5) described above have been performed offline, there is a problem that it takes time to obtain an analysis result. Furthermore, in the work performed by a human hand, there is a possibility that the work may fail and there is a problem that different results may be obtained because the degree of skill in the work varies depending on the analyst. Also, if these multiple operations are performed offline, the analyst needs to be involved in the operations for a long time.

  Therefore, an object of the present invention is to enable a plurality of operations necessary for screening to be performed online.

The screening method of the present invention comprises the following steps (A) to (D) in this order to identify a reaction compound that binds to a target compound.
(A) A primary compound in which a target compound and a reactive compound are injected into a sample injection section as a sample, and the sample injected from the sample injection section is guided to a first dimension analysis column by a mobile phase for first dimension analysis and separated into sample components. Original analysis process,
(B) a fractionation step of fractionating a portion including a reaction product in which a target compound and a reaction compound are combined among separated sample components into a loop channel;
(C) Using the solvent that dissociates the reaction compound from the reaction product as a mobile phase for concentration, selectively capture the reaction compound while dissociating the reaction compound from the reaction product fractionated in the fractionation step. A reaction compound concentration step leading to the trap column; and (D) a second-dimensional analysis step of analyzing the sample captured by the trap column with a mobile phase for second-dimension analysis to the second-dimensional analytical power ram.

In the present invention, after preparing the reaction solution of the target compound and the reaction compound and injecting it from the sample injection part, a plurality of operations (A) to (D) for screening are performed online.
The target of screening in the present invention is a reaction compound that binds to a target compound to form a reaction product. In this case, the “bond” is a covalent bond, an ionic bond, a coordinate bond, a hydrogen bond, or the like. In addition to chemical bonds, this includes physical bonds by inclusions (the inclusion of other compounds in voids inside proteins) and van der Waals forces.
Further, “dissociation” of a reaction compound from a reaction product means that a chemical or physical bonding state is released.

As sample injection method into the sample injection unit, sequentially injected target compounds per se and the reaction compound itself.
In general, a substance that binds to a protein, particularly a bonded state due to an interaction that occurs in a living body is an equilibrium state, in which binding and dissociation are repeated at high speed. Therefore, for example, when a reaction compound is prepared as a sample in a state where it is bound to a protein that is a target compound and injected into a sample injection part of a liquid chromatograph, a mobile phase for transferring the sample to the first-dimensional analysis column is used. When diluted, the equilibrium state is lost, and depending on the mobile phase, the sample in the equilibrium state may be dissociated and may not be bound again. For example, if the reactive compound is p-nitrophenyl-di-N-acetyl-β-chitobioside and the protein is WGA (wheat germ lectin), it is clear that they bind by interaction. It was found that p-nitrophenyl-di-N-acetyl-β-chitobioside and WGA were detected in a dissociated state when injected into the sample injection part of a liquid chromatograph in the state of being reacted. Therefore, it was found that when a component such as p-nitrophenyl-di-N-acetyl-β-chitobioside is a reactive compound, it is difficult to accurately analyze the reactive compound bound to the protein.

  In the form of the screening method of the present invention to enable the present invention to be applied even when the reaction product of the target compound and the reaction compound is diluted in the mobile phase and dissociates, the sample injected into the sample injection portion The target compound itself and the reaction compound having a lower molecular weight are injected into the sample injection section in the order in which the reaction compound is injected first and then the target compound is injected. Use a size exclusion column that is selected so that the elution rate depends on the molecular weight so that the target compound elutes first, so that the target compound injected later will flow out before the reaction compound in the first dimension analysis column. By doing so, the target compound and the reaction compound are reacted in the first-dimensional analysis column.

  The “size exclusion column” means a column capable of separating a component larger than a certain molecular size (molecular weight) and a smaller molecule. In general, in a size exclusion column, a component having a high molecular weight has a high elution rate, and a component having a low molecular weight has a low elution rate. The size exclusion column can set the molecular weight to be separated (exclusion limit molecular weight) to a desired size. For example, based on the molecular weight of a protein, the protein or a component having a higher molecular weight and a smaller size than the protein A component having a molecular weight can be separated. The size exclusion column used in the present invention has an exclusion limit molecular weight equal to or smaller than the size of the target compound. By using such a size exclusion column, the reaction product bound to the target compound and a reaction compound having a molecular weight lower than the exclusion limit molecular weight is eluted as fast as the target compound, and the reaction compound that does not bind to the target compound has a molecular weight that is the exclusion limit. Since it is smaller than the molecular weight, it elutes later than the target compound and reaction product. Thereby, the reaction compound that is bound to the target compound and the reaction compound that is not bound can be separated.

In the reaction compound concentration step, it is preferable that the concentration mobile phase is further mixed when the fractionated reaction product is led to the trap column by the concentration mobile phase.
Although a target compound is not specifically limited, A protein can be mentioned as an example.

The screening apparatus of the present invention includes a first-dimensional analysis flow path for separating a sample injected from a sample injection section into a first-dimensional analysis column by a mobile phase for first-dimensional analysis and separating it into sample components, and a sample in the sample injection section A sample supply device for injecting, a fractionation channel for fractionating a portion of the separated sample component containing a reaction product in which a target compound and a reaction compound are combined into a loop channel, and from the reaction product to the above A reaction compound concentrated flow that leads to a trap column that selectively captures the reaction compound while dissociating the reaction compound from the reaction product fractionated in the fractionation flow path using a solvent that dissociates the reaction compound as a concentration mobile phase. A reactive compound that binds to the target compound with a channel and a second-dimensional analysis channel that guides the sample captured in the trap column to the second-dimensional analytical ram by the mobile phase for second-dimensional analysis You It is intended.
As a result, a plurality of operations necessary for screening can be performed online.

When the reaction product of the target compound and the reaction compound is diluted by the mobile phase and dissociates, the sample supply device separates and reacts the target compound itself and the reaction compound having a lower molecular weight as a sample. Controlled to inject the compound first, followed by the target compound, the first dimension analysis column is selected to have different elution rates depending on molecular weight so that the target compound elutes first than the reaction compound. In this way, the target compound injected later flows out before the reaction compound in the first dimension analysis column so that the target compound reacts with the reaction compound in the first dimension analysis column. can do.
It is preferable that the reaction compound concentration flow path further includes a flow path for mixing the concentrated mobile phase to a flow path for transferring the fractionated reaction product by the concentration mobile phase.

  In the screening method of the present invention, (A) the first dimension analysis step, (B) the fractionation step, (C) the reaction compound concentration step, and (D) the second dimension analysis step are included in this order. Multiple tasks for screening can be performed online.

If the reaction product of the target compound and the reaction compound is dissociated even when diluted by the mobile phase, the reaction compound is injected first, and then the target compound is injected, so that the first dimension analysis column By using a size exclusion column as a reaction between the target compound and the reaction compound in the first dimension analysis column, the reaction product is not diluted by the mobile phase and prevents dissociation. Can do. Thereby, since the reaction product can be fractionated without dissociating, the reaction compound that binds to the target compound can be analyzed accurately.
When a protein is used as a target compound in the screening method of the present invention, a reaction compound that binds to the protein can be identified.

  The screening device of the present invention is configured to inject a sample into a sample injection unit into a sample supply device, and to provide a first-dimensional analysis channel, a fractionation channel, a reaction compound concentration channel, a second-dimensional analysis channel, These are connected to each other, so that a plurality of operations for screening can be performed online.

If the target compound itself and the reaction compound having a lower molecular weight are separately injected as a sample by the sample supply device and the reaction compound is injected first, and then the target compound is injected, then the target compound and the reaction compound Even when the reaction product is dissociated when diluted with the mobile phase, the reaction compound that reacts with the target compound can be separated from the reaction compound that does not react.

  When the fractionated reaction product is introduced into the trap column by the concentration mobile phase, if the concentration mobile phase is further mixed, the reaction product is diluted with the concentration mobile phase and the reaction compound is separated from the target compound. Can promote dissociation.

Hereinafter, embodiments of the screening method of the present invention and a screening two-dimensional liquid chromatograph used for the implementation will be described with reference to the drawings. FIG. 1 is a flow chart schematically showing a reference example of the two-dimensional liquid chromatograph of the present invention.
Here, the target compound is a protein, and a reactive compound that binds to the protein is identified by screening.

As a sample in this reference example, a mixture of protein and some reaction compounds is used. Therefore, in addition to the reaction product produced by binding of the protein and the predetermined reaction compound, the sample contains a compound that does not bind to the protein, and may further contain unreacted protein.

The screening apparatus of this reference example includes a first-dimensional analysis channel A for separating a sample into a protein (including a reaction product to which a reaction compound is bound) and an unreacted reaction compound, and the separated sample components. Among them, a fractionation channel B for fractionating only the reaction product, a concentrated mobile phase feeding channel C for feeding the concentration mobile phase, and a concentrated mobile phase from the protein that has become the reaction product. A reaction compound concentration channel D for guiding the reaction compound to the trap column 24 while dissociating the reaction compound, and a two-dimensional solution for feeding a mobile phase for second-dimensional analysis for transferring the reaction compound trapped in the trap column 24 A two-dimensional liquid comprising an eye analysis mobile phase feeding flow path E and a second dimension analysis flow path F for analyzing a reaction compound transferred by the second dimension second analysis mobile phase in the second dimension analysis column 26. Chromatog And it includes the full. The screening apparatus further includes a sample supply device including a sample injection nozzle 32 in order to inject a sample into the sample injection unit 8 on the first-dimensional analysis flow path A. The flow paths A to F are connected to the respective ports of the 6-port valves 12 and 22 that can selectively connect the flow paths.

The upstream end of the first-dimensional analysis channel A is connected to the tank 2a, and the mobile phase for first-dimensional analysis is accommodated in the tank 2a. On the first-dimensional analysis channel A, a degasser 4, a liquid feed pump 6, a sample injection unit 8, and a first-dimensional analysis column 10 are provided from the upstream side. The degasser 4 is provided to remove bubbles and the like from the mobile phase for first-dimensional analysis sent by the liquid feed pump 6. The sample is injected from the sample injection unit 8 by the sample injection nozzle 32, and the injected sample is guided to the first dimension analysis column 10 by the first dimension analysis mobile phase and separated for each component. .
Here, as the mobile phase for the first-dimensional analysis, for example, a phosphate buffer solution containing 0.8% sodium chloride can be mentioned.

On the fractionation channel B, a loop channel 14 for fractionation is formed so that the component guided to the fractionation channel B can be stored.
The upstream end of the concentration mobile phase liquid flow path C is connected to the tank 2b, and the concentration mobile phase is stored in the tank 2b. The concentration mobile phase is fed via the degasser 4 by the liquid feed pump 16. An example of the concentration mobile phase is 0.1% FTA (trifluoroacetic acid).

A trap column 24 is provided on the reaction compound concentration flow path D so that only the reaction compound can be captured.
As the trap column 24, a macromolecule such as protein is not captured, but only a reaction compound is selectively captured. Here, an internal reverse phase column is used.

  The upstream end of the mobile phase feeding flow path E for second-dimensional analysis is connected to the tanks 2c and 2d. For example, pure water containing 0.1% TFA is stored in the tank 2c, and acetonitrile containing 0.1% TFA is stored in the tank 2d, and these are supplied at a predetermined liquid supply speed by liquid supply pumps 18a and 18b. The solution is fed to the mixer 20, mixed at a predetermined mixing ratio by the mixer, and fed as a mobile phase for second-dimensional analysis. These liquid feed pumps 18 a and 18 b and the mixer 20 constitute a gradient liquid feed mechanism 21.

The second-dimensional analysis flow path F includes a second-dimensional analysis column 26, a detector 28, and a mass spectrometer 30 from the upstream side. The detector 28 is, for example, an ultraviolet detector or a differential refractometer.
In the flow path F, the sample is separated into components by the second-dimensional analysis column 26, detected by the detector 28, and each component can be specified by the mass spectrometer 30.

  The 6-port valve 12 has ports a to f, and the 6-port valve 22 has ports g to l. The downstream end of the first-dimensional analysis channel A is connected to the port a of the 6-port valve 12. The upstream end of the fractionation flow path B is connected between the ports c and f of the 6-port valve 12. The concentration mobile phase liquid flow path C is connected to the ports d and e of the 6-port valve 12, sends the concentrate to the port e, and further mixes the liquid from the port d and the concentration mobile phase. Be able to. In addition, the concentration mobile phase liquid flow path C is connected to the port g of the 6-port valve 22 via the mixing path C ′, and the liquid obtained by mixing the liquid from the port d and the concentration mobile phase is ported. The liquid can be fed to g. The reaction compound concentration channel D is connected between the ports i and l of the 6-port valve 22. The downstream end of the mobile phase feeding flow path E for second-dimensional analysis is connected to the port k of the 6-port valve 22. The upstream end of the second-dimensional analysis flow path F is connected to the port j of the 6-port valve 22. The port b of the 6-port valve 12 and the port h of the 6-port valve are opened to the drains 15 and 25, respectively.

  When the 6-port valve 12 connects the ports ab, cd, and ef, the ports a-f, bc, and dh are blocked and the ports a-f are disconnected. , B-c, and d-e, the ports a-b, cd, and ef are blocked.

  When the 6-port valve 22 connects the ports g-h, i-j, and k-l, the ports g-l, hi-i, and j-k are blocked, and the ports g-l , H-i and j-k, the ports g-h, i-j, and k-l are blocked.

When the 6-port valve 12 is switched to connect between ports af, bc and de, the first-dimensional analysis flow path A is connected to the fractionation flow path B, and the primary The sample components that have passed through the original analysis column 10 are transferred to the loop flow path 14.
When the 6-port valve 12 is switched so that the ports ab, cd, and ef are connected, the downstream end of the first-dimensional analysis flow path A is connected to the drain 15, and the primary The sample passed through the original analysis column is discharged.

  As a result, the 6-port valve 12 is switched at a specific timing to connect the ports a-f, bc, and de, so that the reaction among the sample components separated by the first-dimensional analysis column 10 is performed. Only the protein containing the product can be sent to the loop flow path 14 for fractionation, and the remaining unnecessary components can be discharged to the drain 15.

  The 6-port valve 12 is switched so that the ports a-b, cd, and ef are connected, and the 6-port valve 22 is connected between the ports g-l, hi, and j-k. In such a case, a part of the concentration mobile phase fed by the liquid feed pump 16 is transferred to the port e by feeding the concentration mobile phase through the concentration mobile phase liquid flow path C. And f are led to the loop flow path 14, and the protein containing the reaction product fractionated in the loop flow path 14 is passed through the mixing flow path C ′ via the ports c and d and trapped via the ports g and l. Guided to column 24. At this time, the concentration mobile phase that has not been introduced into the port e among the concentration mobile phases fed by the liquid feed pump 16 is mixed with the mobile phase from the port d at the upstream end of the mixing channel C ′, and the reaction The mobile phase containing the protein containing product is diluted. The reaction product is dissociated into a protein and a reaction compound by contacting the concentration mobile phase. Only the reaction compound is captured in the trap column 24, and the remaining components including the protein are discharged to the drain 15 through the ports i and h.

  Further, when the 6-port valve 22 is switched so that the ports g-h, i-j, and k-l are connected, the second-phase analysis mobile phase liquid flow path E is used for the second-dimensional analysis. When the mobile phase is fed, the mobile phase for second-dimensional analysis is guided to the reaction compound concentration channel D through the ports k and l of the 6-port valve 22. The mobile phase for second-dimensional analysis led to the reaction compound concentration flow path D elutes the reaction compound captured by the trap column 24 and passes through the ports i and j of the 6-port valve 22 for the second-dimensional analysis flow path. Introduced into F. In the second-dimensional analysis channel F, the reaction compound guided by the mobile phase for second-dimensional analysis is separated by the second-dimensional analysis column 26, detected by the detector 28, and further its component is specified by the mass spectrometer 30. Is done.

  Next, an example of the operation when screening is performed using the above two-dimensional liquid chromatograph will be described. The screening method of this example includes the following steps. 2, 3 and 4 are flow charts schematically showing the flow path of the mobile phase at the time of sample analysis. FIG. 2 is a first-dimensional analysis step, FIG. 3 is a reaction compound concentration step, and FIG. Each of the second dimension analysis steps is shown.

[First dimension analysis process]
A solution in which a protein and a reaction compound are mixed is used as a sample. The sample is prepared in advance by the analyst.
As shown in FIG. 2, during the first-dimensional analysis, the first-dimensional analysis mobile phase is fed into the first-dimensional analysis flow path A. As shown in (A), when a sample is injected into the sample injection portion 8 by the sample injection nozzle 32, the sample is transferred to the first dimension analysis column 10 by the mobile phase for first dimension analysis, and the protein and the specific It is separated into a protein portion containing a reaction product produced by reaction with a reaction compound and an unreacted reaction compound. Among these, the protein portion containing the reaction product is switched so that the 6-port valve 12 is connected between ports af, bc, and de, and is sent to the loop flow path 14. . As shown in (B), the sample components other than the protein containing the reaction product are switched to a state in which the 6-port valve 12 is connected between the ports ab, cd, and ef. It is discharged to the drain 15.

  Here, since the flow rate of the mobile phase for the first dimension analysis is constant, the first dimension is calculated from the flow rate of the mobile phase for the first dimension analysis and the distance between the first dimension analysis column 10 and the 6-port valve 12. The time until the protein containing the reaction product separated by the analytical column 10 reaches the 6-port valve 12 can be calculated, and the reaction is generated by switching the 6-port valve 12 based on the calculated time. Only proteins containing substances can be selectively sent to the loop channel 14 for storage.

  Although not shown in this embodiment, a detector is provided between the first-dimensional analysis column 10 and the 6-port valve 12, and the 6-port valve 12 is switched based on the detection signal of the detector. May be.

[concentrated]
Next, the concentration mobile phase is sent to the flow path indicated by the thick line in FIG. The 6-port valve 12 is switched so that the ports a-b, cd, and ef are connected, and the 6-port valve 22 is connected between the ports g-l, hi, and j-k. Is switched to a connected state. The mobile phase for concentration is fed to the loop flow path 14 via the ports e and f of the 6-port valve 12 by the liquid feed pump 16. At this time, a part of the concentration mobile phase flowing through the concentration mobile phase liquid supply channel C is not supplied to the port e, but is mixed with the solution from the port d and flows into the mixing channel C ′. The protein containing the reaction product stored in the loop flow path 14 passes through the mixing flow path C ′ through the port d together with the concentration mobile phase, and flows into the port g of the 6-port valve 22. At this time, the reactive compound comes into contact with the concentrated mobile phase, and the reactive compound bound to the protein is dissociated. The sample introduced into the port g flows into the concentration channel D through the port l, only the reaction compound is captured by the trap column 24, and the remaining components are discharged to the drain 25 through the ports i and h.

[Second dimension analysis]
The 6-port valve 22 is switched to a state where the ports g-h, i-j, and k-l are connected, so that the mobile phase for second-dimensional analysis flows through the flow path shown by the thick line in FIG. In addition, the mobile phase for second-dimensional analysis is introduced into the trap column 24 through the ports k and l of the 6-port valve 22 by the gradient liquid feeding mechanism 21. The reaction compound trapped in the trap column 24 is eluted by the mobile phase for second dimension analysis, and is introduced into the second dimension analysis column 26 through the ports i and j of the 6-port valve 22 together with the mobile phase for second dimension analysis. Separated into components. Each separated component is detected by the detector 28 and specified by the mass spectrometer 30.

According to the two-dimensional liquid chromatograph in this example, after an analyst prepares a sample in which a protein and a reaction compound are mixed, the sample is prepared online with a protein containing a reaction product and an unreacted reaction compound. And fractionating only the protein containing the reaction product, then dissociating the reaction product, concentrating and capturing only the reaction compound, and analyzing the reaction compound. Work can be done online in the same analysis system. Also, online operations are easy to automate.
Furthermore, if work other than sample preparation is automatically performed, a large amount of analysis can be performed under a certain condition without causing a difference in results due to a human error or a difference in skill level.

Target compound, depending on the combination of the reaction compound and the first dimension analysis mobile phase, the method of Reference Example above to be injected into the sample injection part as a reaction solution was premixed reaction compound with a target compound, the sample injection unit 8 In some cases, the reaction solution is diluted in a large amount by the mobile phase up to the first-dimensional analysis column 10, and the already bound target compound and the reaction compound may be dissociated. FIG. 5 shows an embodiment in which the screening of the present invention can be executed even in such a case.

  In this embodiment, the sample supply device including the sample injection nozzle 32a separates the target compound protein itself and the reaction compound having a lower molecular weight separately from the sample injection unit 8 as a sample and the reaction compound first. It is controlled to inject the protein which is the target compound.

  The first-dimensional analysis column 10a is a size exclusion column selected so that the elution rate differs depending on the molecular weight so that the target compound protein elutes first than the reaction compound. Here, a GPC (Gel Permeation Chromatography) column or a GFC (Gel Filtration Chromatography) column is used as the size exclusion column of the first-dimensional analysis column 10a. The exclusion molecular weight of the size exclusion column used in this example is about the molecular weight of the protein as the target compound, and it is possible to separate a protein having a molecular weight equal to or higher than the molecular weight from a reaction compound to be analyzed having a smaller molecular weight. It can be done.

As shown in FIG. 5B, the sample injection nozzle 32a first sucks the protein 34 and then sucks the reaction compound 36. Thereby, in the sample injection nozzle 32a, a layer of the reaction compound 36 is formed on the distal end side, and a layer of the target compound 34 is formed on the proximal end side. When the sample is injected into the sample injection unit 8 in this state, the reaction compound 36 is injected first, and then the protein 34 as the target compound is injected. The sample injected into the sample injection unit 8 is transferred to the first-dimensional analysis column 10a in the order of the reaction compound 36 and the protein 34 in the order of injection.
The analysis operation using the liquid chromatograph of this embodiment is the same as that of the first embodiment shown with reference to FIGS.

  6A to 6C are diagrams showing the movement of the sample in the first-dimensional analysis column 10a. (A) shows a state immediately after the sample is introduced into the first-dimensional analysis column 10a, and the reaction compound 36 is in the front and the protein 34 is in the rear in the order of injection. However, in the first dimension analysis column 10a, the component having a molecular weight exceeding the exclusion limit molecular weight, that is, the protein 34 moves at a high speed, and the reaction compound 36 having a molecular weight smaller than the exclusion limit molecular weight moves at a low speed. Due to the difference in movement speed, the region of the protein 34 overtakes the region of the reaction compound 36 as shown in (B) in the first-dimensional analysis column 10a. At this time, the reaction compound that binds to the protein 34 binds to the protein to become a reaction product 36b and moves with the protein 34 at a high moving speed, but the reaction compound 36a that does not bind to the protein remains at a low moving speed. Move. Eventually, as shown in (C), the region bound to the protein to become the reaction product 36b is separated from the unreacted reaction compound 36a not bound to the protein and eluted from the first-dimensional analysis column 10a. To do.

  As a result, as shown in FIG. 7, a chromatogram consisting of a protein peak containing the reaction product 36b of the reaction compound bound to the protein and a peak of the reaction compound that has not been reacted separated therefrom is obtained. Can do.

  In the screening apparatus of this embodiment, at the timing when the protein portion containing the reaction product is eluted and reaches the 6-port valve 12, the 6-port valve 12 is connected between the ports af, bc, and de. It is switched so as to be in a connected state (see FIG. 2A), and the protein portion containing the reaction product is sent to the loop channel 14 and stored. In the unreacted reaction compound portion 36a eluted after the protein portion containing the reaction product, the 6-port valve 12 is connected between the ports ab, cd, and ef (FIG. 2 ( By switching to (see B), it is discharged to the drain 15.

  FIG. 8 is a diagram showing the results of analyzing the components eluted from the first-dimensional analysis column in the embodiment of FIG. 5, and FIG. 8A shows a sample of a reaction solution obtained by mixing a target compound and a reaction compound in advance and reacting them. When injected into the injection part 8, (B) shows the case where the target compound and the reaction compound are separately injected into the sample injection part 8 according to the same embodiment.

  As a sample, WGA (1 mg / mL) was used as the target compound protein, and p-nitrophenyl-di-N-acetyl-β-chitobioside (0.1 mg / mL) was used as the reaction compound. A 50 mM phosphate buffer (pH 6.8) /0.8% NaCl solution was used as the mobile phase for the first dimension analysis, and was flowed at a flow rate of 0.05 mL / min. Further, as the first-dimensional analysis column 10a, a silica-based DIOL column (product of Shimadzu Corporation), which is a size exclusion column having a length of 150 mm and an inner diameter of 2 mm, was used, and the temperature of the column oven was set to 25 ° C.

  In the measurement of obtaining the chromatogram of (B), 30 μL of protein WGA as a sample was sucked into the sample injection nozzle 32a, and then 5 μL of p-nitrophenyl-di-N-acetyl-β-chitobioside was sucked. Thereafter, a sample was injected into the sample injection portion 8 from the sample injection nozzle 32a.

  8A and 8B, the peak indicated by a broken line indicates a peak when only p-nitrophenyl-di-N-acetyl-β-chitobioside is injected into the sample injection portion. 8 (A) and 8 (B) have different horizontal scales, the peaks of p-nitrophenyl-di-N-acetyl-β-chitobioside are shown at different positions. Their retention times are equal.

  As shown in FIG. 8 (A), the elution time of p-nitrophenyl-di-N-acetyl-β-chitobioside was affected by WGA, but p-nitrophenyl-di-N-acetyl-β- Compared to the case where chitobioside alone was injected, no significant change was observed, indicating that p-nitrophenyl-di-N-acetyl-β-chitobioside was dissociated from the protein WGA. On the other hand, as shown in (B), when the sample was injected according to this example, there was no peak at the elution time position of p-nitrophenyl-di-N-acetyl-β-chitobioside, It can be seen that p-nitrophenyl-di-N-acetyl-β-chitobioside reacted with protein WGA and shifted to the position of WGA elution time.

  From this result, in the case where p-nitrophenyl-di-N-acetyl-β-chitobioside and protein WGA are used as a sample, the reaction solution is prepared by mixing both in advance and then injected into the sample injection unit 8. Although it is diluted by the mobile phase and dissociates, the reaction product cannot be separated, but p-nitrophenyl-di-N-acetyl-β-chitobioside is injected first, and then the protein WGA is It can be seen that p-nitrophenyl-di-N-acetyl-β-chitobioside that reacts with protein WGA can be screened if injected.

  In the above embodiment, the mass spectrometer 30 is provided on the downstream side of the detector 28, but the present invention is not limited to this. For example, the mass spectrometer 30 is provided on the downstream side of the detector 28. A probe for dropping a sample may be provided, and fractionated and collected for each component in a sample container such as a plate.

It is a channel figure showing a reference example of a screening device roughly. It is a figure which shows the flow path of the 1st-dimensional analysis process of the two-dimensional liquid chromatograph in the screening apparatus of the same reference example , (A) is fractionating the protein containing a reaction product, (B) is an unnecessary component. It is a figure which shows each when discharging. It is a flow chart which shows the concentration process of the reaction compound in the reference example . It is a figure which shows the flow path of the 2nd-dimensional analysis process in the reference example . It is a channel figure showing roughly one example of a screening device. It is a figure which shows the motion of the sample in the size exclusion column in the Example. It is a wave form diagram which shows an example of the chromatogram obtained from the eluate from the size exclusion column in the Example. In the screening apparatus of the same Example, it is a figure which shows the result of having analyzed the component eluted from the 1st dimension analysis column, (A) is a sample injection part with the reaction solution which mixed the target compound and the reaction compound beforehand, and was made to react (B) is a case where the target compound and the reaction compound are separately injected into the sample injection portion according to the embodiment.

Explanation of symbols

2a, 2b, 2c, 2d Mobile phase tank 4 Degasser 6, 16, 18a, 18b Liquid feed pump 8 Sample injection part 10, 10a First dimension analysis column 12, 22 6-port valve 14 Fraction loop 15, 25 Drain 20 Mixer 21 Gradient solution feeding mechanism 24 Trap column 26 Second dimension analysis column 28 Detector 30 Mass spectrometer 32, 32a Sample injection nozzle 34 Target compound 36 Reactive compound 36b Reaction product

Claims (5)

(A) A primary compound in which a target compound and a reactive compound are injected into a sample injection section as a sample, and the sample injected into the sample injection section is guided to a first dimension analysis column by a mobile phase for first dimension analysis and separated into sample components. Original analysis process,
(B) a fractionation step of fractionating a portion including a reaction product in which a target compound and a reaction compound are combined among separated sample components into a loop channel;
(C) Using the solvent that dissociates the reaction compound from the reaction product as a concentration mobile phase, the reaction compound is selectively captured while dissociating the reaction compound from the reaction product fractionated in the fractionation step. A reaction compound concentration step leading to the trap column; and (D) a second dimension analysis step for analyzing the sample captured by the trap column by leading to the second dimension analytical power ram by the mobile phase for second dimension analysis,
A screening method for identifying a reactive compound that binds to a target compound,
The sample injected into the sample injection part is the target compound itself and the reaction compound itself having a smaller molecular weight,
Using a size exclusion column selected so that the elution rate differs depending on the molecular weight so that the target compound elutes earlier than the reaction compound as the first dimension analysis column,
Injection into the sample injection section is performed in the order of injecting the reaction compound first, and then injecting the target compound, so that the target compound injected later in the first-dimensional analysis column flows out before the reaction compound. Thus, the target compound and the reaction compound are allowed to react in the first-dimensional analysis column ,
In addition, in the step of injecting into the sample injection portion, one sample injection nozzle is used, and a sample is formed so that the layer of the reactive compound is formed on the tip side of the sample injection nozzle and the layer of the target compound is formed on the base end side. After inhalation , the screening method is characterized in that the sample injection nozzle is inserted into the sample injection section, and the reaction compound and the target compound are sequentially injected into the sample injection section by one sample injection operation .   2. The screening method according to claim 1, wherein in the reaction compound concentration step, the concentration mobile phase is further mixed when the fractionated reaction product is led to the trap column by the concentration mobile phase.   The screening method according to claim 1, wherein the target compound is a protein. A first-dimensional analysis flow path for guiding the sample injected from the sample injection section to the first-dimensional analysis column in the first-dimensional analysis mobile phase and separating it into sample components;
A sample supply device for injecting a sample into the sample injection unit;
A fractionation channel that fractionates a portion of the separated sample component containing a reaction product in which a target compound and a reaction compound are combined into a loop channel;
A trap column that selectively captures the reaction compound while dissociating the reaction compound from the reaction product fractionated in the fractionation flow path using a solvent that dissociates the reaction compound from the reaction product as a mobile phase for concentration. A reaction compound concentration channel that leads to
A second-dimensional liquid chromatograph provided with a second-dimensional analysis flow path for analyzing the sample captured in the trap column by a mobile phase for second-dimensional analysis and guiding it to a second-dimensional analytical force ram; A screening device that binds and identifies a reactive compound having a molecular weight smaller than that of the target compound ,
The first dimension analysis column is a size exclusion column selected so that the elution rate varies depending on the molecular weight so that the target compound elutes earlier than the reaction compound,
The sample supply device uses one sample injection nozzle, and after inhaling the sample so that the reaction compound layer is formed on the tip end side and the target compound layer is formed on the base end side of the sample injection nozzle, The first-dimensional analysis is performed by inserting an injection nozzle into the sample injection portion and controlling the reaction compound and the target compound to be sequentially injected into the sample injection portion by a single sample injection operation. A screening apparatus in which a target compound reacts with a reactive compound in the first-dimensional analysis column when a target compound injected later in the column flows out before the reactive compound.   The screening apparatus according to claim 4, wherein the reaction compound concentration flow path further includes a flow path for mixing the concentrated mobile phase to a flow path for transferring the fractionated reaction product by the concentration mobile phase.

Images