WO2005101452A1 - Procede de preparation destine a la microanalyse de la composition de melanges de substances - Google Patents

Procede de preparation destine a la microanalyse de la composition de melanges de substances Download PDF

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Publication number
WO2005101452A1
WO2005101452A1 PCT/DE2005/000690 DE2005000690W WO2005101452A1 WO 2005101452 A1 WO2005101452 A1 WO 2005101452A1 DE 2005000690 W DE2005000690 W DE 2005000690W WO 2005101452 A1 WO2005101452 A1 WO 2005101452A1
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Prior art keywords
matrix
analyte
analytes
acid
preparation
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PCT/DE2005/000690
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German (de)
English (en)
Inventor
Bernhard Spengler
Werner Bouschen
Daniel Eikel
Dieter Kirsch
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Justus-Liebig-Universität Giessen
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Publication of WO2005101452A1 publication Critical patent/WO2005101452A1/fr

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Classifications

    • 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
    • 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
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/10—Ion sources; Ion guns
    • H01J49/16—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission
    • H01J49/161—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission using photoionisation, e.g. by laser
    • H01J49/164—Laser desorption/ionisation, e.g. matrix-assisted laser desorption/ionisation [MALDI]

Definitions

  • the present invention relates to a method with which substance mixtures can be prepared for mass spectrometry in such a way that they can be examined by means of the spatially resolved microscopic surface analysis with a usable lateral resolution of a few micrometers, as well as the preparations produced with the aid of this method.
  • the preparation method according to the invention With the help of the preparation method according to the invention, the chemical composition of substance mixtures can be reproduced on a much smaller scale than before. At the same time, a larger mass range is accessible for mass spectrometry, so that biologically relevant substances can also be examined.
  • the present invention relates to the fields of chemistry, biology and biochemistry, physics and instrumental analysis.
  • the aim of spatially resolved, microscopic surface analysis is to qualitatively and / or quantitatively map the chemical composition of a sample or a mixture of substances (e.g. a biological cell, a biological tissue or a semiconductor sample) with high resolution.
  • a sample or a mixture of substances e.g. a biological cell, a biological tissue or a semiconductor sample
  • an analytical image shows the quantity distribution of a certain substance within the sample as an image (eg as a grayscale image).
  • SBADI-MS scanning microsonde matrix-assisted laser desorption ionization mass spectrometry
  • MALDI-Imagin ' g is known to be operated only with a usable lateral resolution of about 25-30 ⁇ m, since with this method a chemical matrix substance in dissolved form must be added to also larger molecules (such as peptides and proteins The mixing process and the matrix crystals that form when they dry up limit the usable spatial resolution to the above-mentioned 25 to 30 ⁇ m, since analyte molecules are inevitably displaced spatially on the surface to this extent.
  • the crystals usually have a diameter of 5 ⁇ m to 500 ⁇ m, which also limits the usable spatial resolution
  • Part of the mechanistically based MALDI mass spectrometry lies in the much larger mass range, which can be examined (up to 10 6 u) and which thus makes biologically relevant substances such as proteins, oligosaccharides and lipids accessible.
  • Methods for evaporating samples with different materials are known from electron microscopy, but are not suitable for MALDI-MS, since other matrix materials such as gold are used in electron microscopy to make the surface conductive.
  • the selection of the matrix substance for the MALDI depends on the type of analyte molecules; well over a hundred different matrix substances have now become known.
  • the matrix substances used in high excess have the task, among other things, of separating the analyte molecules as much as possible and attaching them to the sample carrier, transferring them into the gas phase without formation of matrix or other molecules during the laser bombardment by forming a vapor cloud, and finally with protonation or Ionize deprotonation.
  • analyte and matrix A number of different methods have become known for the application of analyte and matrix. The simplest of these is to pipette a solution containing analyte and matrix onto a cleaned, metallic sample carrier.
  • the drop of solution forms a wetting surface on the metal surface (or its oxide layer), the size of which on hydrophilic surfaces corresponds to a multiple of a drop diameter. The size depends on the hydrophilicity and microstructuring of the metal surface and on the properties of the droplet, in particular the solvent.
  • a sample spot of small matrix crystals the size of this wetting area is formed, but as a rule there is no uniform coverage of the wetting area.
  • the crystals of the matrix usually begin to grow on the inner edge of the wetting on the metal plate. They grow towards the inside of the wetting surface. They often form radiation-like crystals, such as 2,5-dihydroxybenzoic acid (2,5-DHB) or 3-hydroxypicolinic acid (HPA), which extend towards the interior of the stain often lift off the carrier plate.
  • 2,5-DHB 2,5-dihydroxybenzoic acid
  • HPA 3-hydroxypicolinic acid
  • the matrix substance is already present on the carrier plate before the solvent droplets, which now only contain the analyte molecules, are applied. If the surface of the sample carrier plate is not hydrophilic but rather hydrophobic, smaller crystal conglomerates are formed, but the droplets tend to migrate in an uncontrollable manner when they dry out. The location of the crystal conglomerates can therefore not be predicted and requires a search in the MALDI process. In addition, there is a great risk that droplets will combine and make separate analysis of the samples impossible.
  • Sample carrier plate is already coated with a matrix layer and many analytes are applied simultaneously - for example with the help of a Multipette. Since the sample droplets released on the matrix with diameters of 200 ⁇ m to 600 ⁇ m are very small, very small amounts of analyte are sufficient. All- However, with this technique it is not possible to examine the surfaces on which the analyte or analytes are located on a microscale.
  • DE 197 54 978 A1 describes a method for producing and loading MALDI sample carriers, in which the sample carrier consists of an electrically conductive material, the surface of which is made hydrophobic.
  • hydrophilic anchor areas for the analytes are arranged in a grid and the hydrophilic solution of the analytes is applied. Even with this method, it is not possible to examine surfaces which contain analyte mixtures on a microscale.
  • the coupling of thin-layer chromatography and mass spectrometry is set out in DE 199 37438 C2: there analytes are separated on a TLC plate, which also serves as a carrier plate for the subsequent MS; following the DC, the carrier plate is provided with matrix solution and examined by mass spectrometry. Since the droplets with matrix solution have diameters between 10 and 50 ⁇ m, they are too large for mass spectrometric methods with a resolution of 1 ⁇ m.
  • DE 44 08 034 C1 describes a method for MALDI analysis of samples that were previously separated by means of 2D gel electrophoresis.
  • sample carrier plates are used, which are coated with an optically smooth, adsorptive matrix layer.
  • the analyte samples are transferred from the moist electrophoresis plate to the matrix by direct contact, preferably by electrophoretic transport, the two-dimensional distribution of the analytes being retained.
  • the matrix layer has a thickness of 300 nm to 600 nm.
  • DE 100 27 794 A1 describes a method for analyzing enzyme-catalyzed reactions using MALDI-TOF-MS, in which a solution containing matrix and analyte is applied to a polished, coated or vapor-coated carrier material with the aid of a nanoplotter.
  • a solution containing matrix and analyte is applied to a polished, coated or vapor-coated carrier material with the aid of a nanoplotter.
  • the production of structured bio sample carriers for MS analysis is disclosed in DE 100 43 042 A1.
  • There solutions from biomolecules and matrix substances are applied to hydro- or lyophobic surfaces, which contain hydrophilic anchor areas as well as affinity sorbents for the biomolecules. Fine crystalline matrix crystals form on the hydrophilic anchor areas, into which at least at least partially biomolecules are involved.
  • homogeneous incorporation of the biomolecules into the matrix cannot
  • DE 198 34 070 A1 describes such a method. Since liquid matrices are used there, the matrix liquid consisting of an ether polyol or a polyether polyol, the investigation of crystalline surfaces is not possible with this method.
  • DE 196 17 011 A1 describes a method for generating ions of high molecular weight analytes using MALDI, in which the matrix consists of at least two components, one component of which is decomposed by the action of the laser radiation (explosive).
  • the method is also suitable for laser foci with diameters in the range between 3 ⁇ m and 10 ⁇ m; within this focus diameter, however, the entire layer is removed at once, except for the sample carrier underneath.
  • the method is therefore not suitable for a three-dimensional analysis.
  • a method for the MALDI analysis of biological samples is disclosed in US Pat. No. 5,808,300.
  • a solution of the molecules to be analyzed is produced and deposited as a linear trace on an electrically conductive carrier material by capillary electrophoresis and dried.
  • the matrix with the analyte material can be dissolved and recrystallized.
  • this method can only be used up to laser foci greater than or equal to 25 ⁇ m and is therefore not suitable for microscopic surface analysis.
  • the present invention For the first time, it enables substance mixtures to be analyzed to be prepared for mass spectrometry in such a way that analyte molecules are incorporated into the matrix in a direction-controlled manner, the spatial redistribution of analyte molecules in the matrix being kept within such narrow limits that the scanning microsensor-matrix-assisted laser desorption 5 ionization mass spectrometry results in a spatial shift of less than or equal to 3 ⁇ m.
  • the object of the present invention is to provide a method for the preparation of analytes for mass spectrometry which avoids the disadvantages of the prior art with regard to the size of the matrix crystals and ensures a direction-controlled incorporation of the analyte molecules into the matrix, with direction-controlled Installation is understood; that the analyte molecules are installed in all three spatial directions essentially in the relative position to one another in which they were before being installed in the matrix.
  • This object is achieved according to the invention by a method in which the analyte is first applied to a sample carrier, then the matrix is deposited on the analyte which is essentially solid at room temperature and standard pressure and o finally the analyte or the analytes by vapor deposition of the matrix layer with a vaporous solvent for the matrix can be incorporated.
  • Another object of the invention is to provide a device for carrying out the method according to the invention. This object is achieved according to the invention by the subject matter of claim 15.
  • the device according to claim 17 having the advantage that the thickness of the matrix can be controlled and the device according to claim 18 having the advantage that the degree of integration of the analyte can be controlled is, so that overall a quality control of the preparation is guaranteed.
  • the device according to claim 19 has the advantage that it enables a high throughput of the preparation to be produced, in which the preparation takes place in a manner comparable to an assembly line production or a production at rotary tables.
  • An analyte is understood to be a mixture of at least one substance, this being at least one substance of chemical and / or biological and / or biochemical origin.
  • a sample carrier for mass spectrometry is a device which receives the analyte or analytes or on which the analyte or the analytes and the matrix are applied.
  • the matrix in connection with the matrix-assisted laser desorption ionization mass spectrometry is a substance whose energy absorption is matched to the wavelength of the desorption laser, so that the matrix is the energy of the Absorbs laser light and desorbs and protonates the analyte (s) embedded in the matrix, which is why the analyte can enter the gas phase and be analyzed without fragmentation.
  • the preparation method according to the invention for micro-range analysis of the composition of substance mixtures avoids the disadvantages of the prior art by applying the analyte or analytes to the sample carrier in a first step, applying the matrix in the second step and incorporating the or the in a third step Analytes into the matrix.
  • the matrix and the incorporation of the analyte are applied sequentially and not simultaneously.
  • analytes and / or matrix substances can be applied to sample carriers. These methods are suitable both for applying the analyte to the sample carrier (first step of the preparation method according to the invention) and for applying the matrix (second step of the preparation method according to the invention), the same or different of these known methods for applying analyte and matrix can be used. These methods known to the person skilled in the art are given below as methods for applying the matrix, it being known to the person skilled in the art that he can also use them for applying the analyte or analytes.
  • the matrix is preferably evaporated as a solid and then deposited on the analyte or analytes. It is obvious to the person skilled in the art that he can also use the vapor deposition method to apply the analyte.
  • the analyte is integrated into the matrix in the method according to the invention by exposing the sample carrier to a solvent vapor after the vapor deposition of the matrix, preference being given to using solvents in which the matrix substance dissolves to generate the vapor.
  • the matrix can optionally be exposed to a gas-steam mixture, for example in the case of analytes sensitive to oxidation or decomposition.
  • This gas-steam mixture consists of an inert carrier gas (“gas”) that cannot be condensed under reaction conditions and a component (“steam”) that can be condensed under reaction conditions.
  • gas inert carrier gas
  • steam component
  • Reaction conditions mean the selected combination of pressure and temperature.
  • the matrix sub- punch in a vacuum chamber, which can be evacuated in a pressure range from atmospheric pressure to 1 * 10 "7 mBar, in a container.
  • the matrix substance can sublime into the gas phase and is deposited on the side of the sample carrier on which the analyte is located with the sample carrier installed at a distance of 5 to 40 mm above the container, and the sample carrier is installed in such a way that the matrix substance is deposited on the analyte or analytes when it is deposited at pressures and temperatures at which the matrix material sublimes from the storage container and is deposited on the sample carrier material without the matrix substance and / or analyte decomposing.
  • a temperature sensor can be attached to the outer wall of the container to control the temperature of the container a pressure range from approx. 100 mbar to approx.
  • the thickness of the deposited matrix layer can be determined, for example, by methods known to the person skilled in the art, such as mass determination (quartz crystal measurement), mechanical scanning (probe cut method), optical interferometry (Tolanski interferometer), electron microscopy, scanning tunneling microscopy, scanning probe microscopy or laser scanning microscopy.
  • This second step of the preparation can also be replaced by other techniques known to the person skilled in the art which do not, or only within the resolution of, the spatial distribution of the analyte on the surface of the sample carrier
  • Modify mass spectrometer This includes methods that spray the matrix in a suitable solvent with a gas stream or with an electrical potential (electrospray), apply the matrix by mechanically distributing the solid, by stamping or printing, thermal transfer process, laser printing process, inkjet printing process, applying the matrix by
  • the matrix In order to spray the matrix by means of a gas stream, the matrix is dissolved in a solvent and finely atomized by means of a gas stream from a solvent reservoir through a nozzle (gas stream).
  • the drops that form in the The micrometer range is deposited on the analyte (s) and dries there immediately.
  • the amount of solvent that reaches the sample and thus also the migration of the analyte on the surface can be determined by the choice of the distance between the nozzle and the sample carrier.
  • the drops can be generated by an electric field between the nozzle and the sample surface.
  • the matrix solvent droplets can also be generated by ultrasonic nebulization known to the person skilled in the art.
  • droplets are released from a reservoir of matrix solution by means of ultrasound from the surface of the matrix solution.
  • Another possibility known to those skilled in the art for applying the matrix is the mechanical distribution of the matrix by means of, for example, a scraper or a spatula, by pouring the matrix onto the sample and finely distributing it using the spatula.
  • the matrix can also be applied to the sample carrier with the aid of a stamp to which the matrix is applied and which is then pressed onto the sample carrier, or other printing methods.
  • Another printing process is the laser printing process, in which the matrix is applied to the sample carrier with a roller and heated with a laser beam in order to adhere to the surface.
  • the matrix can be applied to the sample carrier by thermal transfer or inkjet printing processes. The matrix is either heated briefly in solution in a reservoir and sprayed through the expansion through a nozzle on the sample carrier or with the help of a
  • the sample carriers are exposed to a moist environment in a second step, so that the analyte is adequately incorporated into the Matrix happens and at the same time migration is minimized.
  • This is achieved by exposing the applied matrix layer to a solvent vapor.
  • the sample carrier is placed in a gas-tight, sealed container together with a solvent reservoir in which one or more solvents are located.
  • a solvent or solvent mixture in which the matrix substance is soluble is to be selected.
  • the solvent reservoir is heated so that a solvent vapor is formed in the container and thus also above the sample carrier, heating with solvent vapor until the container atmosphere is saturated.
  • the sample holder can remain in the container for up to 14 days.
  • the extent of the direction-controlled installation of the analyte or analytes is sufficient if the matrix particles have a size which corresponds approximately to the resolution of the desired laser of 0.5 ⁇ m to 3 ⁇ m to be used in the later SMALDI measurement.
  • the adequate integration of the analyte or analytes into the matrix can be checked, for example, by methods known to those skilled in the art, such as polarization microscopy, light microscopy and fluorescence microscopy. Evaporation of the matrix with a solvent vapor probably triggers recrystallization of the matrix layer. When the degree of recrystallization of the matrix is checked by means of polarizing microscopy, the absorption of polarized light is measured.
  • Recrystallized areas of the matrix appear in different colors in the polarizing microscope, since the spectral absorption depends on the direction of radiation and wavelength. Before recrystallization, the matrix layer appears uniformly white or gray in polarized light. The recrystallization is complete when colored areas with dimensions in the range of at least 0.5 micrometers have arisen in polarized light.
  • the degree of recrystallization can be monitored with the aid of fluorescence microscopy by monitoring the lateral uniformity of the microscopic distribution of matrix and analyte, which fluoresce at different wavelengths.
  • matrix substances usually have natural fluorescence
  • peptides or proteins that contain tryptophan can be observed via its natural fluorescence, for example.
  • methods are known for carrying out fluorescent labeling of any peptides or proteins, in order in this way to fluoresce at suitable wavelengths To be able to observe the matrix and proteins.
  • the microscopic intermixing of analyte and matrix can also be checked by only observing the covering matrix layer in reflected light fluorescence microscopy before recrystallization, while after recrystallization both matrix and analyte can be observed via their fluorescence signals ,
  • the action of the solvent vapor on the matrix layer can be monitored using a quartz balance by determining the frequency shift during the moistening phase: the mass of the evaporated matrix layer initially increases due to solvent absorption and then decreases again during the recrystallization of the matrix.
  • the recrystallization and the incorporation of the analyte can be regarded as successful if the uptake of solvent took place over a period of several hours or days, preferably 3 hours to 14 days.
  • the solvent vapor causes the matrix to recrystallize.
  • the analyte is built into the matrix layer on the surface of the sample carrier. Since the matrix layer should have a thickness of 0.5 ⁇ m to a maximum of 3 ⁇ m, the duration of the recrystallization process can limit the migration of the analyte in such a way that the analyte penetrates completely into the layer, but does not penetrate more than approx. Moved 1 ⁇ m to 3 ⁇ m away from its original location (see Fig. 18).
  • the solvent vapor can also be generated by methods known to those skilled in the art for spraying the solvent (see also spraying the matrix) or atomizing by means of ultrasonic atomization (see also ultrasonic atomization of the matrix).
  • the recrystallization of the matrix layer can be influenced by temperature and pressure inside the container. At temperatures in the range from 40 to 80 ° C., recrystallization of the layer can be observed within 1 to 5 days, the recrystallization being checked as described above, for example by means of light, polarization or fluorescence microscopy or with the aid of a quartz balance.
  • the process can be accelerated by increasing the temperature, the temperature being selected so that chemical see reactions and / or decomposition of the analyte (s) to be avoided.
  • the recrystallization can also be accelerated by increasing the pressure in the container.
  • a matrix layer is obtained which both fulfills the necessary requirement for the incorporation of the analyte or analytes into the matrix and leads to a direction-controlled incorporation of the analyte into the matrix.
  • Effective spatial resolution is understood to mean the resolution that can be achieved with the aid of the mass spectrometer (determining the resolution: laser focus) and the preparation of the sample (determining the resolution: migration of the analyte or analytes).
  • the matrix should be present in a molar excess of approximately 100: 1 to 100000: 1 in order to spatially separate the analyte molecules and thereby to minimize the intermolecular interactions between the analyte molecules. If the substances to be examined lie on a surface and / or are bound to this surface, the preparation must ensure that the analyte and the matrix are mixed sufficiently. takes place to ensure the spatial separation of the molecules. Binding to surfaces can take place, for example, but not exhaustively, by means of anchors or beads.
  • Scanning mass spectrometry is understood to mean 5 mass spectrometric methods in which the laser beam or the ion beams or the neutral particle beams on the one hand and the sample surface on the other hand are defined and moved relative to one another in such a way that mass spectra of all areas of the sample surface are recorded and the mass spectra obtained at their respective origin can be assigned on the sample surface.
  • the prepared sample can also be used for microscopic methods of mass spectrometry.
  • Large area is understood to mean a laser beam that shots an area of the sample surface that is larger than the area in which the detector can separate ions with regard to their different origins on the sample surface.
  • a distribution image of the ions can thus be created by reading out individual imaging locations of the detector.
  • analytes can be applied to a sample carrier. This includes, for example, spraying on the dissolved analyte and then drying it. Drying can be carried out according to methods known to the person skilled in the art, such as, for example, but not exhaustively, at room temperature and standard pressure, at temperatures above room temperature and / or at pressures under normal pressure and by lyophilization.
  • biological samples such as, for example, cells, cell components, cell extracts and tissue sections, can be applied to the sample carrier by pressing on, growing on or sticking on.
  • sample carriers whose surface is provided with so-called anchor molecules, these anchors specifically binding certain groups of substances from an analyte mixture, but not binding other groups of substances.
  • sample carriers are known to the person skilled in the art, the surface of which is provided with so-called beads, these beads in turn having anchor molecules on their surface. By depositing the beads with a size of a few nanometers down to the micrometer range, the available surface can be provided with anchor molecules greatly enlarge.
  • solid supports which consist, for example, of beads, needles, combs or wafers.
  • the method according to the invention is suitable for all matrix substances known to the person skilled in the art. These include, but are not limited to: 2-aminobenzoic acid, 3-aminobenzoic acid, 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid), alpha-cyano-4-hydroxycinnamic acid (CHCA), 3-hydroxypicolinic acid (3-HPA), 2 , 5-dihydroxybenzoic acid (2,5-DHB), 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 2,6-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, picolinic acid, 2,4,6- Trihydroxyacetophenone, 2,3,4-trihydroxyacetophenone, nitrobenzyl alcohol, nicotinic acid, ferulic acid, caffeic acid, ellagic acid, cis-o-cumaric acid, trans-o-cumaric acid, cis
  • a condensable component such as, but not exhaustive, water, methanol, ethanol, acetone, acetonitrile, propanol, isopropanol, n-butanol, iso- Butanol, tert-butanol, ethyl acetate, benzene, toluene, 1, 2-dimethylbenzene, 1, 3-dimethylbenzene, 1, 4-dimethylbenzene, cyclohexane, cyclohexanol, dichloromethane, chloroform, trifluoroacetic acid, acetic acid, dimethylformamide, diethylamine , Phenylethylamine can be used.
  • a condensable component such as, but not exhaustive, water, methanol, ethanol, acetone, acetonitrile, propanol, isopropanol, n-butanol, iso- Butanol, tert-butanol
  • solvents for example from the groups of esters, ethers, carboxylic acids, amines, aliphatic, aromatics, araliphatics and haloaliphatics as well as mixtures of at least two solvents are known to the person skilled in the art and can be used without leaving the scope of protection of the patent claims.
  • These solvents can be wholly or partially deuterated, so that D 2 O can be used instead of water, for example, in order to be able to exchange easily removable, acidic protons in the matrix and / or analyte substance for deuterium ions if the atmosphere is to be exchanged is taken care of.
  • Deuteration and exchange of the atmosphere are known to the person skilled in the art and can be found, for example, in B. Spengler, F.
  • an inert gas can optionally be used in addition to the vaporizable solvent, such as air, nitrogen, helium, neon, argon, krypton, xenon, carbon dioxide and mixtures thereof
  • the gas composition of this inert gas can be changed during the vapor deposition, for example by exchanging one gas for another, for example air can be exchanged for nitrogen in order to avoid oxidation reactions.
  • sample carrier materials which are suitable for the application of analytes and matrices for mass spectrometric examinations. These include, but are not limited to, gold, aluminum, steel, silicon, Teflon, quartz, metals, metal alloys, metal oxides, PVDF, cellulose, regenerated cellulose, anionically modified cellulose, cationically modified cellulose, nylon, nitrocellulose and those known to those skilled in the art
  • Functionalized surfaces that can be loaded with beads or anchor molecules, for example. Functionalized surfaces are to be understood as meaning such chemical structures which selectively bind physically and / or chemically and / or matrix molecules and / or react chemically or biochemically with anchor and / or matrix molecules.
  • sample carrier materials that are not electrically conductive are used, they have to connect them to an electrically conductive material in order to obtain a sample carrier suitable for mass spectrometry if the sample carrier is to be the first acceleration electrode within the mass spectrometer .
  • size and shape of the sample carrier (for example round or rectangular) must be selected depending on the respective measuring system and on the samples to be measured.
  • Measureroscopically structured samples are understood to mean those samples whose spatial dimensions can only be resolved with a microscope, that is to say that the spatial dimensions are approximately 1 nm to 200 ⁇ m. According to the present invention, layers which are exclusively uniform are referred to as being sufficiently uniform have such inhomogeneities which are smaller than the laser focus, preferably smaller than 3 ⁇ m.
  • analyte bwz. the analytes and the matrix substance can also be applied by repeating a single method several times and / or by combining several methods for applying analyte or matrix.
  • Solvent reservoirs known to the person skilled in the art are suitable for recrystallization of the matrix layer, for example, but not exhaustively: containers or beakers which are open so that the solvent vapors can escape; Materials that absorb the solvent, such as, but not limited to, impregnated wipes made of paper, textile materials or plastics, and sponges; Containers or beakers from which the solvent is sprayed or pressed through a nozzle or membrane; Containers or cups from which droplets are transported into the gas phase using ultrasound (ultrasonic nebulizers). It is known to the person skilled in the art that these solvent reservoirs can also be used in the preparation of the matrix layer.
  • the analyte is separated and thus migrated according to its hydrophobicity or polarity. Since the analytes, here for example peptides, have a different polarity due to their amino acid composition, their affinity for the different polar solvents is different. In most cases, the matrix is dissolved in a mixture of several solvents (e.g. water: polar solvent, ethanol: weakly polar solvent).
  • solvents e.g. water: polar solvent, ethanol: weakly polar solvent
  • the non-polar solvent predominantly evaporates due to the higher vapor pressure and the hydrophilic components of the analyte accumulate in the liquid rest of the sample in the middle before it also dries up.
  • the same phenomena also take place on a microscale.
  • the rate of crystallization is a decisive factor for the conditions in the crystals.
  • Embodiment 1 Meligration in Dried-droplet Standard Preparation
  • the crystallization procedure of the so-called “Dried-droplet” standard method leads to a circular sample approximately one to two millimeters in size, the edge of which consists of larger crystals and the inner area of which is made of finer crystals.
  • the sample plate (aluminum carrier with 8 ⁇ m gold coating, 20 mm diameter) as described below, to avoid contamination of the plate with other substances, in particular sodium and potassium ions, for this purpose 5 to 10 times 2 to 5 ml of different solvents (eg: acetone (ACS, Merck, Darmstadt), isopropanol (LiChrosolv, Merck, Darmstadt), water (ACS, Sigma-Aldrich, Frankfurt), ethanol (Uvasol, Merck, Darmstadt)) and added with a dust and lint-free paper towel (e.g.
  • the sample plate is dried either in the air for several minutes or under a hot air stream in a few seconds.
  • the size of the crystals formed strongly depends on the speed of drying or crystallization. If the sample plate is dried in air, a distinctive edge of the sample is created, which consists of large, up to several
  • Embodiment 2 shows the distribution images of a peptide mixture of the highly hy • drophilic liptropin 1-10 and the highly hydrophobic anti-inflammatory peptide.
  • a "Dried-droplet" preparation as in Embodiment 1 used. 1 ⁇ l peptide mixture in ethanol / water (1: 1 v / v, 5 * 10 "5 mol / l) was added to the sample carrier (aluminum carrier with 8 ⁇ m gold coating, 20 mm diameter) and with 1 ⁇ l matrix (2, 5-DHB) in ethanol / water (2: 3 v / v,
  • FIG. 5 shows a clear segregation of the two analytes through the different grayscale images, while the intensity of the matrix signal is evenly distributed across the crystal.
  • Embodiment 3 Full MALDI Standard Preparation
  • a modified method means that hardly any segregation effects are observed within the crystals.
  • Figure 6 is a peptide mixture of dynorphin 1-9 (hydrophobic) and vasopressin [Arg ⁇ ] (hydrophilic) in the same solvent ratios and with the same matrix solution as in embodiment 2 with a concentration of 5 * 10 "5 mol / l each.
  • rapid crystallization was now carried out.
  • the grayscale images of the rastered image section (generation as in exemplary embodiment 8) of 100 ⁇ m by 100 ⁇ m show the distribution of the two analytes and the matrix ( Figure 6).
  • the two peptides are largely homogeneously distributed was determined with an Olympus BX41 microscope with integrated scale.
  • Including the third component (matrix) shows an even distribution of analyte and matrix. Since the analytes are only measured at the locations where the matrix or a matrix crystal is also present, the distribution depends on the distribution of the matrix, but the mixture of the peptides is homogeneously distributed within the crystal.
  • Embodiment 4 Standard Preparation of Peptides of Different Sizes
  • a comparison of peptides of different sizes also shows that the analytes can be inhomogeneously distributed within the larger matrix crystals and separate during the preparation. Likewise, the analytes can also be distributed inhomogeneously within microcrystalline matrices. In addition to the time required for the matrix-analyte mixture to dry out, the hydrophobicity or the polarity, the size of the peptides also plays a role. According to Bouschen, W; Flad, T .; Müller, CA, Spengler, B. Characterization of biological samples by Scanning Microprobe MALDI (SMALDI) mass spectrometry with 1 ⁇ m lateral resolution, 50 th American Society for Mass Spectrometry Conference; 2.6.
  • SALDI Scanning Microprobe MALDI
  • Example 7 The sample was prepared as in Example 3, the measurement as described in Example 8.
  • 0.5 ⁇ l analyte and 0.5 ⁇ l are mixed on the sample plate and dried in a warm air stream (approx. 60 ° C.) in approx.
  • the sample shows an edge with large crystals and a finely crystalline inner area of the sample. Again, an area of 100 ⁇ m by 100 ⁇ m was scanned with a step size of 1 ⁇ m, however the spatial analytical resolution of these images is due to an improvement in the optical The quality of the laser focus is noticeably higher than in Figure 5 and Figure 6.
  • the grayscale ( Figure 7) shows an inhomogeneous distribution of the analytes within a crystal, although the difference in hydrophilicity for these peptides is not as great as in the first example ,
  • Embodiment 5 Evaporation
  • the first step for applying the matrix to the sample to be examined consists in evaporating the matrix as a solid and then depositing it on the sample.
  • the sample carriers were vaporized with an evaporation system.
  • the device consists of a glass bell, which is evacuated with an external gas ballast pump (D12, 12 m 3 / h, Leybold AG, Cologne, Germany) and an internal oil diffusion pump (130 l / min).
  • the final pressure when using the oil diffusion pump is MO '5 mbar, with the gas ballast pump alone 1 -10 "3 mbar can be reached.
  • the vacuum is controlled by a vacuum gauge from Pfeiffer Vacuum (Asslar, Germany, Compact Filling Range Gauge) With the help of various valves, the vacuum in the glass bell can be continuously adjusted from atmospheric pressure to 1 • 10 "3 mbar.
  • the glass bell contains two independent electrode systems that can hold different filaments or boats.
  • the electrodes can be supplied with a current of up to 40 A to allow the materials to evaporate.
  • a boat is used in which up to 50 mg material can be heated.
  • a boat is a thin-rolled metal container that is clamped between two electrodes and heated by a current that is passed through the boat ( Figure 11).
  • a thermal sensor which measures the temperature by means of resistance measurement, is attached to the boat to set a constant temperature over a longer period of time.
  • Embodiment 5a Resistance of matrix substances when heated
  • the three matrices 2,5-DHB (melting point: 236 ° C - 238 ° C), sinapic acid (melting point: 203 ° C - 205 ° C) and CHCA (melting point: 252 ° C) were used. All three matrices were first examined for their resistance to heating. For this purpose, a sample holder was mounted 15 mm above the boat in which the matrix was located, with the sample holder side down. The boat was filled with 10 mg to 60 mg of the respective matrix substance. The boat was heated in such a way that the temperature was raised by 10 ° C. per minute from room temperature. It was possible to prepare up to five samples with the stated amounts of matrix substance.
  • Embodiment 5b Mass spectrometric measurement of the matrix layers
  • the grown layers were then measured using the "Lamma 2000" mass spectrometer, the same measurement conditions as in Aus leadership example 7 were chosen.
  • the 2.5-DHB layer was first formed by heating the matrix in the vapor deposition system at 100 ° C. to 140 ° C. for 9 to 15 minutes at atmospheric pressure. Was preferably heated at 130 ° C for 12 minutes at atmospheric pressure.
  • the layer on the support showed a spectrum in the mass spectrometer that was comparable to normal prepared MALDI-MS spectra (embodiment 1, Figure 12, top).
  • Embodiment 5c sublimation of the matrix at reduced pressure
  • Embodiment 5d Evaporation of sample carriers to which analyte was previously applied
  • Embodiment 6 Function of the matrix after vapor deposition
  • Figure 14 shows that the matrix is largely unchanged chemically even after sublimation according to the exemplary embodiments 5 to 5d and that it still fulfills its function in the desorption / ionization process.
  • the sample carrier was loaded with a drop of the peptide mixture according to embodiment 5d. After the drop had dried, the matrix layer (2,5-DHB) was evaporated. The matrix layer produced was then redissolved in the area of the dried drop with 1 ⁇ l ethanol / water 1: 1 (v / v) and dried again using a warm air stream (60 ° C.). The resulting matrix crystals now showed the typical appearance of a normal MALDI preparation according to embodiment 1.
  • Embodiment 7 Moistening the matrix
  • Evaporation and the subsequent dissolution and recrystallization of the matrix layer proved to be a sensible strategy for matrix preparation while maintaining detection sensitivity and spatial resolution.
  • the separation of the preparation into two steps was a crucial point in order to achieve extensive control over the preparation conditions for producing a homogeneous matrix layer with integration of the analyte.
  • the sample carriers were exposed to a moist environment.
  • the fully steamed sample carriers were placed in a closed glass container.
  • a paper towel for example Kimwipes (Kimberly-Clark, Neenah, USA), was placed under the sample holder and soaked in distilled water. The container was then heated from the bottom so that an atmosphere saturated with water formed around the sample holder.
  • the container was placed about 0.5 cm to 2 cm deep, preferably 1 cm deep, in a sand bath with temperature control for heating.
  • the container was wrapped with a sealing film, for example Parafilm M (Karl Hecht KG, Sondheim, Germany) to seal it to prevent it from drying out.
  • a sealing film for example Parafilm M (Karl Hecht KG, Sondheim, Germany) to seal it to prevent it from drying out.
  • the first experiments were carried out with 1 ml of distilled water and a temperature of the sand bath of 65 ° C. Later tests were carried out with 0.5 ml of water and higher temperatures of 80 ° C, since the condensation on the lid could be limited by reducing the amount of water added and the risk of droplets being reduced so far that no more drops from the lid dripped. The temperature was measured with a thermometer at the bottom of the container.
  • Embodiment 7a changing the rate of crystallization
  • the crystallization could be accelerated by increasing the temperature of the humid atmosphere.
  • Experiments with a temperature of 125 ° C in the sand bath led to recrystallization after only 24 hours, and after three days no further change in the sample surface could be found.
  • the changes in the surface were determined by optical inspection using an Olympus BX41 microscope with an integrated scale.
  • a further increase in the temperature no longer seemed sensible due to the melting temperatures of the matrices used of 200 to 260 ° C.
  • Temperatures higher than 70 ° C on the sample plate should be avoided with regard to the samples to be measured, since from these temperatures the denaturation of proteins begins and undesirable changes occur in biological samples.
  • Embodiment 7b control of the spatial distribution of a felt pen line after recrystallization
  • Embodiment 7c Control of the spatial distribution of peptides after recrystallization.
  • the change in the layers by incubation with water also depends on the analyte or analytes themselves.
  • a different crystallization behavior of the matrix was found when comparing the areas within and next to the applied peptide mixture.
  • Figure 17 1 .mu.l of the peptide mixture of substance P, melittin and insulin prepared as described in exemplary embodiment 5d was applied to a gold sample carrier which had previously been coated extensively with a dilute red dye. After the drop had dried, the support was evaporated for five minutes with 2.5-DHB at 48 ° C.
  • Embodiment 7d Measurement of the analyte prepared according to the invention by means of SMALDI mass spectrometry
  • Figure 18 shows that a spatial analytical resolution of approximately 2 ⁇ m can be achieved with this preparation method according to embodiment 7c by means of SMALDI mass spectrometry according to embodiment 8. Two measurements of the same sample site were carried out. A range of 100 ⁇ m by 100 ⁇ m was scanned with a step size of 1 ⁇ m. One laser shot per raster step was emitted on the sample and a mass spectrum was recorded. Figure 18 shows the intensity distributions for three masses.
  • the distribution of the individual components shows that both the matrix and the red dye are evenly distributed. Since exactly the edge of the dried drop was scanned, substance P as an analyte of the peptide mixture is not evenly distributed. A border in the upper area of the image of approx. 3 ⁇ m width can clearly be seen, in which the intensity of the substance P mass signal drops to zero.
  • the intensities achieved in these measurements are significantly more intense compared to the spray method. This suggests a much better incorporation of the analyte from the surface into the resulting matrix crystals.
  • a second measurement of the same sample location and the same measurement conditions as for the first measurement proves that the matrix layer is relatively thin (less than 2 ⁇ m), since it has already been partially removed. In order to achieve a low spatial migration of the analyte, these thin layers are necessary. Installation in much thicker matrix layers with usable signal intensities inevitably results in higher migration. The expected dependence of the intensity of the peptide signal on the matrix signal can also be seen.
  • the measurements illustrate the possibility of processing complex biological or synthetic surfaces with the preparation method described.
  • Embodiment 8 Measurement of the analyte by scanning mass spectrometry
  • Embodiment 8a Components of the measuring device
  • a flight tube - a field grating, a detector consisting of a double microchannel plate with an active diameter of 40 mm,
  • Vacuum pumps oil rotary pump to generate the fore vacuum (pump capacity 16m 3 / h, Leybold AG, Cologne), turbomolecular pump to generate the High vacuum (360 l / s, Leybold AG, C perfume), pressure in the vacuum chamber after pumping out approx. 5 * 10 "7 mbar
  • Embodiment 8b Selected distances within the measuring device • Pinhole - sample surface: 3.9 mm
  • Embodiment 8c Selected potentials
  • Embodiment 8d Scanning the sample carrier Scan area: 100 ⁇ m x 100 ⁇ m Scan step size: 1 ⁇ m scan speed: 10 pixels per second 10,000 mass spectra per pixel
  • Embodiment 8e pre-focusing
  • an Nd: YLF laser for example Model 421 QD (ADLAS, Lübeck), can be used, output energy 100 ⁇ J per nanosecond pulse at 524 nm.
  • a quadrupling the frequency of this laser 5 is achieved externally by a temperature-controlled BBO crystal (barium laboratory to generate the second nonlinear harmonic).
  • the final pulse energy is approx. 15 ⁇ J at 262 nm.
  • the Nd: YLF laser beam has a strictly elliptical shape after being quadrupled by the BBO crystal.
  • a special optical correction is necessary to ensure a high numerical aperture at the entrance of the focusing objective lenses.
  • the ratio of the large and small diameter of the ellipse is approximately 1: 8 when using this laser.
  • the beam After prefocusing with a spherical lens, the beam therefore fills a narrow strip only in the input lens of the focusing lens.
  • a special optical unit5 was developed for the circularization, which pre-focuses the two beam axes differently (see FIG. 4).
  • the x-axis is the small, the y-axis the large ellipse axis.
  • Both axes are 28-29-focused by cylindrical lenses, both one-dimensional foci being adjusted to the same focal plane and a circular beam profile is obtained in this way.
  • the pre-focusing of the Nd: YLF laser beam has already been published by Spengler and Hubert (B. Spengler and M.
  • Embodiment 8g Peak Detection and Statistical Evaluation The individual spectra obtained per sample position were summed, then the centroids 23 were calculated and mass, peak areas 22 and location coordinates were assigned. The frequency distribution of all detected masses was then determined. The maxima were determined in the histogram and the mass windows for the images were determined. For each image, the center of gravity 24 was calculated by weighting the individual masses with the peak areas, and the relative measurement uncertainty Si was determined.
  • Embodiment 8h image creation
  • the peak areas of each detected mass were converted into 16 bit gray levels.
  • the entire group of images was scaled for maximum contrast in order to be able to compare all of the images obtained from 2,5-DHB, substance P, melittin and insulin.
  • individual images were scaled to get maximum contrast for the distribution of each individual substance.
  • Embodiment 8i Alternative scanning of the analytes to be examined
  • the X-Y-Z shift table was not moved in this experiment, but the laser was moved relative to the shift table.
  • a laser with a focus diameter of 0.3 ⁇ m to 0.6 ⁇ m was used, the scan step size was also 1 ⁇ m (not shown).
  • Embodiment 9 Analytes at risk of oxidation or decomposition
  • Exemplary embodiment 10 Preferred method for applying the matrix
  • Illustration 1
  • the middle column shows the size of the migration of the analyte on the surface.
  • the right column shows the size of the integration of the analyte from the surface into the matrix layer.
  • the laser beam 7 is directed through a lens with a central bore 5 onto the piezo table 6 with the sample carrier.
  • the emitted ions are accelerated by means of the ion-optical arrangement 4 through the ion guide channel 1 and through the flight tube 2 to the detector 3.
  • Light from a light source for sample observation 11 is likewise directed onto the sample carrier via dichroic mirrors 10; a CCD camera 8 generates optical images of the examined sample area.
  • Figure 4 Comparison between edge and center of a Dried-Droplet preparation: a) measurement of large crystals of the edge (30 measurements averaged) b) measurement of fine crystalline center (30 measurements averaged)
  • Figure 5 Comparison between edge and center of a Dried-Droplet preparation: a) measurement of large crystals of the edge (30 measurements averaged) b) measurement of fine crystalline center (30 measurements averaged)
  • Figure 5 Comparison between edge and center of a Dried-Droplet preparation: a) measurement of large crystals of the edge (30 measurements averaged) b) measurement of fine crystalline center (30 measurements averaged)
  • Figure 5 Comparison between edge and center of a Dried-Droplet preparation: a) measurement of large crystals of the edge (30 measurements averaged) b) measurement of fine crystalline center (30 measurements averaged)
  • Dried-droplet preparation with 2,5-DHB as a matrix Slow crystallization (approx. 10 minutes) results in large crystals with different analyte distributions. (100 ⁇ m x 100 ⁇ m, 1 ⁇ m raster) a) distribution pattern 2,5-DHB, matrix, m / z 137.1 b) distribution pattern anti-inflammatory peptide, m / z 1084.5, hydrophobic c) distribution pattern lipotropin 1-10, m / z 950.5, hydrophilic
  • Rapid crystallization produces small crystals with a homogeneous distribution of the analyte. (100 ⁇ m x 100 ⁇ m, 1 ⁇ m raster) a) distribution pattern 2.5-DHB, matrix, m / z 154.1 b) distribution pattern vasopressin [Arg8], m / z 1137.7, hydrophilic c) distribution pattern dynorphin 1-9, m / z 1084.5, hydrophobic
  • Edge of a third-droplet preparation with 2.5-DHB as a matrix (100 ⁇ m x 100 ⁇ m, 1 ⁇ m raster)
  • Fine crystalline crystals inside a third-droplet preparation with 2.5-DHB as a matrix 100 ⁇ m x 100 ⁇ m, 1 ⁇ m step size
  • the current control 15 of the electrodes 13 is required for temperature control 16 of the boat 19 (not visible).
  • the pressure control 17 is controlled via the valve 22.
  • the sample plate 20 is clamped in the sample holder 18 for vapor deposition. Between the electrodes 13, the boat H® is filled with matrix and over the Current control 15 (not visible) heated.
  • the temperature control 16 (not visible) can be carried out via a temperature sensor 21.
  • Figure 11 Microscopic image of a vaporized sample holder: gold sample holder with dye from a felt pen (upper part of the picture) and vaporization 2,5-DHB (left part of the picture) a) 650 x 500 ⁇ m b) 65 x 50 ⁇ m
  • Figure 18 Ion distribution images of the boundary of a prepared peptide mixture (100 x 100 ⁇ m, 1 ⁇ m increment) a) 2,5-DHB, m / z 273 b) Red felt tip pen color, m / z 450 c) Substance P, m / z 1348
  • Figure 19 Container 23 for moistening the sample carrier with analyte 20 a) Container 23 with lid 24 b) Container 23 without lid with sample carrier 20 on sample support 25

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Abstract

L'invention concerne un nouveau procédé de préparation de surfaces destiné à la spectrométrie de masse MALDI (SMALDI) à balayage à haute résolution. Tout d'abord l'analyte ou les analytes puis la matrice sont déposés sur la surface du porte-échantillon, la matrice ayant une épaisseur de couche de 0,5 µm à 3 µm. Ensuite la matrice est vaporisée dans une atmosphère de solvant et donc est recristallisée et intègre l'analyte. Le procédé de vaporisation selon l'invention permet la migration des molécules d'analyte pendant la recristallisation de la matrice et l'intégration des analytes simultanée seulement dans une mesure correspondant à l'épaisseur de couche de la matrice, c'est-à-dire d'environ 0,5 µm à 3 µm. Les surfaces préparées selon l'invention sont donc bien adaptées à la spectrométrie de masse MALI à balayage à résolution locale, puisque le lieu d'origine et donc les données locales des molécules d'analytes sont largement conservées pendant la préparation. L'invention concerne également un dispositif permettant d'effectuer le procédé selon l'invention.
PCT/DE2005/000690 2004-04-16 2005-04-15 Procede de preparation destine a la microanalyse de la composition de melanges de substances WO2005101452A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2446251A (en) * 2006-12-18 2008-08-06 Bruker Daltonik Gmbh Preparation of a matrix layer for mass spectrometry
US7667196B2 (en) 2006-04-27 2010-02-23 Bruker Daltonik Gmbh Sample preparation for mass spectrometric imaging
EP2975393A4 (fr) * 2013-04-04 2016-03-23 Shimadzu Corp Procédé de préparation d'échantillon pour maldi et dispositif de préparation d'échantillon

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102017110476B4 (de) 2017-05-15 2019-03-07 Bruker Daltonik Gmbh Präparation biologischer Zellen auf massenspektrometrischen Probenträgern für eine desorbierende Ionisierung

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5854486A (en) * 1996-12-27 1998-12-29 R. T. Hodgson Method and apparatus for MALDI mass spectrometry
WO2003071263A1 (fr) * 2002-02-19 2003-08-28 Genome Institute Of Singapore, National University Of Singapore Dispositif de focalisation isoelectrique
DE10207615A1 (de) * 2002-02-22 2003-09-18 Sunyx Surface Nanotechnologies Ultraphobe Oberfläche mit einer Vielzahl reversibel erzeugbarer hydrophiler und/oder oleophiler Bereiche

Family Cites Families (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2235529B (en) * 1989-08-23 1993-07-28 Finnigan Mat Ltd Method of preparing samples for laser spectrometry analysis
GB2235528B (en) * 1989-08-23 1993-07-28 Finnigan Mat Ltd Method of preparing samples for laser spectrometry analysis
GB2236184B (en) * 1989-09-12 1993-07-28 Finnigan Mat Ltd Method of preparing samples for laser spectrometry analysis
DE4143071C2 (de) * 1991-12-27 1995-05-24 Finnigan Mat Gmbh Verfahren zur Bereitstellung eines Analytes für eine Untersuchung
DE4408034C1 (de) * 1994-03-10 1995-07-13 Bruker Franzen Analytik Gmbh Verfahren zur massenspektrometrischen Analyse von Proben aus 2D-Gel-Elektrophoreseplatten mit matrixunterstützter ionisierender Laser-Desorption
DE19617011C2 (de) * 1996-04-27 2000-11-02 Bruker Daltonik Gmbh Matrixkomponentengemisch für die matrixunterstützte Laserdesorption und Ionisierung sowie Verfahren zur Zubereitung eines Matrixkomponentengemisches
DE19618032C2 (de) * 1996-05-04 2000-04-13 Bruker Daltonik Gmbh Lagerfähig vorpräparierte Maldi-Probenträger
US5808300A (en) * 1996-05-10 1998-09-15 Board Of Regents, The University Of Texas System Method and apparatus for imaging biological samples with MALDI MS
DE19628178C1 (de) * 1996-07-12 1997-09-18 Bruker Franzen Analytik Gmbh Verfahren zum Beladen von Probenträgern für Massenspektrometer
DE19754978C2 (de) * 1997-12-11 2000-07-13 Bruker Daltonik Gmbh Probenträger für die MALDI-Massenspektrometrie nebst Verfahren zur Herstellung der Platten und zum Aufbringen der Proben
DE19834070B4 (de) * 1998-07-29 2007-02-15 Bruker Daltonik Gmbh Ionisierung hochmolekularer Substanzen durch Laserdesorption aus flüssigen Matrices
DE19923761C1 (de) * 1999-05-21 2001-02-08 Bruker Daltonik Gmbh Aufreinigende Probenträger für die MALDI-Massenspektrometrie
DE19949735A1 (de) * 1999-10-15 2001-05-10 Bruker Daltonik Gmbh Prozessieren von Proben in Lösungen mit definiert kleiner Wandkontaktfläche
DE19937438C2 (de) * 1999-08-07 2001-09-13 Bruker Daltonik Gmbh Kopplung Dünnschicht-Chromatographie und Massenspektrometrie (TLC/MS)
DE10027794A1 (de) * 2000-06-07 2001-12-13 Basf Ag Verfahren zur Analyse Enzym-katalysierter Umsetzungen mit MALDI-TOF-Massenspektrometrie
DE10043042C2 (de) * 2000-09-01 2003-04-17 Bruker Daltonik Gmbh Verfahren zum Belegen eines Probenträgers mit Biomolekülen für die massenspektrometrische Analyse
WO2003040715A1 (fr) * 2001-11-05 2003-05-15 Irm, Llc. Procedes de preparation d'echantillons pour la spectrometrie de masse maldi

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5854486A (en) * 1996-12-27 1998-12-29 R. T. Hodgson Method and apparatus for MALDI mass spectrometry
WO2003071263A1 (fr) * 2002-02-19 2003-08-28 Genome Institute Of Singapore, National University Of Singapore Dispositif de focalisation isoelectrique
DE10207615A1 (de) * 2002-02-22 2003-09-18 Sunyx Surface Nanotechnologies Ultraphobe Oberfläche mit einer Vielzahl reversibel erzeugbarer hydrophiler und/oder oleophiler Bereiche

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7667196B2 (en) 2006-04-27 2010-02-23 Bruker Daltonik Gmbh Sample preparation for mass spectrometric imaging
GB2446251A (en) * 2006-12-18 2008-08-06 Bruker Daltonik Gmbh Preparation of a matrix layer for mass spectrometry
GB2446251B (en) * 2006-12-18 2012-05-23 Bruker Daltonik Gmbh Preparation of a matrix layer for mass spectrometry
US8237113B2 (en) 2006-12-18 2012-08-07 Bruker Daltonik Gmbh Preparation of a matrix layer for spectrometry
EP2975393A4 (fr) * 2013-04-04 2016-03-23 Shimadzu Corp Procédé de préparation d'échantillon pour maldi et dispositif de préparation d'échantillon
JPWO2014163179A1 (ja) * 2013-04-04 2017-02-16 株式会社島津製作所 Ｍａｌｄｉ用試料調製方法及び試料調製装置

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