WO2005101452A1

WO2005101452A1 - Preparation method or the microscopic analysis of the composition of substance mixtures

Info

Publication number: WO2005101452A1
Application number: PCT/DE2005/000690
Authority: WO
Inventors: Bernhard Spengler; Werner Bouschen; Daniel Eikel; Dieter Kirsch
Original assignee: Justus-Liebig-Universität Giessen
Priority date: 2004-04-16
Filing date: 2005-04-15
Publication date: 2005-10-27
Also published as: DE102004019043A1; DE102004019043B4

Abstract

The invention relates to a novel method for the preparation of surfaces for high resolution scanning MALDI mass spectrometry (SMALDI). The analyte or analytes are firstly applied to the sample support surface and then the matrix, whereby the matrix has a layer thickness of 0.5 µm to 3 µm. The matrix is then vapour-treated with a solvent atmosphere, recrystallising the same and thus integrating the analytes. By means of said vapour-treatment method the analyte molecules migrate during the recrystallisation and the resultant integration of the analyte on a scale corresponding to just the layer thickness of the matrix, in other words 0.5 µm to 3 µm. Surfaces prepared by said method are thus suitable for the spatially-resolved, scanning MALDI-mass spectrometry, as the location of origin and thus spatial information about the analyte molecule during the preparation are largely retained. The invention further relates to a device for carrying out said method.

Description

Preparation method for micro-range analysis of the composition of substance mixtures

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. 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. [Description and Introduction of the General Field of the Invention]

The present invention relates to the fields of chemistry, biology and biochemistry, physics and instrumental analysis.

[State of the art]

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. Unlike an optical microscope image, such an analytical image shows the quantity distribution of a certain substance within the sample as an image (eg as a grayscale image). Up to now, this has been possible in two-dimensional images, among other things, with the aid of laser-assisted, mass spectrometric methods (laser microprobe mass spectrometry) with a spatial resolution of 0.5 to 1 μm and an upper mass limit of approx. 500 to 800 u. In contrast to laser microsonde mass spectrometry, in which a sample is scanned directly without pretreatment with the help of a highly focused laser beam, the so-called "scanning microsonde matrix-assisted laser desorption ionization mass spectrometry" (SMALDI-MS)), also under the The term "MALDI-Imagin ^' g" is known to be operated only with a usable lateral resolution of about 25-30 μm, 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 μm, since analyte molecules are inevitably displaced spatially on the surface to this extent. Furthermore, the crystals usually have a diameter of 5 μm to 500 μm, 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. For this task it has proven to be advantageous to incorporate the analyte molecules individually into the crystals of the matrix substances during their crystallization or at least finely distributed in the interface regions between the small balls. It seems essential to separate the analyte molecules from each other, i.e. not to allow clusters of analyte molecules in the applied sample.

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. After the solution has dried on, 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. In aqueous solutions, 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. When using 2,5-DHB, the center of the spot is often empty or covered with fine crystals, which are often of little use for MALDI ionization due to their high concentration of alkali salts. The loading of analyte molecules is very uneven. This type of assignment therefore requires a visual observation of the sample carrier surface through a video microscope during MALDI ionization, which can be found in most commercially available mass spectrometers for this type of analysis. The ion yield and mass resolution fluctuate in the sample spot from place to place. It is often a tedious process to find a favorable spot on the sample spot with good analyte ion yield and good mass resolution, and so far only experience and trial and error have helped here. There are control programs for mass spectrometers with algorithms for automatically finding the best spots for MALDI ionization, but these methods, with their many attempts and evaluations, are necessarily quite slow.

In other application methods, 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.

The prior art knows numerous methods with which even the smallest analyte quantities can be made accessible to a MALDI analysis and which allow MALDI sample carrier plates to be loaded with a large number of analytes in a short time and to be made accessible to automatic MS. DE 196 28 178 C1 describes a method in which a MALDI

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 μm to 600 μm 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. Subsequently, 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 μm, they are too large for mass spectrometric methods with a resolution of 1 μm. DE 44 08 034 C1 describes a method for MALDI analysis of samples that were previously separated by means of 2D gel electrophoresis. Here, 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. However, this leads to an inhomogeneous distribution of the analyte in the sample drop deposited on the matrix. 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. However, homogeneous incorporation of the biomolecules into the matrix cannot be achieved in this way.

Furthermore, the prior art knows methods for ionizing high-molecular analytes. 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). As a result, the method is also suitable for laser foci with diameters in the range between 3 μm and 10 μm; 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. Optionally, the matrix with the analyte material can be dissolved and recrystallized. However, this method can only be used up to laser foci greater than or equal to 25 μm and is therefore not suitable for microscopic surface analysis. DE 102 07 615 A1 describes a method for producing ultraphobic surfaces on which hydrophilic and / or oleophilic areas can be reversibly produced. Solutions of MALDI matrices are first deposited on these surfaces and then analyte solutions are applied. By dissolving and recrystallizing the matrix, the analytes are built into the MALDI matrix.

In all methods of the prior art with the exception of US Pat. No. 5,808,300, the solution of the analyte is either applied to a sample carrier which has already been prepared with matrix, or the matrix and analyte solution are applied together. With these procedures, the analyte molecules inevitably migrate in the matrix layer. The three-dimensional structure of the analyte layer is changed in any case: analyte molecules in front of the Installation in the matrix, for example in the direction of the x coordinate in the order A, B, C, may be present in the order, for example, in the order B, C, A after installation in the matrix. This applies to all three spatial directions, so that with the methods of the prior art it is not possible to check the direction in which the analytes are incorporated into the matrix. The technique described in US Pat. No. 5,808,300 for applying matrices to analytes in the form of tissue sections does not allow such a directional control of the incorporation of the analytes into the matrix and therefore does not permit spatial resolutions in the range of 1 μm in the MALDI measurement. 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 μm.

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.

Advantageous embodiments of the device result from subclaims 16 to 19, 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.

It is known to the person skilled in the art that 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.

It is known to the person skilled in the art that the matrix in connection with the matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) 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.

Numerous methods are known to the person skilled in the art with which 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. In the preparation method according to the invention, 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. Inert is to be understood as meaning gases which are not soluble in the condensed steam. Reaction conditions mean the selected combination of pressure and temperature.

In order to apply the matrix to the sample carrier on which the analyte or analytes are already located using the vapor deposition process, 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. In this pressure range, 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. 1 * 10 ^"2 mbar and a temperature range from 20 ° C to 130 ° C so that an amorphous layer of matrix is deposited over the analyte or analytes on the sample carrier within 2-15 minutes. 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

Molecular beam epitaxy, electrodiffusion or electrochemical deposition. 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. It is known to the person skilled in the art that 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. Alternatively, the drops can be generated by an electric field between the nozzle and the sample surface.

The methods are described, for example, in Herbert Budzikiewicz: mass spectrometry. An introduction. 4th edition, VCH, Weinheim, 1998 or F. Lottsspeich, H. Zorbas: Bioanalytik, Spektrum Akademischer Verlag, 1st edition 1998. Furthermore, the matrix solvent droplets can also be generated by ultrasonic nebulization known to the person skilled in the art. Here, 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. Furthermore, 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

Piezo crystal sprayed by applying an electrical voltage to the sample holder. The printing processes mentioned are known to the person skilled in the art and can be found, for example, in “Der Brockhaus. Naturwissenschaft und Technik ", Version 3.0, CD-ROM, Bibliographisches Institut & F.A. Brockhaus AG, Mannheim; Spectrum Akademischer Verlag GmbH, Heidelberg, 2003.

In order to achieve an improved integration of the analyte into the evaporated matrix layer, 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. For this purpose, 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 μm to 3 μm 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. While conventional matrix substances usually have natural fluorescence, peptides or proteins that contain tryptophan can be observed via its natural fluorescence, for example. Furthermore, 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. In addition to the lateral uniformity, 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 ,

Furthermore, 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.

Due to the good solubility of the matrix substance in the solvent or in the solvent mixture, the solvent vapor causes the matrix to recrystallize. In this process, 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 μm to a maximum of 3 μm, 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 μm to 3 μm 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. As an alternative or in addition to the temperature increase, the recrystallization can also be accelerated by increasing the pressure in the container.

Through both steps of the preparation, i.e. by the application of the matrix and the subsequent recrystallization, 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.

In the spatially resolved analysis of biological or synthetic samples, the preparation of the sample is a decisive step that limits the sensitivity that can be achieved, the mass resolution and the effective spatial resolution of the measurement. 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).

There is a fundamental difference between a standardized MALDI-MS preparation according to the state of the art and the preparation of a surface for mass spectrometric analysis. In the case of the preparation according to a method according to the prior art, the analyte or the analytes are generally in dissolved form or are brought into a dissolved form. This considerably eases the necessary requirements for a MALDI preparation, since in this case the sample can be mixed with the matrix or one

Mixing of the analyte or analytes and matrix takes place on the sample plate before crystallization. As a result, the individual analyte molecules are separated from one another by the matrix. It is known to the person skilled in the art that 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. It is known to the person skilled in the art that mixing with the matrix is in principle only possible if the analytes are present on the surface in the solid phase 5, so that the matrix must act on the surface in dissolved form and dissolve the analyte or analytes. At the same time, this means that in a MALDI preparation according to the prior art, the spatial localization of the substances is completely lost by this step, since the liquid remains on the surface for up to several minutes before the solvent evaporates. The information gained, which is achieved by the high spatial resolution of a device with a highly focused laser beam and a resulting spatial resolution in the range of the possible maximum focus of the laser beam, is then lost in advance through the preparation (see Spengler B., Hubert, M .: Scanning microprobe matrix-assisted laser5 desorption ionization (SMALDI) mass spectrometry: Instrumentation for sub-micrometer resolved LDI and MALDI surface analysis, Journal of the American Society of Mass Spectrometry; 13 (2002) 735-748).

In the method according to the invention, care should be taken to ensure that the extent of the analyte's movement away from its respective application location remains less than the diameter of the laser or ion beam focus of the mass spectrometer, and at the same time that the analyte molecules are incorporated into the matrix in order to to be able to use the prepared sample in scanning mass spectrometry. 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.

Furthermore, the prepared sample can also be used for microscopic methods of mass spectrometry. This means that ions are brought into the gas phase from the sample with the help of a large-area laser beam and with the aid of ion optics, these ions are imaged on a detector in such a way that the spatial origin of the ions and the imaging location can be assigned to one another. 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.

Numerous methods are known to the person skilled in the art, with the aid of which 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. Furthermore, it is known to the person skilled in the art that 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. It is also possible to grow cells on the sample carrier by methods known to the person skilled in the art, the person skilled in the art knowing that he has to choose a sample carrier material on which the cell line to be grown adheres. The application of biological samples is described, for example, in L. Li, R.W. Garden, E.V. Romanova, and J.V. Sweedler: "In situ sequencing of peptides from biological tissues and single cells using MALDI-PSD / CID analysis.", Analytical Chemistry; 71 (1999) 5451-5458.

Furthermore, the person skilled in the art is familiar with 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. Furthermore, 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. In addition, the person skilled in the art knows 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-p-cumaric acid, trans-p-cumaric acid, 6-aza-2- thiothymidine, urea, succinic acid, adipic acid, malonic acid and malononitrile.

For the production of the solvent vapor for the recrystallization of the matrix layer and for the production of this matrix layer, known to the person skilled in the art as 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. Other 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. Lützenkirchen, R. Kaufmann: “On-target deuteration on for Peptide Sequencing by Laser Mass Spectrometry ", Organic Mass Spectrometry, 28 (1993), 1482-1490. To produce the gas-steam mixture, solvents or solvent mixtures in which the selected matrix substance is soluble should be selected. In the case of analytes which are, for example, sensitive to oxidation or easily decomposable, 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 person skilled in the art is aware that the gas composition of this inert gas (carrier 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.

The person skilled in the art knows numerous 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. It is known to the person skilled in the art that if 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 , It is known to the person skilled in the art that the 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. “Microscopically 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 μm. 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 μm.

The person skilled in the art is aware that the 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.

If the standard methods for sample preparation are examined with regard to crystal size, migration and behavior of the analyte when incorporated into the crystal, it can be seen that 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). When the sample crystallizes out, 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. At the same time, the rate of crystallization is a decisive factor for the conditions in the crystals. If the individual crystals are now examined for the distribution of the analyte, the results found are in contrast to the studies carried out on matrix crystals from Horneffer, V .; Forsmann, A .; Strupat, K .; Hillenkamp, F .; Kubitscheck, U; Localization of analyte molecules in MALDI preparations by confocal laser scanning microscopy, analytical chemistry; 73 (2001) 1016-1022. The images taken from crystals using confocal microscopy do not indicate a different distribution of the analytes within the crystal. For these investigations, however, the crystals have been grown over a much longer period and are considerably larger, so that crystallization from the chemical equilibrium can be assumed. In MALDI matrix preparation, an inhomogeneous distribution is apparently only caused by the rapid drying of the sample. This is also indicated by the measurements in Figure 6.

It is readily apparent to the person skilled in the art that the method according to the invention for the preparation of analytes is suitable for the automated production of preparations for analytical measurements, preferably for mass spectrometric measurements.

[Embodiments]

Embodiment 1 ("Migration 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. Before preparation, the sample plate (aluminum carrier with 8 μm 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. KimWipes, Kimberly-Clark, Neenah, USA), then 2 to 5 ml of ethanol are dropped onto the sample holder and under a hot air stream using a hair dryer dried at approx. 80 ° C. 0.5 μl of the analyte mixture solution (typical concentrations: 1 * 10 ^"7 mol / l to 5 * 10 ^{" 5} mol / l) are added dropwise to the sample carrier, which is still warm to the touch. Then 0.5 μl of 2.5-DHB solution (10 mg / ml in ethanol / 0.1% aqueous TFA, 60:40 V / V) is added to the drop and mixed. Depending on the desired duration of the crystallization process, 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

100 μm long crystals. By increasing the water content in the matrix solution up to 100%, this effect, given the much lower vapor pressure of the water, can be intensified. Since the inner part of the sample is still covered with liquid during the crystallization of the edge, the crystals in the inner area only form shortly before the sample dries up. A layer of many very small crystals is formed in a short time. This layer can be clearly seen from the edge of the sample, usually through a narrow area between the edge and the finely crystalline interior, in which there is almost none Crystals are present, differentiate. It can thus be shown in a very simple manner that the analyte is separated by the way the crystallization process takes place within the sample. Already the comparison between 30 and 40 accumulated mass spectra according to example 7 from the edge or from the inner area of the sample shows (Figure 4) that the almost inevitable contamination of a sample by sodium or potassium ions is more localized inside the sample than on Edge.

Embodiment 2 ("Slow MALDI Standard Preparation") Figure 5 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 μl peptide mixture in ethanol / water (1: 1 v / v, 5 * 10 ^"5 mol / l) was added to the sample carrier (aluminum carrier with 8 μm gold coating, 20 mm diameter) and with 1 μl matrix (2, 5-DHB) in ethanol / water (2: 3 v / v,

20 μg / ml) mixed on the plate. The sample then crystallized in the room air for about five minutes. The slow crystallization resulted in large 2.5 DHB crystals with a size of up to 500 μm at the edge of the sample. A range of 100 μm by 100 μm was scanned with a resolution of 1 μm. The grayscale distribution images (see Example 8 and Figure 5) showed a different distribution of the peptide signals within a large crystal. The left side of the pictures is dominated by a crystal that is larger than the image section. In the middle you can see a narrow needle with a width of approx. 10 μm. The size of the crystals was determined using an Olympus BX41 microscope with an integrated scale. While the hydrophilic peptide dominates the upper right part of the large crystal, the hydrophobic peptide can be found in the lower left area. Figure 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 ("Fast MALDI Standard Preparation")

In contrast to example 2, a modified method means that hardly any segregation effects are observed within the crystals. In Figure 6 is a peptide mixture of dynorphin 1-9 (hydrophobic) and vasopressin [Argδ] (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. However, rapid crystallization was now carried out. By heating the sample plate to 65 ° C. before the sample preparation to 75 ° C., preferably to 70 ° C., or by drying the sample under a hot air stream at 70 ° C. to 90 ° C., preferably 80 ° C., a very rapid crystallization can be achieved in about 5 to 10 seconds The grayscale images of the rastered image section (generation as in exemplary embodiment 8) of 100 μm by 100 μm show the distribution of the two analytes and the matrix (Figure 6). Inside the crystals , which have a size of 10 μm to 50 μm, 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 μm lateral resolution, 50 ^th American Society for Mass Spectrometry Conference; 2.6. - 06.06.2002, Orlando, Florida, clearly shown in Figure 7 and Figure 8. The sample was prepared as in Example 3, the measurement as described in Example 8. A peptide mixture of substance P, melittin and human insulin (individual concentration each 5 ^* 10 ^"5 mol / l, in the ratio substance P: melittin: human insulin = 1: 2: 4 mixed) in water / ethanol (1: 1 v / v) with 2.5-DHB (20 mg / ml, water / Ethanol, 6: 4 v / v) as in Example 1. 0.5 μl analyte and 0.5 μl are mixed on the sample plate and dried in a warm air stream (approx. 60 ° C.) in approx. 1 minute The sample shows an edge with large crystals and a finely crystalline inner area of the sample. Again, an area of 100 μm by 100 μm was scanned with a step size of 1 μm, 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 vacuum evaporation system JEE-4B from Joel (Japan) (Figure 9), originally used for the preparation of samples for electron microscopy, is used here for the MALDI preparation of biological materials.

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. When evaporating matrix, 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). For temperature control (up to 300 ° C), 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. Even the first experiments showed that it was not necessary to heat the matrices to the melting point, since a layer was already deposited on the sample carrier by sublimation at normal pressure at lower temperatures. Even at temperatures in the range of 80 ° C to 130 ° C, enough material sublimated to form a fine crystalline layer on the sample carrier (Figure 11, right photo). When heating the matrix substance for the first time, make sure that the temperature is increased by a maximum of 5 ° C per minute, because when heated more intensely, the crystal water contained in the matrix substance evaporates too quickly, causing the matrix material to sublime abruptly and such large matrix crystals during subsequent sublimation arise that the matrix crystals do not adhere to the sample carrier.

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). Degradation products of 2,5-DHB were found in low intensity in the low mass range from m / z = 46 u to 137 u, whereby low intensity means that the signal intensities were only a factor of 2 to 5 above the background noise. In contrast to the mass spectra of a MALDI preparation, more 2,5-DHB clusters in the mass range from m / z = 300 u to 600 u could be seen in the spectrum.

Embodiment 5c: sublimation of the matrix at reduced pressure

Further experiments showed that the formation of degradation products and clusters could be suppressed if the sublimation of the matrix was carried out at a reduced pressure and thus also at a reduced temperature (Figure 12, lower spectrum). The optimum value for the sublimation of 2.5-DHB was a pressure range of 5 * 10 ^"1 mbar to 5 mbar, preferably a pressure of 1 mbar. This resulted in a temperature range of 40 to 60 ° C. for 2.5-DHB , preferably from 48 ° C. for sublimation The sample carrier was steamed for 5 minutes The two other matrices, sinapic acid and CHCA, showed a similar behavior: Sinapic acid sublimates at normal pressure from about 90 ° C. at a reduced pressure of 1 mbar 45 ° C. CHCA sublimates at about 110 ° C. under normal pressure and at 1 mbar at 53 ° C. The mass spectra also contained degradation products and clusters of the matrices. The layers which were evaporated onto the sample supports turned out to be not very stable against mechanical stress (Figure 11, left photo) Even the bombardment with the highly focused nitrogen laser beam was sufficient to break larger parts out of the layers, especially with sinapic acid and CHCA Want.

With laser energy close to the threshold of ion detection, the damage to the layers could be reduced in such a way that measurements with a focus diameter and an analytical resolution of 0.5 to 5 μm were possible. With a laser focus of 0.8 μm, this laser energy was close to that Threshold of ion detection approx. 1 * 10 ¹⁰ W / cm ² ; with a laser focus of 15 μm approx. 1 * 10 ⁷ W / cm ² . The red dye of the felt-tip pen previously applied to the sample carrier with the aid of a felt-tip pen made it clear that by applying the matrix in accordance with this exemplary embodiment, a substance applied before the preparation does not migrate beyond the analytical resolution. The clear boundary between the colored line and the gold surface is retained even after vapor deposition with the matrix and shows no migration in the resolution range from 0.5 to 3 μm. The pictures were taken with an Olympus BX41 microscope with an integrated scale.

Embodiment 5d: Evaporation of sample carriers to which analyte was previously applied

In order to check the matrix layers produced with regard to their ability to operate MALDI mass spectrometry of surfaces with them, a mixture of the three peptides substance P, melittin and insulin was applied to the sample holder according to embodiment 1 before the evaporation according to this embodiment. The individual components were each dissolved at a concentration of 1 * 10 ^"5 mol / l in ethanol / water 1: 1 (v / v) and then mixed in a ratio of 1: 2: 4 (substance P: melittin: insulin). This mixture showed a mass spectrum with good, approximately equally high signal intensity for all three components in the mass spectrum for MALDI preparation according to embodiment example 1. 1 μl of the mixture was applied to the sample holder, and the drop was completely dry after about 10 minutes at room temperature and standard pressure , so that vapor deposition with matrix could be carried out according to this exemplary embodiment, after the vapor deposition of the sample carrier with matrix

(2,5-DHB), the mass spectra initially showed only very low signal intensities for the three substances. At irradiance levels of approximately 1 * 10 ⁷ W / cm ² to approximately 1 ^* 10 W / cm with a laser focus of approximately 1 μm, which are usually used in MALDI measurements with the device described in application example 8, no mass signals could be obtained of the peptides are detected. Weak mass signals were only recognizable when the irradiance was increased five to ten times in the range from 5 * 10 ¹⁰ W / cm ² to 10 ^* 10 ¹⁰ W / cm ² (application example 8) and the sum of 200 to 400 individual spectra (Figure 13). Also for devices with laser foci from 50 μm to 200 μm, in which the radiation strengths in the range from 1 ^* 106 W / cm2 to 1 ^* 101 W / cm2, only a five to ten-fold increase in the irradiance leads to a measurement signal. The low intensity of the peptide signals (Figure 13) can be explained by the poor mixing of the analytes with the matrix. The incorporation of the analyte is the matrix crystal is for measurement according to Horneffer, V .; Dreisewerd, K; Ludemann, H. C; Hillenkamp, F .; Lage, M .; Strupat, K .; Is the incorporation of analyzes into matrix crystals a prerequiste for matrix-assisted laser desorption / ionization mass spectrometry? A study of five positional isomers of dihydroxybenzoic acid; International Journal of Mass Spectrometry; 187 (1999) 859-870, not absolutely necessary, however, due to the small laser focus, the area from which the matrix and analyte are desorbed at the same time is very small. When summing up the individual spectra, it was observed that the signal intensities of the peptides fluctuate widely within several individual measurements. If the same sample point is bombarded several times, only the matrix in the mass spectrum is visible. After 5-10 laser shots, the analytes are visible in the spectrum for 2-3 shots. The signal intensity for this sample site then decreased overall. This phenomenon indicated poor integration of the analyte into the matrix. Only when the vaporized layer had almost been removed were the analyte and matrix desorbed together for a few laser shots. The sample was then used up at this point.

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. For this purpose, 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 μl 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. The resulting mass spectra according to embodiment 7 did not differ from the typical mass spectra of a normal MALDI preparation. Because of the possible reduction now Due to the better incorporation of the analytes into the matrix, the irradiance was not observed in this spectrum. It is noteworthy that no degradation products of the matrix were now visible (Figure 14, section).

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 (vapor deposition of the matrix and integration of the analyte) 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. In order to achieve an improved integration of the analyte into the evaporated matrix layer according to embodiment 5d, the sample carriers were exposed to a moist environment.

For this purpose, 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. When choosing the container, it should be noted that the condensed water formed on the lid and on the edge of the container did not drip onto the sample. 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. In order to maintain a supersaturated atmosphere over several days, 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. 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. In the Initial experiments showed a recognizable recrystallization of the matrix layers depending on the choice of matrix after two to three days of incubation (Figure 15). While 2,5-DHB showed visually significant changes, the changes in sinapic acid and CHCA were barely visible. The recrystallization was checked by optical inspection with an Olympus BX41 microscope with an integrated scale.

The incubated layers of 2,5-DHB recrystallized much faster. The first optical changes became visible after 1 to 3 hours and the process was completely completed after 14 days. The different behavior of the three matrices is due to their different water solubilities. While sinapic acid and CHCA dissolve very poorly in water, 2,5-DHB is readily water-soluble. The 2.5-DHB layer became much more stable due to the recrystallization, and the removal of large parts by mechanical loads or by laser bombardment was prevented.

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

The boundary of a felt pen line in Figure 16 (left photo) shows both before and after vapor deposition with the matrix according to embodiment 5c and incubation with water (right photo) according to this embodiment still a sharp line between paint and untreated aluminum sample holder. Despite the change due to the preparation, this type of treatment based on optical assessment using an Olympus BX41 microscope with an integrated scale can be assumed to maintain the spatial distribution of the analytes on the surface of the sample within the resolution of the scanning laser according to embodiment 7 remains.

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. Already when applying peptide mixtures to the surface according to application example 1 and subsequent preparation according to embodiment 5d and this embodiment (7c), 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. in the vapor deposition unit. It was then incubated with water at a sand bath temperature of 125 ° C. for 24 hours. The limit of the previously dried drop was clearly recognizable after vapor deposition with the matrix and also after incubation of the analytes with water (Figure 17). A recrystallization of the matrix was also observed. Nevertheless, the sharp limit of the previously applied peptide mixture was retained after optical inspection using an Olympus BX41 microscope with an integrated scale. This example shows that preparation is possible without a noticeable migration of the peptide mixture. 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 μm 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 μm by 100 μm was scanned with a step size of 1 μm. 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 top row shows the mass signal of the applied matrix 2,5-DHB ([2 * 2,5-DHB-2 * H ₂ O + H] ⁺ = 273 u), the middle row belongs to the previously applied diluted red dye ( m / z = 450 u) and the bottom row belongs to the peptide substance P (m / z = 1348.6 u). 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 μm 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 μm), 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. With this preparation method, a method is available for the first time with which SMALDI mass spectrometry can be carried out with a spatial analytical resolution of 2 μm. Embodiment 8 ("Measurement of the analyte by scanning mass spectrometry")

A sample mixture containing 2,5-dihydroxybenzoic acid (2,5-DHB), substance P (a peptide), melittin and insulin was examined by SMALDI time-of-flight MS. The sample matrix consisted of 2,5-DHB.

Embodiment 8a: Components of the measuring device

A "LAMMA 2000" mass spectrometer (Figure 2) was used, comprising: a nitrogen laser (N ₂ laser): VSL 337 N D laser (Laser Science Inc., Cambridge, Massachusetts, USA), λ = 337 nm, Pulse repetition rate 10 Hz, pulse duration 3 ns, lateral resolution = focus diameter of the laser beam: 0.6-1.5 μm, - an ion source, containing a pinhole with an opening diameter of 7 mm and a grid, an ion guide channel, opening 4mm diameter (internal dimension), which is conical expanded over a distance of 8 mm to an internal dimension of 6 mm,

- a flight tube, - a field grating, a detector consisting of a double microchannel plate with an active diameter of 40 mm,

- X-Y-Z piezo table (Graf Mikrotechnik, Wertingen), on which the sample holder was attached and by moving the sample holder was scanned with a stationary laser beam. Before the start of the measurement, the sliding table was moved once in the z direction to focus the sample carrier once. During the measurement, the movement was only in the x and y directions. Screen size: 100 μm x 100 μm, minimum step size 0.25 μm

- Microprocessor system for controlling the sliding table (Motorola 68000 microporcessor, Motorola Inc., Schaumburg, Illinois)

- 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, Cologne), 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

• Pinhole - grid: 8.6 mm,

• Grid - ion guide channel opening: 1.9 mm,

• ion guide channel opening - field termination grid in front of detector: 1306 mm and

• Field termination grid - detector surface: 20 mm.

Embodiment 8c: Selected potentials

• Pinhole: fixed at +10000 V,

• sample plate: +13261 V,

• grid: -1810 V, • detector surface: -1850 V,

• ion guide channel, flight tube and field termination grid were at ground potential (0V)

Embodiment 8d: Scanning the sample carrier Scan area: 100 μm x 100 μm Scan step size: 1 μm scan speed: 10 pixels per second 10,000 mass spectra per pixel

Embodiment 8e: pre-focusing

The laser beam was pre-focused outside the vacuum in the submicrometer range. Suprasil® quartz lenses known to those skilled in the art were used for prefocusing with astigmatic correction, which prefocused the laser beam to approximately 10 μm. Embodiment 8f: Use of an Nd: YLF laser As an alternative to the nitrogen laser (N ₂ laser), an Nd: YLF laser, for example Model 421 QD (ADLAS, Lübeck), can be used, output energy 100 μJ 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 μJ at 262 nm. The Nd: YLF laser beam has a strictly elliptical shape after being quadrupled by the BBO crystal. In this case, 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. After prefocusing with a spherical lens, the beam therefore fills a narrow strip only in the input lens of the focusing lens. For this reason, 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. Hubert, Scanning Microprobe matrix-Assisted Laser Desorption lonization (SMALDI) Mass Spectrometry: Instrumentation for Sub-Micrometer Resolved LDI and MALDI Surface Analysis, Journal of the American Society of Mass Spectrometry 2002, 13, 735-748) .5 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. In parallel, 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 μm to 0.6 μm was used, the scan step size was also 1 μm (not shown).

Embodiment 9: Analytes at risk of oxidation or decomposition

If the method according to the invention is used for the preparation of analytes which contain one or more substances which are easily oxidizable and / or decompose easily, it is advisable to additionally use an inert carrier gas according to claim 5 when treating the matrix with solvent vapor (without illustration) ).

Exemplary embodiment 10: Preferred method for applying the matrix In the method according to the invention for the preparation of surfaces for micro-range analysis, it is particularly advantageous to apply the matrix using the vapor deposition method according to claim 12, wherein layer thicknesses of the matrix of 0.5 μm to 3 μm are preferred be (without picture). [Legends of Figures and List of Reference Numbers] List of Reference Numbers

19 drawings are listed below.

1. ion guide channel

2. Flight tube with vacuum housing

3. Detector

4. ion-optical arrangement 5. lens with central bore

6. X-Y-Z piezo table and X-Y-Z stepper motor table

7. Laser

8.CCD camera (Charge Coupied Device camera)

9. Pre-focusing with astigmatic correction. 10. Dichroic mirrors

11. Light source for sample observation

12. Evaporation plant

13. Electrodes for connecting the boat

14. Vacuum container 15. Current control for electrodes

16. Temperature control

17. Pressure control

18. Sample holder

19. Shuttle 20. Sample carrier with analytes

21. Temperature sensor

22.Valve

23. Container with solvent reservoir

24. Lid of the container 25. Sample support Figure legends

Illustration 1 :

Different methods for surface preparation: a) standard preparation b) electrospray c) spraying method d) vapor deposition method

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.

Figure 2:

After astigmatic correction and pre-focusing 9 and deflection via dichroic mirrors 10, 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 3:

Representation of the images of the surface distributions of 2,5-DHB, substance P, melittin and insulin, as well as of different isotopomers, derivatives and addition products thereof, obtained by automatic image processing without pre-scan or without specifying masses.

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:

Dried-droplet preparation with 2,5-DHB as a matrix: Slow crystallization (approx. 10 minutes) results in large crystals with different analyte distributions. (100 μm x 100 μm, 1 μm 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

Figure 6:

Third-droplet preparation with 2,5-DHB as a matrix: Rapid crystallization (approx. 10 seconds) produces small crystals with a homogeneous distribution of the analyte. (100 μm x 100 μm, 1 μm 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

Figure 7:

Edge of a third-droplet preparation with 2.5-DHB as a matrix (100 μm x 100 μm, 1 μm raster)

Figure 8:

Fine crystalline crystals inside a third-droplet preparation with 2.5-DHB as a matrix (100 μm x 100 μm, 1 μm step size)

Figure 9:

Evaporation plant Jeol JEE-4B 12 with vacuum container 14. 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.

Figure 10:

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 μm b) 65 x 50 μm

Figure 12:

Mass spectra of thermally sublimed and recondensed matrix a) 2,5-DHB sublimates at 130 ° C under normal pressure in 12 minutes b) 2,5-DHB sublimates at 48 ° C at 1 mBar in 5 minutes

Figure 13:

MALDI - mass spectra of a peptide mixture after vapor deposition with thermally sublimed and recondensed matrix (2,5-DHB) with a) high and b) medium laser intensity (300 spectra added up)

Figure 14:

MALDI mass spectrum of 2,5-DHB / peptide mix after the evaporation was dissolved with ethanol / water 1: 1 (30 spectra added up)

Figure 15:

Incubation of 2.5-DHB layer with water a) support vaporized with 2.5-DHB b) support vaporized with 2.5-DHB and moistened with water for 3 days c) vaporized support with 2.5-DHB and 14 days moistened with water

Figure 16:

Felt pen stroke on gold test carrier a) before and b) after steaming with 2.5-DHB and moistening (650 x 500 μm) Figure 17:

Change of the edge of the peptide mixture drop on gold carrier (650 x 500 μm) a) After evaporation with 2,5-DHB b) After incubation with water

Figure 18: Ion distribution images of the boundary of a prepared peptide mixture (100 x 100 μm, 1 μm 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

Claims

[Expectations]

1. Process for the preparation of analytes for mass spectrometry, characterized in that in a first step the analyte or analytes are applied to the sample carrier, then the matrix is deposited on the essentially solid analyte or analytes and finally the analyte by treating the matrix with a vapor of a solvent of the matrix is incorporated into the matrix in a direction-controlled manner.

2. A method for the preparation of analytes for mass spectrometry according to claim 1, characterized in that for the purpose of the directionally controlled incorporation of the analyte or analytes into the matrix, the following steps are carried out in succession:

- Transfer the sample holder into a substantially closed container, the container containing a solvent reservoir or a device for receiving a solvent reservoir, - Filling the solvent reservoir with a solvent or a solvent mixture and, if it is not a solvent reservoir integrated in the container, move it the filled solvent reservoir in the container,

- heat the solvent reservoir until the container atmosphere is saturated with solvent vapor,

- Leave the sample holder in the container under the saturated solvent vapor atmosphere for 3 hours to 14 days until the integration of the analyte or analytes into the matrix layer has ended,

- Remove the sample carrier containing the matrix layer and analyte from the container.

3. A method for the preparation of analytes for mass spectrometry according to claim 2, characterized in that the solvent or solvents for filling the solvent reservoir are selected from the group consisting of water, methanol, ethanol, acetone, acetonitrile, propanol, isopropanol and 5 n-butanol , isobutanol, 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, esters, ethers, carboxylic acids, amines, aliphatics, aromatics, araliphatics, haloalipates. 4. Process for preparation of analytes for mass spectrometry according to claim 2 or 3, characterized in that the solvent is wholly or partially deuterated, which is preferably D ₂ O.

5. A method for the preparation of analytes for mass spectrometry according to one of claims 2 to 4, characterized in that the atmosphere 5 contains a carrier gas in addition to the solvent vapor when the analyte or analytes are integrated into the matrix layer, selected from the group consisting of air, nitrogen and helium , Neon, argon, krypton, xenon, carbon dioxide.

6. Process for the preparation of analytes for mass spectrometry according to one of claims 2 to 5, characterized in that the composition of the carrier gas is changed during the integration of the analyte or analytes into the matrix layer.

7. Process for the preparation of analytes for mass spectrometry according to one of claims 2 to 6, characterized in that the material of the sample holder is selected from metals, preferably gold or aluminum, 5 metal alloys, steel, silicon, Teflon, quartz, PVDF, cellulose rain rated cellulose, anionically modified cellulose, cationically modified cellulose, nylon, nitrocellulose, surfaces modified with beads or anchor molecules.

8. A method for the preparation of analytes for mass spectrometry according to 5 one of claims 2 to 7, characterized in that the method for applying the analyte or analytes to the sample carrier is selected from spraying and subsequent drying, binding of the analyte or analytes to anchor molecules or Beads or beads provided with anchor molecules, binding to solid supports, binding to beads, binding to needles, binding to combs, binding to wafers, spraying in a suitable solvent using a gas stream, spraying in a suitable solvent using an electrical potential (electrospray -Method), mechanical distribution of the solid analyte or analytes, stamping, printing, laser printing processes, thermal transfer processes, ink jet printing processes, Mo-5 lecular beam epitaxy, electrodiffusion, electrochemical deposition, ultrasonic nebulization, sublimation and subsequent deposition, and - if it concerns An alyten of biological origin - pressing, growing or gluing. 9. A method for the preparation of analytes for mass spectrometry according to one of claims 2 to 8, characterized in that the method for applying the matrix is selected from spraying in a suitable solvent using a gas stream, spraying in a suitable solvent using a electrical potential (electrospray process), mechanical distribution of the solid analyte or analytes, stamping, 5 printing, laser printing process, thermal transfer process, ink jet printing process, molecular beam epitaxy, electrodiffusion, electrochemical deposition, ultrasonic nebulization, sublimation and subsequent precipitation.

10. A method for the preparation of analytes for mass spectrometry according to one of claims 2 to 9, characterized in that the application of the analyte or analytes and / or the application of the matrix substance is carried out by vapor deposition of the sample carrier surface, the following steps being carried out in succession:

- bring the analyte (s) or the matrix substance into a container of a vacuum chamber,

- Secure the sample holder at a distance of 5 mm to 40 mm to this container in the vacuum chamber so that the side of the sample holder to be evaporated faces the analyte or the matrix substance, - Evacuate the vacuum chamber, the pressure inside the chamber decreasing evacuation is preferably 100 mbar to 1 * 10 ^"2 mbar,

- heat the container until the analyte (s) or the matrix substance sublime and then maintain this temperature,

- deposit the analyte (s) or the matrix substance on the sample carrier surface, preferably within 2 to 15 minutes,

- stop the supply of heat and ventilate the vacuum chamber until the pressure inside corresponds to the ambient pressure,

- Remove the vaporized sample holder.

11. A method for the preparation of analytes for mass spectrometry according to one of claims 2 to 10, characterized in that the application the matrix substance is carried out according to the method of claim 10, layer thicknesses of 0.5 μm to 3 μm being preferably produced. 12. A method for the preparation of analytes for mass spectrometry according to one of claims 2 to 11, characterized in that the matrix substance is selected from 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-p-cumaric acid, trans-p-cumaric acid, 6-aza-2-thiothymidine, urea, succinic acid, adipic acid, malonic acid, malononitrile. 13. A method for the preparation of analytes for mass spectrometry according to one of claims 2 to 12, characterized in that the analyte or the analytes and / or the matrix substance by repeated repetition of a method and / or by combining several methods for applying analyte and / or matrix substance can be applied. 14. Use of the method according to one of claims 2 to 13 for the analysis of substances of chemical, biological and / or biochemical origin, preferably for the analysis by means of the high-resolution scanning MALDI mass spectrometry. 15. Device for performing the method according to one of claims 1 to 13, comprising - a holder (18) for a carrier (20),

- and means for applying one or more analytes to the support (20), and means (19) for depositing a matrix on the analyte or analytes,

5 characterized in that the device further comprises means (23) for supplying a vapor of a solvent for the matrix. 16. The device according to claim 15, characterized in that the means (23) for supplying a vapor of a solvent for the matrix are designed in the form of a heatable liquid bath. 17. The device according to one of claims 15 to 16, characterized in that the The device further comprises means for determining the thickness of the matrix during the deposition of the matrix on the analyte, for example in the form of a quartz crystal balance, which is arranged in such a way that the means for depositing the matrix on the analyte have essentially the same ratio to the Weighing surface of the quartz crystal balance must be at the surface of the analyte. 18. Device according to one of claims 15 to 17, characterized in that the device further comprises means for determining the degree of integration of the analyte in the matrix, for example in the form of means for determining the volume of the matrix, which during of the integration process swells, or in the form of a microscope or in the form of a fluorescence microscope in the case of fluorescence-labeled analytes or in the form of a polarization microscope in the event that the integration of the analyte triggers a recrystallization of the matrix.

9. Device according to one of claims 15 to 18, characterized in that the holder for one or more carriers (20) is designed to be movable relative to the means for applying an analyte and the means (19) for depositing a matrix on the analyte and the arrangement is such that the carrier or carriers

- initially with the means for applying an analyte to the carrier (s) (20),

- thereafter with the means (19) for depositing a matrix on the analyte or analytes and the associated means for determining the thickness of the matrix,

- and then interacts with the means (23) for supplying a steam with the associated means for determining the degree of integration of the analyte in the matrix for automation of the preparation.