JP4284104B2 - Atmospheric pressure laser ionization mass spectrometer - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a mass spectrometer that performs ionization using a laser under atmospheric pressure.
[0002]
[Prior art]
Conventionally, laser ionization mass spectrometers such as MALDI (Matrix-assisted Laser desorption) have been used as devices for performing DNA and proteome analysis.
There is a mass spectrometer (hereinafter referred to as MALDI / MS) using ionization as an ion source. The laser ionization mass spectrometer is advantageous over MS using an electrospray ionization (ESI) ion source in that it can analyze sensitivity and high molecular weight proteins.
[0003]
However, the conventional laser ionization mass spectrometer is disadvantageous in terms of workability because it takes time to set the sample and the like, and it is difficult to continuously measure many samples because the ion source itself is in a high vacuum. . For example, an example of preprocessing for MALDI / MS is described in Patent Document 1 below.
[0004]
In addition, as a configuration for continuously ionizing a large number of samples, for example, an example as in Patent Document 2 below can be considered. However, the configuration for introducing a sample from a liquid chromatograph (LC) under atmospheric pressure to an ionization part with high vacuum is complicated, and ionization in high vacuum directly measures a biological sample. Not suitable for doing. Therefore, it is desirable to ionize under atmospheric pressure.
[0005]
Therefore, in recent years, an atmospheric pressure laser ionization mass spectrometer that performs ionization using a laser at atmospheric pressure has been proposed. This apparatus is introduced in, for example, Patent Document 2, Patent Document 3, Patent Document 4, Non-Patent Document 1, Non-Patent Document 2, and the like.
[0006]
[Patent Document 1]
JP 2002-365177 A
[Patent Document 2]
Japanese Patent Application Laid-Open No. 64-65763
[Patent Document 3]
Special Table 2002-517886
[Patent Document 4]
EP0964427A2
[Non-Patent Document 1]
Analytical Chemistry, Vol.72, No.4, 652-657 (2000)
[Non-Patent Document 2]
Analytical Chemistry.2000, Vol.72, 5239-5243 (2000)
[0007]
[Problems to be solved by the invention]
All the devices that perform ionization under atmospheric pressure are fixed on a sample stage and ionized by irradiating the sample stage with a laser, and have a structure of a sample stage on which only one sample is placed. Yes. Therefore, no consideration has been given to the measurement of a plurality of samples placed on a sample stage and the ionization of continuous data. Therefore, when there are many kinds and many samples, there is a problem that the measurement efficiency and throughput are poor.
[0008]
An object of the present invention is to provide an atmospheric pressure laser ionization mass spectrometer that measures a large amount of sample efficiently and with high throughput.
[0009]
[Means for Solving the Problems]
In order to achieve the above object, the present invention is characterized in that an ion source that irradiates a sample with a laser at atmospheric pressure to perform ionization, a mass analyzer that performs mass analysis of ions generated by the ion source, and an apparatus In the laser ionization mass spectrometer having a data processing unit having a display unit for setting the measurement conditions and the like and displaying the measurement result, the ion source unit includes a nozzle for continuously dropping a sample to be measured, and the dropping A movable sample stage on which the sample is placed, a cover member that is shaped to cover the sample stage and to which a voltage is applied, and a spray unit that sprays an inert gas, and the sample with respect to the laser irradiation position The ionization is performed continuously by moving the table.
[0010]
With the above configuration, a plurality of samples can be continuously laser ionized and subjected to mass analysis even under atmospheric pressure.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
An embodiment of the present invention will be described below with reference to FIG.
[0012]
First, the configuration of the apparatus of the present invention will be described below. An outlet 301 of the conical cover 310 is disposed in the vicinity of the first pore 102 in the first pore electrode 101 that is an ion introduction port of the mass spectrometer main body 100. The cover 310 and the first pore electrode 101 are shown on the same central axis in FIG. 1, but this is not always necessary. The angle at which the central axis of the cover 310 and the central axis of the first pore electrode 101 intersect may be an oblique angle or a right angle. It is arranged in the vicinity that the flow lines 431 and 432 after the flow of the non-volatile gas flow line 420 containing the ions 411 and 412 enter the first pore 102 as much as possible. It is important for the introduction of ions into the mass spectrometer main body 100 that reduces the diffusion of air in the atmosphere and is efficient.
[0013]
Here, the non-volatile gas (for example, nitrogen, argon, etc.) is configured to spray the sample 402 from the gas pipe 421 for non-volatile gas. Further, the gas pipe 421 is provided with a heater 422 for heating the gas, and the temperature adjusting device 423 controls and adjusts the temperature of the nonvolatile spray gas. Further, the gas flow rate adjusting device 424 controls and adjusts the flow rate of the nonvolatile gas. Temperature adjusting device 423 and gas flow rate adjusting device
424 is controlled by a computer connected via a gas temperature control signal line 425 and a gas flow rate control line 426, respectively.
[0014]
A heater 741 is connected to the cover 310 in the vicinity where ions 411 and 412 flow, and the cover 310 is controlled to be heated in a range of, for example, about 50 to 200 degrees. A power source 740 is connected to the heater 741 and is controlled and adjusted by a control signal from a computer via a control signal line 742.
[0015]
A laser for ionization is generated on the disc-shaped sample stage 500 through a transparent body 202 that transmits a laser beam generated by the laser source 200 and passes through a lens 201 (for example, a convex lens) that converges the laser, and further passes through a laser beam attached to the cover 310. The sample 402 is irradiated to ionize the sample.
[0016]
In addition, the sample dispenser 600 drops the liquid sample held by the sample holding split portion 601 onto the sample stage 500. The sample dispenser 600 is connected to a liquid chromatograph, a capillary electrophoresis apparatus, or the like, and is in a state where samples are continuously supplied. Further, the disk-shaped sample stage 500 rotates around a rotation center axis 501 shown in the drawing. Therefore, the sample 401 dropped from the sample dispenser 600 is configured to sequentially move to the ionization position where the sample 402 is shown as the disk-shaped sample stage 500 rotates.
[0017]
Next, the operation of this embodiment will be described below.
[0018]
The sample 401 dropped by the sample dispenser 600 is ionized by moving the disk-shaped sample stage 500 to the ionization position sequentially. Since the laser passes through the transparent body 202 through which the laser beam passes, there is no hole in the cover 310. Therefore, it does not promote ion diffusion. At this time, the sample 402 is sprayed with a non-volatile gas so that the sample is not ionized or a chemical reaction such as oxidation does not occur in the atmosphere. The flow rate of the sprayed gas is controlled to a flow rate that reduces the diffusion of ions corresponding to the shape of the cover 310 and efficiently pushes out ions from the cover outlet end 301. For example, when the cover outlet end 301 is more constricted, the force of pushing back by the fluid resistance and the electric field is applied to the ions, so that the ions can be detected with higher sensitivity when the gas flow rate is increased. Further, in order to reduce contamination on the inner surface of the cover 310 due to various ions generated during ionization and to prevent contamination, the gas is heated by the heater 422 and the cover 310 is further heated by the heater 741. This reduces noise due to contamination. Ions in the atmosphere are pushed by the non-volatile gas linear flow 420, and diffusion is reduced by the cover 310 and introduced into the first pores 102 of the mass spectrometer main body 100 like ions 413. The introduced ions 413 are further introduced into the interior like the ions 414 by the electric field force 430 applied after the first pore 102.
[0019]
The sensitivity of the mass spectrometer is determined by the number of ions introduced per time, that is, the concentration of ions in the atmosphere introduced into the first pores 102. Therefore, reducing the diffusion of ions in the atmosphere is a very important problem. However, when the cover 310 is not provided, the ions 412 diffuse three-dimensionally, so that the concentration of target ions for measurement in the atmosphere decreases. Moreover, it becomes easy to be influenced by the change of the position of a sample and a non-volatile gas flow rate, and possibility that it will become unstable becomes high. In this embodiment, the above problem is solved by providing the cover 310.
[0020]
Another embodiment of the present invention will be described with reference to FIG.
[0021]
The feature of the present embodiment is a case where the cover 315 is formed of a cylindrical electrode. As shown in FIG. 2, the position of the sample 402 to be ionized is arranged on the mass spectrometer side from the center of the electrode. Further, a voltage is applied to the cover 315 via a conductor 701 by a high voltage power source 700. The applied voltage is controlled by a computer via the control signal line 702.
[0022]
The operation of this embodiment will be described. As the voltage applied to the cover 315, a positive voltage is applied if the measurement target ion is a positive ion, and a negative voltage is applied if it is a negative ion. In order to position the sample 402 to be ionized closer to the mass spectrometer than the center of the electrode, an electric field that generates a force for moving the ions 411 and 412 to the mass spectrometer is applied to the cover 315. Furthermore, since a voltage having the same polarity as that of the ion for measurement is applied to the cover 315, a force repelling the cover 315 is generated in the ion for measurement. Therefore, it is possible to reduce a decrease in sensitivity due to adsorption of ions for measurement to the cover 315 or the like.
[0023]
Another embodiment of the present invention will be described with reference to FIG.
[0024]
The present embodiment is an example developed by combining FIG. 1 and FIG. 2 already described.
[0025]
First, the configuration of the present embodiment will be described. In this example, the conical cover 320 is cut into a ring shape, and the inlet side cover 321 and the outlet side cover 322 are electrically divided into two by an insulator 323. Here, an example in which the image is divided into two is shown, but the number of divisions may be increased in the same manner as three divisions, four divisions, and the like. Further, an upper electrode 331 and a lower electrode 332 are provided on the entrance side of the cover 320. Furthermore, power supplies 710, 720, and 730 for applying a high voltage are provided, and a high voltage is applied to each cover and electrode via conductors 711, 721, and 731. Each power source is connected to a computer via signal lines 712, 722, 732 and controlled. Here, the electrodes 331 and 332 and the inlet side cover 321 apply a voltage having a higher absolute value than the outlet side cover 322. Each voltage is simultaneously applied with a positive or negative voltage in synchronization.
[0026]
Next, the operation of this embodiment will be described.
[0027]
When the measurement target ions 411 ionized by the laser are positive ions, a positive high voltage of about several kilovolts (kV) is applied to the inlet side and outlet side covers 321, 322, and electrodes 331, 332. At this time, the entrance side cover 321 has more high A voltage is applied and a high voltage is applied so that the lowest electric field is generated at the outlet end 301 of the cover 320. The ions 411 and 412 are pushed out to the cover outlet end 301 by the force of the electric field. Further, the ions are pushed out to the cover outlet end 301 by the inert spray gas flow 420. At this time, the ions 411, 412 and the cover 320 are electrically of the same polarity, so that the ions receive a repulsive force from the cover 320. For this reason, ions are efficiently introduced into the mass spectrometer with reduced diffusion.
[0028]
Further, since a high voltage is applied to the cover 320, a high electric field is generated in the cover. Of course, the closer to the cover, the higher the electric field. Therefore, the ions for measurement receive a repulsive force from the cover 320, and the ions are not adsorbed on the cover 320 to reduce the number of ions, and contamination such as contamination can be greatly reduced.
[0029]
Also in this embodiment, the sample stage can be rotated.
[0030]
Another embodiment of the present invention will be described with reference to FIG.
[0031]
First, the configuration of the present embodiment will be described. In the present embodiment, the sample stage is provided with a disk-shaped sample stage 500, and a rotation center axis 501 is provided with a mechanism 510 that moves back and forth in the same direction as the traveling direction of the ion flow, for example. In addition, a moving mechanism for finely adjusting the sample stage to the left and right 512 and the upper and lower 513 with respect to the traveling direction of the ion flow is also provided.
[0032]
Next, the operation will be described. For example, when a normal matrix is added to a sample for laser ionization and dried, the sample is fixed concentrically as a sample arrangement line on a disc-shaped sample stage 500 like a center 431, an outer side 432, and an inner side 433. When ionizing each sample arrangement line, by moving the central axis 501 of the sample stage, for example, the sample arrangement lines 432 and 432 are sequentially arranged.
Ionization is performed with 431 and 433. As a result, many samples can be placed on the sample stage, and laser ionization can be performed efficiently and with high throughput. FIG. 4 shows a state in which the outer array sample 432 has been ionized.
[0033]
It is also possible to perform sample dripping and ionization simultaneously. In this case, if sample dripping is performed on the outer 432 array line, ionization ionizes the inner 433 array line, and if sample dripping is performed on the inner 433 array line, Ionization ionizes the outer 432 alignment lines. When the sample is dropped at the center 431 array line, the ionization is also at the center 431 array line. Thereby, ionization can be performed more efficiently.
[0034]
Another embodiment of the present invention will be described with reference to FIG.
[0035]
In this embodiment, the sample array on the sample stage 500 is formed in a spiral shape instead of a concentric circle. This can be performed by continuously moving the central axis from 502 to the central axes 501 to 503 while rotating the disk-shaped sample stage 500. In this example as well, the sample dropping and laser ionization can be performed simultaneously with high efficiency and high efficiency as described above.
[0036]
Furthermore, in this embodiment, the sample can be dropped continuously by connecting the sample outlet of the liquid chromatograph to the sample dispenser 600, for example. Therefore, a TIC (total ion chromatogram) can be obtained by laser ionization in the same manner as in the case of a combination of a normal atmospheric pressure ionization (such as electrospray ionization) mass spectrometer and a liquid chromatograph.
[0037]
Another embodiment of the present invention will be described with reference to FIG.
[0038]
First, the configuration of the present embodiment will be described. In this embodiment, a square sample stage 520 is provided, and a mechanism for moving the sample stage to the front and rear 511 and the left and right 512 is provided. Further, a moving mechanism for fine adjustment in the vertical direction 513 of the sample table is also provided.
[0039]
Next, the operation of the present invention will be described.
[0040]
For example, when a normal matrix is added to a sample for laser ionization and dried, the sample is dropped onto a sample arrangement line corresponding to the movement of the front, rear, left and right sample stands 520 shown in FIG. When ionizing each array line, the sample stage 520 is sequentially moved back and forth corresponding to the position of the sample spot, and then sequentially moved to the left and right to be ionized like the sample 402. Thereby, laser ionization can be performed efficiently and with high throughput.
[0041]
Further, when an atmospheric pressure laser is used for ionization and the sample dispenser 600 is provided outside the cover 310 as described above, a rectangular sample base is used.
520 is sequentially ionized by moving back and forth corresponding to the position of the sample spot. At the same time, the sample is dropped at the position 401 by the sample dispenser 600 on the opposite side. As described above, the sample can be dropped and ionized at the same time, and the sample can be dropped and laser ionized at the same time efficiently and with high throughput.
[0042]
Another embodiment of the present invention will be described with reference to FIG.
[0043]
In this embodiment, the sample arrangement on the sample stage 520 is not dropped by the sample spot but continuously dropped by the sample dispenser 600.
[0044]
This can be obtained by sequentially moving the square sample table 520 left and right from a certain front and rear position as shown in FIG. In this example as well, the laser ionization can be performed at the position 404 while dropping the sample at the position 405 with high throughput as efficiently as described above.
[0045]
Furthermore, in this embodiment, the sample can be dropped continuously by connecting the sample outlet of the liquid chromatograph to the sample dispenser 600, for example. Accordingly, a TIC (total ion chromatogram) can be obtained by laser ionization, as in the case of a combination of a normal atmospheric pressure ionization (such as electrospray ionization) mass spectrometer and a liquid chromatograph.
[0046]
Another embodiment of the present invention will be described with reference to FIG.
First, the configuration will be described with reference to the drawings. In the above-described atmospheric pressure laser ionization, a sample stage 520 that is movable via a signal line 530 controlled from the data processing apparatus 900 is provided, an ion diffusion prevention cover 310 is provided, and the sample stage 520 is further observed. Means, for example, a microscope 800 with a CCD is provided. The microscope 800 is provided so as to penetrate the cover 300. This may be a microscope that can be used under atmospheric pressure, such as an optical microscope or a fluorescence microscope. Further, the microscope 800 is connected to the data processing device 900 through the signal line 801, and the data taken from the microscope 800 is transferred to the data processing device 900 to perform image processing. In the example of FIG. 8, since the microscope 800 is provided directly above, the position of the laser source 200 is shifted and the laser is irradiated from an oblique direction.
[0047]
Next, the operation will be described.
[0048]
A sample 402 to be measured by laser ionization on the sample stage 520 (or an area to be measured by laser ionization) is measured by the microscope 800 and displayed as a microscope image on the data processing device 900, and the operator performs an ionization area from this image. Is specified. The data processing apparatus 900 controls the sample stage 520 via the signal line 530, and moves the sample stage 520 so that the designated region is a position where laser ionization is performed. Thereafter, the specified region is laser ionized, ions are taken into the mass spectrometer, and mass spectrometry is performed. At this time, the sample position information, ionization order information, mass spectrometer measurement data, measurement condition information, laser intensity, irradiation time, number of irradiations, wavelength, etc. associated with the microscope image are associated. To the data processing apparatus 900. In addition, the information of the ionized region is stored as a figure in the image information of the microscope stored in the data processing apparatus 900, the position is designated by a cursor operated by a pointing device, and the measurement data and measurement conditions of the mass spectrometer are specified. , Position information, laser irradiation conditions, and other information display functions. Of course, when this display data is large, it may be automatically jumped to another screen.
[0049]
Therefore, the operator can easily specify the sample and region to be measured from the microscope image, the operation efficiency is improved, and it is not necessary to ionize an unnecessary region, so that the lifetime of the laser source 204 can be extended. Is possible. Furthermore, the position information of the measured region, the measurement conditions of the laser and the mass spectrometer, and the measurement data can be easily obtained from the microscope image data stored in the data processing device 900, and the operation efficiency is greatly improved.
[0050]
An application example using the configuration of the above-described embodiment will be described below with reference to FIG.
[0051]
In this example, a gel after fractionating a protein by two-dimensional electrophoresis is used as a measurement target. Since the configuration of the ion source of the present invention is atmospheric pressure ionization, a gel for two-dimensional electrophoresis can be directly set on a sample stage and ionized.
[0052]
The example of FIG. 9 is an example of a microscope image displayed on the display screen of the data processing apparatus 900. An image 901 shows a whole image of a gel of two-dimensional electrophoresis (or a product to which a protein or the like is transferred), and the fractionated protein spots are 451 to 455. A scale 920 and its size 921 are displayed on the same screen corresponding to the magnification of the microscope 800. In addition, the size 930 and the size 931 of the designated laser spot are also displayed. The laser spot diameter can be set to a minimum of about 0.25 μm by narrowing the laser beam with the lens 201. In this example, an example of 0.1 mm is shown. Of course, the smaller the laser spot diameter, the more accurately the measurement area can be ionized, but the number of times of laser irradiation increases and measurement time is required.
[0053]
In the data processing apparatus 900, the irradiation position of the laser beam is automatically and optimally arranged with respect to the ionization region 910 corresponding to the protein spot designated by the operator. The enlarged view in FIG. 9 shows an example in which the laser irradiation positions 911 to 915 are arranged with respect to the protein spot 451. The data processing apparatus 900 controls and moves the sample stage 520 according to this arrangement to perform laser ionization.
[0054]
In this example, the operator can specify an appropriate laser spot diameter 930 from the information of the scale 920 and the size and shape of the sample spots 451 to 455, thereby improving the operation efficiency. In the usual laser ionization under a low vacuum, a sample spot of two-dimensional electrophoresis is fractionated and taken out, and further, only the protein is dried and ionized from the fraction on the sample stage. Therefore, such troublesome time-consuming work can be eliminated, and analysis with high throughput becomes possible.
[0055]
In addition, by designating the spot 451 that is the ionization region of the sample as described above, the data of the designated spot that has already been measured and accumulated, for example, the coordinates of the center position of the sample spot, the spectrum data of the MS, and the measurement of the MS Conditions, laser measurement conditions, and the like can be automatically displayed as a sub-screen 902. As a result, data with the measurement spot of interest can be easily obtained, and the operation efficiency is greatly improved.
[0056]
Next, another application example will be described with reference to FIG.
[0057]
FIG. 10 is an example of a microscope image of an animal cell 460 displayed on the screen of the data processing apparatus 900. The size of a normal animal cell is about several tens of μm to several hundreds of μm. For example, if a phase contrast optical microscope is used, the structure of the cell can be observed to some extent without staining the cell. Even in the case of staining, any substance that does not inhibit laser ionization and does not contain salt or the like may be used as a staining agent.
[0058]
The microscopic image of the cell 460 includes a cell nucleus 461, a nucleolus 462, a mitochondria 463 and 464. For example, when the constituent material of the nucleus 461 is examined, a region 916 to be laser ionized with respect to the nucleus 461 is designated. Further, for example, if it is desired to examine a substance constituting the mitochondria 463 and 464, a region to be laser ionized is designated on the screen using a pointing device or the like as 917 and 918. Thereafter, as described above, the sample spot size is set to 10 for example.
If μm is designated, the ionization region is automatically arranged at the designated laser spot, and the sample table 520 is moved and controlled to perform laser ionization. The sample stage 520 can have a moving accuracy of about 0.5 μm.
[0059]
In conventional laser ionization under low vacuum, it was impossible to ionize the sample as it was due to evaporation of the sample. However, in the present invention, multiple types of living proteins are measured continuously. I can do it. Since proteins in cells are denatured by heat, drying, etc., it is very significant to analyze live and live proteins. Furthermore, since only the sample to be measured is laser ionized in a specified region, it is possible to reduce noise of unnecessary impurities and improve the sensitivity of the sample to be measured.
[0060]
Next, an embodiment of the moving mechanism will be described with reference to FIG.
[0061]
First, the configuration will be described. A sample table 520 is provided on the moving table 521, and a sample solution 470 containing cells, for example, is placed thereon. As shown in the figure, the portion on which the sample table 520 is placed has a small shape that can move within the cover 310 for preventing ion diffusion.
[0062]
The moving table 521 is installed on the base 504 via a ball bearing 505 that can move in the front-rear direction 511 and the left-right direction 512. Further, the moving table 521 on which the sample table is placed includes a protruding plate 522 having a ball screw shaft 541. The ball screw shaft 541 includes a ball screw nut portion 540 and a gear 540 fixed thereto, and meshes with a gear 551 fixed to the shaft of the pulse motor 550. Further, the pulse motor 550 is connected to the data processing device 900 via the control signal line 552. Thereby, by rotating the pulse motor 550, the movable table
521 can be moved in the front-rear direction 511. The moving mechanism in the left-right direction 512 is exactly the same as described above.
[0063]
With the above configuration, according to the configuration of FIG. 11, the front-rear direction 511 and the left-right direction 512 can be independently moved by a control signal from the data processing device 900. Of course, for more precise position confirmation, position coordinates may be detected by laser light, and feedback may be performed for fine adjustment of the position. Further, marking may be provided on the sample table 520, a precise position may be detected from a microscope image, and feedback may be applied to finely adjust the position. When a stepping motor, a ball screw (or screw screw), and a gear are used as in this example, a driving step of 0.1 μm and a feeding accuracy of 0.3 μm can be realized.
[0064]
Next, an example of measurement in the present invention will be described with reference to FIGS.
[0065]
Here, an example in which proteins are separated into spots by two-dimensional electrophoresis will be described with reference to FIG. Of course, the sample is not a protein as long as it is on the sample stage. In this example, as shown in FIG. 9, the protein spots 451 to 455 are separated, and the protein spot 451 is designated as the ionization region 910.
[0066]
When measuring by manually specifying the region of laser ionization, it is as follows. The operator designates the ionization range 450, the laser ionization line step 903, the laser spot size 930, the laser ionization region 910, and the like from the data processing apparatus 900. Correspondingly, the data processing apparatus 900 optimally arranges and displays the laser spots 911 to 915 on the laser ionization line 904 from the microscope image. As shown in the enlarged view.
[0067]
When automating the above operations, there are the following problems.
[0068]
Proteins actually extracted from a living body include impurities and various unknown proteins, and even if they are separated into spots by two-dimensional electrophoresis, the background is dirty, that is, unnecessary signals exist. For this reason, unnecessary regions are ionized to reduce measurement efficiency, or an ionization region for a spot is set too wide, and an unnecessary noise component is increased to reduce S / N.
[0069]
Therefore, when the ionization region is automatically selected, a threshold value (threshold value) is set for the data for each pixel of the microscope image, and a region equal to or greater than the threshold value is set as the ionization region. Specifically, the color density (shading information) of each pixel is used as an index. An embodiment in this case will be described with reference to FIGS.
[0070]
FIG. 13 shows signal values (vertical axis indicates color density) obtained from a microscope image on a laser ionization line 904 including sample spots 451, 454, and 455. The peak of the signal profile of the sample spots 451, 454, 455 is 456, 457, 458, respectively. Between the peaks of the signal profile, there is a higher noise than the baseline 905 shown. Therefore, the operator sets threshold setting lines 906 and 907 for automatically setting the ionization region so that the target sample can be efficiently ionized. In the setting line 906, a large signal value in the sample area can be obtained, but the separation of the signal values of the sample spots 454 and 455 becomes worse. In the setting line 907, the signal value of the sample region is lowered, but the separation of the signal values of the sample spots 454 and 455 is improved.
[0071]
FIG. 14 shows an example of a setting line 908 having an inclination. This is suitable when the signal profile 465 of the background impurity is raised and raised on the left or right side as shown.
[0072]
FIG. 15 shows an example in which the value of the threshold setting line is changed halfway. The lower part of FIG. 15 shows two-dimensional electrophoresis spots of proteins. The upper part shows the signal profile on the lower laser ionization line 905. Here, the sample spot 459 is outside the measurement target, but when the sample spots 456, 457, and 458 are to be measured, the threshold value setting lines 9091, 9092, and 9093 for designating laser ionization are used. Set. As described above, by setting the threshold level independently according to the purpose, laser ionization can be performed efficiently and throughput can be improved.
[0073]
In the lower part of FIG. 15, the threshold setting line on the laser ionization line 905 is not designated, but the threshold is designated on the surface on the microscopic image. In this example, the threshold level is represented by the display density of each region. It is set so that the higher the threshold level, the darker the display. Of course, the reverse setting is also acceptable. Designation of the area and display density of each region can be arbitrarily performed by an operator using a pointing device such as a mouse. If a display density serving as a threshold is set in advance, the display density of each area is compared with the display density set as the threshold, and the ionized area can be automatically recognized. Designation of shading of each area can be arbitrarily set by an operator. In this example, the background removal threshold level can be specified as a surface instead of as a line, so that ionization is performed on samples that are two-dimensionally formed on a surface such as two-dimensional electrophoresis. The threshold level of the area can be specified efficiently, and the operation efficiency can be increased.
[0074]
Next, the operation when setting the threshold values of FIGS. 13 to 15 will be described with reference to FIG.
[0075]
First, a microscopic image of a sample is measured and loaded into the data processing device 900. The measurement target region and dimensions are confirmed from this image. Next, the location of the sample to be measured is confirmed, and the range of laser ionization is designated. Next, the size of the sample area is judged from the actual size scale displayed in the image, and a laser spot size with an efficient number of times of laser irradiation is designated and displayed. Next, the size of the sample area is judged from the actual size scale displayed in the image, and an efficient laser line step is designated. Next, the threshold of the image signal value to be laser ionized is designated. Also, as described above, when specifying by a signal profile, a threshold setting line is arbitrarily specified. In addition, when specifying on the microscope image, it is specified by setting the ionization region surface using the display density on the image.
[0076]
Based on the above specification and setting, the data processing device 900 extracts the ionization region and displays it on the image. Here, if it is inappropriate, the setting items are arbitrarily returned.
[0077]
Next, laser ionization conditions such as laser irradiation time and number of irradiations, and measurement conditions for mass spectrometry are set. At the time of measurement, the data processing apparatus 900 moves the sample stage according to the specified conditions, and ionizes the automatically extracted laser irradiation region. At the same time, the sample spot position information is held, and the sample spot is stored in correspondence with the laser ionization conditions, MS conditions and measurement data.
[0078]
Finally, the measured MS data is identified by searching a protein database or the like.
[0079]
As described above, it is possible to automatically ionize only the target sample by simply performing simple designation, and the measurement efficiency can be improved. Furthermore, since other than the target object is not ionized, the signal value is improved and the S / N is improved. In addition, since unnecessary laser irradiation is not performed, there is an effect of prolonging the life of a laser having a limited life.
[0080]
An example of measuring a sample in which proteins are not separated by liquid chromatogram or two-dimensional electrophoresis will be described. FIG. 17 is an example of an observation screen on a microscope in which a fluorescent protein 472 (for example, red) and a fluorescent material 474 (for example, blue) are combined with a sample 471 and 473, respectively, and lighted. . In this example, two kinds of proteins 471 and 473 are mixed as a measurement target. Naturally, proteins 475 and 476 which are not to be measured and other impurities are also mixed.
[0081]
In addition, for binding a fluorescent substance to a protein molecule to be measured, a fluorescent labeling ligand obtained by chemically binding a fluorescent dye to a ligand that specifically binds to the protein molecule is prepared, and this is bound to the protein to cause redness. , Blue, yellow, etc. are known. Further, not only proteins but also chromosomes, nucleotides, mRNAs and the like can be illuminated with fluorescence. (Details are described in detail in pages 47 to 92, written by Takashi Funatsu, edited by Japan Photobiology Association, "New Optical Technology that Opens Life".)
Next, an example in which an ionization region is automatically set in order to analyze each protein 471, 473 will be described with reference to FIGS. FIG. 18 shows an image example when an image taken from the microscope is transmitted through the data processing apparatus 900 by filtering only red in the data processing. Therefore, the protein 471 is not observed as shown by the broken line, but the fluorescent substance 472 bonded thereto is observed. In addition, no other substances not subject to measurement are observed. Therefore, a sample that is not a measurement target is not set as an ionization region.
[0082]
The ionization region can be set by the method shown in FIG. 16 as described above. However, the ionized region 901 in FIG. 18 is enlarged by a specified coefficient magnification to a size including a sample after selecting a fluorescent portion with a threshold. This ionization region 910 is automatically set so as to be covered by irradiating a laser spot 911 a plurality of times.
[0083]
FIG. 19 is an analysis example of another protein 473 mixed at the same time. In this microscopic image, a blue fluorescent substance 473 is bonded to the protein 473 and blue is transmitted by data processing. The automatic setting process is as described above. Therefore, it is possible to analyze a plurality of proteins simultaneously from one sample by using fluorescent materials of different colors.
[0084]
From the above, even if the liquid chromatogram is not used for accurate separation, the specific component (protein, etc.) to be measured is automatically selected and ionized by illuminating it with a specific color fluorescent substance. Therefore, the signal-to-noise ratio can be improved and the analysis can be performed more efficiently. This example is effective even when multiple proteins are bound by interaction, and only one component (protein) is bound to a fluorescent substance to easily measure only the target component. Can do. In general, proteins selectively interact with and bind to various types of proteins in cells to perform specific functions in the body, so it is necessary to analyze complex proteins that are bound by protein interactions in this way. Is very important for physiology and drug discovery.
[0085]
【The invention's effect】
In the present invention, measurement can be performed with high sensitivity in an atmospheric pressure laser ionization mass spectrometer. In addition, a large amount of sample can be automatically and efficiently measured with high throughput.
[Brief description of the drawings]
FIG. 1 is a diagram showing an embodiment of an ion source unit according to the present invention.
FIG. 2 is a diagram showing an embodiment of an ion source unit according to the present invention.
FIG. 3 is a diagram showing an embodiment of an ion source unit according to the present invention.
FIG. 4 is a diagram showing an embodiment of an ion source unit of the present invention.
FIG. 5 is a diagram showing an embodiment of an ion source unit according to the present invention.
FIG. 6 is a diagram showing an embodiment of an ion source unit according to the present invention.
FIG. 7 is a diagram showing an example of an ion source unit according to the present invention.
FIG. 8 is a diagram showing an example in which a gel of a two-dimensional electrophoresis apparatus is used as a sample.
FIG. 9 is a diagram showing an example using animal cells as samples.
FIG. 10 shows another example of a captured image in which a microscope is combined in the present invention.
FIG. 11 is a diagram showing an example of a sample table moving mechanism;
FIG. 12 is a diagram for explaining a setting example at the time of measurement according to the present invention.
FIG. 13 is a diagram for explaining a setting example at the time of measurement according to the present invention.
FIG. 14 is a diagram for explaining a setting example at the time of measurement according to the present invention.
FIG. 15 is a diagram for explaining a setting example at the time of measurement according to the present invention.
FIG. 16 is a flowchart illustrating an operation during measurement according to the present invention.
FIG. 17 is a diagram showing an example in which two types of proteins in a biological sample are illuminated with different fluorescence.
FIG. 18 is a diagram showing an example in which only a certain protein is fluoresced.
FIG. 19 is a diagram showing an example in which only a certain protein is illuminated with fluorescence.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 100 ... Mass spectrometer main body, 101 ... 1st pore electrode, 102 ... 1st pore, 200 ... Laser source, 201 ... Lens, 202 ... Transparent body, 310, 320 ... Cover, 421 ... Gas capillary, 422, 741 ... heater, 423 ... temperature adjustment device, 424 ... flow rate adjustment device, 451-455 ... spot, 460 ... cell, 461 ... cell nucleus, 462 ... nuclear body, 463,464 ... mitochondria, 500,520 ... sample stage, 521 ... Moving platform, 522 ... Protrusion plate, 550 ... Pulse motor, 600 ... Sample dispenser, 700,710,720,730,740 ... Power source, 800 ... Microscope, 900 ... Data processing device.

Claims (9)

An ion source that performs ionization by irradiating a sample with laser at atmospheric pressure, a mass analyzer that performs mass analysis of ions generated by the ion source, and a display that sets the measurement conditions of the device and displays the measurement results In an atmospheric pressure laser ionization mass spectrometer equipped with a data processing unit having a unit,
The ion source unit includes a nozzle that continuously drops a sample to be measured, a movable sample stage on which the dropped sample is placed, a conical or cylindrical cover member that covers the sample stage, and the cover A power source that applies a voltage of the same polarity as the ions to the member, and a spray unit that sprays an inert gas from the inlet side of the cover member to the outlet side of the cover member,
The mass spectrometer includes an ion inlet, and the outlet of the cover member is disposed in the vicinity of the ion inlet and spaced from the ion inlet. In claim 1,
The atmospheric pressure laser ionization mass spectrometer is characterized in that the cover member has a lower absolute value of the voltage applied toward the outlet side. In claim 1 or 2,
An atmospheric pressure laser ionization mass spectrometer characterized in that ionization is continuously performed by moving the sample stage with respect to a laser irradiation position. In claim 1 or 2,
An atmospheric pressure laser ionization mass spectrometer characterized in that the cover member includes a transparent body that transmits laser light. In claim 1 or 2,
The atmospheric pressure laser ionization mass spectrometer characterized in that the sample stage has a disc shape and has a rotation axis, and is configured to rotate around the rotation axis. In claim 1 or 2,
The atmospheric pressure laser ionization mass spectrometer according to claim 1, wherein the sample stage includes an operation unit movable in the front-rear direction and the left-right direction with respect to the ion traveling direction. In claim 1 or 2,
An atmospheric pressure laser ionization mass spectrometer characterized in that the ion source is provided with a microscope for observing the surface of the sample table, and an image obtained by the microscope is displayed on the display unit. In claim 1 or 2,
An atmospheric pressure laser ionization mass spectrometer characterized in that a threshold value is set for color density data obtained by the microscope, and an area on a sample stage having a density equal to or higher than the threshold value is set as an ionization area. In claim 1 or 2,
An atmospheric pressure laser ionization mass spectrometer comprising heating means for heating the cover member.

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