JP2018087760A - Pretreatment method for imaging mass analysis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a pretreatment method for imaging mass analysis which enables even a sample, such as a biological sample, hard to be ionized to be subjected to appropriate imaging mass analysis.SOLUTION: There is provided a pretreatment method for imaging mass analysis by which a sample placed on a sample platform is subjected to acid vapor treatment. The sample is a tissue section containing amyloid β. Acid is formic acid. The acid vapor treatment is carried out at 60°C-80°C.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a preprocessing method for imaging mass spectrometry.

  Imaging mass spectrometry is a technique for examining the distribution of substances having a specific mass by performing mass analysis on each of a plurality of measurement points in a two-dimensional region of a sample such as a biological tissue section. The imaging mass spectrometer includes a sample introduction unit, an ion source, a separation analysis unit, and a data processing unit (Patent Documents 1 and 2).

  Imaging mass spectrometry is mainly aimed at small molecules such as lipids, but imaging mass spectrometry is applied to brain pathological research to elucidate the mechanism of the onset of Alzheimer's disease in Japan, which has reached a super-aging society. There is expected. In addition, the application of this technology to general neurological diseases in general and to biological tissues in general is expected to have a synergistic effect with conventional pathological research.

  However, in imaging mass spectrometry of biological samples such as peptides and proteins, ionization is difficult because the measurement target is a relatively large molecule.

JP 2007-157353 A JP 2014-215043 A

  The present invention has been made in view of such problems, and provides a preprocessing method for imaging mass spectrometry that enables appropriate imaging mass spectrometry even for samples that are difficult to ionize, such as biological samples. With the goal.

  The pretreatment method of imaging mass spectrometry according to the present invention is characterized in that a sample placed on a sample stage is treated with acid vapor.

  According to the present invention, appropriate imaging mass spectrometry is possible even for molecules that are difficult to ionize, such as biological samples.

It is a conceptual diagram of the imaging mass spectrometer concerning this embodiment. It is a photograph figure which shows the state which installed the slide glass on the vial. It is a photograph figure which shows the state which covered the petri dish and covered the incubator. It is a photograph figure of the tissue section by MALDI-TOFMS measurement, the left side is a comparative example which does not perform acid vapor treatment, and the right side is this example which performed acid vapor treatment.

  Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings. However, the embodiments are for facilitating understanding of the principle of the present invention, and the scope of the present invention is as follows. The present invention is not limited to the embodiments, and other embodiments in which those skilled in the art appropriately replace the configurations of the following embodiments are also included in the scope of the present invention.

  As shown in FIG. 1, an imaging mass spectrometer 900 includes a sample introduction unit 100, an ion source 200 such as MALDI (Matrix Assisted Laser Desorption Ionization), and a time-of-flight mass spectrometer TOF-MS (Time-MS). A separation analysis unit 300 that is of-flight mass spectrometry) and a data processing unit 400 are included. A sample is introduced into the apparatus by the sample introduction unit 100, the sample is ionized by the ion source 200, ions are separated and detected by the difference in mass by the separation analysis unit 300, and data processing is performed by the data processing unit 400.

(1) Pretreatment step The sample is a non-ionizable sample having a property of being difficult to ionize, for example, a biological tissue section. A biological tissue section is a tissue section containing protein aggregates. The protein forming the aggregate is, for example, amyloid β, tau, α-synuclein, Huntington, TDP-43 (TAR DNA-binding protein 43 kDa).

  Biological tissue sections are prepared in the same way as the section preparation method for microscopes, but the components of OCT (Optimal Cutting Temperature) compound used when preparing frozen tissue sections contain a synthetic polymer, which prevents ionization. Therefore, a tissue section is prepared without embedding with a compound. The prepared tissue slices prevent the ionization of lipids in the tissue, so that the lipids are washed away with ethanol water, fixed in tissue with Carnoy's solution (anhydrous ethanol: chloroform: (ice) acetic acid = 6: 3: 1), and vacuumed. After being dried, it is placed on the sample stage.

  The sample stage is, for example, a slide glass. Since ions generated by the ion source 200 are introduced into the separation / analysis unit 300 by a voltage gradient, a voltage is applied to the slide glass holding the sample, and the slide glass is preferably an ITO-coated slide glass. .

  In the invention according to this embodiment, the sample placed on the sample stage is treated with acid vapor as a pretreatment for ionization of the sample before the sample to be measured is introduced into the apparatus by the sample introduction unit 100. The

  The acid vapor treatment is a treatment in which a sample placed on a sample stage is placed in a sealed container, a gaseous acid volatilized by heat is filled in the sealed container, and the sample is exposed to a gaseous acid. Although not necessarily bound by such theory, ionization is promoted by reducing the pH of the sample and increasing the basicity of the gaseous acid by the acid vapor treatment.

  The acid used for the acid vapor treatment is not particularly limited as long as it can accelerate the ionization of the sample. For example, formic acid, sulfuric acid, acetic acid, citric acid and the like are preferable, but formic acid is preferable. The temperature of the acid vapor treatment is not particularly limited as long as the vaporized gaseous acid can be filled in the sealed container, but for example, 50 ° C to 90 ° C, preferably 60 ° C to 80 ° C. It is possible to do in. The temperature of the acid vapor treatment is, for example, 1 minute to 30 minutes, preferably 5 minutes to 10 minutes.

(2) Sample introduction step Next, a uniform thin-film matrix is applied to the acid vapor-treated sample by, for example, an airbrush, and crystallized by drying. The matrix is not particularly limited as long as it can mediate laser energy transfer. For example, sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid), CHCA (α-cyano-4-hydroxy) Hydroxycinnamic acid), ferulic acid (trans-4-hydroxy-3-methoxycinnamic acid), gentisic acid (2,5-dihydroxybenzoic acid), HPA (3-hydroxypicolinic acid), disranol (1,8- Dihydroxy-9,10-dihydroanthracen-9-one) can be used, and sinapinic acid is preferred.

  The sample introduction unit 100 includes, for example, an ITO-coated slide glass and a slide glass adapter that is a support for supporting the slide glass. The sample introduction unit 100 introduces a sample into the apparatus.

(3) Ionization step The ion source 200 can be appropriately selected depending on the physical properties of the sample, and is, for example, MALDI. MALDI is an ionization method suitable for analysis of biopolymers such as proteins. The MALDI ionization method is a very soft ionization because the matrix-derived H ⁺ (protons) generate pseudo-molecular ions added to the sample, and the spectrum is easy to interpret because it is difficult to generate multivalent ions. It is possible to obtain Moreover, even a hardly soluble sample can be ionized by mixing with a matrix in a solid phase. The laser of the ionization source is, for example, a nitrogen laser or a YAG laser.

  The ion source is not limited to MALDI, and for example, ESI, EI, FAB or the like is also possible.

(4) Separation and analysis process In the time-of-flight mass spectrometer TOF-MS, the material ionized by the laser receives energy from a certain electric field and begins to fly. In the drift region, a small mass has a high speed and a large mass has a slow speed.Therefore, a difference occurs in the arrival time at the detector, and the mass spectrum is obtained by measuring the time difference and converting it to a mass. It is done.

  The separation / analysis unit is not limited to the TOF, and may be, for example, IT, Sector, Q-pole, or the like.

(5) Data processing step Data analysis is performed on the acquired mass spectrum with software dedicated to imaging, and visualized as an image.

  An ITO-coated slide glass (Glass Slides for MALDI Imaging: Part No. 237001) and a slide glass adapter target (MTP slide-adapter II: Part No. 235380) were used. MALDI was used as the ion source. The separation analysis unit used TOF-MS. The software used in the data processing section was Bruker Daltonics flexImaging.

  The sample used was a human Alzheimer's disease donor tissue section. The sample was washed with 70% ethanol for 30 seconds, then washed with 100% ethanol for 30 seconds to remove the lipid, and then 3 times with Carnoy's solution (anhydrous ethanol: chloroform: (ice) acetic acid = 6: 3: 1). The tissue was fixed by partial treatment, then washed with 100% ethanol for 30 seconds, washed with 0.1% TFA for 30 seconds, vacuum-dried, and placed on an ITO-coated slide glass.

  A petri dish with a lid was used as an airtight container used for acid vapor treatment. A plurality of glass vials were placed in the petri dish to raise the bottom and heated to 60 ° C. Next, 5 ml of 100% formic acid was placed in a petri dish and heated to 60 ° C. After the temperature was fixed, the sample placed on the ITO-coated slide glass was placed on the vial. This state is shown in FIG. Next, the petri dish was covered and the incubator was covered, and acid vapor treatment was performed in a formic acid gas phase at 60 ° C. for 5 minutes. This state is shown in FIG.

  Next, two sets of air brushes were used to alternately apply a matrix and a hydration solution to the acid vapor-treated sample. In application with an air brush, care was taken to quantitatively control the particle size and the amount of scattering discharged from the spray. The matrix was prepared by dissolving 200 mg sinapinic acid in acetonitrile. Hydration solution was prepared with 90% methanol and 2% formic acid.

  The results of MALDI-TOFMS measurement on tissue sections are shown in FIG. The left is an imaging image of human brain amyloid β1-42 that was not subjected to acid vapor treatment and the right was subjected to acid vapor treatment. Obviously, the acid vapor treatment made it possible to visualize the distribution of the peptide in the brain parenchyma.

  It can be used for imaging mass spectrometry of biological tissue sections.

DESCRIPTION OF SYMBOLS 100: Sample introduction part 200: Ion source 300: Separation analysis part 400: Data processing part 900: Imaging mass spectrometer

Claims (6)

  A pretreatment method for imaging mass spectrometry, comprising subjecting a sample placed on a sample stage to acid vapor treatment.   The pretreatment method for imaging mass spectrometry according to claim 1, wherein the sample is hardly ionizable.   The pretreatment method for imaging mass spectrometry according to claim 1 or 2, wherein the sample is a tissue section containing protein aggregates.   The pretreatment method for imaging mass spectrometry according to any one of claims 1 to 3, wherein the sample is a tissue section containing amyloid β.   The pretreatment method of imaging mass spectrometry according to any one of claims 1 to 4, wherein the acid is formic acid.   The pretreatment method for imaging mass spectrometry according to claim 1, wherein the acid vapor treatment is performed at 60 ° C. to 80 ° C. 6.