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WO2009054078A1 - Procédé de préparation d'un échantillon pour une analyse de masse par élimination/ionisation laser assistée par matrice utilisant une technique de microdistribution, et procédé d'analyse de masse par élimination/ionisation laser assistée par matrice - Google Patents

Procédé de préparation d'un échantillon pour une analyse de masse par élimination/ionisation laser assistée par matrice utilisant une technique de microdistribution, et procédé d'analyse de masse par élimination/ionisation laser assistée par matrice Download PDF

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Publication number
WO2009054078A1
WO2009054078A1 PCT/JP2007/071353 JP2007071353W WO2009054078A1 WO 2009054078 A1 WO2009054078 A1 WO 2009054078A1 JP 2007071353 W JP2007071353 W JP 2007071353W WO 2009054078 A1 WO2009054078 A1 WO 2009054078A1
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WO
WIPO (PCT)
Prior art keywords
matrix
sample
mass spectrometry
assisted laser
ionization mass
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2007/071353
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English (en)
Japanese (ja)
Inventor
Tsuyoshi Nakanishi
Masaru Furuta
Kiyohiro Sugiyama
Naoya Endo
Rintaro Yamamoto
Eiji Ando
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Shimadzu Corp
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Shimadzu Corp
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Priority to JP2009537879A priority Critical patent/JP5012904B2/ja
Priority to PCT/JP2007/071353 priority patent/WO2009054078A1/fr
Publication of WO2009054078A1 publication Critical patent/WO2009054078A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G01N27/626—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using heat to ionise a gas
    • G01N27/628—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using heat to ionise a gas and a beam of energy, e.g. laser enhanced ionisation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
    • H01J49/0409—Sample holders or containers
    • H01J49/0418—Sample holders or containers for laser desorption, e.g. matrix-assisted laser desorption/ionisation [MALDI] plates or surface enhanced laser desorption/ionisation [SELDI] plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
    • G01N2001/282—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on with mapping; Identification of areas; Spatial correlated pattern
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
    • G01N2001/2826—Collecting by adsorption or absorption

Definitions

  • the present invention relates to a method for preparing a matrix-assisted laser desorption / ionization mass spectrometry sample and a matrix-assisted laser desorption / ionization mass spectrometry method using a micro-dispensing technique.
  • the present invention belongs to the field of matrix-assisted laser desorption / ionization (MALDI) mass spectrometry.
  • MALDI matrix-assisted laser desorption / ionization
  • the present invention also belongs to biological fields such as cell biology, biochemistry, and molecular biology, and medical fields such as clinical science, diagnostics, and pathology.
  • M A L D I method has established a soft ionization technique for living organisms, and research using M A L D I mass spectrometry is rapidly developing in various technical fields including the proteomics field.
  • Patent Document 1 Japanese Patent Laid-Open No. 2003-98154
  • Patent Document 2 Japanese Patent Laid-Open No. 2005-283123.
  • techniques for directly measuring specimens such as cells and biological tissues have been developed and developed. For example, by directly measuring a biological tissue specimen, the biological molecules (eg, lipids, peptides, proteins, etc.) contained in the biological tissue specimen are detected while maintaining the position information in the living body. Is possible.
  • MALD I mass spectrometry direct measurement of biomolecules contained in biological tissue specimens has also been performed using the MA LD I mass spectrometer. In such a technique, for example, a thin section of a biological tissue specimen is used as a sample, a matrix solution is supplied to a plurality of positions on the thin section, and a mass spectrum is obtained for each position.
  • Examples of supplying the matrix solution include spraying of the matrix solution using a spray technique, and a minute dispensing of the matrix solution using an ink jet technique using a piezo element or the like.
  • Patent Document 3 Japanese Patent Application Publication No. 2004-347594 and Non-Patent Document 1: Anal ytica! Chemistry (1997) vol.69, pp.4751-4760, dispensing minute amounts of matrix using ink jet technology.
  • an acoustic microdispenser One example is micro-dispensing using an acoustic microdispenser.
  • Non-Patent Document 2 Analytical Chemistry (2006) vol.78, pp.6448-6456 reports that MALD I mass spectrometry was performed directly on living tissues using an acoustic microdispenser.
  • MALD I mass spectrometry is also performed directly on the target substance on the membrane on which the target substance is immobilized (plotted). For example, a measurement target substance is plotted on a membrane, an enzyme treatment solution and a matrix solution are dispensed in a minute amount using ink jet technology, and then the target substance is directly analyzed on the membrane (for example, Patent Document 4: International Publication No. 98-47006, pamphlet, Patent Document 5: Japanese Special Table 2001-521623, and Non-Patent Document 3: Molecular & Cellular Proteomics (2002) vol.1, pp.490-499). Inkjet technology has been widely used in the printing industry.
  • coloring components selected from dyes or pigments, branched alcohols having a mono- or divalent carbon number of 5 to 10 carbon atoms.
  • An aqueous ink composition containing at least a acetylene glycol surfactant, a humectant, a resin emulsion, and water is used (see, for example, Patent Document 6: Japanese Patent Laid-Open No. 2006-257361).
  • Patent Document 1 Japanese Unexamined Patent Publication No. 2003-98154
  • Patent Document 2 Japanese Laid-Open Patent Publication No. 2005-283123
  • Patent Document 3 Japanese Unexamined Patent Publication No. 2004-347594
  • Patent Document 4 Pamphlet of International Publication No. 98 47006
  • Patent Document 5 Japan Special Table 2001-521623 Publication
  • Patent Document 6 Japanese Unexamined Patent Publication No. 2006-257361
  • Non-Patent Document 1 Analytical Chemistrv (1997) vol.69, pp.4751-4760
  • Non-Patent Document 2 Analytical Chemistry (2006) vol. 78, pp. 6448-6456
  • Non-Patent Document 3 Molecular & Cellular Proteomics (2002) vol. 1, pp. 490-499
  • the matrix solvent is an aqueous solution of acetonitrile / trifluoroacetic acid (as a specific example, 50 (v / v)% acetonitrile 0.1
  • acetonitrile / trifluoroacetic acid as a specific example, 50 (v / v)% acetonitrile 0.1
  • a matrix solution can be prepared by dissolving sinapinic acid as a matrix in a solvent having such a composition, for example, if it can analyze a wide mass range.
  • matrices other than sinapinic acid are used for direct MALDI-MS analysis on tissue sections.
  • 2,5-dihydroxybenzoic acid which is less hydrophobic than sinapinic acid, is used as the matrix.
  • 2,5 nidihydroxybenzoic acid is used as the matrix.
  • Acetonitrile monotrifluoroacetic acid aqueous solution is used.
  • 2,5-dihydroxybenzoic acid is less useful than sinapinic acid in that the mass value range that enables MS analysis is limited to the low molecular weight side.
  • conventional matrix solutions tend to limit the number of matrices that can be usefully used, and as a result, there is a problem that the mass value range that allows MS analysis is limited. Therefore, there is a need for a method that enables stable minute dispensing even when using a matrix that can analyze a wider range of mass values, such as sinapinic acid.
  • the present inventors need to overcome the difficulty of conventional micro-dispensing due to viscosity and surface tension. I focused on that.
  • a matrix solvent having the same composition as before has low viscosity and surface tension. Such physical properties are inappropriate for stable minute dispensing using a piezo element or the like.
  • the mechanical properties of such matrix solutions are not paid attention to, those having the same composition as before have long been used.
  • the ink composition has mechanical properties for realizing stable continuous dispensing. Attachment to grant Additives are being studied. Such additives impart favorable physical properties to the ink composition, but cannot be used in the matrix solution.
  • An object of the present invention is a method for preparing a sample for matrix-assisted laser desorption / ionization mass spectrometry using a micro-dispensing technique, which enables stable micro-dispensing to be performed continuously and a wide range of matrices. It is to provide a method applicable to this.
  • Another object of the present invention is to provide a matrix-assisted laser desorption / ionization mass spectrometry capable of measuring a wide mass range and analyzing with high sensitivity and accuracy.
  • the present inventors have found that the object of the present invention can be achieved by imparting appropriate viscosity and surface tension to the matrix solution, and have completed the present invention.
  • the present invention includes the following inventions.
  • a matrix solution that should be used for mass spectrometry by matrix-assisted laser desorption / ionization, with a viscosity of 0.85 m Pa's (23 ° C) or higher and 25-30 m NZm Disperse the matrix solution with a surface tension of (23 ° C) into a sample to be measured using a minute amount dispensing method that dispenses 1 f I to obtain a sample for mass spectrometry
  • the upper limit of the viscosity is not particularly limited.
  • the maximum allowable viscosity that can be dispensed by a microdispensing device having an ink jet mechanism is set to 50 mPas. (23 ° C).
  • the matrix solution has, as a solvent, a linear or branched alcohol having 3 to 10 carbon atoms, and preparation of a sample for matrix-assisted laser single desorption ionization mass spectrometry according to (1) Law.
  • Alcohol having a straight chain or branched chain having 3 to 10 carbon atoms is 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-1 monopropanol, t- Butanol, 1-pentanol, 2-pentanol, 2, 2-dimethyl-1-propanol, 2-methyl-1-butanol, 1-hexanol, 2-hexanol, 3,3-dimethyl-2-butanol, 1 1-Heptanol, 2-Heptanol, 2, 3-Dimethylol 1-Pentanol, 1-Octanol, 2-Octanol, 2, 2-Dimethizole, 3- Hexanol, 2, 5-Dimethyl 2-hexanol , 1-nonanol, 2-nonanol, 3-nonanol, 2,3-dimethyl-3-heptanol, 3,6-dimethyl-3-heptanol, 1-decanol, 3-decan
  • Matrix is ⁇ -cyano 4-hydroxycynamic acid, 2,5-dihydroxybenzoic acid, 2- (4-hydroxyphenylazo) benzoic acid, sinapinic acid, ferulic acid, 3-hydroxypicolinic acid, and The method for preparing a sample for matrix-assisted laser desorption / ionization mass spectrometry according to any one of (1) to (4), which is selected from the group consisting of dithranol.
  • the sample to be subjected to the measurement includes a biomolecule selected from the group consisting of peptides, sugar chains, lipids, nucleic acids, derivatives thereof, and complexes, (1) to (5) Preparation of Sampsole for matrix-assisted laser desorption / ionization mass spectrometry.
  • the sample immobilized on the support is prepared by a step including dropping a biomolecule preparation onto the support and drying, or
  • a matrix-assisted laser desorption in which the sample for mass spectrometry obtained by the method according to any one of (1) to (13) is subjected to measurement using a matrix-assisted laser desorption / ionization mass spectrometer. Separation mass spectrometry.
  • a method for preparing a sample for matrix-assisted laser desorption / ionization mass spectrometry using a microdispensing means which can continuously perform stable microdispensing and can be applied to a wide range of matrices. Applicable methods can be provided.
  • Figure 1 shows the matrix that can be used in the matrix solution of the present invention in Experimental Example 1. After the various matrix solvents (a) to G) containing the solvent (f) (g) (h) (i) G) are dropped on the stainless steel plate, the spread of each droplet is reduced. It is a photograph shown.
  • Figure 2 shows an image of water sensitive paper and a magnified image (zoomed image 1 and zoomed image 2) after continuous dispensing of the previous matrix solution (A) onto water sensitive paper in Experimental Example 4. ).
  • FIG. 3 is an image of water sensitive paper after continuous dispensing of the matrix solution (G) of the present invention onto water sensitive paper in Experimental Example 5.
  • FIG. 4 is a graph showing a state in which a conventional matrix solution (A) and the matrix solutions (K), (J) and (I) of the present invention were formed on a frozen section of mouse brain tissue in Example 1. This is a picture of the matrix spot.
  • Fig. 5 shows tissue MS analysis of frozen sections of mouse brain tissue in Example 1 using the conventional matrix solution (A) and the matrix solutions (K), (J) and (I) of the present invention. It is a mass spectrum obtained by going.
  • FIG. 6 is a diagram showing a method for measuring the viscosities of the matrix solvent and the matrix solution in the experimental examples and examples.
  • FIG. 7 is a diagram showing a method for measuring the surface tension of the matrix solvent and the matrix solution in the experimental examples and examples.
  • the matrix solution has a viscosity of 0.85 mPa ⁇ s (23 ° C) or more and a surface tension of 25 to 30 mN Zm (23 ° C).
  • the upper limit of the viscosity is not particularly limited. From the viewpoint that the present invention uses a microdispensing means, for example, the maximum allowable viscosity that can be dispensed by a microdispensing apparatus equipped with an ink jet mechanism as a microdispensing means is 50 m Pa ⁇ s (2 3 ° C).
  • the viscosity of the matrix solution and the surface tension are linked to some extent, that is, the surface tension decreases if the viscosity increases. There is a trend. From this viewpoint, the viscosity at which the surface tension is the above 25 m N m can be set as a preferable upper limit value. Specifically, it depends on the type and amount of the additive.
  • the diffusion of the matrix solution increases.
  • the dispensed matrix solution is spread over the measurement sample, or the measurement sample and the matrix are evenly distributed. This is inadequate for MS analysis because it inhibits crystallization formation.
  • the viscosity will be too low. If the viscosity becomes too small, dispensing becomes unstable, causing matrix crystals to precipitate at the liquid discharge port in the dispensing means, and in the extreme case, the discharge port will become completely jammed.
  • the matrix solution of the present invention is assumed to be composed of a matrix substance as a solute and components other than the matrix as a solvent (hereinafter sometimes referred to as a matrix solvent).
  • a matrix solvent a solvent that is positioned as one of the components constituting the solvent.
  • the matrix material that can be used in the present invention (hereinafter simply referred to as a matrix) is not particularly limited. That is, in the method of the present invention, for example, undesired precipitation of the matrix at the liquid discharge port of the dispensing nozzle of the ink jet mechanism can be suppressed, and stable dispensing can be continuously performed. Even a highly precipitated matrix that could not be stably dispensed when used in the present invention can be used effectively.
  • the matrix can be selected from a wide range according to the biological material to be measured. Therefore, specific examples of the matrix are not particularly limited. For example, -Cyano 4-hydroxycain acid, 2,5-dihydroxybenzoic acid, 2- (4-hydroxyphenylazo) benzoic acid , Sinapinic acid, ferulic acid, 3-hydroxypicolinic acid, dithranol, and glyceose.
  • sinapinic acid having a wide mass range to be measured can be used effectively.
  • the concentration of the matrix in the matrix solution is not particularly limited, and can be appropriately determined by those skilled in the art according to the type of matrix. For example, it can be used at a concentration of 0.1 mg / m I to saturation.
  • the matrix material itself is a high viscosity (eg, glycerol, etc.)
  • its concentration can be appropriately determined by those skilled in the art by considering the viscosity of the matrix material itself and the viscosity of the entire matrix solution. .
  • an additive for imparting the physical properties described in item 1 above is usually included in the matrix solution. Since the matrix solution of the present invention has a viscosity higher than that of the conventional matrix solution, the additive is usually a liquid having a certain viscosity per se and added to the conventional matrix solution. According to the method, one that can increase the viscosity of the matrix solution and does not hinder the mass spectrometry itself (that is, can obtain the same signal information as before) is appropriately selected by those skilled in the art.
  • an alcohol having a linear or branched chain having 3 to 3 carbon atoms can be used as the additive.
  • Typical examples of such an alcohol include 1 propanol, 2 — Propanol, 1-butanol, 2-butanol, 2-methyl-11 propanol, t-butanol, 1-pentanol, 2-pentanol, 2, 2-dimethyl-1, 1-propanol, 2-methyl-1-1-butanol, 1 Hexanol, 2-hexanol, 3,3-dimethyl-2-butanol, 1-heptanol, 2-heptanol, 2,3-dimethyl-1-pentanol, 1-octanol, 2-octanol, 2,2-dimethyl-3 —Hexanol, 2,5-dimethyl-2-hexanol, 1-nonanol, 2-nonanol, 3-nonanol, 2,3-dimethyl-3-heptanol, 3, 6 Dimethyl 3
  • the additive is preferably an alcohol having a straight or branched chain having 3 to 6 carbon atoms, an alcohol having a straight or branched chain having 3 to 5 carbon atoms, and further having 3 to 3 carbon atoms.
  • Alcohols having 4 straight or branched chains are preferred.
  • the solubility in water is 0.1 g 1 OO m I (20 ° C) to infinity, and moreover 1 g Z 1 OO m I (20 ° C) ⁇ Infinite alcohol is preferred.
  • these alcohols can be used singly or in combination.
  • a person skilled in the art can appropriately adjust the viscosity and surface tension to desired values by adding an appropriate amount of additives at the stage of solvent preparation, and then dissolve the matrix in the prepared solvent.
  • a highly viscous matrix material when used, it itself affects the viscosity of the matrix solution. Therefore, in this case, the person skilled in the art uses the theoretical value of the viscosity inherent in the matrix substance in addition to the theoretical value of the viscosity inherent in the additive, so that the matrix solution as a whole, not the solvent, is used. The viscosity can be adjusted as appropriate.
  • the specific viscosities contained in the matrix solution vary depending on the type of substance selected as the additive because the inherent viscosity of the substance selected as the additive varies, but as a guideline, It can be 5-30 (v / v)% or 5-20 (v / v) o / 0 . Below the above range, the viscosity tends to be too small. If the viscosity is too small, matrix crystals may precipitate at the liquid discharge port of the dispensing means, and in extreme cases, the discharge port may be completely clogged. Lack. Above the above range, the surface tension tends to decrease excessively. If the surface tension is too low, the dispensed matrix solution is likely to spread on the measurement sample, or it may be difficult to form uniform crystallization between the measurement sample and the matrix, which may be inappropriate for MS analysis. .
  • solvents in conventional matrix solutions may be used.
  • it can be selected from the group consisting of water, acetonitrile, trifluoroacetic acid, formic acid, methanol, ethanol, aceton, and ⁇ trahydrofuran.
  • other solvent constituents and their compositions can be determined appropriately by those skilled in the art.
  • the solubility of the above-mentioned additives in the other solvent constituents can be considered.
  • at least water, acetonitrile, and trifluoroacetic acid are used as other solvent constituents. That is, the matrix solvent preferably contains the additive, water, acetonitrile, and trifluoroacetic acid.
  • Trifluoroacetic acid is often used in an amount of about 0.1 to 0.5 (v / v)% in a solvent.
  • the amount of acetonitrile is not particularly limited and various amounts are acceptable. For example, it can be used in such an amount that the total amount of additives and acetonitrile is 30 to 80 (V / V) o / o in the solvent.
  • matrix solvents include 5 to 30 (v / v)% C3-C10 linear or molecular alcohol, 20 to 45 (v / v)% acetonitrile, and 0 . 1 (v / v)% trif
  • An aqueous solution containing chloroacetic acid can be mentioned.
  • the sample to be subjected to the measurement in the present invention includes any sample that can be handled in a study using matrix-assisted laser desorption / ionization mass spectrometry (MALDI) mass spectrometry, and is not limited at all.
  • MALDI matrix-assisted laser desorption / ionization mass spectrometry
  • a sample to be subjected to measurement usually contains a biomolecule as an object to be detected.
  • the biomolecule to be detected is not particularly limited. Examples include peptides, sugar chains, lipids, nucleic acids, derivatives and complexes thereof. Peptides include oligopeptides and proteins. Nucleic acids include DNA and RNA. Derivatives include fusion peptides, chemically modified peptides, and post-translationally modified peptides. The complex includes glycopeptides and glycolipids.
  • detection targets are included in the specimens shown in item 3-1, and in preparations containing biomolecules shown in item 3-2.
  • the specimen includes those containing specimens such as biological cells and tissues, that is, cell specimens and tissue specimens.
  • the specimen is usually provided as embedded in a suitable embedding medium.
  • the specimen may be derived from any organism. In the case of animals, amphibians, reptiles, birds and mammals are widely accepted, and those derived from mammals are particularly preferable. Among these, those derived from mice and humans are more preferable.
  • a tissue specimen it is usually provided as a slice specimen that retains the structure in vivo.
  • Such specimens include specimens that are research targets in any analysis including morphological, immunohistochemical and enzymatic histochemical analyses.
  • the specimen is a pathological specimen
  • the disease affecting the living body may be cancer, Alzheimer's disease, Parkinson's disease, ischemic brain disease, or ischemic heart disease.
  • the specimen described above may be a specimen for analyzing pharmacokinetics.
  • Specimens for analyzing pharmacokinetics are specimens for verifying the potential as drugs from the viewpoint of pharmacokinetics (absorption, distribution, metabolism, and excretion). Specifically, drugs were administered. It is a specimen derived from a living body. In such specimen analysis, for example, the presence of a drug that has reached the target site is examined by detecting a biomolecule to which the drug binds.
  • the sample embedding medium is not particularly limited, and examples thereof include water, paraffin, celloidin, carbowax, gelatin, albumin, agarose, epoxy resin, polyester resin, and water-soluble resin such as glycol methacrylate. It is done.
  • a frozen section using water as an embedding medium and a paraffin section using paraffin as an embedding medium are often used as section samples.
  • Frozen sections may be subjected to the present invention by returning to room temperature, but other section specimens may be appropriately subjected to appropriate processing to enable mass spectrometry (eg, processing to remove embedding media). And can be used as an exposed specimen. Exposure means that the embedding medium is eluted from the specimen in which the biological sample is embedded in the embedding medium, and the biological sample is exposed. A section specimen of a biological sample in such a state can be used in the present invention.
  • the localization of the biomolecule to be subjected to mass spectrometry can be morphologically verified as the same as in the living body. That is, the target biomolecule can be detected by mass spectrometry while maintaining the position information in the living body, and further mass spectrometry imaging is possible.
  • Preparations containing biomolecules include any sample other than the above specimens that can be handled in studies using MALDI mass spectrometry. Specific examples include, but are not limited to, collected body fluids, cell extracts, cell-free synthetic products, enzyme-treated products, synthetic peptides, nucleic acid amplification products, synthetic nucleic acids, and purified products thereof. None.
  • the purified product includes a purification process well known to those skilled in the art and a product developed by electrophoresis. When electrophoretic treatment is performed, the preparation has a form contained in a medium such as a gel, but in other cases, the preparation has a form of a solution or a suspension. Yes.
  • Samples to be measured such as the above preparations containing specimens and biomolecules, are either fixed directly on the membrane (specifically, transcription, etc.) and then fixed on a suitable support. (Specifically, transferred or pasted) and used for measurement.
  • the support is not particularly limited.
  • the material of the support include glass, paper, wood, metal, magnetic particles, polymer, and a combination of these.
  • Polymers include synthetic resins and polysaccharides.
  • Membranes can also be positioned as an embodiment of the polymer support.
  • the membrane material include synthetic resins such as polyvinylidene difluoride (PVDF), nitrocellulose, polyamide, and polyethylene, and derivatives thereof.
  • the polyamide include nylon. Transfer to the membrane has the advantage that the sample can be stored for a long time.
  • a plurality of types of supports having different materials can be used.
  • the present invention is directed to mass spectrometry Therefore, at least a support including a material having electrical conductivity is used.
  • an electrically conductive support for example, a sample plate for mass spectrometry, which is a metal support, is often used.
  • the present invention is not limited to this, and a support coated with an electrically conductive material is also used. be able to.
  • the material of the coated support is not particularly limited, and specific examples include those exemplified above.
  • ITO indium tinoxide
  • More specific examples of the support coated with an electrically conductive material include indium tin oxide coated slide glass and indium tin oxide coated sheet.
  • the method for immobilization can be appropriately determined by those skilled in the art and includes immobilization by transfer or pasting.
  • Examples of the transfer method include electrical transfer, thermal transfer, and vacuum transfer.
  • a conductive double-sided adhesive tape can be used for attachment onto the support.
  • it is fixed by attaching it directly to the support, or by transferring it onto the membrane and then attaching or transferring the biomolecule transferred onto the membrane onto the solid support. It can be performed.
  • immobilization can be performed by dropping the preparation directly on a support.
  • the dispensing technique described in Item 6 below can be used.
  • An efficient analysis can be performed by arranging a large number of spots on the support.
  • the sample to be subjected to the measurement may have been appropriately processed for detection by mass spectrometry.
  • Such treatment is not particularly limited, and examples thereof include enzyme degradation treatment and treatment for forming an immunological complex. These processes are appropriately performed by those skilled in the art depending on the type of molecule to be detected.
  • the matrix solutions shown in items 1 and 2 above are dispensed to the samples to be measured shown in items 3 and 5 above using a micro-dispensing means.
  • micro-dispensing refers to dispensing micro-droplets from 1 f I to "! I.
  • micro-dispensing means manual (for example, micropipetter) and automatic (for example, automatic dispensing device) Any device or apparatus capable of forming such a minute droplet can be used without any particular limitation, and the present invention can stabilize a minute dispensing of a matrix solution. Therefore, it is particularly useful when using an automatic dispensing device that performs continuous dispensing as a minute amount dispensing means, and also useful when using a device that dispenses a smaller amount of liquid droplets.
  • an apparatus capable of performing minute dispensing those equipped with various discharge mechanisms can be used without particular limitation.
  • the ink jet mechanism include an ink jet mechanism including a liquid storage container having a liquid discharge port and a piezo element provided in the storage container, and an ink jet mechanism including a liquid storage container having a liquid discharge port and a heating mechanism of the storage container.
  • an ink jet mechanism is used as a minute amount dispensing means, it is possible to dispense a minute amount of droplets of about 50 pI to 1 ⁇ I.
  • Examples of such an apparatus include International Publication No. 98 No. 0 4 7 0 06 Pamphlet, Japanese Patent Application Laid-Open No.
  • a mechanism capable of forming a very small amount of ink jet droplets for example, a droplet forming mechanism using electrostatic attraction force may be used.
  • An example of such a device is the pamphlet of International Publication No. 0 3/0 2 0 4 1 8.
  • the present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.
  • the amount expressed in% is based on volume unless otherwise specified.
  • acetonitrile is abbreviated as MeCN
  • trifluoroacetic acid as TFA
  • butanol as BuOH
  • methanol as MeOH
  • propanol as PrOH
  • sinapinic acid as SA.
  • the horizontal axis represents mass / charge (Mass / Charge)
  • the vertical axis represents relative intensity (% lnt).
  • the surface tension was measured using an automatic contact angle meter “Drop Shape Analysis System” DSA10 (manufactured by KRUSS).
  • the liquid sample was collected in a syringe, and a pendant drop was formed at the tip of the injection needle as shown in FIG. From the contour shape and density of the hanging drop, by image processing, Equation 1 (where g is the gravitational acceleration, p is the density of the liquid sample, de is the maximum diameter of the hanging drop, and ds is de above the lower end of the hanging drop.
  • the surface tension was calculated by fitting the diameter of the hanging drop, 1 / H at the position of (1), to the correction term calculated from ds / de. Specifically, as shown in FIG. 7, after automatically measuring the de of the hanging drop hanging from the tip of the injection needle, ds was calculated from the value, and the surface tension ⁇ was calculated from the value by Equation 1. By this method, the surface tension ⁇ was calculated 10 times or more, and the value obtained by averaging them was used as the final surface tension value.
  • the addition of alcohol does not exceed a certain amount (that is, 20% for 2-BuOH and 30% for t-BuOH) to prevent diffusion of the matrix solution and reduce sensitivity in MS analysis. It turns out that it suppresses.
  • the upper limit of addition differs depending on the alcohol, This seems to be due to the difference in surface tension. Therefore, in order to investigate in more detail the characteristics of the solvent for the matrix added with alcohol, the viscosity and surface tension of each solution were confirmed in Experimental Example 2 below.
  • Matrix solvents with various compositions were prepared, and their respective viscosities and surface tensions were measured. Viscosity was measured using V Type V former RO VISCOMETER (A & D company ⁇ td) when the matrix solvent was stable at 23 ° C. Table 1 below shows the viscosity and surface tension in examples of matrix solvents using alcohols having 3 or 4 carbon atoms and examples of matrix solvents having other compositions.
  • Viscosity Surface tension Matrix solvent (Aqueous solution) (mPa-s) (mN / m)
  • a matrix solvent (a) comprising 50% MeCN and 0.1% TFA aqueous solution was prepared.
  • sinapinic acid (SA) was dissolved at a commonly used concentration of 5 mg / mL to prepare a matrix solution (A).
  • the viscosity and surface tension of the matrix solvent (a) and the matrix solution (A) were measured. The results are shown in Table 2.
  • the preparation of the matrix solution of the present invention having the desired viscosity and surface tension is not limited to the method in which the entire matrix solution is strictly adjusted to the desired viscosity and surface tension, but the matrix solvent is previously prepared. It is permissible to adjust the viscosity and surface tension to the desired values and then dissolve the matrix substance in the matrix solvent.
  • a matrix solvent (a) which is an aqueous solution containing 50% MeC and 0.1% TFA, a-cyan-4-hydroxycacin acid is dissolved at a concentration of 5 mg / ml, and a conventionally used matrix solution (A ) was prepared.
  • This matrix solution (A) was continuously dispensed onto the support.
  • water sensitive paper is used as the support.
  • the water-sensitive paper is a special paper that turns from yellow to blue when it comes into contact with water, and was used to accurately evaluate the dispensing position.
  • Dispensing was performed continuously for 4 hours under the condition of 100 pL per drop and 5 drops per spot on the support. At this time, the distance between each dispensing position is 500 ⁇ ⁇ .
  • a chemical printer CHIP-1000 manufactured by Shimadzu Corporation having an ink jet mechanism was used.
  • Figure 2 shows the results of continuous dispensing of water-sensitive paper using the matrix solution that has been generally used in the past as described above.
  • Figure 2 shows an image of water sensitive paper after dispensing, and an enlarged image (zoomed image 1 and zoomed image 2).
  • Sinapic acid (SA) was dissolved at a concentration of 5 mg / ml in a matrix solvent (g), which is an aqueous solution containing 40% MeCN, 10% 2-BuOH, and 0.1% TFA, and the matrix solution (G ) was prepared.
  • This matrix solution (G) was continuously dispensed onto water sensitive paper.
  • Dispensing was performed continuously for 2 hours under the condition of 100 pL per drop and 10 drops per spot on the support. At this time, the distance between each dispensing position is about 400 ⁇ .
  • the same chemical printer CHIP-1000 manufactured by Shimadzu Corp.
  • Experimental Example 4 was used as a dispensing device.
  • Figure 3 shows the results of continuous dispensing of water-sensitive paper using the matrix solution of the present invention as described above. Show.
  • tissue MS analysis using the matrix solution of the present invention was performed.
  • the ITO sheet on which the tissue section was placed was cut out to an appropriate size with scissors, and the tissue was washed with an alcohol solution. Thereafter, the tissue sections were completely dried by attaching them to a stainless steel plate through a conductive double-sided tape.
  • a matrix solution having the following composition using sinapinic acid (SA) as a matrix was prepared.
  • each matrix solution was poured onto the tissue section using a chemical printer CHIP-1000 (manufactured by Shimadzu Corporation).
  • CHIP-1000 manufactured by Shimadzu Corporation.
  • 10 drops of 100 pL / 1 drop were applied continuously, and this was repeated 40 times.
  • the distance between each dispensing position was 800 ⁇ .
  • the matrix solution of the present invention contains an additive for stabilizing dispensing (that is, t-BuOH in this example). Regardless of the sinapinic acid crystals on the tissue section, the conventional matrix solution (A) As in the case, it is obtained as having a good crystallization state.
  • mass analysis for each dispensing position was performed in linear mode / positive mode using MALDI-TOF MS and AXIMA-TOF 2 (Shimadzu Corporation).
  • the calibration of the mass value was performed by an external standard method using a commercially available standard compound.
  • the measurement method was performed by raster analysis (169 points at 250x250 mm, 25 mm pitch, 5shots, 169 profiles) with respect to the center of the matrix spot.
  • the matrix solution of the present invention is different from the conventional matrix solution (A), and can perform long-term dispensing very stably. enable. For this reason, it is clear that a large number of matrix spots arranged in an orderly manner can be obtained in the same crystallization state even within a certain range on the tissue section. Therefore, when the matrix solution of the present invention is used, unlike the case where the conventional matrix solution is used, the MS analysis result showing the good analysis sensitivity and resolution as obtained in Example 1 is obtained. It is clear that it can be obtained at any position in the range as well.
  • the matrix solution of the present invention makes it possible to stably carry out a continuous minute dispensing, so that a large number of the same spots can be formed in the entire specific range, that is, the spots can be uniformly formed. It becomes possible to form.
  • good analysis sensitivity and resolution can be obtained without causing matrix crystallization inhibition. It becomes possible.
  • the present invention can be applied to a matrix solution having a composition other than the above as long as it has a specific viscosity and surface tension.
  • the present invention is also applicable to a matrix solution containing a matrix other than sinapic acid, an additive other than t-butanol, and / or other matrix solvent constituents other than the above.
  • the present invention provides a dispensing technique other than the ink jet technique, and

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Abstract

Cette invention porte sur un procédé pour préparer un échantillon pour une analyse de masse par élimination/ionisation laser assistée par matrice utilisant une technique de microdistribution, lequel procédé permet de conduire en continu une microdistribution de façon stable et peut être appliqué à des matrices extensives. Le procédé comprend la distribution d'une solution de matrice, qui doit être utilisée dans une analyse de masse par un procédé d'élimination/ionisation laser assistée par matrice et a une viscosité non inférieure à 0,85 mPa·s (23°C) et une tension superficielle de 25 à 30 mN/m (23°C), dans un échantillon, devant être mesuré, à l'aide de moyens de microdistribution pour une distribution de 1 fl à 1 µl, afin d'obtenir un échantillon pour une analyse de masse.
PCT/JP2007/071353 2007-10-26 2007-10-26 Procédé de préparation d'un échantillon pour une analyse de masse par élimination/ionisation laser assistée par matrice utilisant une technique de microdistribution, et procédé d'analyse de masse par élimination/ionisation laser assistée par matrice Ceased WO2009054078A1 (fr)

Priority Applications (2)

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JP2009537879A JP5012904B2 (ja) 2007-10-26 2007-10-26 微量分注技術を用いたマトリクス支援レーザー脱離イオン化質量分析用サンプルの調製法及びマトリクス支援レーザー脱離イオン化質量分析法
PCT/JP2007/071353 WO2009054078A1 (fr) 2007-10-26 2007-10-26 Procédé de préparation d'un échantillon pour une analyse de masse par élimination/ionisation laser assistée par matrice utilisant une technique de microdistribution, et procédé d'analyse de masse par élimination/ionisation laser assistée par matrice

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PCT/JP2007/071353 WO2009054078A1 (fr) 2007-10-26 2007-10-26 Procédé de préparation d'un échantillon pour une analyse de masse par élimination/ionisation laser assistée par matrice utilisant une technique de microdistribution, et procédé d'analyse de masse par élimination/ionisation laser assistée par matrice

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JP7331233B1 (ja) 2022-11-22 2023-08-22 株式会社プロトセラ 質量分析用試料の調製方法
EP3712922B1 (fr) * 2019-03-20 2025-09-24 Ricoh Company, Ltd. Procédé, dispositif et support informatique pour la préparation d'un échantillon de mesure pour spectrométrie de masse maldi, échantillon pour spectrométrie de masse maldi, et procédé de spectrométrie de masse maldi

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CN119678039A (zh) * 2022-08-12 2025-03-21 株式会社岛津制作所 基质溶液、质量分析方法、存储介质、判别方法

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JP2013134102A (ja) * 2011-12-26 2013-07-08 Shimadzu Corp 質量分析用マトリックスの添加剤
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