WO2007030849A1 - Procede de detection de composes organiques - Google Patents

Procede de detection de composes organiques Download PDF

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Publication number
WO2007030849A1
WO2007030849A1 PCT/AT2006/000376 AT2006000376W WO2007030849A1 WO 2007030849 A1 WO2007030849 A1 WO 2007030849A1 AT 2006000376 W AT2006000376 W AT 2006000376W WO 2007030849 A1 WO2007030849 A1 WO 2007030849A1
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WO
WIPO (PCT)
Prior art keywords
acid
chloro
methyl
cellulose
butene
Prior art date
Application number
PCT/AT2006/000376
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German (de)
English (en)
Inventor
Günther Bonn
Isabel Feuerstein
Christian Huck
Guenther Stecher
Matthias Rainer
Original Assignee
Austria Wirtschaftsservice Gesellschaft mit beschränkter Haftung
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Application filed by Austria Wirtschaftsservice Gesellschaft mit beschränkter Haftung filed Critical Austria Wirtschaftsservice Gesellschaft mit beschränkter Haftung
Publication of WO2007030849A1 publication Critical patent/WO2007030849A1/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
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry

Definitions

  • the present invention relates to a method for the detection of organic compounds, in particular peptides or proteins, from biological sample solutions comprising the steps
  • biomarkers eg high molecular weight, biochemical, macromolecular compounds, such as polypeptides, ribonucleic acids, carbohydrates, lipids etc., but sometimes also low molecular weight, simple molecules
  • biomarkers eg high molecular weight, biochemical, macromolecular compounds, such as polypeptides, ribonucleic acids, carbohydrates, lipids etc., but sometimes also low molecular weight, simple molecules
  • biomarkers organic compounds
  • ELISA enzyme linked immunosorbent assay
  • RIA radio immunoassay
  • chromatographic separation processes are often extremely expensive, expensive and sometimes too less effective.
  • gel electrophoresis methods are currently used in particular (eg SDS-polyacrylamide gel electrophoresis or agarose gel electrophoresis), whereby simple one-dimensional gel electrophoresis methods are relatively inaccurate and the quantitative determination is nevertheless time-consuming and relatively poorly reproducible.
  • conventional one-dimensional gel electrophoresis method only perform a separation of the organic compounds by molecular weight
  • two-dimensional electrophoresis in addition to the separation of different mass also a separation according to different charge distribution in the molecule by means of isoelectric focusing is performed.
  • Two-dimensional electrophoresis methods are also very complex and can only be performed by trained personnel. In addition, such methods are relatively time consuming and you can always analyze only a single sample per gel in two-dimensional gel electrophoresis, which significantly increases the space requirements and also the effort and material requirements.
  • EP 0 700 521 presents a technique in which a solution is applied to a mass spectrometer sample carrier which has been functionalized so that the organic compounds to be investigated can bind to the functionality of the surface.
  • the unbound substances are washed away and then a matrix is applied to the sample carrier.
  • Solvents are evaporated until crystallization of the matrix into which the to be examined Molecule is embedded.
  • the surface of the matrix is then bombarded with a laser, and ionized parts of the molecule are released from the matrix and can be detected by a mass spectrometer.
  • US Pat. No. 6,225,047 also suggests a method in which the sample to be analyzed is incubated by means of functionalized essentially spherical macroscopic particles (eg beads or ellipsoids), summarized as so-called beads, for example cellulose beads. Subsequently, the beads are centrifuged off and the supernatant of the solution is discarded. The beads may then be applied to a (mostly functionalized, i.e., derivatized) mass spectrometer sample carrier, and by applying a matrix, the conventional MALDI method is used.
  • a functionalized i.e., derivatized i.e., derivatized
  • Object of the present invention is therefore to provide a method of the type mentioned with improved properties.
  • the functionalized cellulose beads have an average diameter of ⁇ 20 microns, preferably from about 8-10 microns.
  • the invention is based on the surprising knowledge that functionalized cellulose beads having an average diameter of ⁇ 20 .mu.m, preferably 8-10 .mu.m, have particularly good separation and analysis properties in MALDI-TOF MS, specifically in a mass range of those to be investigated Connections of about 500
  • cellulose beads having a mean diameter of about 500 - 1500 microns.
  • Such cellulose beads have for the first time been characterized by Chen and Zhao (Biotechnology and
  • the cellulose beads are porous.
  • the cellulose beads should have a specific surface area of 1m 2 / g to 30m 2 / g, preferably about 9.7m 2 / g. Due to the porosity of the cellulose beads, the surface is significantly increased and there is a larger area for the adsorption of macromolecular organic compounds. The larger the porosity, the larger the surface area of the cellulose beads.
  • the average specific surface should not become too large (which would correspond to a large number of small pores, for example), since in too small pores macromolecules no longer bind sufficiently or macromolecules, due to steric constraints, no longer penetrate into the pores can penetrate.
  • the average pore diameter of the cellulose beads is about 0.2 ⁇ m to 1 ⁇ m, preferably about 0.3 ⁇ m to 0.4 ⁇ m.
  • Diameter range of the pores can adsorb molecules of the above sizes particularly well. With a pore size of about 0.1 ⁇ m, as described by Chen and Zhao, the ability of cellulose particles to adsorb macromolecules is significantly worse.
  • the size of the pores and thus the adsorption capacity for biomolecules or generally for organic compounds thus depends on the ratio of the diameter of the molecule to be adsorbed to the pore size.
  • the structure of, for example, proteins or polypeptides may be both more fibrous and more spherical.
  • a diameter of up to a few nanometers can be expected in the given mass range (0.5 kDa to about 80 kDa). Consequently, it has been found to be favorable if the pore diameter of the cellulose beads is about. 50 to 500, preferably about one hundred times greater than the molecular diameter of the organic compounds to be investigated.
  • complex bonds with free OH groups, NH groups, SH groups, ether groups or carboxyl groups can be formed by the loading with metal ions. It has proved to be advantageous if the metal ions from the group Fe 2+ , Fe 3+ , Al 3+ , Bi 3+ , Cu + , Cu 2+ , Mn 2+ , Ca 2+ , Mg 2+ , Zn 2+ , Cd 2+ , Au 2+ , Pd 2+ , Cr 3+ . In the preferred variant, Cu 2+ ions are used.
  • a particular advantage in the use of functionalized cellulose beads is that the immobilization of the organic compounds takes place in suspension. Thereby, a particularly easy separation of the remaining in solution organic compounds, of those which bind to the cellulose beads, possible, for example by centrifugation.
  • the residue after a centrifugation step (under mild centrifugation conditions) forms a kind of suspension in which the cellulose beads are present with bound organic compounds.
  • These are then applied to the mass spectrometer sample carrier, for example pipetted. It has proved to be particularly advantageous if the mass spectrometer sample carrier is free of functionalities. Free of functionalities here means that the surface is not covered by a additional derivatization step is chemically altered.
  • the functionalization of the mass spectrometer sample carrier is particularly complex and expensive, but this is often required in the prior art. Therefore, the main advantage of this method is that the functionalities for binding the organic compounds no longer have to be coupled to the mass spectrometer sample carrier by complex derivatization methods, but that the derivatization of the Beads can be simply in solution or suspension.
  • the mass spectrometer sample carrier does not need to be further functionalized.
  • mass spectrometer sample carrier made of conductive material, preferably made of stainless steel, titanium or graphite.
  • a derivatized mass spectrometer sample carrier can be used to further improve the separation of the process.
  • a matrix is applied and crystallized.
  • the matrix used is mainly sinapinic acid and / or ⁇ -cyano-4-hydroxycinnamic acid.
  • the matrix is pipetted, for example, as a solution, and the, preferably volatile, solvent is evaporated before the ionization step.
  • other matrix substances known from the literature can also be used.
  • This method is particularly suitable when the organic compounds have a mass of from 500 Da to 80 kDa, preferably from 2 kDa to 20 kDa. Especially in this mass range, the separation of the individual compounds is particularly good.
  • the biological samples can be obtained from different areas.
  • the biological sample solutions may be from the group of blood, blood serum, blood plasma, urine, tissue fluid, cell extracts, tissue extracts, organ extracts or cerebrospinal fluid.
  • the cellulose beads at least one
  • Linker which is located between cellulose and the chemical functionalization.
  • a linker is understood to mean a compound which is covalently bound to a free OH group of cellulose and has, for example, a free functionality which is suitable for another derivatization can be used. But it is also possible that the linker itself has the chemical functionalization that is used to bond the organic compound.
  • the chemical functionalization could be a free carboxyl group that binds a metal ion, which subsequently serves as a complexing agent for an organic compound.
  • the linker comprises at least one binding functionality.
  • the binding functionality selected from the group consisting of carbon bonds, epoxides, halogens, amino groups, hydroxy groups, acid groups, acid chlorides, cyanide groups, aldehyde groups, sulfate groups, sulfonate groups, phosphate groups, metal-complexing groups, antibodies against protein A, antibodies against protein G, thioethers, biotin, thiols and mixtures thereof.
  • the linker comprises an epoxide group and is preferably selected from the group consisting of glycidyl methacrylate, epichlorohydrin, 3,4-epoxybutyl acrylate, 2-methyl-2-propenyl oxirane carboxylic acid ester, 3- ( 2-methyloxiranyl) -2-propenoic acid methyl ester, dihydro-4- (2-propenyloxy) -2 (3H) -furanone, 2-methyl-2-propenoic acid ⁇ oxiranylmethyl ester, tetrahydro-3-furanyl-2 Propenoic acid esters, oxiranylmethyl-2-butenoic acid esters, 1-methylethenyl-oxiraneacetic acid esters, oxiranylmethyl-3-butenoic acid esters, (3
  • Crotonic acid esters tetrahydro-2-furanyl-2-propenoic acid esters, (2-methyloxiranyl) -methyl-2-propenoic acid esters, 2-methyl-2-propenoic acid 3-oxetanyl esters, and mixtures thereof.
  • the linker is a chemical group selected from the group consisting of iminodiacetic acid, nitrilotriacetic acid, N-carboxy- ⁇ -alanine, aspartic acid, 2-amino-2-methyl-propanedi acid, 2-furanacetic acid, 5-ethyl-3-hydroxy-4-methyl-2 (5H) -furanone, tetrahydro-4-methylene-3-furanacetic acid, asparagine acid, 2-butenedi acid, methylenepropandiic acid, and mixtures thereof.
  • the linker contains a carboxylic acid group and preferably selected from the group consisting of 2-butenedioic acid,
  • Cellulose type A is microcrystalline cellulose, the particles having a diameter of about 20-75 microns.
  • the shape of the particles is undefined, that is, the particles can have different three-dimensional structures.
  • This type of cellulose is also from Sigma Aldrich.
  • Cellulose Type B This is spherical cellulose made according to the instructions of Chen and Zhao. Subsequently, the thus prepared cellulose particles were frozen with liquid nitrogen and crushed. The cellulose particles thus prepared were substantially spherical and had a diameter of 20-30 ⁇ m.
  • Cellulose Type C These are spherical cellulose, ie cellulose beads that are already functionalized with triacetate (triacetate cellulose) and that have a particle diameter of 6 - 8 ⁇ m. The mean pore diameter of the particles is about 0.19 ⁇ m. The particles are from Chisso Corporation, Japan and were renatured before use, ie the triacetate was hydrolytically cleaved.
  • Cellulose Type D These are cellulose beads according to the invention or spherical cellulose having particularly good properties, the average diameter of the particles being about 8-10 ⁇ m and the average pore diameter being about 0.34 ⁇ m. The particles are also from Chisso Corporation, Japan.
  • cellulose For the derivatization or functionalization of cellulose, 5 g of the respective types A, B, C or D were dispersed with vigorous stirring in 230 ml of water for 2 minutes. The suspension of cellulose thus obtained was mixed with 328 mM monomeric glycidyl methacrylate, 6.5 mM ammonium persulfate and 8.6 mM sodium thiosulfate as redox catalyst and for
  • the final product was filtered under vacuum applied, washed with deionized water and saturated with Cu 2+ ions by incubating for 2 hours the derivatized cellulose with a 50 mM Cu 2+ solution at about 23 ° C.
  • Cu 2+ solution for example, CuSO 4 comes into question.
  • 3 ⁇ g of the suspended IDA-Cu 2+ cells were placed in a 0.5 ml centrifuge tube and activated with 50 mM sodium acetate buffer at room temperature and then centrifuged at 13,000 rpm for about 1 minute. After the centrifugation step the supernatant was discarded and the residue was equilibrated twice with 200 ⁇ l of PBS buffer.
  • Fig. 1 the result of the thus examined four different types of cellulose (A, B, C, D) is shown graphically.
  • the abscissa shows the mass to charge ratio (M / Z) of the ionized molecules, in a mass range of 2000 Da to 10,000 Da.
  • the ordinate describes the signal size of the mass spectrometric analysis according to MALDI-TOF-MS, whereby primarily the relative result of the signal heights to one another is to be considered. It is readily apparent that particularly in the case of cellulose type D, a particularly good separation occurs in almost all mass ranges, in particular in the range 4000-5000 Da and 7000-9000 Da. The results were also virtually identical to samples stored for three weeks.
  • the spherical functionalized cellulose B-type beads still gave good results in mass spectrometric analysis, although the signal intensity was already lower than in type A. Primarily, this is due to the deteriorated surface accessibility of the organic compounds on the functionalized cellulose beads.
  • the surface accessibility and thus the adsorption capacity of the organic compounds on the functionalized cellulose beads depends on the one hand on the average pore diameter and on the other hand on the particle size of the cellulose beads.
  • the investigations showed that the most favorable pore diameter range is about 0.34 ⁇ m. But above all, the particle size is critical, which has been found here that a particle size between 8 and 10 microns is particularly well suited. These particles are easy to process, both during derivatization and during subsequent pipetting onto the mass spectrometer sample carrier. In particular, the larger particles (type B and type A) were much more difficult to handle in pipetting and work up.
  • Fig. 2 once again schematically enumerates the important steps (A) of the cellulose derivatization by means of an example:
  • First step the free OH groups of the cellulose (not derivatized) are mixed with glycidyl methacrylate.
  • cellulose is bound with glycidyl methacrylate and a free epoxide group.
  • iminodiacetic acid (IDA) and Na 2 CO 3 is added.
  • IDA iminodiacetic acid
  • Cu 2+ is added, whereby the carboxylate groups complex the Cu 2+ and, for example, can enter into a fourfold complex with proteins.
  • Fig. 3 the sample preparation is shown, where first derivatized cellulose beads are added to the blood serum samples.
  • step 2 the incubation and binding of the organic compounds to the functionalized cellulose beads is carried out, followed by a washing and centrifuging step in step 3.
  • step 4 the sample (ie the cellulose beads with the bound organic compounds in the form of a suspension) on the Mass spectrometer sample carrier pipetted on. After addition of the matrix (not shown here), direct analysis of the particles is carried out by MALDI-TOF-MS. (Step 5)

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
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  • Urology & Nephrology (AREA)
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  • Biochemistry (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Optics & Photonics (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

L'invention concerne un procédé de détection de composés organiques, notamment de peptides ou protéines, de solutions échantillons biologiques. Ce procédé consiste à incuber les solutions échantillons biologiques avec au moins une sorte de cellulose (billes) sensiblement sphérique et chimiquement fonctionnalisée à la surface ; à séparer les substances, demeurant en solution, des billes cellulosiques incubées, fonctionnalisées, de préférence par centrifugation, application des billes cellulosiques incubées fonctionnalisées sur un support d'échantillon spectromètre de masse (cible) et à analyser les composés organiques liés aux billes cellulosiques par spectrométrie de masse MS désorption/ionisation laser assistée par matrice à temps de vol (MALDI-TOF MS). Les billes cellulosiques fonctionnalisées ont un diamètre moyen < 20 µm, de préférence d'environ 8-10 µm.
PCT/AT2006/000376 2005-09-12 2006-09-11 Procede de detection de composes organiques WO2007030849A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ATA1494/2005 2005-09-12
AT14942005A AT502369A1 (de) 2005-09-12 2005-09-12 Verfahren zur detektion organischer verbindungen

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CN114134322B (zh) * 2020-09-04 2023-03-14 苏州博萃循环科技有限公司 一种从含铜锰钙锌混合溶液中分离铜锰的方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003077851A2 (fr) * 2002-03-11 2003-09-25 Hk Pharmaceuticals, Inc. Composes et procedes pour analyser le proteome

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003077851A2 (fr) * 2002-03-11 2003-09-25 Hk Pharmaceuticals, Inc. Composes et procedes pour analyser le proteome

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
FEUERSTEIN ET AL: "Material-Enhanced Laser Desorption/Ionization (MELDI)-A New Protein Profiling Tool Utilizing Specific Carrier Materials for Time of Flight Mass Spectrometric Analysis", JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY, ELSEVIER SCIENCE INC, US, vol. 17, no. 9, 12 June 2006 (2006-06-12), pages 1203 - 1208, XP005658517, ISSN: 1044-0305 *
FEUERSTEIN I ET AL: "Detection of prostate cancer using serum proteomics pattern in a histological confirmed population", EUROPEAN UROLOGY SUPPLEMENTS, vol. 4, no. 3, March 2005 (2005-03-01), & 20TH ANNUAL MEETING OF THE EUROPEAN-ASSOCIATION-OF-UROLOGY; ISTANBUL, TURKEY; 20050317,, pages 156, XP005007077, ISSN: 1569-9056 *
FEUERSTEIN ISABEL ET AL: "Derivatized cellulose combined with MALDI-TOF MS: A new tool for serum protein profiling", JOURNAL OF PROTEOME RESEARCH, vol. 4, no. 6, 18 November 2005 (2005-11-18), pages 2320 - 2326, XP002416436, ISSN: 1535-3893 *
FEUERSTEIN ISABEL ET AL: "Phosphoproteomic analysis using immobilized metal ion affinity chromatography on the basis of cellulose powder", PROTEOMICS, vol. 5, no. 1, January 2005 (2005-01-01), pages 46 - 54, XP002416435, ISSN: 1615-9853 *
FEUERSTEIN ISABEL:: "Development of cellulose based stationary phases for the analysis of biomolecules", UNIVERSITÄT INNSBRUCK,, DISSERTATION., 17 March 2005 (2005-03-17), Innsbruck,AT, pages I - 152, XP002416442 *
RASKA CHRISTINA S ET AL: "Direct MALDI-MS/MS of phosphopeptides affinity-bound to immobilized metal ion affinity chromatography beads.", ANALYTICAL CHEMISTRY. 15 JUL 2002, vol. 74, no. 14, 15 July 2002 (2002-07-15), pages 3429 - 3433, XP002416437, ISSN: 0003-2700 *
TANG N ET AL: "CURRENT DEVELOPMENTS IN SELDI AFFINITY TECHNOLOGY", MASS SPECTROMETRY REVIEWS, JOHN WILEY AND SONS, NEW YORK, NY, US, vol. 23, January 2004 (2004-01-01), pages 34 - 44, XP009047591, ISSN: 0277-7037 *
UNI-PRESSESTELLE: "Unterwegs", UBT-AKTUELL, vol. 5, July 2004 (2004-07-01), Bayreuth, pages 1 - 25, XP002416550, Retrieved from the Internet <URL:http://www.uni-bayreuth.de/presse/ubtaktuell/cinque-04.pdf> [retrieved on 20061206] *

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