JP2004101477A - Two-dimensional high-performance liquid chromatograph apparatus and protein analyzer using the same - Google Patents

Abstract

An object of the present invention is to provide a high-performance liquid chromatograph having a high resolution and capable of automatically performing high-precision analysis, and a protein analyzer using the same.
A pre-stage liquid supply means for supplying a gradient eluent at a fixed amount of 200 μl / min or less, an autosampler for introducing a sample and a gradient eluate into a liquid supply flow path, and a pre-stage separation column. A first-stage separation unit provided with a desalting unit for performing desalination treatment in the trap column 20; a second-stage liquid sending means 22 for sending a gradient eluent at a fixed amount of 100 μl / min or less; and a second stage provided with a second-stage separation column 24 A separation unit; a mass analysis unit 26 connected to the post-stage separation unit; and a control unit 28 for controlling the operation of introducing the liquid into the flow path, the operation of the liquid feeding unit, and the switching operation of the liquid feeding flow path by the autosampler. A two-dimensional high-performance liquid chromatograph apparatus comprising: and a protein analyzer using the same.
[Selection diagram] Fig. 1

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a two-dimensional high-performance liquid chromatograph and a protein analyzer using the same, and more particularly to automation in separation and analysis of proteins and improvement in analysis accuracy.
[0002]
[Prior art]
By clarifying the relationship between the vast amount of genetic information revealed with the progress of genetic information analysis projects such as the genome project in recent years, and various proteins that interact in a complex manner in cells, It is required to carry out functional analysis of. Proteome analysis is an attempt to comprehensively capture the relationships among various proteins that support cell functions. However, current analysis techniques require a great deal of time and effort to analyze proteins, and comprehensively and quickly grasp changes in the proteome, which is a collection of such diverse proteins. Is required. Conventionally, a two-dimensional electrophoresis method or the like is generally used for proteome analysis.
[0003]
[Problems to be solved by the invention]
When a high-performance liquid chromatograph is applied to this proteome analysis to separate a mixture of various components, the resolution of the conventional high-performance liquid chromatograph is limited by the relationship between the characteristics of the column used and the characteristics of each component. was there. That is, for example, when a large number of components are mixed as components, depending on the properties such as the polarity of each component and column characteristics, some components are well separated, but a group of components that are not sufficiently separated even through a column also exist at the same time. There are cases.
[0004]
In addition, electrophoresis, which has been generally performed as a conventional separation analysis of proteins, has a problem that, although separation can be performed with high resolution, automation is difficult and reproducibility and quantitativeness are difficult to secure.
The present invention has been made in view of the above-mentioned problems of the prior art, and has as its object a high-performance liquid chromatograph apparatus having high resolution and capable of automatically performing high-precision analysis, and a protein analyzer using the same. Is to provide.
[0005]
[Means for Solving the Problems]
In order to achieve the above object, a two-dimensional high-performance liquid chromatograph of the present invention is a high-performance liquid chromatograph that detects each component in a sample separated by a separation column by gradient elution,
A first-stage liquid sending means for sending a gradient eluent at a fixed amount of 200 μl / min or less;
An autosampler for introducing the sample and the gradient eluent into the liquid sending flow path,
A pre-stage separation column for separating the sample into components,
A pre-stage separation unit with
A desalting unit comprising a column for trapping each of the separated components separated in the pre-stage separation unit, and performing a desalting treatment on each of the separated components by flowing a washing liquid through the column by a pump.
A second-stage liquid sending means for sending the gradient eluent at a fixed amount of 100 μl / min or less;
A post-stage separation column that further separates each of the separation components,
A post-separation unit with
A mass spectrometry unit connected to the post-stage separation unit,
A control unit is provided for controlling the operation of introducing the liquid into the flow channel by the autosampler, the operation of the liquid feeding means, and the switching operation of the liquid feeding flow channel.
[0006]
Further, in the device,
The pre-stage separation column is an ion exchange column,
The post-separation column is a reversed-phase column,
One gradient eluent from the autosampler is introduced into the ion exchange column to trap the separated components in the column of the desalting section, the separated components are desalted in the desalting section, and the reversed phase is separated in the subsequent separation section. Perform separation and sample detection in the mass spectrometer,
Subsequently, it is preferable to similarly perform the operation in each of the other gradient eluents prepared in the autosampler.
[0007]
The protein analyzer of the present invention uses the above device,
Using a mixture containing the peptide, which has been pre-processed to decompose the protein to be analyzed into peptides in advance, as a sample,
Separating each peptide from the mixture by separation at each separation section,
The mass spectrometry section performs mass spectrometry on the separated peptide to identify an amino acid sequence of the peptide.
[0008]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
FIG. 1 is a schematic view of a two-dimensional high-performance liquid chromatograph according to an embodiment of the present invention. The apparatus 10 shown in FIG. 1 includes a pre-separation unit including a liquid sending unit 12, an autosampler 14, and an ion exchange column 16, a desalination unit including a pump 18 and a trap column 20, a gradient liquid sending unit 22, and a reverse phase. A post-stage separation unit including the column 24 and a mass spectrometric unit 26 connected to the post-stage separation unit are provided.
The analysis operation in each of these units is controlled by a control unit including a computer 28.
[0009]
Hereinafter, the present apparatus will be described according to the measurement procedure.
Pre-stage separation unit In the present embodiment, a syringe pump filled with the first gradient eluent is provided as the liquid sending means 12 of the pre-stage separation unit, and the eluate is quantitatively ion-exchanged by drive control from the computer 28. The solution is sent to the column 16. The flow rate of the eluent in the pre-separation section is preferably 200 μl / min or less in order to enable separation of a very small amount of sample with high resolution. The flow rate of the eluent in the pre-stage separation section is determined in consideration of the sample load so that a sample composed of many components can be well separated.
Further, as the stationary phase of the ion exchange column 16, various ion exchangers used as the stationary phase of the ion exchange chromatograph are appropriately used depending on the properties of the sample and the like.
[0010]
A sample solution containing a sample to be analyzed is introduced from the autosampler 14. Further, gradient eluents having different ionic strengths are also introduced from the autosampler 14. The autosampler used here has been conventionally used in HPLC, and includes those commercially available as devices for automatically and continuously analyzing a plurality of samples, but those having the same functions as those described above. In addition, a commercially available autosampler obtained by improving the measurement performance according to the purpose is also included.
[0011]
As such an autosampler, one having the configuration shown in FIG. 2 is exemplified. In FIG. 2, a sample solution is sealed in vial A, and gradient eluents having different ionic strengths are sealed in vials B, C, and D (in the conventional usage, vials B, C, and D contain other samples). The solution is sealed) and arranged on a rack.
A needle 32 for collecting a sample solution or each gradient eluate from the vial is connected to an injector valve 36 via a loop 34. The needle is held by a drive mechanism (not shown), and can move between the vial, the washing port 38, and the injection port 40. The valve 42 is a rotary six-way valve that switches the flow path of the liquid that is sucked and discharged by a plunger 44 that is configured to reciprocate by mechanical force. 46 is a cleaning liquid bottle. The injector valve 36 is connected to the liquid sending flow path of the liquid chromatograph device 10, and introduces the sample solution or the subsequent gradient eluent into the first flow of the eluent filled in the syringe pump.
[0012]
An example of an operation for introducing a sample or a gradient eluent by the autosampler configured as described above is as follows. The valve 42 inserts the needle 32 into the vial A at a position where the port 0-a communicates as shown in the figure, and pulls the plunger 44 to suck up a predetermined amount of the sample solution. The sucked sample solution remains in the loop 34 and does not reach the valve 42 or the plunger 44. Next, the needle 32 is removed from the vial A and moved to the injection port 40. By operating the injector valve 36 and introducing the sample solution in the loop 34 into the liquid sending flow path following the ion exchange column, the first separation by the eluent from the syringe pump is started. Then, when separating the sample with the eluent sealed in the vial B, the needle 32 is moved to the vial B, and the above operations are repeated. Subsequently, the same operation is repeated when the sample is separated using the eluent sealed in the vials C and D.
[0013]
In this way, the autosampler is used not only for introducing the sample but also for introducing and sending each gradient eluent. There is an advantage that a system can be constructed.
[0014]
A part of the sample introduced from the autosampler is separated by the first eluent in the ion exchange column 16 of FIG. 1, flows out of the column, and is separated into the trap column 20 connected via the six-way valve 30. It is trapped in the state that it was. The separated sample in the trap column 20 is analyzed through a series of operations of desalting, reversed-phase gradient separation, and mass spectrometry described later. Subsequently, the sample remaining in the ion exchange column 16 is partly separated by the eluent from the vial B in FIG. 2 and flows out of the ion exchange column 16, is trapped in the trap column 20, and is similarly desalted. The analysis is performed through a series of operations such as reverse phase gradient separation and mass spectrometry. Further, the sample remaining in the ion exchange column 16 with the eluent from the vial C and then the vial D is separated and trapped in the trap column 20, and similarly analyzed through the subsequent steps.
[0015]
Desalting section In the pre-stage separating section, it is possible to separate to a certain extent based on the ionicity of each component in the sample, but it may not always be possible to perform sufficient separation. In particular, when a mixed component containing various peptides is used, such as a sample in protein analysis, each component separated by ion exchange separation is not separated into a single peptide, but is separated into a plurality of peptide components. There may be. In order to perform analysis using a mass spectrometer as a subsequent detector, it is necessary to separate a single component.
Therefore, in the present apparatus, the second-stage separation is performed by further performing the reverse-phase separation in the subsequent-stage separation unit.
[0016]
However, when each of the separated components is directly introduced into a reversed-phase separation system, there is a problem that salts contained in the components are mixed into a subsequent mass spectrometer and affect the performance thereof.
Therefore, in the present invention, a desalting unit for desalting the components separated from the ion exchange column 16 is provided.
After the separation component from the ion exchange column 16 is trapped in the trap column 44 following the pre-separation section via the six-way valve 30, a cleaning liquid such as an aqueous solution of an organic acid is sent from the pump 18 to the trap column 20. Then, each separated component is desalted in the trap column 20. Further, in some cases, other impurities are simultaneously washed away from the separated components to obtain a concentrated separated component.
[0017]
Post-separation part After desalting each of the separated components in the trap column 20, the six-way valve 30 is rotated to switch the flow path. Then, each separated component is sent to the reversed phase column 24, and is subjected to gradient separation by an eluent for reversed phase separation.
[0018]
In the liquid sending means 22 of the second-stage separation unit, as shown in FIG. 3, the two types of eluent components are pumped in fixed amounts from the two syringe pumps 50a and 50b, and the components are mixed by the mixer 52. An eluent in which each eluent component is mixed at an arbitrary composition ratio is prepared and introduced into the reverse phase column 24.
The composition ratio of the eluent is changed stepwise under the control of the computer 28, and the eluent is introduced into the reversed-phase column 24 as a series of eluents having an elution intensity gradient to perform gradient separation. Each of the separated components is further separated into components based on the elution strength of the eluent.
The flow rate of the eluent in the second-stage separation section is preferably 100 μl / min or less in order to enable separation of a very small amount of sample with high resolution.
[0019]
In addition, when the liquid is sent at an eluent flow rate on the order of nl / min, particularly when the eluent is sent at a flow rate of 500 nl / min or less, it is preferable to use the liquid sending means shown in FIG. In the liquid sending means shown in the figure, as the eluent to be introduced into the reversed-phase column, a plurality of eluents having different elution intensities for the respective separated components are adjusted, and the branch flow paths 60, 60.・ ・ It is stored in advance. Each eluent is pressure-fed by a metering pump 62, passes through a port of a valve 64, and is connected to a 10-port manifold 66 (for example, Z10M1 manufactured by VICI).・ It is stored in each.
[0020]
The number, shape, and the like of the branch channels 60 are not particularly limited, and are appropriately selected according to the purpose of analysis. In the present embodiment, the ten branch channels each have an inner diameter of 0.25 mm and a length of 0.25 mm. It is formed of a 200 mm, 10 μl PEEK tube, and stores a predetermined amount of each eluent.
By switching the opening and closing of the port 68 of the valve 64, each of the branch flow paths 60 can be filled with the eluent directly from the syringe 70. This eliminates the need for adjusting the piping each time to exchange or replenish the eluent.
The filling of each of the eluents into the branch channels 60, 60,... Specifically, for each of the branch channels 60, 60,..., An eluent of a predetermined composition is automatically prepared and automatically sent to and stored in each of the branch channels 60, 60,. To do. When this means is employed, the filling accuracy of the eluent can be increased, so that the analysis accuracy can be improved.
[0021]
The rotary type valve 72 functions as a means for selecting each eluent temporarily stored in each of the branch flow paths 60, 60..., And opens a predetermined port 74 to supply a desired eluent. Can be flowed to subsequent columns. A controller (not shown) is connected to the outlet of the valve 72, and the selection of the valve position is controlled by an electric signal from the computer 28 in FIG.
[0022]
Each eluent stored in a predetermined amount in each of the branch channels 60, 60... Is successively introduced into the reversed-phase column 24 sequentially for each predetermined amount. That is, the ten types of eluents having different elution intensities stored in the ten branch channels are introduced into the reversed-phase column 24 as a series of eluents having ten stages of elution intensity gradients, and gradient separation is performed. . Then, each of the components separated by ion exchange separation is further separated into components.
[0023]
As the eluent for the reverse phase separation, a reverse phase solvent containing a volatile acid is suitably used, and is appropriately adjusted depending on the properties of the sample and the reverse phase column.
As the stationary phase of the reversed-phase column, various stationary phases used as the stationary phase of the reversed-phase chromatography are appropriately used depending on the properties of the eluent and the sample.
[0024]
Mass spectrometry unit In this way, after being separated into single components by two-stage separation, qualitative and quantitative analysis of each component is performed by the mass analyzer 26 directly connected to the subsequent separation unit. As an interface between the high performance liquid chromatograph and the mass spectrometer, ESI (electrospray ionization), APCI (atmospheric pressure chemical ionization) and the like are used. Further, as the mass spectrometer, a configuration such as a time-of-flight type or an ion trap type may be applied.
[0025]
Control unit In the present embodiment, a series of measurement operations such as injection of the sample and the eluent into the flow channel by the autosampler, operation of each liquid feeding means, and switching of the flow channel by the six-way valve 30 are shown in FIG. , And the analysis with high resolution can be performed continuously and automatically.
[0026]
Protein analysis device Hereinafter, protein analysis using the two-dimensional high-performance liquid chromatograph according to the above embodiment will be described.
In the case of performing protein analysis, a mixture containing the protein to be analyzed, which has been degraded into a number of peptides by enzymatic digestion in advance, is used as a sample.
[0027]
In FIG. 1, the sample introduced into the ion-exchange column 16 of the pre-stage separation section is separated to some extent mainly based on ionicity. However, the sample contains various peptides, and each separated component is not separated into a single peptide, but contains multiple peptide components.
Therefore, reverse phase separation is performed in the subsequent separation unit through the desalting unit, and further, the second stage separation is performed for each separated component.
[0028]
In this way, after separation into each peptide component to be measured in the sample by the two-stage separation, the amino acid sequence of each peptide component is identified in the directly connected mass spectrometer 26.
The amino acid sequence of the peptide fragment identified by mass spectrometry is translated into a gene coding sequence based on the correlation between each amino acid and the gene code. Furthermore, in the entire gene coding sequence of a chromosome whose sequence is already known, a portion corresponding to the sequence is identified, and the peptide fragment is used as a component from the sequence around the sequence portion, and the specific protein in the sample is The presence and its amino acid sequence are revealed.
[0029]
【Example】
Using the apparatus of the present invention shown in FIG. 1, a two-dimensional nano-liquid chromatogram of a trypsin digest of serum albumin (HSA) or Escherichia coli protein trypsin in the human body was measured. An anion exchange column (QA 10 μm 0.8 mm ID × 3 mm L 10 μl / min) was used as a separation column in the former separation unit, and a reversed phase column (C18 3 μm 0.15 mm ID × 45 mm L 100 nl / min) was used as a separation means in the latter separation unit. In addition, a small trap column (C18 3 μm 0.5 mmID × 1 mmL) was provided in the desalination section, and the separated components after ion exchange separation were washed with a formic acid aqueous solution and then separated on a reverse phase column, and then the mass spectrometer (ESI-TOF) was used. MS: Q-tof micro capillary voltage 1500 V).
[0030]
A syringe pump was used as a liquid sending means of the preparatory separation section, and the syringe was filled with the first gradient eluent, solution A (25 mM Tris-HCl pH 8.0). The sample (HSA 100 fmol / μl 10 μl or Escherichia coli protein 1 μg / 10 μl) is placed in the first vial of the autosampler, and the gradient eluate (second: A + 15 mM NaCl 15 μl, third: A + 15 mM NaCl 15 μl) in the second to fourth vials. Fourth: A + 400 mM NaCl (40 μl) was previously sealed, and introduced into the liquid feed channel in this order to perform gradient separation.
[0031]
As the eluent to be introduced into the reversed phase column, a mixture of solution C (0.1% formic acid aqueous solution) and solution D (0.1% acetonitrile aqueous solution of formic acid) was used. / D was mixed at a rate of / min to perform gradient elution.
[0032]
FIG. 5 shows the measurement results using HSA as a sample, and FIG. 6 shows the measurement results using Escherichia coli protein as a sample. FIG. 7 shows the results of a collision-induced dissociation analysis measurement (sample: E. coli protein) using a TOF type mass spectrometer. The measurement operation was performed reproducibly, and the amino acid sequence of the E. coli protein fragment could be automatically separated and identified.
[0033]
【The invention's effect】
As described above, according to the two-dimensional high-performance liquid chromatograph of the present invention, the separation sections having different separation characteristics with respect to the sample are provided in the former stage and the latter stage, so that analysis with high resolution is possible.
In addition, since a desalination unit is provided between each separation unit, it is possible to measure by connecting each separation unit, and it is also possible to connect with a mass spectrometry unit and to perform high precision measurement. .
In addition, a control unit that controls a series of measurement operations such as injection of the sample and the eluent into the flow path by the autosampler, operation of each liquid feeding means, and switching of the liquid feeding flow path is provided, so that high resolution is achieved. Analysis can be performed continuously and automatically.
In addition, by performing protein analysis using the two-dimensional high performance liquid chromatograph of the present invention, amino acid sequence identification of a peptide can be automatically and accurately performed for a complex peptide mixture.
[Brief description of the drawings]
FIG. 1 is a schematic configuration diagram of a two-dimensional high-performance liquid chromatograph according to an embodiment of the present invention.
FIG. 2 is a schematic configuration diagram illustrating an example of an autosampler provided in the apparatus of the present invention.
FIG. 3 is a schematic configuration diagram illustrating an example of a second-stage liquid sending unit.
FIG. 4 is a schematic configuration diagram illustrating an example of a second-stage liquid sending section.
FIG. 5 is a measurement result of an example.
FIG. 6 is a measurement result of an example.
FIG. 7 is a measurement result of an example.
[Explanation of symbols]
10: Two-dimensional high-speed chromatograph apparatus 12: Liquid sending means 14: Autosampler 16: Ion exchange column 18: Pump 20: Trap column 22: Gradient liquid sending means 24: Reversed phase column 26: Mass analyzer 28: Computer 30: Hex valve 32: Needle 34: Loop 36: Injector valve 38: Wash port 40: Injection port 42: Valve 44: Plunger 46: Wash liquid bottle 50a, 50b: Syringe pump 52: Mixer 60: Branch channel 62: Metering pump 64: Valve 66: Manifold 68: Port 70: Syringe 72: Valve 74: Port

Claims (3)

In a high-performance liquid chromatograph that detects each component in a sample separated by a separation column by gradient elution,
A first-stage liquid sending means for sending a gradient eluent at a fixed amount of 200 μl / min or less;
An autosampler for introducing the sample and the gradient eluent into the liquid sending flow path,
A pre-stage separation column for separating the sample into components,
A pre-stage separation unit with
A desalting unit comprising a column for trapping each of the separated components separated in the pre-stage separation unit, and performing a desalting treatment on each of the separated components by flowing a washing liquid through the column by a pump.
A second-stage liquid sending means for sending the gradient eluent at a fixed amount of 100 μl / min or less;
A post-stage separation column that further separates each of the separation components,
A post-separation unit with
A mass spectrometry unit connected to the post-stage separation unit,
A two-dimensional high-speed liquid chromatograph apparatus comprising: a control unit that controls an operation of introducing the liquid into the flow channel by the autosampler, an operation of the liquid feeding unit, and a switching operation of the liquid feeding channel. The device of claim 1,
The pre-stage separation column is an ion exchange column,
The post-separation column is a reversed-phase column,
One gradient eluent from the autosampler is introduced into the ion exchange column to trap the separated components in the column of the desalting section, the separated components are desalted in the desalting section, and the reversed phase is separated in the subsequent separation section. Perform separation and sample detection in the mass spectrometer,
The two-dimensional high-performance liquid chromatograph apparatus according to claim 1, wherein each of the other gradient eluents prepared in the autosampler is operated in the same manner. Using the device according to claim 1 or 2,
Using a mixture containing the peptide, which has been pre-processed to decompose the protein to be analyzed into peptides in advance, as a sample,
Separating each peptide from the mixture by separation at each separation section,
A protein analyzer, wherein the mass spectrometry unit performs mass spectrometry on the separated peptide to identify an amino acid sequence of the peptide.

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