EP1573776B1 - Procédé de production d'un support d'échantillons pour spectromètrie de masse de type maldi - Google Patents

Procédé de production d'un support d'échantillons pour spectromètrie de masse de type maldi 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
European Patent Office
Prior art keywords
maldi matrix
points
maldi
layer
sample carrier
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP03795893.1A
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German (de)
English (en)
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EP1573776A2 (fr
Inventor
Karsten Reihs
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Qiagen GmbH
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Qiagen GmbH
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Publication date
Application filed by Qiagen GmbH filed Critical Qiagen GmbH
Publication of EP1573776A2 publication Critical patent/EP1573776A2/fr
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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.

Claims (12)

  1. Procédé de fabrication d'un produit plat, de préférence un porte-échantillons, qui présente plusieurs points ou points partiels (6) de matrice MALDI,
    dans lequel un produit plat en plusieurs couches présentant une première couche (2) dotée d'une surface ultraphobe (3) et d'une couche de support (4) est formé,
    la première couche (2) étant appliquée de manière réversible sur la couche de support (4) et les points ou points partiels (6) de matrice MALDI étant déposés sur le porte-échantillons par dépôt d'une substance de matrice MALDI en phase gazeuse, de préférence par sublimation,
    les points ou points partiels (6) de matrice MALDI constituant des parties hydrophiles complètement entourées par des parties ultraphobes et
    l'écart local maximum du produit plat par rapport à la planéité étant < 100 µm sur une longueur de 100 mm.
  2. Procédé selon la revendication 1, caractérisé en ce que pendant le dépôt en phase gazeuse, le porte-échantillons est recouvert par une plaque qui présente des perforations dont la superficie de la section transversale correspond à la superficie de la section transversale des points de matrice MALDI.
  3. Procédé selon la revendication 2, caractérisé en ce que la plaque présente au moins une autre perforation par laquelle une information est transférée par dépôt de la substance de matrice MALDI en phase gazeuse sur le porte-échantillons.
  4. Procédé selon la revendication 3, caractérisé en ce que l'information contient la composition de la substance de matrice MALDI et/ou des points d'ajustement.
  5. Procédé selon l'une des revendications précédentes, caractérisé en ce que les points de matrice MALDI sont disposés suivant un motif.
  6. Procédé selon l'une des revendications précédentes, caractérisé en ce que les points de matrice MALDI présentent des sous-structures.
  7. Procédé selon la revendication 6, caractérisé en ce que les points de matrice MALDI sont divisés en plusieurs points partiels de préférence isolés les uns des autres.
  8. Procédé selon l'une des revendications précédentes, caractérisé en ce que différentes substances de matrice MALDI sont appliquées sur un porte-échantillons.
  9. Procédé selon la revendication 6, caractérisé en ce qu'au moins plusieurs points ou points partiels de matrice MALDI sont formés d'une substance de matrice MALDI.
  10. Procédé selon l'une des revendications précédentes, caractérisé en ce que la substance de matrice MALDI utilisée est l'acide α-cyano-4-hydroxycinnamique.
  11. Procédé selon l'une des revendications précédentes, caractérisé en ce que la première couche (2) est collé sur la couche de support (4).
  12. Procédé selon la revendication 11, caractérisé en ce qu'un contact électrique est établi entre la première couche (2) et la couche de support (4).
EP03795893.1A 2002-12-13 2003-12-15 Procédé de production d'un support d'échantillons pour spectromètrie de masse de type maldi Expired - Lifetime EP1573776B1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10258674 2002-12-13
DE10258674A DE10258674A1 (de) 2002-12-13 2002-12-13 Verfahren zur Herstellung eines Probenträgers für die MALDI-Massenspektrometrie
PCT/EP2003/014230 WO2004055857A2 (fr) 2002-12-13 2003-12-15 Procede de production d'un support d'echantillons pour spectrometrie de masse de type maldi

Publications (2)

Publication Number Publication Date
EP1573776A2 EP1573776A2 (fr) 2005-09-14
EP1573776B1 true EP1573776B1 (fr) 2017-12-06

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EP03795893.1A Expired - Lifetime EP1573776B1 (fr) 2002-12-13 2003-12-15 Procédé de production d'un support d'échantillons pour spectromètrie de masse de type maldi

Country Status (6)

Country Link
US (1) US20060121599A1 (fr)
EP (1) EP1573776B1 (fr)
JP (1) JP2006514738A (fr)
AU (1) AU2003298183A1 (fr)
DE (1) DE10258674A1 (fr)
WO (1) WO2004055857A2 (fr)

Families Citing this family (10)

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Publication number Priority date Publication date Assignee Title
DE102005022823A1 (de) * 2005-05-02 2006-11-09 Qiagen Gmbh Bioprobenträger für massenspektroskopische Analysen
JP5020742B2 (ja) * 2007-08-27 2012-09-05 日本電子株式会社 Ｍａｌｄｉイオン源を備えた質量分析装置およびｍａｌｄｉイオン源用サンプルプレート
WO2010014512A2 (fr) * 2008-07-30 2010-02-04 The Brigham And Women's Hospital, Inc. Préparation de plaques de test pour désorption-ionisation laser assistée par matrice
EP2157432A1 (fr) 2008-08-15 2010-02-24 Qiagen GmbH Procédé d'analyse d'un échantillon complexe par spectrométrie de masse
EP2452200A1 (fr) * 2009-07-09 2012-05-16 Koninklijke Philips Electronics N.V. Revêtement de surface pour spectrométrie de masse par ionisation et désorption laser de molécules
WO2011027819A1 (fr) * 2009-09-02 2011-03-10 公益財団法人野口研究所 Procédé de spectrométrie de masse
JP5359924B2 (ja) * 2010-02-18 2013-12-04 株式会社島津製作所 質量分析装置
US9211542B2 (en) * 2010-05-21 2015-12-15 Eidgenossische Technische Hochschule Zurich High-density sample support plate for automated sample aliquoting
WO2014162557A1 (fr) * 2013-04-04 2014-10-09 株式会社島津製作所 Procédé de préparation d'un échantillon pour maldi et dispositif associé
DE102015003440A1 (de) * 2015-03-16 2016-09-22 Friedrich-Schiller-Universität Jena Verfahren zur MALDI-MSI Analytik von Objekten, insbesondere biologischen Gewebeproben, und Target zur Analytik sowie dessen Herstellung

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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 (de) * 1997-12-11 2000-07-13 Bruker Daltonik Gmbh Probenträger für die MALDI-Massenspektrometrie nebst Verfahren zur Herstellung der Platten und zum Aufbringen der Proben
DE19834070B4 (de) * 1998-07-29 2007-02-15 Bruker Daltonik Gmbh Ionisierung hochmolekularer Substanzen durch Laserdesorption aus flüssigen Matrices
CA2356178C (fr) * 1998-12-24 2010-04-13 Sunyx Surface Nanotechnologies Gmbh Surface ultraphobe
EP1181705A2 (fr) * 1999-04-29 2002-02-27 Ciphergen Biosystems, Inc. Support d'echantillons a revetement hydrophobe pour spectrometre de masse en phase gazeuse
DE19923761C1 (de) * 1999-05-21 2001-02-08 Bruker Daltonik Gmbh Aufreinigende Probenträger für die MALDI-Massenspektrometrie
DE10005600A1 (de) * 2000-02-09 2001-08-16 Bayer Ag Ultraphobes Flächengebilde mit einer Vielzahl von hydrophilen Bereichen
DE10043042C2 (de) * 2000-09-01 2003-04-17 Bruker Daltonik Gmbh Verfahren zum Belegen eines Probenträgers mit Biomolekülen für die massenspektrometrische Analyse

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

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