WO2006058919A1 - Procede pour concentrer des biomolecules - Google Patents

Procede pour concentrer des biomolecules Download PDF

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Publication number
WO2006058919A1
WO2006058919A1 PCT/EP2005/056414 EP2005056414W WO2006058919A1 WO 2006058919 A1 WO2006058919 A1 WO 2006058919A1 EP 2005056414 W EP2005056414 W EP 2005056414W WO 2006058919 A1 WO2006058919 A1 WO 2006058919A1
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WO
WIPO (PCT)
Prior art keywords
crystalline structure
biomolecules
solvent
chip
ultraphobic
Prior art date
Application number
PCT/EP2005/056414
Other languages
German (de)
English (en)
Inventor
Karsten Dr. Reihs
Original Assignee
Qiagen Gmbh
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Qiagen Gmbh filed Critical Qiagen Gmbh
Publication of WO2006058919A1 publication Critical patent/WO2006058919A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

Definitions

  • the present invention relates to a method for concentrating biomolecules near the surface of a crystalline structure comprising at least biomolecules and another substance. Furthermore, the present invention relates to chips and a method for performing a mass spectrometric analysis.
  • 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
  • a drop of liquid is usually applied to a cleaned sample carrier, for example pipetted, and then analyzed. Often, however, the analyzes are of poor quality.
  • the object is achieved with a method for concentrating biomolecules in the vicinity of the surface of a crystalline structure having at least biomolecules and a further substance in which preferably the crystalline structure is at least once briefly acted upon by a solvent reversibly.
  • biomolecules are enriched in the vicinity of the surface of a crystalline structure which comprises at least biomolecules and a further substance.
  • a biomolecule in the sense of the present patent application is any molecule which is produced by it during the life cycle of any virus or monocellular or multicellular organism.
  • Biomolecules contain at least one oxygen, nitrogen, sulfur, and / or phosphorus atom.
  • Exemplary of biomolecules may be mentioned: game gels, aptamers, ribozymes, peptides, polypeptides, proteins, antibodies, nucleic acids, nucleic acid analogs, DNA, double-stranded DNA, RNA, double-stranded RNA / DNA, vitamins, carbohydrates, hormones, glycopeptides, glycoproteins, lipids, Fatty acids and cholesterol.
  • they can also be large amounts of the same or different biomolecules. These can be unorganized next to each other or build functional units due to interactions. Examples include protein complexes, genomes, cell nuclei, ribosomes, cells, cell aggregates, tissues or whole organisms.
  • the crystalline structure has a further substance.
  • this substance is a MALDI matrix which is required for carrying out the MALDI mass spectrometry, which will be explained in more detail below.
  • Preferred MALDI matrices 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.
  • MALDI Matrix as preferably crystalline Structure exists.
  • a biomolecule is embedded, which, however, often deposit in comparatively deep layers and not on the surface and thus are insufficiently available for a mass spectrometer analysis.
  • the biomolecules are concentrated near the surface; i.e. the biomolecules are enriched near the surface of the crystalline structure.
  • This enrichment preferably takes place in that the crystalline structure is at least once briefly subjected to a reversible reaction with a solvent.
  • a solvent is preferably applied to the crystalline structure for a short period of time, preferably for one to ten seconds, and then again preferably completely removed.
  • the residence time of the solvent on the crystalline structure it penetrates into the crystalline structure, with the concentration of the solvent being naturally largest at the surface and then decreasing.
  • Suitable solvents are any solvent which, on the one hand, can penetrate into the crystalline structure, whether it dissolves and / or dissolves the crystalline structure, or diffuses into the crystalline structure.
  • the solvent should be such that the biomolecules preferentially accumulate in it.
  • the solvent is water, acetonitrile and / or their mixture. As soon as the biomolecules have accumulated in the solvent, the solvent is removed as abruptly as possible, preferably evaporated, leaving the biomolecules enriched therein on the crystalline structure.
  • the crystalline structure is charged with a gaseous or liquid solvent.
  • the solvent is in the form of vapor, which precipitates on the crystalline structure.
  • the solvent After the solvent has been applied to the crystalline structure and has remained there for a short time, it is again removed as completely as possible, which can be done in any manner known to those skilled in the art. Preferably, however, the solvent is removed by raising the temperature and / or by applying a sufficient vacuum in the region of the crystalline structure.
  • the temperature of the crystalline structure is changed.
  • the crystalline structure is particularly preferably cooled and reheated at least once.
  • cooling preferably very rapid cooling, for example with a pelitic element, particularly preferably to temperatures of colder -5 ° C., for example, the solvent vapor precipitates on the crystalline structure as a frost.
  • the frost melts and the solvent can penetrate into the crystalline structure.
  • the removal is then preferably carried out by applying a sufficient vacuum and optionally a further increase in the temperature of the crystalline structure. This process sequence is preferably repeated several times.
  • the loading of the solvent and its removal takes place at least once, but more preferably several times.
  • the following process steps are carried out: exposure of the crystalline structure with solvent, retention of the solvent on the crystalline structure for a short period of time, wherein the solvent penetrates into the crystalline structure and as complete as possible removal of the solvent.
  • the method according to the invention is particularly suitable for enriching the biomolecules in crystalline structures of chips.
  • Chips are sample carriers that have a variety of spots including biological material. These spots usually have a diameter between 1 ⁇ m 2 and 10 mm 2 . Preferably, a solvent amount of 0.05 to 2 .mu.l per spot is applied to the crystalline structure.
  • biomolecules are enriched near the surface, they are more amenable to mass spectrometric analysis than in the prior art. With the chip, therefore, better, in particular more accurate, analyzes can be achieved than with chips according to the prior art.
  • the chip is obtainable according to the invention by the method according to the invention.
  • the chip has a crystalline structure which comprises biomolecules and at least one further substance, the biomolecules being enriched in the vicinity of the surface.
  • the crystalline structure includes a MALDI matrix.
  • This preferred embodiment of the chip is particularly suitable for MALDI mass spectrometry, which will be described below.
  • the chip has an ultraphobic surface, particularly preferably with hydrophilic anchor points.
  • 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.
  • ultraphobic surfaces are described, for example, in WO 98/23549, WO 96/04123, WO 96/21523, WO 99/10323, WO 99/10324, WO 99/101 11, WO 99/101 13, WO 99/101 12 and WO 96/34697, which are hereby incorporated by reference and thus part of the disclosure.
  • Such an ultraphobic surface is described in international patent application WO 99/10322, which is hereby incorporated by reference and thus forms part of the disclosure.
  • Hydrophilic and / or oleophilic anchor points in the context of the invention are areas on which a drop of water or oil can be deposited; i.e. a drop of water or oil, which is brought into contact with the hydrophilic and / or oleophilic area suspended from a pipetting system, remains attached to it and thus separates from the pipetting system.
  • a water or oil drop with a volume of 10 .mu.l on the hydrophilic and / or oleophilic areas assumes a contact angle ⁇ 120.degree., Preferably ⁇ 1.degree., Very preferably ⁇ 90.degree., And / or the contact angle of this droplet exceeds 10.degree ,
  • the surface of the hydrophilic and / or oleophilic areas is completely enclosed by an ultraphobic area.
  • the surface is substantially ultraphobic and has a plurality of hydrophilic and / or oleophilic anchor points.
  • the hydrophilic and / or oleophilic anchor points can be produced on the ultraphobic surface, for example by chemical and / or mechanical removal of at least part of the layer thickness of the ultraphobic layer, preferably by means of laser.
  • the hydrophilic and / or oleophilic regions are produced by a modification of only the uppermost molecular layer of the ultraphobic surface.
  • this modification is a mechanical and / or thermal ablation, but preferably only a maximum of one molecule layer of the ultraphobic surface is removed.
  • the modification is carried out by the thermal or chemical change of the ultraphobic surface but without removal, as described for example in DE 199 10 809 A1, which is hereby incorporated by reference and thus applies as part of the disclosure.
  • this modification of the ultraphobic surface whose layer thickness remains essentially unchanged.
  • the hydrophilic and / or oleophilic regions are reversibly producible on parts of the ultraphobic surface.
  • Such surfaces or processes for the reversible hydro- or oleophilization of parts of the ultraphobic surfaces are described in the German patent application DE 102 07 615, which is hereby introduced as a reference and thus applies as part of the disclosure.
  • the hydrophilic and / or oleophilic areas may be of any shape and size. However, they preferably have an area of 1 ⁇ m 2 - 10 mm 2 . On such a surface, a drop of liquid with a diameter of 5 microns - 5 mm settle and preferably anchored so that it does not dissolve itself hanging down from the surface.
  • the ultraphobic surface is electrically conductive.
  • This embodiment of the present invention is particularly advantageous for MALDI mass spectrometry.
  • sample carriers are preferably made of glass, plastic or metal.
  • a further subject of the present invention is a method for carrying out a mass spectrometric analysis, in which the molecules to be analyzed are enriched in the vicinity of the surface of a crystalline structure before or during the mass spectrometric analysis of the biomolecules.
  • mass spectrometric analysis is MALDI mass spectrometry.
  • biomolecules are preferably mixed with a matrix substance and this sample is then metered onto sample carriers with preferably hydro- and / or oleophilic areas, for example pipetted. This sample is then dried on the surface. The resulting crystals are analyzed, for example, with a MALDI-TOF mass spectroscope in linear or in reflector operation. Details of this procedure can 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 plan science research, Oxford University Press, (1996) page 86-101 which is hereby incorporated by reference and thus forms part of the disclosure.
  • MALDI method it is possible to record mass spectra of high quality.
  • the person skilled in the art knows that first of all the MALDI matrix can be metered onto the sample carrier and dried there, and then the sample to be analyzed is applied to the dried MALDI matrix and again dried and then analyzed.
  • the present invention is particularly useful in the field of drug discovery and biotechnology.
  • the uses of the inventions in this field is also an object of the present invention.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

L'invention concerne un procédé pour concentrer des biomolécules à proximité de la surface d'une structure cristalline contenant au moins une biomolécule et une autre substance. La présente invention porte également sur une puce et un procédé pour réaliser une analyse par spectrométrie de masse.
PCT/EP2005/056414 2004-12-03 2005-12-02 Procede pour concentrer des biomolecules WO2006058919A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102004058555.5 2004-12-03
DE200410058555 DE102004058555A1 (de) 2004-12-03 2004-12-03 Verfahren zur Aufkonzentrierung von Biomolekülen in der Nähe der Oberfläche einer kristallinen Struktur

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WO2006058919A1 true WO2006058919A1 (fr) 2006-06-08

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DE (1) DE102004058555A1 (fr)
WO (1) WO2006058919A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040197921A1 (en) * 2000-09-01 2004-10-07 Bruker Daltonik Gbmh Structured biosample support plates for mass spectroscopic analyses and procedures for manufacturing and use

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2235529B (en) * 1989-08-23 1993-07-28 Finnigan Mat Ltd Method of preparing samples for laser spectrometry analysis
DE19617011C2 (de) * 1996-04-27 2000-11-02 Bruker Daltonik Gmbh Matrixkomponentengemisch für die matrixunterstützte Laserdesorption und Ionisierung sowie Verfahren zur Zubereitung eines Matrixkomponentengemisches
DE19618032C2 (de) * 1996-05-04 2000-04-13 Bruker Daltonik Gmbh Lagerfähig vorpräparierte Maldi-Probenträger
DE19710166C1 (de) * 1997-03-12 1998-12-10 Bruker Franzen Analytik Gmbh Zwei-Schritt-Verfahren der DNA-Amplifikation für MALDI-TOF-Messungen
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
DE19923761C1 (de) * 1999-05-21 2001-02-08 Bruker Daltonik Gmbh Aufreinigende Probenträger für die MALDI-Massenspektrometrie
DE19949735A1 (de) * 1999-10-15 2001-05-10 Bruker Daltonik Gmbh Prozessieren von Proben in Lösungen mit definiert kleiner Wandkontaktfläche
DE19937438C2 (de) * 1999-08-07 2001-09-13 Bruker Daltonik Gmbh Kopplung Dünnschicht-Chromatographie und Massenspektrometrie (TLC/MS)
DE10108453B4 (de) * 2001-02-22 2005-10-20 Bruker Daltonik Gmbh Massenspektrometrische Mutationsanalyse mit photolytisch spaltbaren Primern
DE10112387B4 (de) * 2001-03-15 2004-03-25 Bruker Daltonik Gmbh Massenspektrometrische Genotypisierung
GB0120131D0 (en) * 2001-08-17 2001-10-10 Micromass Ltd Maldi target plate

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040197921A1 (en) * 2000-09-01 2004-10-07 Bruker Daltonik Gbmh Structured biosample support plates for mass spectroscopic analyses and procedures for manufacturing and use

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ALLWOOD D A ET AL: "PREPARATION OF 'NEAR' HOMOGENEOUS SAMPLES FOR THE ANALYSIS OF MATRIX-ASSISTED LASER DESORPTION/IONISATION PROCESSES", APPLIED SURFACE SCIENCE, ELSEVIER, AMSTERDAM, NL, vol. 103, 1996, pages 231 - 244, XP002910711, ISSN: 0169-4332 *
ERICSON C ET AL: "AN AUTOMATED NONCONTACT DEPOSITION INTERFACE FOR LIQUID CHROMATOGRAPHY MATRIX-ASSISTED LASER DESORPTION/IONIZATION MASS SPECTROMETRY", ANALYTICAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY. COLUMBUS, US, vol. 75, no. 10, 15 May 2003 (2003-05-15), pages 2309 - 2315, XP001170906, ISSN: 0003-2700 *
GOBOM J ET AL: "Alpha-cyano-4-hydroxycinnamic acid affinity sample preparation. A protocol for MALDI-MS peptide analysis in proteomics.", ANALYTICAL CHEMISTRY. 1 FEB 2001, vol. 73, no. 3, 1 February 2001 (2001-02-01), pages 434 - 438, XP002365016, ISSN: 0003-2700 *
KUSSMANN M ET AL: "Matrix-assisted laser desorption/ionization mass spectrometry sample preparation techniques designed for various peptide and protein analytes", JOURNAL OF MASS SPECTROMETRY 1997 UNITED KINGDOM, vol. 32, no. 6, 1997, pages 593 - 601, XP002365017, ISSN: 1076-5174 *
NORDHOFF E ET AL: "Sample preparation protocols for MALDI-MS of peptides and oligonucleotides using prestructured sample supports", INTERNATIONAL JOURNAL OF MASS SPECTROMETRY, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 226, no. 1, 15 March 2003 (2003-03-15), pages 163 - 180, XP004413199, ISSN: 1387-3806 *
NUWAYSIR LYDIA M ET AL: "Matrix-assisted laser desorption by Fourier transform mass spectrometry", PROC SPIE INT SOC OPT ENG; PROCEEDINGS OF SPIE - THE INTERNATIONAL SOCIETY FOR OPTICAL ENGINEERING 1991 PUBL BY INT SOC FOR OPTICAL ENGINEERING, BELLINGHAM, WA, USA, vol. 1437, 1991, pages 112 - 123, XP002365018 *

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