EP1573776B1 - Method for producing a sample carrier for maldi-mass spectrometry - Google Patents

Method for producing a sample carrier for maldi-mass spectrometry Download PDF

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Publication number
EP1573776B1
EP1573776B1 EP03795893.1A EP03795893A EP1573776B1 EP 1573776 B1 EP1573776 B1 EP 1573776B1 EP 03795893 A EP03795893 A EP 03795893A EP 1573776 B1 EP1573776 B1 EP 1573776B1
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EP
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Prior art keywords
maldi matrix
points
maldi
layer
sample carrier
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EP03795893.1A
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German (de)
French (fr)
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EP1573776A2 (en
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Karsten Reihs
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Qiagen GmbH
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Qiagen GmbH
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    • 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

Definitions

  • the present invention relates to a method for producing a sample carrier having a plurality of MALDI matrix dots, a sheet obtainable by the method according to the invention and a long-term stable sheet.
  • mass spectrometry For the analysis of samples, for example in drug chemistry or in biological research and production, mass spectrometry has become increasingly popular. For the analysis of biomolecules in the samples, mass spectrometry with ionization by matrix-assisted laser desorption and ionization (MALDI) is preferably used.
  • MALDI matrix-assisted laser desorption and ionization
  • Sample carriers with MALDI points are for example from the WO 00/67293 known.
  • the MALDI matrix dots are produced according to the prior art by applying the matrix substance as a solution in the form of a drop of liquid to a sample carrier and drying it there. Particularly in the case of series tests in which in some cases more than 200 MALDI matrix points have to be applied to the sample carrier, this method is very complicated despite automation techniques. In addition, the MALDI matrix points are not uniform in shape and are not homogeneous in themselves. Furthermore, the position of the matrix points on the sample carrier is relatively inaccurate. In order to prevent a running into each other of two adjacent points, their distance must be selected to be correspondingly large.
  • the object is achieved according to the invention with a process for producing a sheet, preferably a sample carrier, with a plurality of MALDI matrix points, in which the MALDI matrix points applied by precipitation of the MALDI matrix substance from the gas phase on the sample carrier become.
  • the MALDI matrix points can have any shape. They are very reproducible to produce, very homogeneous and have a surface structure, which can be achieved with very good mass spectra.
  • a sheet in the context of the invention is any desired shaped body with an arbitrarily shaped surface.
  • the sheet is a plate with a flat surface, most preferably a sample carrier, but preferably has no indentations.
  • the sheet of the invention is a film.
  • a MALDI matrix point in the sense of the invention essentially consists of at least one MALDI matrix substance known to the person skilled in the art.
  • Preferred MALDI matrix substances are 3-hydroxypicolinic acid, ⁇ -cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, sinapinic acid, 2,4,6-trihydroxyacetophenone, nitrobenzyl alcohol, nicotinic acid, ferulic acid, caffeic acid, 2-aminobenzoic acid, picolinic acid, 3-aminobenzoic acid, 2,3,4-trihydroxyacetophenone, 6-aza-2-thiothymidine, urea, succinic acid, adipic acid, malonic acid or a mixture thereof.
  • the MALDI matrix substance is ⁇ -cyano-4-hydroxycinnamic acid.
  • the MALDI matrix substance selected is a compound which sublimes on the sheet and is visible to the human eye.
  • the MALDI matrix points are generated by precipitation of the MALDI matrix substance from the gas phase.
  • Precipitation from the gas phase in the context of the invention is any method in which the MALDI matrix substance is transferred from the gas phase to the sheet. Examples include condensation or sublimation.
  • the application of the MALDI matrix dots to the sheet occurs by sublimation.
  • Sublimation in the sense of the invention involves the MALDI matrix substance being transferred as a solid into the gas phase and / or deposited in the form of a solid from the gas phase on the sheet.
  • the sublimation preferably takes place in vacuo. Particularly preferably, the solid is heated to sublimation.
  • a plurality of MALDI matrix substances are more preferably used in parallel or sequentially.
  • the MALDI matrix substances can be used to produce different MALDI matrix points.
  • a MALDI matrix point it is also conceivable for a MALDI matrix point to have a substructure, for example subdots which are present separately from one another, each of which is composed of a different MALDI matrix substance.
  • a substructure may also be, for example, concentric circular rings, each consisting of a different MALDI matrix substance.
  • the sample carrier is preferably covered by the gaseous phase, particularly preferably the sublimation, by a shaped body, a so-called mask, which has through-cut recesses. Only in the area of these recesses does the MALDI matrix substance settle on the sheet, forming a MALDI matrix point or partial point.
  • This mask can have any number of recesses, which can have any shape.
  • the recesses may be round, rectangular, square, triangular or oval, to name but a small number of the possible shapes.
  • the shape can be used to distinguish the respective MALDI matrix substances on a sheet.
  • the sheet may also be originally covered by multiple masks, which are then removed one after the other be applied, for example, to different areas of the sheet according to the invention different MALDI matrix substances.
  • the mask has further recesses with which information can be transmitted to the fabric.
  • the MALDI matrix substance settles on the sheet, so that the information is visible there.
  • Examples of information in the context of the invention are the labeling of the rows and columns of a grid, abbreviation for the MALDI matrix substance used but also adjustment points that allow an exact adjustment of the fabric in the corresponding analyzer.
  • the MALDI matrix points preferably have an area of 1 ⁇ m 2 -10 mm 2 . On such a surface, a drop of liquid can settle and preferably anchored so that it does not dissolve itself hanging down from the sheet according to the invention.
  • the MALDI matrix points are arranged along an exact grid, which allows easy control of metering and / or analyzing machines.
  • the MALDI matrix points can have any shape. Examples of possible shapes are rectangular, square, triangular or oval.
  • the shape of the MALDI matrix points can be used to distinguish them, because the shape can be seen, for example, under a microscope in the mass spectrometer during the analysis. For example, a particular form may be associated with a particular MALDI matrix substance.
  • a MALDI matrix point has a substructure. This substructure may consist of a plurality of mutually isolated partial points, which preferably each consist of a different MALDI matrix substance. The substructure may also have a plurality of concentric circular rings.
  • the embodiment having a plurality of partial points has the advantage that a single drop of a substance to be analyzed, which is brought into contact with the MALDI matrix point, simultaneously wets several partial points and thus at a MALDI matrix point investigations with several different matrix Substances can be performed.
  • the MALDI matrix dots or partial dots represent regions which are more wettable than their environment and which are each completely enclosed by a less wettable, preferably ultraphobic, region.
  • a less wettable, preferably ultraphobic, region By this embodiment, it is possible to deposit a drop of liquid at a very specific location and anchor there comparatively firmly.
  • the crystalline structure of the MALDI matrix points or partial points preferably has a crystallite size ⁇ 1 ⁇ m.
  • This preferred embodiment of the present invention has the advantage, for example, that the MALDI matrix points are dissolved very well and uniformly by the substance to be tested and / or that a very good signal results.
  • Ultraphob in the sense of the invention means that the contact angle of a drop of water and / or oil lying on an ultraphobic surface is more than 150 °, preferably more than 160 ° and most preferably more than 170 ° and / or the rolling angle 10 ° does not exceed.
  • the roll-off angle is understood to be the angle of inclination of a basically flat but structured surface against the horizontal, in which a stationary drop of water and / or oil with a volume of 10 ⁇ l is moved due to gravity at an inclination of the surface.
  • Such ultraphobic surfaces are for example in WO 98/23549 . WO 96/04123 . WO 96/21523 . WO 00/39369 . WO 00/39368 . WO 00/39239 . WO 00/39051 . WO 00/38845 and WO 96/34697 which are hereby incorporated by reference and thus considered part of the disclosure.
  • Such an ultraphobic surface is in the international Patent Application WO 99/10322 which is hereby incorporated by reference and therefore forms part of the disclosure.
  • the inventive method is particularly suitable for the production of fabrics, with a variety of MALDI matrix points. This sheet is therefore also an object of the present invention.
  • the sheet is designed as a disposable item.
  • a multilayer sheet having a first layer with an ultraphobic surface and a carrier layer is suitable, the first layer being reversibly applied to the carrier layer and the maximum local deviation of the sheet from the flatness being ⁇ 100 ⁇ m, more preferably ⁇ 20 ⁇ m on a length of 100 mm.
  • This sheet has the advantage that the first layer with the ultraphobic surface can be detached from the carrier layer after a single or multiple use and can be replaced by a new first layer, so that it is precluded that this first layer has been contaminated by previous experiments ,
  • the first layer with the ultraphobic surface is particularly inexpensive to produce as a disposable article.
  • the flatness defined according to the invention ensures that the fabric can be used in all conventional mass spectrometric and / or optical analysis devices.
  • the first layer is adhered to the carrier layer.
  • the sheet according to the invention can be used in a variety of ways, but it is preferably suitable for mass spectroscopic and / or optical analyzes.
  • the mixture was then pre-pickled in H 3 PO 4 (100 g / l) for 20 seconds, rinsed in distilled water for 30 seconds and in a mixture of HCl / H 3 BO 3 ( 4 g / l each) at 35 ° C. and 120 mA / cm 2 at 35 V AC electrochemically pickled.
  • the thus treated sheet was coated with a about 50nm thick gold layer by sputtering under high vacuum. Finally, the sample was immersed for 24 hours in a closed vessel by immersion in a solution of the thiol CF 3 - (CF 2 ) 7 - (CH 2 ) 2 -SH in benzotrifluoride (pa, 1g / l) at room temperature coated monolayer, then rinsed with benzotrifluoride (pa) and dried.
  • the surface has a static contact angle of 178 ° for water. If the surface inclines by ⁇ 2 °, a water drop of 10 ⁇ l volume rolls off.
  • the temperature of the solid is controlled in the powder bed with a thermocouple to 180 ° C.
  • the layer thickness of the deposited film of ⁇ -cyano-4-hydroxycinnamic acid is determined by means of a quartz crystal film thickness gauge previously calibrated with an absolute layer thickness determination (e.g., atomic force microscope). The sublimation is stopped at a layer thickness of 1 ⁇ m.
  • FIGS. 1 to 5 The following are the FIGS. 1 to 5 described.
  • FIG. 1 shows the sheet 1, which consists of a first layer 2 with an ultraphobic surface 3 and a support layer 4.
  • the first layer 2 is fixed on the carrier layer by means of an adhesive layer 5.
  • the person skilled in the art recognizes that the adhesive layer 5 does not necessarily have to be present.
  • the adhesive layer 5 consists of an electrically conductive material, so that there is an electrical contact between the first layer 2 and the carrier material.
  • FIG. 2 shows a sheet according to the invention, to which a plurality of MALDI matrix points 6 were sublimated grid-shaped.
  • the MALDI matrix points in the respective rows 1-4 have different shapes that symbolize the user that different MALDI-Marix substances were used in each row.
  • the MALDI matrix point 2D is shown enlarged, so that it can be seen that it consists of four sub-points 8. These partial points 8 are composed of different MALDI matrix substances, so that four different analyzes can be performed at this MALDI matrix point.
  • an inscription 7 has additionally been applied to the fabric, informing the user about the MALDI matrix substance used in this series.
  • the fabric has two centering crosses 9.
  • the lettering and the centering crosses are sublimated onto the web by placing a mask over the web having corresponding recesses.
  • rows 1 to 4 of the grid were produced one after the other.
  • FIG. 3 shows the mask with 8 x 8 openings 12 in diameter of 0.8 mm with a distance of the centers of 2.25 mm (not to scale).
  • FIG. 4 shows a photomicrograph of a matrix spot coming out of the mask FIG. 3 was obtained.
  • mixtures of substances are first separated by chromatography before a MALDI examination.
  • examples are mixtures of peptides generated by enzymatic (e.g., trypsin) peptide degradation.
  • the fractions of the eluate are then applied to the MALDI matrix points 6 of the sample carrier 1.
  • MALDI matrix points 6 are used which are very close to each other but are still completely enclosed by an ultraphobic region.
  • the distances (measured from center to center) of the MALDI matrix points are 1.5 times their diameter.
  • MALDI matrix points generated by sublimation over masks their location and size are very well defined. In this way it can be achieved that the liquid can flow out of the opening 11 of the LC column 10 for application to the MALDI matrix points continuously. Separate collecting of the fractions in vessels with subsequent pipetting onto the matrix points or a discontinuous generation of fractions by lingering the outlet opening over a MALDI matrix point and subsequent rapid movement to the next MALDI matrix point is eliminated.
  • the sample carrier 1 is moved at a constant speed under the outlet opening 11 away.
  • Each MALDI matrix point now captures a constant volume of liquid dispensed from the exit orifice.
  • the process is started at point A and runs meandering over all points to point E.
  • the MALDI matrix points then contain the entire eluate of the chromatography on the MALDI matrix points A to E in fractions which correspond to the constant volume, which each MALDI matrix point picks up. By changing the speed with which the carrier 1 is moved under the opening 11, this volume can be changed.

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  • Optics & Photonics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Description

Die vorliegende Erfindung betrifft ein Verfahren zur Herstellung eines Probenträgers mit einer Vielzahl von MALDI-Matrix-Punkten, ein Flächengebilde erhältlich mit dem erfindungsgemäßen Verfahren sowie ein langzeitstabiles Flächengebilde.The present invention relates to a method for producing a sample carrier having a plurality of MALDI matrix dots, a sheet obtainable by the method according to the invention and a long-term stable sheet.

Für die Analyse von Proben beispielsweise in der Wirkstoffchemie oder in der biologischen Forschung und Produktion hat sich vermehrt die Massenspektrometrie durchgesetzt. Für die Analyse von in den Proben befindlichen Biomolekülen wird vorzugsweise die Massenspektrometrie mit Ionisierung durch Matrix-unterstützte Laserdesorption und Ionisierung (MALDI) eingesetzt.For the analysis of samples, for example in drug chemistry or in biological research and production, mass spectrometry has become increasingly popular. For the analysis of biomolecules in the samples, mass spectrometry with ionization by matrix-assisted laser desorption and ionization (MALDI) is preferably used.

Bei dem MALDI-Verfahren werden insbesondere Biomoleküle und/oder biologisches Material in Form eines Flüssigkeitstropfens auf einen sogenannten MALDI-Matrix Punkt dosiert, beispielsweise pipettiert, und getrocknet Die sich dabei bildenden Kristalle werden beispielsweise mit einem MALDI-TOF Massenspektrometer im linearen- oder im Reflektor-Betrieb untersucht Details zu diesem Verfahren können Nordhoff et. al. " MALDI-MS as a new method for the analysis of nucleic acid (DNA and RNA) with molecular masses up to 150,000 Dalton, Application of modern mass spectrometric methods to plant science research, Oxford University press, (1996) Seite 86-101 entnommen werden, das hiermit als Referenz eingeführt wird und somit als Teil der Offenbarung gilt.In the MALDI method, in particular biomolecules and / or biological material are metered in the form of a liquid drop to a so-called MALDI matrix point, for example, pipetted, and dried. The resulting crystals are linear or in the reflector, for example, with a MALDI-TOF mass spectrometer Operation examines details about this procedure Nordhoff et. al. "MALDI-MS as a new method for the analysis of nucleic acid (DNA and RNA) with molecular mass up to 150,000 Dalton, Application of modern mass spectrometric methods to plant science research, Oxford University press, (1996) page 86-101 which is hereby incorporated by reference and thus forms part of the disclosure.

Probenträger mit MALDI-Punkten sind beispielsweise aus der WO 00/67293 bekannt.Sample carriers with MALDI points are for example from the WO 00/67293 known.

Die MALDI-Matrix-Punkte werden gemäß dem Stand der Technik erzeugt, indem die Matrix-Substanz als Lösung in Form eines Flüssigkeitstropfens auf einen Probenträger aufgebracht und dort getrocknet wird. Insbesondere bei Serienversuchen, bei denen teilweise mehr als 200 MALDI-Matrix-Punkte auf den Probenträger aufgebracht werden müssen, ist dieses Verfahren trotz Automationstechniken sehr aufwendig. Darüber hinaus sind die MALDI-Matrix-Punkte in ihrer Form nicht gleichmäßig und in sich nicht homogen. Weiterhin ist die Position der Matrix-Punkte auf dem Probenträger relativ ungenau. Um ein Ineinanderlaufen zweier benachbarter Punkte zu verhindern, muß deren Abstand entsprechend groß gewählt werden.The MALDI matrix dots are produced according to the prior art by applying the matrix substance as a solution in the form of a drop of liquid to a sample carrier and drying it there. Particularly in the case of series tests in which in some cases more than 200 MALDI matrix points have to be applied to the sample carrier, this method is very complicated despite automation techniques. In addition, the MALDI matrix points are not uniform in shape and are not homogeneous in themselves. Furthermore, the position of the matrix points on the sample carrier is relatively inaccurate. In order to prevent a running into each other of two adjacent points, their distance must be selected to be correspondingly large.

Es stellt sich deshalb die Aufgabe ein Verfahren zur Herstellung eines Flächengebildes mit einer Vielzahl von MALDI-Matrix-Punkten zur Verfügung zu stellen, das die Nachteile des Standes der Technik nicht aufweist.It is therefore an object to provide a method for producing a sheet with a plurality of MALDI matrix points, which does not have the disadvantages of the prior art.

Gelöst wird die Aufgabe erfindungsgemäß mit einem Verfahren zur Herstellung eines Flächengebildes, vorzugsweise eines Probenträgers, mit einer Vielzahl von MALDI-Matrix-Punkten, bei dem die MALDI-Matrix-Punkte durch Niederschlag der MALDI-Matrix-Substanz aus der Gasphase auf den Probenträger aufgetragen werden.The object is achieved according to the invention with a process for producing a sheet, preferably a sample carrier, with a plurality of MALDI matrix points, in which the MALDI matrix points applied by precipitation of the MALDI matrix substance from the gas phase on the sample carrier become.

Es war für den Fachmann überaus erstaunlich und nicht zu erwarten, dass es mit dem erfindungsgemäßen Verfahren gelingt, eine beliebige Anzahl von MALDI-Matrix-Punkten gleichzeitig zu erzeugen. Die MALDI-Matrix-Punkte können eine beliebige Form aufweisen. Sie sind sehr gut reproduzierbar herstellbar, sehr homogen und weisen eine Oberflächenstruktur auf, mit der sich sehr gute Massenspektren erzielen lassen.It was extremely surprising for the person skilled in the art and it would not be expected that the method according to the invention would succeed in producing any number of MALDI matrix points simultaneously. The MALDI matrix points can have any shape. They are very reproducible to produce, very homogeneous and have a surface structure, which can be achieved with very good mass spectra.

Ein Flächengebilde im Sinne der Erfindung ist jeder beliebige Formkörper mit einer beliebig gestalteten Oberfläche. Vorzugsweise ist das Flächengebilde jedoch eine Platte mit einer ebenen Oberfläche, ganz besonders bevorzugt ein Probenträger, der jedoch vorzugsweise keine Einbuchtungen aufweist. Am meisten bevorzugt ist das erfindungsgemäße Flächengebilde eine Folie.A sheet in the context of the invention is any desired shaped body with an arbitrarily shaped surface. Preferably, however, the sheet is a plate with a flat surface, most preferably a sample carrier, but preferably has no indentations. Most preferably, the sheet of the invention is a film.

Ein MALDI-Matrix-Punkt im Sinne der Erfindung besteht im wesentlichen aus mindestens einer dem Fachmann geläufigen MALDI-Matrix-Substanz. Bevorzugte MALDI-Matrix-Substanzen sind 3-Hydroxypicolinsäure, α-Cyano-4-hydroxy-zimtsäure, 2,5-Dihydroxybenzoesäure, Sinapinsäure, 2,4,6-Trihydroxyacetophenon Nitrobenzylalkohol, Nikotinsäure, Ferulasäure, Kaffeesäure, 2-Aminobenzoesäure, Picolinsäure, 3-Aminobenzoesäure, 2,3,4-Trihydroxyacetophenon, 6-Aza-2-thiothymidin, Harnstoff, Bernsteinsäure, Adipinsäure, Malonsäure oder deren Mischung. Ganz besonders bevorzugt ist die MALDI-Matrix-Substanz α-Cyano-4-hydroxyzimtsäure.A MALDI matrix point in the sense of the invention essentially consists of at least one MALDI matrix substance known to the person skilled in the art. Preferred MALDI matrix substances are 3-hydroxypicolinic acid, α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, sinapinic acid, 2,4,6-trihydroxyacetophenone, nitrobenzyl alcohol, nicotinic acid, ferulic acid, caffeic acid, 2-aminobenzoic acid, picolinic acid, 3-aminobenzoic acid, 2,3,4-trihydroxyacetophenone, 6-aza-2-thiothymidine, urea, succinic acid, adipic acid, malonic acid or a mixture thereof. Most preferably, the MALDI matrix substance is α-cyano-4-hydroxycinnamic acid.

Vorzugsweise wird als MALDI-Matrix-Substanz eine Verbindung gewählt, die auf dem Flächengebilde sublimiert und für das menschliche Auge sichtbar ist.Preferably, the MALDI matrix substance selected is a compound which sublimes on the sheet and is visible to the human eye.

Erfindungsgemäß werden die MALDI-Matrix-Punkte durch Niederschlag der MALDI-Matrix-Substanz aus der Gasphase erzeugt. Niederschlag aus der Gasphase im Sinne der Erfindung ist jedes Verfahren, bei dem die MALDI-Matrix-Substanz aus der Gasphase auf das Flächengebilde übertragen wird. Beispielhaft seien Kondensation oder Sublimation genannt. Vorzugsweise erfolgt die Auftragung der MALDI-Matrix-Punkte auf das Flächengebilde jedoch durch Sublimation. Sublimation im Sinne der Erfindung beinhaltet, dass die MALDI-Matrix-Substanz als Feststoff in die Gasphase überführt und/oder aus der Gasphase auf dem Flächengebilde feststoffförmig niedergeschlagen wird. Die Sublimation findet vorzugsweise im Vakuum statt. Besonders bevorzugt wird der Feststoff zur Sublimation erwärmt. Vorzugsweise werden bei dem Niederschlag aus der Gasphase, besonders bevorzugt der Sublimation, mehrere MALDI-Matrix-Substanzen besonders bevorzugt parallel oder sequentiell eingesetzt. Die MALDI-Matrix-Substanzen können zur Herstellung unterschiedlicher MALDI-Matrix-Punkte eingesetzt werden. Es ist aber auch denkbar, dass ein MALDI-Matrix-Punkt eine Substruktur, beispielsweise separat voneinander vorliegende Teilpunkte, aufweist, die jeweils aus einer unterschiedlichen MALDI-Matrix-Substanz aufgebaut sind. Eine Substruktur können aber auch beispielsweise konzentrische Kreisringe sein, die jeweils aus einer unterschiedlichen MALDI-Matrix-Substanz bestehen.According to the invention, the MALDI matrix points are generated by precipitation of the MALDI matrix substance from the gas phase. Precipitation from the gas phase in the context of the invention is any method in which the MALDI matrix substance is transferred from the gas phase to the sheet. Examples include condensation or sublimation. Preferably, however, the application of the MALDI matrix dots to the sheet occurs by sublimation. Sublimation in the sense of the invention involves the MALDI matrix substance being transferred as a solid into the gas phase and / or deposited in the form of a solid from the gas phase on the sheet. The sublimation preferably takes place in vacuo. Particularly preferably, the solid is heated to sublimation. Preferably, in the precipitate from the gas phase, particularly preferably the sublimation, a plurality of MALDI matrix substances are more preferably used in parallel or sequentially. The MALDI matrix substances can be used to produce different MALDI matrix points. However, it is also conceivable for a MALDI matrix point to have a substructure, for example subdots which are present separately from one another, each of which is composed of a different MALDI matrix substance. However, a substructure may also be, for example, concentric circular rings, each consisting of a different MALDI matrix substance.

Vorzugsweise wird der Probenträger während des Niederschlags aus der Gasphase, besonders bevorzugt der Sublimation, von einem Formkörper, einer sogenannten Maske, bedeckt, die durchgehende Ausnehmungen aufweist. Nur im Bereich dieser Ausnehmungen schlägt sich die MALDI-Matrix-Substanz auf dem Flächengebilde nieder und bildet dort einen MALDI-Matrix-Punkt oder Teilpunkt. Diese Maske kann eine beliebige Anzahl von Ausnehmungen aufweisen, die eine beliebige Form haben können. Beispielsweise können die Ausnehmungen rund, rechteckig, quadratisch, dreieckig oder oval sein, um nur eine kleine Anzahl der möglichen Formen zu nennen. Die Form kann zur Unterscheidung der jeweiligen MALDI-Matrix-Substanzen auf einem Flächengebilde herangezogen werden. Das Flächengebilde kann auch ursprünglich von mehreren Masken bedeckt sein, die dann nacheinander entfernt werden, um beispielsweise auf unterschiedliche Bereiche des erfindungsgemäßen Flächengebildes unterschiedliche MALDI-Matrix-Substanzen aufzutragen.During the precipitation, the sample carrier is preferably covered by the gaseous phase, particularly preferably the sublimation, by a shaped body, a so-called mask, which has through-cut recesses. Only in the area of these recesses does the MALDI matrix substance settle on the sheet, forming a MALDI matrix point or partial point. This mask can have any number of recesses, which can have any shape. For example, the recesses may be round, rectangular, square, triangular or oval, to name but a small number of the possible shapes. The shape can be used to distinguish the respective MALDI matrix substances on a sheet. The sheet may also be originally covered by multiple masks, which are then removed one after the other be applied, for example, to different areas of the sheet according to the invention different MALDI matrix substances.

In einer besonders bevorzugten Ausführungsform der vorliegenden Erfindung weist die Maske weitere Ausnehmungen auf, mit denen Informationen auf das Flächengebilde übertragen werden können. Im Bereich dieser Ausnehmungen schlägt sich die MALDI-Matrix-Substanz auf dem Flächengebilde nieder, so dass die Information dort sichtbar wird. Beispiele für Informationen im Sinne der Erfindung sind die Beschriftung der Reihen und Spalten eines Rasters, Kürzel für die verwendete MALDI-Matrix-Substanz aber auch Justierpunkte, die eine exakte Justierung des Flächengebildes in dem entsprechenden Analysegerät erlauben.In a particularly preferred embodiment of the present invention, the mask has further recesses with which information can be transmitted to the fabric. In the area of these recesses, the MALDI matrix substance settles on the sheet, so that the information is visible there. Examples of information in the context of the invention are the labeling of the rows and columns of a grid, abbreviation for the MALDI matrix substance used but also adjustment points that allow an exact adjustment of the fabric in the corresponding analyzer.

Vorzugsweise haben die MALDI-Matrix-Punkte eine Fläche von 1µm2-10 mm2. Auf einer derartigen Fläche läßt sich ein Flüssigkeitstropfen absetzen und vorzugsweise so verankern, dass er sich selbst nach unten hängend nicht von dem erfindungsgemäßen Flächengebilde löst.The MALDI matrix points preferably have an area of 1 μm 2 -10 mm 2 . On such a surface, a drop of liquid can settle and preferably anchored so that it does not dissolve itself hanging down from the sheet according to the invention.

Weiterhin bevorzugt sind die MALDI-Matrix-Punkte entlang eines exakten Rasters angeordnet, das ein einfaches Ansteuern von Dosier- und/oder Analysierautomaten ermöglicht. Die MALDI-Matrix-Punkte können eine beliebige Form aufweisen. Beispiele für mögliche Formen sind rechteckig, quadratisch, dreieckig oder oval. Die Form der MALDI-Matrix-Punkte kann zu deren Unterscheidung herangezogen werden, weil die Form beispielsweise unter einem Mikroskop im Massenspektrometer bei der Analyse erkennbar ist. Beispielsweise kann eine bestimmte Form einer bestimmten MALDI-Matrix-Substanz zugeordnet werden. Weiterhin bevorzugt weist ein MALDI-Matrix-Punkt eine Substruktur auf. Diese Substruktur kann aus mehreren von einander isolierten Teilpunkten bestehen, die vorzugsweise jeweils aus einer unterschiedlichen MALDI-Matrix-Substanz bestehen. Die Substruktur kann aber auch mehrere konzentrische Kreisringe aufweisen. Insbesondere die Ausführungsform mit mehreren Teilpunkten hat den Vorteil, dass ein einziger Tropfen einer zu analysierenden Substanz, der mit dem MALDI-Matrix-Punkt in Kontakt gebracht wird, gleichzeitig mehrere Teilpunkte benetzt und somit an einem MALDI-Matrix-Punkt Untersuchungen mit mehreren verschiedenen Matrix-Substanzen durchgeführt werden können.Further preferably, the MALDI matrix points are arranged along an exact grid, which allows easy control of metering and / or analyzing machines. The MALDI matrix points can have any shape. Examples of possible shapes are rectangular, square, triangular or oval. The shape of the MALDI matrix points can be used to distinguish them, because the shape can be seen, for example, under a microscope in the mass spectrometer during the analysis. For example, a particular form may be associated with a particular MALDI matrix substance. Further preferably, a MALDI matrix point has a substructure. This substructure may consist of a plurality of mutually isolated partial points, which preferably each consist of a different MALDI matrix substance. The substructure may also have a plurality of concentric circular rings. In particular, the embodiment having a plurality of partial points has the advantage that a single drop of a substance to be analyzed, which is brought into contact with the MALDI matrix point, simultaneously wets several partial points and thus at a MALDI matrix point investigations with several different matrix Substances can be performed.

Vorzugsweise stellen die MALDI-Matrix-Punkte oder Teilpunkte Bereiche dar, die besser benetzbar als ihre Umgebung sind und die jeweils von einem schlechter benetzbaren, vorzugsweise ultraphoben Bereich vollständig umschlossen sind. Durch diese Ausführungsform ist es möglich, einen Flüssigkeitstropfen an einem ganz bestimmten Ort abzulegen und dort vergleichsweise fest zu verankern.Preferably, the MALDI matrix dots or partial dots represent regions which are more wettable than their environment and which are each completely enclosed by a less wettable, preferably ultraphobic, region. By this embodiment, it is possible to deposit a drop of liquid at a very specific location and anchor there comparatively firmly.

Vorzugsweise weist die kristalline Struktur der MALDI-Matrix-Punkte oder Teilpunkte eine Kristallit-größe < 1 µm auf. Diese bevorzugte Ausführungsform der vorliegenden Erfindung hat beispielsweise den Vorteil, dass die MALDI-Matrix-Punkte von der zu testenden Substanz sehr gut und gleichmäßig angelöst werden und/oder dass ein sehr gutes Signal resultiert.The crystalline structure of the MALDI matrix points or partial points preferably has a crystallite size <1 μm. This preferred embodiment of the present invention has the advantage, for example, that the MALDI matrix points are dissolved very well and uniformly by the substance to be tested and / or that a very good signal results.

Ultraphob im Sinne der Erfindung bedeutet, dass der Kontaktwinkel eines Wasser- und/oder Öltropfens, der auf einer ultraphoben Oberfläche liegt, mehr als 150°, vorzugsweise mehr als 160° und am meisten bevorzugt mehr als 170° beträgt und/oder der Abrollwinkel 10° nicht überschreitet. Als Abrollwinkel wird der Neigungswinkel einer grundsätzlich planen aber strukturierten Oberfläche gegen die Horizontale verstanden, bei dem ein stehender Wasser- und/oder Öltropfen mit einem Volumen von 10 µl aufgrund der Schwerkraft bei einer Neigung der Oberfläche bewegt wird. Solche ultraphoben Oberflächen sind zum Beispiel in der WO 98/23549 , WO 96/04123 , WO 96/21523 , WO 00/39369 , WO 00/39368 , WO 00/39239 , WO 00/39051 , WO 00/38845 und WO 96/34697 offenbart, die hiermit als Referenz eingeführt werden und somit als Teil der Offenbarung gelten.Ultraphob in the sense of the invention means that the contact angle of a drop of water and / or oil lying on an ultraphobic surface is more than 150 °, preferably more than 160 ° and most preferably more than 170 ° and / or the rolling angle 10 ° does not exceed. The roll-off angle is understood to be the angle of inclination of a basically flat but structured surface against the horizontal, in which a stationary drop of water and / or oil with a volume of 10 μl is moved due to gravity at an inclination of the surface. Such ultraphobic surfaces are for example in WO 98/23549 . WO 96/04123 . WO 96/21523 . WO 00/39369 . WO 00/39368 . WO 00/39239 . WO 00/39051 . WO 00/38845 and WO 96/34697 which are hereby incorporated by reference and thus considered part of the disclosure.

In einer bevorzugten Ausführungsform weisen die ultraphoben Bereiche eine Oberflächentopographie auf, bei der die Ortsfrequenz der einzelnen Fourierkomponenten und deren Amplitude a (f) ausgedrückt durch das Integral S (log(f)) = a(f).f errechnet zwischen den Integrationsgrenzen log (f1/µm-1) = -3 und log (f2/µm-1) = 3 mindestens 0,3 beträgt und die aus einem hydrophoben oder insbesondere oleophoben Material besteht oder mit einem haltbar hydrophobierten und/oder insbesondere haltbar oleophobierten Material beschichtet sind. Eine solche ultraphobe Oberfläche ist in der internationalen Patentanmeldung WO 99/10322 beschrieben, die hiermit als Referenz eingeführt wird und somit als Teil der Offenbarung gilt.In a preferred embodiment, the ultraphobic regions have a surface topography in which the spatial frequency of the individual Fourier components and their amplitude a (f) expressed by the integral S (log (f)) = a (f) .f calculated between the integration limits log ( f 1 / μm -1 ) = -3 and log (f 2 / μm -1 ) = 3 is at least 0.3 and which consists of a hydrophobic or especially oleophobic material or coated with a durable hydrophobicized and / or durable oleophobated material are. Such an ultraphobic surface is in the international Patent Application WO 99/10322 which is hereby incorporated by reference and therefore forms part of the disclosure.

Das erfindungsgemäße Verfahren eignet sich insbesondere zur Herstellung von Flächengebilden, mit einer Vielzahl von MALDI-Matrix-Punkten. Dieses Flächengebilde ist deshalb ebenfalls Gegenstand der vorliegenden Erfindung.The inventive method is particularly suitable for the production of fabrics, with a variety of MALDI matrix points. This sheet is therefore also an object of the present invention.

In einer bevorzugten Ausführungsform wird das Flächengebilde als Einmalartikel gestaltet. Für diese Ausführungsform ist insbesondere ein mehrschichtiges Flächengebilde mit einer ersten Schicht mit einer ultraphoben Oberfläche und einer Trägerschicht geeignet, wobei die erste Schicht auf der Trägerschicht reversibel aufgebracht ist und die maximale lokale Abweichung des Flächengebildes von der Planheit <100 µm, besonders bevorzugt < 20 µm auf einer Länge von 100 mm beträgt.In a preferred embodiment, the sheet is designed as a disposable item. For this embodiment, in particular a multilayer sheet having a first layer with an ultraphobic surface and a carrier layer is suitable, the first layer being reversibly applied to the carrier layer and the maximum local deviation of the sheet from the flatness being <100 μm, more preferably <20 μm on a length of 100 mm.

Dieses Flächengebilde hat den Vorteil, dass die erste Schicht mit der ultraphoben Oberfläche nach einmaliger oder mehrmaliger Verwendung von der Trägerschicht abgelöst werden kann und durch eine neue erste Schicht ersetzt werden kann, so dass ausgeschlossen ist, dass diese erste Schicht durch vorhergehende Experimente kontaminiert worden ist. Die erste Schicht mit der ultraphoben Oberfläche ist als Einmalartikel besonders günstig herzustellen. Durch die erfindungsgemäß definierte Planheit ist sichergestellt, dass das Flächengebilde in allen gängigen massenspektrometrischen und/oder optischen Analysegeräten einsetzbar ist.This sheet has the advantage that the first layer with the ultraphobic surface can be detached from the carrier layer after a single or multiple use and can be replaced by a new first layer, so that it is precluded that this first layer has been contaminated by previous experiments , The first layer with the ultraphobic surface is particularly inexpensive to produce as a disposable article. The flatness defined according to the invention ensures that the fabric can be used in all conventional mass spectrometric and / or optical analysis devices.

In einer bevorzugten Ausführungsform des Flächengebildes ist die erste Schicht auf die Trägerschicht aufgeklebt.In a preferred embodiment of the fabric, the first layer is adhered to the carrier layer.

Weiterhin bevorzugt besteht zwischen der ersten Schicht und der Trägerschicht ein elektrischer Kontakt. Diese Ausführungsform ist insbesondere bei massenspektroskopischen Analysen von Vorteil.Further preferably, there is an electrical contact between the first layer and the carrier layer. This embodiment is particularly advantageous in mass spectroscopic analyzes.

Das erfindungsgemäße Flächengebilde ist mannigfaltig einsetzbar, vorzugsweise eignet es sich jedoch bei massenspektroskopischen und/oder optischen Analysen.The sheet according to the invention can be used in a variety of ways, but it is preferably suitable for mass spectroscopic and / or optical analyzes.

Im folgenden wird die Erfindung anhand von Beispiel 1 und Figur 1 - 5 erläutert. Diese Erläuterungen sind lediglich beispielhaft und schränken den allgemeinen Erfindungsgedanken nicht ein.

Figur 1
zeigt den Querschnitt eines erfindungsgemäßen mehrschichtigen Flächengebildes.
Figur 2
zeigt die Oberfläche eines erfindungsgemäßen Flächengebildes.
Figur 3
zeigt eine Maske zur Durchführung des Verfahrens zur Herstellung eines erfindungsgemäßen Flächengebildes.
Figur 4
zeigt einen MALDI-Matrix-Punkt erhältlich mit dem erfindungsgemäßen Verfahren.
Figur 5
zeigt die Verwendung des erfindungsgemäßen Flächengebildes bei der Kopplung von Flüssigkeitschromatographie und MALDI-Untersuchungen.
In the following the invention with reference to Example 1 and Figure 1-5 explained. These explanations are merely exemplary and do not limit the general inventive concept.
FIG. 1
shows the cross section of a multilayer sheet according to the invention.
FIG. 2
shows the surface of a fabric according to the invention.
FIG. 3
shows a mask for carrying out the method for producing a sheet according to the invention.
FIG. 4
shows a MALDI matrix point obtainable by the method according to the invention.
FIG. 5
shows the use of the fabric according to the invention in the coupling of liquid chromatography and MALDI investigations.

Beispiel 1:Example 1:

Ein Probenträger wurde wie folgt hergestellt:

  • Ein walzpoliertes AI-Blech (99,9%) mit einer Fläche von 26x76mm2 und einer Dicke von 0,15 mm wurde bei Raumtemperatur mit Chloroform (p.a.) anschließend 20s in wässriger NaOH (5g/l) bei 50°C entfettet.
A sample carrier was made as follows:
  • A roll-polished Al sheet (99.9%) having an area of 26 × 76 mm 2 and a thickness of 0.15 mm was degreased at room temperature with chloroform (Pa) for 20 seconds in aqueous NaOH (5 g / liter) at 50 ° C.

Danach wurde 20s in H3PO4 (100g/l) vorgebeizt, 30s in destilliertem Wasser gespült und 90s in einer Mischung von HCl/H3BO3 (je 4g/l) bei 35°C und 120mA/cm2 bei 35V Wechselspannung elektrochemisch gebeizt.The mixture was then pre-pickled in H 3 PO 4 (100 g / l) for 20 seconds, rinsed in distilled water for 30 seconds and in a mixture of HCl / H 3 BO 3 ( 4 g / l each) at 35 ° C. and 120 mA / cm 2 at 35 V AC electrochemically pickled.

Nach 30s Spülung in Wasser und 30s alkalischer Spülung in wässiger NaOH (5g/l) wurde erneut 30s in destilliertem Wasser gespült und anschließend 90s in H2SO4 (200g/l) bei 25°C mit 30mA/cm2 bei 50V Gleichspannung anodisch oxidiert.After 30s rinsing in water and 30s alkaline rinse in aqueous NaOH (5g / l) was rinsed again for 30s in distilled water and then 90s in H 2 SO 4 (200g / l) at 25 ° C with 30mA / cm 2 at 50V DC anodic oxidized.

Danach wurde 30s in destilliertem Wasser, dann 60s bei 40°C in NaHCO3 (20 g/l), dann wieder 30s in destilliertem Wasser gespült und 1 Stunde bei 80°C im Trockenschrank getrocknet.Thereafter, it was rinsed in distilled water for 30 seconds, then in NaHCO 3 (20 g / l) for 60 seconds at 40 ° C., then again in distilled water for 30 seconds and dried in a drying oven at 80 ° C. for 1 hour.

Das so behandelte Blech wurde mit einer etwa 50nm dicken Goldschicht durch Kathodenzerstäubung im Hochvakuum beschichtet. Schließlich wurde die Probe 24 Stunden durch Tauchen in eine Lösung des Thiols CF3-(CF2)7-(CH2)2-SH in Benzotrifluorid (p.a., 1g/l) bei Raumtemperatur in einem geschlossenem Gefäß mit einer Monolage beschichtet, anschließend mit Benzotrifluorid (p.a.) gespült und getrocknet.The thus treated sheet was coated with a about 50nm thick gold layer by sputtering under high vacuum. Finally, the sample was immersed for 24 hours in a closed vessel by immersion in a solution of the thiol CF 3 - (CF 2 ) 7 - (CH 2 ) 2 -SH in benzotrifluoride (pa, 1g / l) at room temperature coated monolayer, then rinsed with benzotrifluoride (pa) and dried.

Die Oberfläche weist für Wasser einen statischen Randwinkel von 178° auf. Bei einer Neigung der Oberfläche um < 2° rollt ein Wassertropfen des Volumens 10µl ab.The surface has a static contact angle of 178 ° for water. If the surface inclines by <2 °, a water drop of 10μl volume rolls off.

Auf den Probenträger wurden Matrix-Punkte wie folgt sublimiert:

  • In einer Hochvakumverdampfungsanlage (Edwards E306) werden 0,5 g α-Cyano-4-hydroxyzimtsäure in einen heizbaren Quarztiegel mit einer nach oben gerichteten Öffnung von 10 mm Durchmesser gefüllt. Im Abstand von 150 mm wird ein Probenträger montiert, der mit einer Maske gemäß Figur 3 bedeckt ist. Nach Abpumpen auf einen Basisdruck < 10-5 mbar wird der Quarztiegel mit Hilfe von außen liegenden Wicklungen aus Wolframdraht beheizt.
On the sample carrier, matrix points were sublimated as follows:
  • In a high-vacuum evaporation unit (Edwards E306), 0.5 g of α-cyano-4-hydroxycinnamic acid is charged into a heatable quartz crucible with an opening of 10 mm in the upward direction. At a distance of 150 mm, a sample carrier is mounted, which is equipped with a mask according to FIG. 3 is covered. After pumping down to a base pressure <10 -5 mbar, the quartz crucible is heated by means of external windings made of tungsten wire.

Die Temperatur des Feststoffs wird in der Pulverschüttung mit einem Thermoelement auf 180°C geregelt. Die Schichtdicke des abgeschiedenen Films aus α-Cyano-4-hydroxyzimtsäure wird mit Hilfe eines Schwingquarz-Schichtdickenmessgerätes bestimmt, das zuvor mit einer absoluten Schichtdickenbestimmung (z.B. Rasterkraftmikroskop) geeicht wird. Die Sublimation wird bei einer Schichtdicke von 1 µm abgebrochen.The temperature of the solid is controlled in the powder bed with a thermocouple to 180 ° C. The layer thickness of the deposited film of α-cyano-4-hydroxycinnamic acid is determined by means of a quartz crystal film thickness gauge previously calibrated with an absolute layer thickness determination (e.g., atomic force microscope). The sublimation is stopped at a layer thickness of 1 μm.

Die erhaltenen MALDI-Matrix-Punkte sind beispielhaft in Figur 4 gezeigt. Ein Massenspektrum dieses MALDI-Matrix-Punkt es wurde wie folgt erhalten:

  • MALDI Massenspektrum der einfach protonierten Peptide (1-7): Humanes Angiotensin I und II, Substanz P-methylester, Neurotensin (Aminosäuren 1-11), Neurotensin, ACTH (Aminosäuren 1-17) und ACTH (Aminosäuren 18 - 39), aufgenommen von einem präpariertem MALDI-Matrix-Punkt mit einem Durchmesser von 800 µm. Die Peptide wurden für die massenspkektrometrische Analyse wie folgt präpariert: 0.5µl einer wäßrigen Lösung, die jeweils 5 fmol der Peptide 1-6, 1 fmol des Peptids 7 sowie ein Volumenprozent Trifluoressigsäure und 1 mM des nichtionischen Detergenz n-Octyl-β-D-glukopyranosid enthielt, wurden auf den MALDI-Matrix-Punkt aufpipettiert. Nach dem das Lösungsmittels vollständig abgedampft war, wurde die so präparierte Probe einmal gewaschen. Hierzu wurde der gesamte Probenträger für 2 Sekunden in eine 0.1 %ige Trifluoressigsäure eingetauscht und unmittelbar danach, für die Abtrennung verbleibender Flüssigkeitsreste, für 10 Sekunden in einen Stickstoffgasstrom (2,5 bar) gehalten. Das Massenspektrum positiver Molekülionen wurde in einem MALDI Flugzeitmassenspektrometer der Firma Bruker Daltonik, Bremen (Scout-MTP Autoflex) im Reflektorbetriebsmodus und mit zeitlich verzögerter Ionenextraktion (Verzögerungszeit: 70 Nanosekunden) und 20 kV Beschleunigungsspannung aufgenommen. Zur Verbesserung des Signal/Rausch-Verhältnisses wurden 100 Einzelschussspektren aufsummiert.
The obtained MALDI matrix points are exemplary in FIG. 4 shown. A mass spectrum of this MALDI matrix point was obtained as follows:
  • MALDI Mass spectrum of monoprotonated peptides (1-7): human angiotensin I and II, substance P-methyl ester, neurotensin (amino acids 1-11), neurotensin, ACTH (amino acids 1-17) and ACTH (amino acids 18-39) from a prepared MALDI matrix point with a diameter of 800 μm. The peptides were prepared for mass spectrometric analysis as follows: 0.5 μl of an aqueous solution containing 5 fmoles each of peptides 1-6, 1 fmol of peptide 7 and one volume percent trifluoroacetic acid and 1 mM of the nonionic detergent n-octyl-β-D- glucopyranoside were pipetted onto the MALDI matrix point. After the solvent completely was evaporated, the thus prepared sample was washed once. For this purpose, the entire sample carrier was exchanged for 2 seconds in a 0.1% trifluoroacetic acid and immediately thereafter, for the separation of remaining liquid residues, held for 10 seconds in a nitrogen gas stream (2.5 bar). The mass spectrum of positive molecular ions was recorded in a MALDI time-of-flight mass spectrometer from Bruker Daltonik, Bremen (Scout-MTP Autoflex) in reflector operating mode and with time-delayed ion extraction (delay time: 70 nanoseconds) and 20 kV acceleration voltage. To improve the signal-to-noise ratio, 100 single-shot spectra were added.

Das Ergebnis ist in der folgenden Graphik dargestellt.

Figure imgb0001
The result is shown in the following graph.
Figure imgb0001

Im folgenden werden die Figuren 1 bis 5 beschrieben.The following are the FIGS. 1 to 5 described.

Figur 1 zeigt das Flächengebilde 1, dass aus einer ersten Schicht 2 mit einer ultraphoben Oberfläche 3 und einer Trägerschicht 4 besteht. Die erste Schicht 2 ist auf der Trägerschicht mittels einer Klebschicht 5 fixiert. Der Fachmann erkennt, dass die Klebschicht 5 nicht notwendigerweise vorhanden sein muß. Die Klebschicht 5 besteht aus einem elektrisch leitenden Material, so dass ein elektrischer Kontakt zwischen der ersten Schicht 2 und dem Trägermaterial besteht. FIG. 1 shows the sheet 1, which consists of a first layer 2 with an ultraphobic surface 3 and a support layer 4. The first layer 2 is fixed on the carrier layer by means of an adhesive layer 5. The person skilled in the art recognizes that the adhesive layer 5 does not necessarily have to be present. The adhesive layer 5 consists of an electrically conductive material, so that there is an electrical contact between the first layer 2 and the carrier material.

Figur 2 zeigt ein erfindungsgemäßes Flächengebilde, auf das mehrere MALDI-Matrix-Punkte 6 rasterförmig sublimiert wurden. Die MALDI-Matrix-Punkte in den jeweiligen Reihen 1-4 weisen unterschiedliche Formen auf, die dem Anwender symbolisieren, dass in jeder Reihe unterschiedliche MALDI-Marix-Substanzen verwendet wurden. Der MALDI-Matrix-Punkt 2D ist vergrößert dargestellt, so dass erkennbar ist, dass er aus vier Teilpunkten 8 besteht. Diese Teilpunkte 8 sind aus jeweils unterschiedlichen MALDI-Matrix-Substanzen aufgebaut, so daß an diesem MALDI-Matrix-Punkt vier unterschiedliche Analysen vorgenommen werden können. Unterhalb des MALDI-Matrix-Punktes 4D ist zusätzlich eine Beschriftung 7 auf das Flächengebilde aufgetragen worden, die den Benutzer über die in dieser Reihe verwendete MALDI-Matrix-Substanz informiert. Zusätzlich weist das Flächengebilde zwei Zentrierkreuze 9 auf. FIG. 2 shows a sheet according to the invention, to which a plurality of MALDI matrix points 6 were sublimated grid-shaped. The MALDI matrix points in the respective rows 1-4 have different shapes that symbolize the user that different MALDI-Marix substances were used in each row. The MALDI matrix point 2D is shown enlarged, so that it can be seen that it consists of four sub-points 8. These partial points 8 are composed of different MALDI matrix substances, so that four different analyzes can be performed at this MALDI matrix point. Below the MALDI matrix point 4D, an inscription 7 has additionally been applied to the fabric, informing the user about the MALDI matrix substance used in this series. In addition, the fabric has two centering crosses 9.

Die Beschriftung und die Zentrierkreuze werden wie die MALDI-Matrix-Punkte auf das Flächengebilde sublimiert, indem eine Maske über das Flächengebilde gelegt wird, die entsprechende Ausnehmungen aufweist. Der Fachmann erkennt, dass die Reihen 1 - 4 des Rasters nacheinander hergestellt wurden.The lettering and the centering crosses, like the MALDI matrix dots, are sublimated onto the web by placing a mask over the web having corresponding recesses. The person skilled in the art recognizes that rows 1 to 4 of the grid were produced one after the other.

Figur 3 zeigt die Maske mit 8 x 8 Öffnungen 12 im Durchmesser von 0,8 mm mit einem Abstand der Mittelpunkte von 2,25 mm (nicht maßstabsgetreu). FIG. 3 shows the mask with 8 x 8 openings 12 in diameter of 0.8 mm with a distance of the centers of 2.25 mm (not to scale).

Figur 4 zeigt eine lichtmikroskopische Aufnahme eines Matrixspots, der mit der Maske aus Figur 3 erhalten wurde. FIG. 4 shows a photomicrograph of a matrix spot coming out of the mask FIG. 3 was obtained.

Die sehr vorteilhafte Verwendung der erfindungsgemäßen Probenträger bei der Kopplung von Flüssigkeitschromatographie und MALDI Untersuchung (LC-MALDI Kopplung) zeigt die Figur 5 .The very advantageous use of the sample carrier according to the invention in the coupling of liquid chromatography and MALDI examination (LC-MALDI coupling) shows the FIG. 5 ,

Häufig werden Substanzgemische vor einer MALDI Untersuchung zunächst chromatographisch getrennt. Beispiel sind Gemische von Peptiden, die durch einen enzymatischen (z.B. Trypsin) Peptidabbau erzeugt wurden. Nach der chromatographischen Trennung werden die Fraktionen des Eluates dann auf die MALDI-Matrix-Punkte 6 des Probenträgers 1 aufgebracht.Frequently, mixtures of substances are first separated by chromatography before a MALDI examination. Examples are mixtures of peptides generated by enzymatic (e.g., trypsin) peptide degradation. After the chromatographic separation, the fractions of the eluate are then applied to the MALDI matrix points 6 of the sample carrier 1.

In dem vorliegenden Beispiel werden dazu MALDI-Matrix-Punkte 6 verwendet, die sehr eng aneinander liegen, jedoch noch von einem ultraphoben Bereich vollständig umschlossen sind. Vorzugsweise betragen die Abstände (gemessen von Mittelpunkt zu Mittelpunkt) der MALDI-Matrix-Punkte das 1,5 fache von deren Durchmesser.In the present example, MALDI matrix points 6 are used which are very close to each other but are still completely enclosed by an ultraphobic region. Preferably, the distances (measured from center to center) of the MALDI matrix points are 1.5 times their diameter.

Durch Verwendung von MALDI-Matrix-Punkten, die durch Sublimation über Masken erzeugten wurden, ist deren Ort und deren Größe sehr genau definiert. Auf diese Weise kann erreicht werden, dass die Flüssigkeit aus der Auftrittsöffnung 11 der LC Säule 10 zum Aufbringen auf die MALDI-Matrix-Punkte kontinuierlich auslaufen kann. Ein gesondertes Sammeln der Fraktionen in Gefäßen mit anschließendem Aufpipettieren auf die Matrix-Punkte oder ein diskontinuierliches Erzeugen von Fraktionen durch Verweilen der Austrittsöffnung über einem MALDI-Matrix-Punkt und anschließendes schnelles Weiterbewegen zum nächsten MALDI-Matrix-Punkt entfällt.By using MALDI matrix points generated by sublimation over masks, their location and size are very well defined. In this way it can be achieved that the liquid can flow out of the opening 11 of the LC column 10 for application to the MALDI matrix points continuously. Separate collecting of the fractions in vessels with subsequent pipetting onto the matrix points or a discontinuous generation of fractions by lingering the outlet opening over a MALDI matrix point and subsequent rapid movement to the next MALDI matrix point is eliminated.

Der Probenträger 1 wird in einer konstanten Geschwindigkeit unter der Austrittsöffnung 11 hinweg bewegt. Jeder MALDI-Matrix-Punkt fängt nun ein konstantes Volumen der Flüssigkeit ein, die aus der Austrittsöffnung abgegeben wird. Gestartet wird der Prozess am Punkt A und verläuft mäanderförmig über alle Punkte hinweg zum Punkt E. Die MALDI-Matrix-Punkte enthalten anschließend das gesamte Eluat der Chromatographie auf den MALDI-Matrix-Punkten A bis E in Fraktionen, die dem konstanten Volumen entsprechen, das jeder MALDI-Matrix-Punkt aufnimmt. Durch Änderung der Geschwindigkeit mit der der Träger 1 unter der Auftrittsöffnung 11 verschoben wird, kann dieses Volumen geändert werden.The sample carrier 1 is moved at a constant speed under the outlet opening 11 away. Each MALDI matrix point now captures a constant volume of liquid dispensed from the exit orifice. The process is started at point A and runs meandering over all points to point E. The MALDI matrix points then contain the entire eluate of the chromatography on the MALDI matrix points A to E in fractions which correspond to the constant volume, which each MALDI matrix point picks up. By changing the speed with which the carrier 1 is moved under the opening 11, this volume can be changed.

Claims (12)

  1. Method for producing a surface formation, preferably a sample carrier, with a multitude of MALDI matrix points or sub-points (6), wherein a multilayered surface formation comprising a first layer (2) with an ultraphobic surface (3) and a carrier layer (4) is formed, wherein the first layer (2) is reversibly applied on the carrier layer (4) and the MALDI matrix points or sub-points (6) are applied to the sample carrier by precipitation of a MALDI matrix substance from the gas phase, preferably sublimation, wherein the MALDI matrix points or sub-points (6) represent hydrophilic regions that are completely enclosed by ultraphobic regions, wherein the maximum local deviation of the surface formation from planarity is <100 µm, over a length of 100 mm.
  2. Method according to Claim 1, characterized in that, during the precipitation from the gas phase, the sample carrier is covered by a plate that has through-holes of which the cross-sectional area respectively corresponds to the cross-sectional area of the MALDI matrix points.
  3. Method according to Claim 2, characterized in that the plate has at least one further through-hole, by which information is transferred to the sample carrier by precipitation of the MALDI matrix substance from the gas phase.
  4. Method according to Claim 3, characterized in that the information comprises the composition of the MALDI matrix substance and/or adjusting points.
  5. Method according to one of the preceding claims, characterized in that the MALDI matrix points are arranged along a grid.
  6. Method according to one of the preceding claims, characterized in that the MALDI matrix points have substructures.
  7. Method according to Claim 6, characterized in that the MALDI matrix points are divided into a number of sub-points that are preferably isolated from one another.
  8. Method according to one of the preceding claims, characterized in that different MALDI matrix substances are applied to a sample carrier.
  9. Method according to Claim 8, characterized in that at least a number of MALDI matrix points or sub-points are respectively built up from a MALDI matrix substance.
  10. Method according to one of the preceding claims, characterized in that α-cyano-4-hydroxycinnamic acid is used as a MALDI matrix substance.
  11. Method according to one of the preceding claims, characterized in that the first layer (2) is adhesively attached to the carrier layer (4).
  12. Method according to Claim 11, characterized in that an electrical contact is established between the first layer (2) and the carrier layer (4).
EP03795893.1A 2002-12-13 2003-12-15 Method for producing a sample carrier for maldi-mass spectrometry Expired - Lifetime EP1573776B1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10258674 2002-12-13
DE10258674A DE10258674A1 (en) 2002-12-13 2002-12-13 Manufacturing sample carrier with points for matrix-assisted laser desorption and ionization, forms MALDI matrix points by gas phase sublimation
PCT/EP2003/014230 WO2004055857A2 (en) 2002-12-13 2003-12-15 Method for producing a sample carrier for maldi-mass spectrometry

Publications (2)

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EP1573776A2 EP1573776A2 (en) 2005-09-14
EP1573776B1 true EP1573776B1 (en) 2017-12-06

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US (1) US20060121599A1 (en)
EP (1) EP1573776B1 (en)
JP (1) JP2006514738A (en)
AU (1) AU2003298183A1 (en)
DE (1) DE10258674A1 (en)
WO (1) WO2004055857A2 (en)

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DE102005022823A1 (en) * 2005-05-02 2006-11-09 Qiagen Gmbh Sample carrier with an ultraphobic surface, useful for isolating and analyzing biomolecules, has an affinity zone and a waste zone and/or a zone covered with a matrix-assisted laser desorption/ionization matrix
JP5020742B2 (en) * 2007-08-27 2012-09-05 日本電子株式会社 Mass spectrometer equipped with MALDI ion source and sample plate for MALDI ion source
EP2318819A2 (en) * 2008-07-30 2011-05-11 The Brigham and Women's Hospital, Inc. Preparation of test plates for matrix assisted laser desorption ionization
EP2157432A1 (en) 2008-08-15 2010-02-24 Qiagen GmbH Method for analysing a complex sample by mass spectrometry
CN102472755A (en) * 2009-07-09 2012-05-23 皇家飞利浦电子股份有限公司 Surface coating for laser desorption ionization mass spectrometry of molecules
JPWO2011027819A1 (en) * 2009-09-02 2013-02-04 公益財団法人野口研究所 Mass spectrometry
JP5359924B2 (en) * 2010-02-18 2013-12-04 株式会社島津製作所 Mass spectrometer
US9211542B2 (en) * 2010-05-21 2015-12-15 Eidgenossische Technische Hochschule Zurich High-density sample support plate for automated sample aliquoting
WO2014162557A1 (en) * 2013-04-04 2014-10-09 株式会社島津製作所 Maldi sample preparation method and sample preparation device
DE102015003440A1 (en) * 2015-03-16 2016-09-22 Friedrich-Schiller-Universität Jena Method for MALDI-MSI analysis of objects, in particular biological tissue samples, and target for analysis and its production

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GB9518429D0 (en) * 1995-09-08 1995-11-08 Pharmacia Biosensor A rapid method for providing kinetic and structural data in molecular interaction analysis
US5854486A (en) * 1996-12-27 1998-12-29 R. T. Hodgson Method and apparatus for MALDI mass spectrometry
NZ516848A (en) * 1997-06-20 2004-03-26 Ciphergen Biosystems Inc Retentate chromatography apparatus with applications in biology and medicine
DE19754978C2 (en) * 1997-12-11 2000-07-13 Bruker Daltonik Gmbh Sample holder for MALDI mass spectrometry along with the process for producing the plates and applying the samples
DE19834070B4 (en) * 1998-07-29 2007-02-15 Bruker Daltonik Gmbh Ionization of high molecular weight substances by laser desorption from liquid matrices
MXPA01006488A (en) * 1998-12-24 2002-06-04 Sunyx Surface Nanotechnologies Ultraphobic surface.
CA2371738A1 (en) * 1999-04-29 2000-11-09 Jody Beecher Sample holder with hydrophobic coating for gas phase mass spectrometers
DE19923761C1 (en) * 1999-05-21 2001-02-08 Bruker Daltonik Gmbh Processing of sample molecules of liquids, involves making the sample droplets stand or suspend from lyophilic or lyophobic anchors on flat support surfaces
DE10005600A1 (en) * 2000-02-09 2001-08-16 Bayer Ag Ultraphobic fabric with a variety of hydrophilic areas
DE10043042C2 (en) * 2000-09-01 2003-04-17 Bruker Daltonik Gmbh Method for loading a sample carrier with biomolecules for mass spectrometric analysis

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Publication number Publication date
AU2003298183A1 (en) 2004-07-09
US20060121599A1 (en) 2006-06-08
JP2006514738A (en) 2006-05-11
WO2004055857A3 (en) 2005-12-22
EP1573776A2 (en) 2005-09-14
DE10258674A1 (en) 2004-06-24
WO2004055857A2 (en) 2004-07-01

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