JP2018036100A - MALDI mass spectrometer and matrix observation apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a matrix observation apparatus capable of searching for an irradiation position of a laser beam having high ionization efficiency among sample spots arranged on a sample plate. SOLUTION: A stage 31 on which a sample plate 20 on which a sample is arranged is mounted, a light source unit 40 for irradiating the sample plate 20 with ultraviolet light for observation, and light from the sample plate 20 are detected to create an optical image. The image acquisition unit 50 is provided, and the sample is configured to contain a matrix that absorbs ultraviolet light for observation. [Selection diagram] Fig. 1

Description

  The present invention relates to a matrix-assisted laser desorption ionization (MALDI) mass spectrometer that irradiates a sample mixed with a matrix with laser light, thereby vaporizing or ionizing the sample, and relates to a MALDI mass spectrometer or the like. The present invention relates to a matrix observation apparatus used.

MALDI (Matrix Assisted Laser Desorption / Ionization) is a solvent containing an ionization aid such as DHB (2,5-dihydroxybenzoic acid) and CHCA (α-cyano-4-hydroxycinnamic acid) called “matrix”. As a sample mixed with a small amount of analyte (protein, etc.) is irradiated with laser light, the heat of the irradiated laser light is absorbed and a part of the matrix that is heated is rapidly heated and vaporized. This is a method for vaporizing or ionizing an analysis target substance.
When performing analysis with such a mass spectrometer equipped with a MALDI ion source, for example, a sample is dropped in the form of a spot on the upper surface of a sample plate, and the solvent is evaporated by drying and then placed in a vacuum chamber. Then, the analysis is started by starting the operation of the vacuum pump in order to evacuate the vacuum chamber. Usually, a plurality of samples are arranged on the upper surface of the sample plate so as to be arranged in M rows and N columns, and each sample is moved by moving the sample plate to the irradiation position of the laser light by moving the sample plate. It is ionized one after another.
As a mass spectrometer equipped with a MALDI ion source, for example, MALDI-TOFMS is known, and the generated ions are extracted by an electric field having a predetermined intensity and introduced into the flight space to perform mass analysis. The speed of each ion flying in the flight space depends on its mass-to-charge ratio, and the smaller the mass-to-charge ratio, the greater the speed.Therefore, depending on the flight time to reach the detector, various ions Separately detect each.

4 and 5 are diagrams showing a configuration example of a conventional MALDI-TOFMS. One direction horizontal to the ground is defined as an X direction, a direction horizontal to the ground and perpendicular to the X direction is defined as a Y direction, and a direction (vertical direction) perpendicular to the X direction and the Y direction is defined as a Z direction.
The MALDI-TOFMS 201 includes a mass spectrometer 10, a sample plate 20, a sample stage 31 on which the sample plate 20 is placed, a stage driving unit 32 that moves the sample stage 31, and visible light for observation on the upper surface of the sample plate 20. A visible light source unit (light source unit) 240, an image acquisition device (image acquisition unit) 250 that acquires an image of the upper surface of the sample plate 20, a laser irradiation unit 5 that emits a laser beam for ionization on the sample, And a computer 260 that controls the entire MALDI-TOFMS 201.

The mass analysis unit 10 includes a free flight space 12 in which ions freely fly without being affected by an electric field, an ion transport optical system, a mass analyzer, and an ion detector 11. Electromagnetic lenses and multipole high-frequency ion guides are used, and quadrupole analyzers, ion traps, time-of-flight analyzers, magnetic sector analyzers, etc. are used as mass analyzers. It is done.
Further, an aperture 3 that shields diffusing ions and an Einzel lens 2 as an ion transport optical system for transporting ions to the mass analyzer 10 are provided between a sample plate 20 and a mass analyzer 10 described later. It is arranged. Of course, ion transport optical systems having various configurations other than the Einzel lens 2 may be used.
According to such a mass analyzer 10, ions emitted from the sample by laser irradiation pass through the aperture 3 and the Einzel lens 2 and are sent to the mass analyzer via the ion transport optical system. The various ions are separated according to the mass to charge ratio. When the separated ions reach the ion detector 11, the ion detector 11 outputs a sample signal corresponding to the amount of ions reached to the computer 260.

  The sample plate 20 is made of a conductive metal plate (for example, 8 cm × 3 cm × 0.2 cm), and a circular well having a diameter of, for example, about 3 to 5 mm is formed in M rows on the upper surface of the plate. The sample solution is formed side by side in N rows, and the sample solution is dropped into the well and dried to arrange the sample.

  The MALDI-TOFMS 201 includes a sample stage 31 on which such a sample plate 20 is placed, and a stage driving unit 32 including a motor or the like. Accordingly, the computer 260 is placed by moving the sample stage 31 in the desired X direction and Y direction by outputting a necessary drive signal to the stage drive unit 32 from a stage control unit 61a described later. The sample plate 20 is moved in a desired direction (X, Y direction).

The visible light source unit 240 includes a halogen lamp 241 that emits visible light for observation, and a reflecting mirror 42. The visible light emitted from the halogen lamp 241 is reflected by the reflecting mirror 42 and then irradiated to a predetermined range in the upper surface of the sample plate 20.
Note that the “predetermined range” is an arbitrary range determined in advance by a designer or the like, and is, for example, an area determined so that a part of the well is not lost.

  The image acquisition device 250 includes a visible light camera 251 that acquires a visible light image (optical image) and a reflecting mirror 52. Then, the visible light reflected on the upper surface of the sample plate 20 is detected by the visible light camera 251 after being reflected by the reflecting mirror 52, whereby a visible light image that is an image in a predetermined range on the upper surface of the sample plate 20 is acquired.

  The laser irradiation unit 5 includes a reflecting mirror 7 and a nitrogen laser 6. According to such a laser irradiation unit 5, 337 nm laser light (ultraviolet light) emitted from the nitrogen laser 6 is emitted toward the sample of the sample plate 20 through the reflecting mirror 7. At this time, the irradiation diameter of the laser light irradiated onto the sample is, for example, a minute diameter of 1 μm to several tens of μm.

  The computer 260 includes a CPU 261, an input unit 62, and a display unit 63. Further, the functions processed by the CPU 261 will be described as a block. The stage control unit 61 a that controls the stage driving unit 32 based on the input signal from the input unit 62, the visible light source unit 240, and the image acquisition device 250. A display control unit 261b that captures the acquired visible light image and displays it on the display unit 63, and an analysis control unit that controls the nitrogen laser 6 and digitizes the sample signal from the ion detector 11 to execute appropriate data processing. 61c.

By the way, in the MALDI-TOFMS 201 described above, a sample solution is dropped into a circular well and dried, but the sample is not necessarily arranged at the center of the well, but at a position shifted from the center of the well. May be placed. In addition, crystals are obtained by drying the mixed sample of the matrix and the analyte, but non-uniform large crystals are generated, and the distribution of the analyte is not necessarily uniform, and the sample is not in the center of the well. Even if they are arranged, the optimum measurement site in the sample (hereinafter referred to as “sweet spot”) is not necessarily the center of the well.
Therefore, when performing analysis using the MALDI mass spectrometer 201, the operator moves the sample stage 31 using the input unit 62 while observing the visible light image displayed on the display unit 63, and is suitable for ionization. The position is searched so as to exist within the laser light irradiation range. Alternatively, the sweet spot may be searched by confirming the measurement data by irradiating the laser beam.
In addition, a MALDI mass spectrometer that identifies a region in a sample where an analysis target substance exists from a captured image using a luminance threshold value and mass spectrum data is also disclosed (see, for example, Patent Document 1). In Patent Document 1, the light source used to acquire a captured image is unknown, and naturally, the type (wavelength range) of observation light and the irradiation angle are not described.

Japanese Patent Laid-Open No. 2014-212068

However, in a conventional mass spectrometer equipped with a MALDI ion source, the operator determines the irradiation position of the laser beam by looking at the visible light image obtained by irradiating visible light, but the state of the matrix is ionized. Therefore, there is a problem that the characteristic position considered to be preferable is not always coincident with the sweet spot where the sample signal is strongly output.
There is also a method of determining the irradiation position suitable for ionization using measurement data and mass spectrum data, but it is very difficult to repeat measurement data and mass spectrum data to determine the irradiation position of laser light. It was a hassle.

The applicant examined a method for searching for an irradiation position of laser light with good ionization efficiency among sample spots arranged on the upper surface of the sample plate 20 in the MALDI ion source.
Nitrogen lasers and solid-state lasers are widely used as light sources for ionization in MALDI. The laser light emitted from the nitrogen laser has a wavelength of 337 nm, and the solid-state laser has a wavelength of 355 nm. In order to be ionized by the laser beam, the matrix needs to absorb the energy of the laser beam. Therefore, a substance having a light absorption band (specific wavelength range) in the vicinity of the wavelength, which is the same as the wavelength of the laser beam, is matrix. As a sample. Therefore, when acquiring an optical image, the matrix (crystal) that is observed by observing the optical image acquired by irradiating light close to the wavelength of the laser beam for ionization or the absorption wavelength range of the matrix It was thought that there is a correlation between the distribution state of and the region with good ionization efficiency.

  That is, the matrix observation apparatus of the present invention is an optical image obtained by detecting a light from a stage on which a sample plate on which a sample is arranged, a light source unit that irradiates the sample plate with light for observation, and light from the sample plate. A matrix observation device comprising: an image acquisition unit that creates a matrix; wherein the sample includes a matrix that absorbs light in a specific wavelength range, and the wavelength range of light emitted from the light source unit is the specific wavelength range. It overlaps with the wavelength range.

  According to the matrix observation apparatus of the present invention, the matrix absorbs or reflects the observation light when the wavelength range of the observation light irradiated on the sample overlaps the absorption wavelength range (specific wavelength range) of the matrix. Therefore, it is possible to accurately observe the distribution of the matrix (crystal) and determine a position suitable for ionization by MALDI. Note that the wavelength range of the observation light preferably coincides exactly with the absorption wavelength range of the matrix, but a part of the wavelength range of the observation light overlaps with a part of the absorption wavelength range of the matrix. Then, since the correlation is obtained between the distribution state of the matrix observed in the present invention and the ionization efficiency, the effect of the present invention can be obtained.

(Means and effects for solving other problems)
In the invention described above, the specific wavelength region may be an ultraviolet region or an infrared region.
In the above invention, the light source unit emits ultraviolet light or infrared light from a direction that is equal to or less than a predetermined angle with respect to a vertical direction, and the image acquisition unit is configured to emit ultraviolet light reflected on the upper surface of the sample plate or It may be an ultraviolet light camera or an infrared light camera that detects an infrared light and creates an optical image.

Here, “below the predetermined angle” is an arbitrary angle determined in advance by a designer or the like, and a deep angle at which ultraviolet light or infrared light reflected by the upper surface of the sample plate is incident on the image acquisition unit. It is.
According to the matrix observation apparatus of the present invention, the irradiated irradiation light is absorbed at a location where the matrix is distributed, and thus is observed as a black shadow in the optical image.

In the above invention, the light source unit emits ultraviolet light or infrared light from a direction that is a predetermined angle or more with respect to a vertical direction, and the image acquisition unit is from a sample disposed on the upper surface of the sample plate. You may make it be a visible light camera which detects the emitted visible light and produces an optical image.
Here, “more than a predetermined angle” is an arbitrary angle determined in advance by a designer or the like, and visible light emitted from a sample disposed on the upper surface of the sample plate is incident on the image acquisition unit. It is a shallow angle.
According to the matrix observation apparatus of the present invention, the ultraviolet light or infrared light absorbed by the matrix is emitted as visible light, so that the distribution location of the matrix in the optical image becomes bright.

In the above invention, the matrix may be DHB or CHCA.
In particular, since DHB does not produce crystals uniformly, the matrix observation apparatus of the present invention is particularly effective.
Furthermore, in the above invention, a plurality of samples may be arranged on the upper surface of the sample plate.

  The MALDI mass spectrometer of the present invention includes a matrix observation apparatus as described above, a laser irradiation unit for irradiating the sample with laser light, and a vaporized sample emitted from the sample irradiated with the laser light. Alternatively, a mass analyzing unit that performs mass analysis of ions is provided. In particular, it is preferable that the wavelength range of the observation light includes the wavelength irradiated by the laser irradiation unit used for ionization.

The block diagram which shows 1st embodiment of this invention. The block diagram which shows 1st embodiment of this invention. The block diagram which shows 2nd embodiment of this invention. The block diagram which shows an example of the conventional MALDI-TOFMS. The block diagram which shows an example of the conventional MALDI-TOFMS.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. The present invention is not limited to the embodiments described below, and includes various modes without departing from the spirit of the present invention.

<First embodiment>
1 and 2 are configuration diagrams showing a MALDI-TOFMS according to the first embodiment of the present invention. In addition, about the thing similar to MALDI-TOFMS201 mentioned above, description is abbreviate | omitted by attaching | subjecting the same code | symbol.
The MALDI-TOFMS 1 includes a mass analysis unit 10, a sample plate 20, a sample stage 31 on which the sample plate 20 is placed, a stage driving unit 32 that moves the sample stage 31, and ultraviolet light for observation on the upper surface of the sample plate 20. An ultraviolet light source unit (light source unit) 40 that irradiates the sample plate 20, an image acquisition device (image acquisition unit) 50 that acquires an image of the upper surface of the sample plate 20, a laser irradiation unit 5 that emits a laser beam for ionization to the sample, And a computer 60 that controls the entire MALDI-TOFMS 1.

The ultraviolet light source unit 40 includes an ultraviolet LED 41 that emits ultraviolet light for observation and a reflecting mirror 42. Then, the ultraviolet light emitted from the ultraviolet LED 41 is reflected by the reflecting mirror 42 and then irradiated to a predetermined range in the upper surface of the sample plate 20 from a direction having a set angle α with respect to the Z direction (vertical direction). It is like that.
The wavelength range of the ultraviolet light source unit 40 for observation is preferably a wavelength range including a wavelength of 337 nm of a nitrogen laser or a wavelength of 355 nm of a solid laser. Alternatively, the center wavelength of the ultraviolet light is preferably close to the center wavelength of the laser light emitted from the laser irradiation unit, and more preferably within ± 20 nm of the center wavelength of the laser light emitted from the laser irradiation unit. . The set angle α is preferably 45 ° or less (a predetermined angle or less).

  The image acquisition device 50 includes an ultraviolet light camera 51 that acquires an ultraviolet light image (optical image) and a reflecting mirror 52. Then, the ultraviolet light reflected in the direction of 45 ° on the upper surface of the sample plate 20 is detected by the ultraviolet light camera 51 after being reflected by the reflecting mirror 52, so that the ultraviolet light that becomes an image in a predetermined range on the upper surface of the sample plate 20 is obtained. An image is acquired.

  The computer 60 includes a CPU 61, an input unit 62, and a display unit 63. Further, the function processed by the CPU 61 will be described as a block. The stage control unit 61a that controls the stage driving unit 32 based on the input signal from the input unit 62, the ultraviolet light source unit 40, and the image acquisition device 50 are used. A display control unit 61b that captures the acquired ultraviolet light image and displays it on the display unit 63, and an analysis control unit that controls the nitrogen laser 6 and digitizes the sample signal from the ion detector 11 to execute appropriate data processing. 61c.

  According to such MALDI-TOFMS1, the operator places a sample in the well on the upper surface of the sample plate 20 by dropping and drying a sample solution obtained by mixing DHB, CHCA, etc. and the substance to be analyzed. Then, after placing the sample plate 20 on the sample stage 31, the sample stage 31 is moved using the input unit 62 while observing the ultraviolet light image before starting the analysis, and a position that seems to be suitable for ionization is searched. The sample is aligned on the upper surface of the sample plate 20 so as to be within the laser light irradiation range, but the ultraviolet light is absorbed at the portion where the matrix is distributed and observed as a black shadow in the ultraviolet light image. Therefore, the distribution of the matrix (crystal) can be observed accurately.

<Second embodiment>
FIG. 3 is a configuration diagram showing a MALDI-TOFMS according to the second embodiment of the present invention. In addition, about the thing similar to MALDI-TOFMS1,201 mentioned above, description is abbreviate | omitted by attaching | subjecting the same code | symbol.
The MALDI-TOFMS 101 includes a mass spectrometer 10, a sample plate 20, a sample stage 31 on which the sample plate 20 is placed, a stage drive unit 32 that moves the sample stage 31, and ultraviolet light for observation on the upper surface of the sample plate 20. An ultraviolet light source unit (light source unit) 140 that irradiates the sample plate 20, an image acquisition device (image acquisition unit) 250 that acquires an image of the upper surface of the sample plate 20, a laser irradiation unit 5 that emits laser light for ionization to the sample, And a computer 160 that controls the entire MALDI-TOFMS 101.

The ultraviolet light source unit 140 includes an ultraviolet light LED 141 that emits ultraviolet light for observation. And the ultraviolet light radiate | emitted from ultraviolet light LED141 is irradiated to the predetermined range in the sample plate 20 upper surface from the direction used as the setting angle (beta) with respect to Z direction (vertical direction).
The wavelength range of the ultraviolet light emitted from the ultraviolet light source 140 is preferably a wavelength range including a wavelength of 337 nm of a nitrogen laser or a wavelength of 355 nm of a solid laser. Furthermore, the center wavelength of the ultraviolet light is preferably close to the center wavelength of the light emitted from the laser irradiation unit, and more preferably within ± 20 nm of the center wavelength of the light emitted from the laser irradiation unit. The set angle β is preferably 45 ° or more (a predetermined angle or more).

  The computer 160 includes a CPU 161, an input unit 62, and a display unit 63. Further, the functions processed by the CPU 161 will be described as a block. The stage control unit 61a that controls the stage drive unit 32 based on the input signal from the input unit 62, the ultraviolet light source unit 140, and the image acquisition device 250. A display control unit 161b that captures the acquired visible light image and displays it on the display unit 63, and an analysis control unit that controls the nitrogen laser 6 and digitizes the sample signal from the ion detector 11 to execute appropriate data processing. 61c.

  According to such MALDI-TOFMS 101, the operator places a sample in the well on the upper surface of the sample plate 20 by dropping and drying a sample solution obtained by mixing DHB, CHCA, etc. and the substance to be analyzed. Then, after placing the sample plate 20 on the sample stage 31, the sample stage 31 is moved using the input unit 62 while observing a visible light image before starting the analysis, and a position that seems to be suitable for ionization is searched. The sample is aligned on the upper surface of the sample plate 20 so as to be within the laser light irradiation range, but ultraviolet light absorbed by the matrix is emitted as visible light, so that in the visible light image. Since the matrix distribution location in FIG. 3 appears bright, the matrix (crystal) distribution can be observed accurately.

<Other embodiments>
(1) In the above-described embodiment, the MALDI-TOFMS 1 and 101 have been described as an example. However, the present invention is a matrix observation apparatus for searching for an irradiation position of a laser beam with good ionization efficiency, and an analysis including a MALDI ion source. It can also be applied to all devices. Further, it can be applied to both vacuum MALDI and atmospheric pressure MALDI.
(2) In the MALDI-TOFMS 1 and 101 described above, the configuration including the laser irradiation unit 5 having the nitrogen laser 6 and the ultraviolet light source units 40 and 140 is shown. However, IR (infrared) that emits infrared light for ionization is shown. It is good also as a structure provided with the laser irradiation part which has a laser, and the infrared light source part which radiate | emits the infrared light for observation.
At this time, the sample is placed by dripping and drying the sample solution in which the matrix of the substance having an absorption band in the infrared region and the analysis target substance are mixed. For example, when urea, DHB, succinic acid, sinapinic acid, or the like is used as a matrix, the ionization state varies depending on the irradiation position, so that the present invention is particularly effective.

  The present invention can be suitably used for a MALDI mass spectrometer and the like.

1 MALDI-TOFMS (MALDI mass spectrometer)
20 Sample plate 31 Sample stage 40 Ultraviolet light source (light source)
50 Image acquisition device (image acquisition unit)

Claims (7)

A stage on which a sample plate on which a sample is placed is mounted;
A light source unit for irradiating the sample plate with light for observation;
A matrix observation device comprising an image acquisition unit that detects light from the sample plate and creates an optical image,
The sample contains a matrix that absorbs light in a specific wavelength range,
The matrix observation apparatus, wherein a wavelength range of light emitted from the light source unit overlaps with the specific wavelength range.   The matrix observation apparatus according to claim 1, wherein the specific wavelength region is an ultraviolet region or an infrared region. The light source unit emits ultraviolet light or infrared light from a direction that is a predetermined angle or less with respect to the vertical direction,
The said image acquisition part is an ultraviolet light camera or infrared light camera which detects the ultraviolet light or infrared light reflected on the upper surface of the said sample plate, and produces an optical image, The Claim 2 characterized by the above-mentioned. Matrix observation device. The light source unit emits ultraviolet light or infrared light from a direction that is a predetermined angle or more with respect to the vertical direction,
The matrix observation apparatus according to claim 2, wherein the image acquisition unit is a visible light camera that detects visible light emitted from a sample disposed on an upper surface of the sample plate and creates an optical image. .   The matrix observation apparatus according to claim 1, wherein the matrix is DHB or CHCA.   The matrix observation apparatus according to any one of claims 1 to 5, wherein a plurality of samples are arranged on an upper surface of the sample plate. The matrix observation apparatus according to any one of claims 1 to 6,
A laser irradiation unit for irradiating the sample with laser light;
A MALDI mass spectrometer comprising: a mass analyzing unit that performs mass analysis on a vaporized sample or ions emitted from the sample irradiated with the laser light.

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