EP1334353A1 - Procede de separation et de detection de proteines par electrophorese - Google Patents

Procede de separation et de detection de proteines par electrophorese

Info

Publication number
EP1334353A1
EP1334353A1 EP01996736A EP01996736A EP1334353A1 EP 1334353 A1 EP1334353 A1 EP 1334353A1 EP 01996736 A EP01996736 A EP 01996736A EP 01996736 A EP01996736 A EP 01996736A EP 1334353 A1 EP1334353 A1 EP 1334353A1
Authority
EP
European Patent Office
Prior art keywords
proteins
separation step
separation
protein
electrophoresis
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.)
Withdrawn
Application number
EP01996736A
Other languages
German (de)
English (en)
Inventor
Markus Hammermann
Herbert Platsch
Gerhard Weber
Heinz Eipel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BASF SE
Dr Weber GmbH
Original Assignee
BASF SE
Dr Weber 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
Priority claimed from DE2000156838 external-priority patent/DE10056838A1/de
Priority claimed from DE2001120803 external-priority patent/DE10120803A1/de
Priority claimed from DE2001135497 external-priority patent/DE10135497A1/de
Application filed by BASF SE, Dr Weber GmbH filed Critical BASF SE
Publication of EP1334353A1 publication Critical patent/EP1334353A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
    • G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44773—Multi-stage electrophoresis, e.g. two-dimensional electrophoresis
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2550/00—Electrophoretic profiling, e.g. for proteome analysis

Definitions

  • the invention relates to a method for separating and detecting proteins in order to accelerate proteome analysis.
  • a proteome is the term used to describe and quantify all proteins of an organism, a cell, an organelle or a body fluid at a defined point in time and under precisely defined conditions.
  • proteins are examined to determine which proteins play which role in biological processes and which proteins are of particular importance in interaction with other proteins.
  • the question of the extent to which chemicals, active substances and other external factors (environmental influences, heat, cold, lack of water, pH value, etc.) influence cellular protein expression is investigated in the context of proteome analysis.
  • proteome analysis also tries to find out which proteins in which protein constellations can be responsible for which side effects.
  • the question is examined whether the protein expression of microorganisms can be influenced in such a way that the space-time yields of fermentative production processes can be improved.
  • sample preparation poses a significant problem.
  • the course is set to separate and identify even complex protein patterns. It has been found that the solubility has a significant influence on the separation of proteins. Proteins that dissolve quickly in water generally do not pose any problems with regard to separability.
  • Proteins with stable secondary or tertiary structures that are sparingly water-soluble are stabilized in terms of their solubility behavior by adding chaotropic substances such as guanidine hydrochloride or urea.
  • chaotropic substances such as guanidine hydrochloride or urea.
  • undesired reactions of individual proteins can occur, whereby a protein originally present in one form changes into several forms and thus further increases the heterogeneity of the sample already present.
  • Membrane proteins the natural environment of which are lipid membranes and which tend to agglomerate and become insoluble again when they are isolated from one another, are particularly difficult to handle. These hydrophobic proteins can only be kept in the dissolved state if detergents are added, which, however, can frequently impair the subsequent stages of protein separation.
  • High protein concentrations are highly desirable for most separation and evaluation procedures, but are associated with the risk that aggregations will form.
  • low protein concentrations which have a positive effect on the solubility behavior, are associated with additional preparation steps before the actual separation.
  • Proteins have the character of zwitterions and can therefore be positively or negatively charged. Using electrophoresis methods, individual components can be separated according to their mobility in the electrical field. The electrophoretic
  • Mobility of each protein is a characteristic value.
  • proteome analysis two electrophoresis methods are used, isoelectric focusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
  • SDS sodium dodecyl sulfate
  • This method provides for the separation of the proteins in the first dimension with the isoelectric focusing according to their isoelectric point.
  • the second dimension is a sodium dodecyl sulfate-polyacrylamide gel electrophoresis to separate the proteins according to their size.
  • This method is currently the only procedure with which complex protein mixtures can be separated with high resolution.
  • the advantages of the 2D-PAGE process are the two mutually complementary separation principles, namely IEF and SDS-PAGE processes according to load and molecular weight. In principle, this technique can be used with all proteins.
  • the resolution of this method can be increased significantly by using narrow pH gradients in isoelectric focusing ("zoom gels").
  • Staining is carried out, for example, with dyes such as Coomassie blue, with colloidal silver, with zinc / imidazole or with fluorescent dyes such as Sypro ® Ruby, Sypro ® Orange or Sypro ® Red. All dyeing methods used have in common that the bond between protein and dye reagent is not covalent , but on the basis of ionic, hydrophobic or Van der Waal interactions. After staining, the gels are usually digitized using a scanner or a fluorescence scanner.
  • US 6,043,025 and US 6,127,134 describe a process and a kit with which one can detect differences in two or more protein samples.
  • the protein extracts from different samples are covalently labeled with different, positively charged fluorescent dyes, combined and subjected to 2D PAGE. Identical proteins from the different samples can then be detected and quantified on the basis of their different fluorescence wavelengths in the same gel.
  • the proteins of a first cell are prepared using known treatment techniques, the first cell originating from a first group of cells.
  • the proteins are covalently labeled with a first dye from a pair of dyes.
  • a second cell is prepared using known treatment techniques that were taken from a second group of cells.
  • the proteins of this second cell are covalently labeled with a second dye.
  • radioactive methods can also be used.
  • the cells are mixed with certain isotope-labeled compounds such as 35 S-cysteine or 32 PO 4 3 " .
  • a phosphor imager is usually used for digitization in these cases.
  • image evaluation programs such as MELANIE, PDQUEST, IMAGEMASTER or The protein spots are then detected, quantified and assigned in the digitized gels obtained.
  • the entire procedure from electrophoresis to staining, detection and quantification according to the 2D-PAGE method is very complex.
  • the object of the invention is to develop a separation method for proteins which reduces the use of gels, is quick and easy to carry out and can be automated, and allows simple quantification of the separated proteins.
  • this object is achieved by a method for the separation and detection of proteins, the protein samples being contained in a separation buffer solution and the following method steps being carried out:
  • the protein samples are broken down into individual fractions according to free-flow electrophoresis with isoelectric focusing (IEF) or isotachophoresis and with a
  • the protein fractions are separated in one or more capillaries according to capillary electrophoresis, at least one labeling substance being detected in the individual capillary (s).
  • protein fractions obtained after the first separation step are linked with a reactive fluorescent dye.
  • a reactive fluorescent dye This can be done, for example, by coupling the N-hydroxysuccinimide esters (NHS esters) or isothiocyanates of fluorescent dyes to free amino groups of the proteins. Free amino groups are preferably the N-termini or lysine selected as coupling sites.
  • the dyes can be covalently bound to the individual proteins in the process proposed according to the invention.
  • protein fractions obtained after the first separation step are mixed with a marking substance.
  • a marking substance e.g. Sypro® Ruby, Sypro Orange or Sypro Red.
  • the fluorescent dyes Sypro Ruby, Sypro Orange or Sypro®Red can be bound by adsorption, e.g. by hydrophobic, ionic or Van der Waal's forces.
  • capillaries In a second separation step, capillaries can be used which are either provided with a polyacrylamide gel or which do not contain this substance, i.e. are empty.
  • the individual proteins are detected in the second separation step using laser-induced fluorescence. Sensitive fluorescence detection ensures a wide dynamic range and high sensitivity.
  • sodium dodecyl sulfate can be added to the separation buffer. Due to the high separation performance of capillary electrophoresis, a significantly better resolution in the second dimension is achieved compared to the 2D-PAGE gel process; Furthermore, the high degree of automation of both separation steps, the FFE and the capillary electrophoresis, enables a significantly higher throughput and thus a better statistical validation of the results obtained. In order to ensure the parallel processing of a higher number of protein samples after free-flow electrophoresis, all wells of a microtiter plate can be detected and quantified separately in parallel by as many capillaries. This can be achieved, for example, by using a commercially available device for DNA sequencing.
  • several different fluorescent dyes can be detected simultaneously in each capillary in the second separation step.
  • the sensitive fluorescence detection ensures a large dynamic range and high sensitivity. Due to the high separation performance of capillary electrophoresis, a significantly better resolution in the second dimension is achieved compared to the 2D PAGE gel process. Due to the high degree of automation of both separation steps, FFE and capillary electrophoresis, a significantly higher throughput and thus a better statistical validation of the measured results is expected.
  • the separation of the protein samples in the first separation step can e.g. in an advantageous manner in a microtiter plate, a microtiter plate being used, the number of wells of which corresponds to that of the separated protein fractions.
  • the number of capillaries used in the second separation step according to capillary electrophoresis advantageously corresponds to the number of sample fractions introduced into the microtiter plate.
  • two different free-flow electrophoresis methods can be used for the method proposed according to the invention: isoelectric focusing and isotachophoresis.
  • isotachophoresis can be used in the first separation step.
  • a potential gradient is formed in the electrical field in a discontinuous buffer system consisting of the lead and secondary electrolytes.
  • the field strength is higher in the area of ions with low mobility than in the area of more mobile ions. Since all ions must migrate at the same speed, pure zones of the individual proteins are formed from the protein sample mixture. In the equilibrium state, the ion with the highest mobility follows the lead ion of the lead electrolyte, the one with the lowest mobility moves in front of the follow-up electrolyte, the others move in between in the order of decreasing mobility.
  • interval isotachophoresis is performed (Gerhard Weber and Petr Bocek in Electrophoresis 1998, 19, 3090 - 3093). After the protein sample and the electrolytes have been applied to the free-flow electrophoresis chamber, high voltage is applied for 2 minutes to separate the proteins, then the separated fractions are conveyed without tension via a series of tubes into the individual wells of a microtiter plate.
  • individual protein fractions are obtained in microtiter plates in this electrophoresis device.
  • the proteins in these fractions can be linked both to a marker, for example a fluorescent dye such as Sypro®Orange, Sypro®Red or Sypro®Ruby, and to at least one reactive fluorescent dye.
  • a marker for example a fluorescent dye such as Sypro®Orange, Sypro®Red or Sypro®Ruby
  • Derivatives such as the N-hydroxysuccinimide esters (NHS esters) or the isothiocyanates of fluorescent dyes, which are coupled to free amino groups of the proteins, are suitable for this. N-termini or lysine of the proteins or the protein fractions are particularly suitable.
  • the second separation step e.g. Separate covalently fluorescence-labeled proteins using capillary electrophoresis.
  • the one or more capillary tubes used can be filled on the one hand with a polyacryamide gel, on the other hand the use of unloaded, i.e. empty capillary tube possible.
  • Detection preferably follows with laser-induced fluorescence, and sodium dodecyl sulfate can be added to the separation buffer to improve the running behavior of the proteins in capillary electrophoresis.
  • all wells of the microtiter plate are separately detected and quantified in parallel in as many capillaries.
  • a commercially available device for DNA sequencing is used, for example a Mega-BACE from Amersham Pharmacia or another device of a similar design.
  • An advantage over conventional 2D gel electrophoresis with subsequent non-covalent staining with image evaluation for quantification is that the electropherograms obtained by the method described here can be easily quantified using commercially available software.
  • several different marking substances such as, for example, fluorescent dyes
  • only a fluorescent dye or fluorescent substance can be detected just as well.
  • the multiple samples analyzed in one run are proteins from different cells or from cells in different stages of development or from cells that were exposed to different external conditions (such as heat, cold, active substances, chemicals, etc.).
  • Dissolution of the protein samples or the protein samples of cellular origin in the second Dimension compared to the 2D PAGE method can be achieved. Due to the significantly better automatability of both methods, free-flow electrophoresis and capillary electrophoresis, a significantly higher throughput and thus a better statistical validation of the results will take place.
  • a program sequence for evaluating the electropherograms is to be created; furthermore, in order to implement the method proposed according to the invention, a free-flow electrophoresis apparatus (“Octopus” from Dr. Weber GmbH), for example, and, for example, a Mega-BACE sequencer from Amersham or a similar device are required.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Hematology (AREA)
  • Electrochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé de séparation et de détection de protéines ou d'échantillons de protéines d'origine cellulaire, les protéines étant contenues dans une solution tampon de séparation, caractérisé en ce que, dans une première étape de la séparation, les échantillons de protéines sont séparés en fractions individuelles par électrophorèse à écoulement libre ou par focalisation isoélectrique (IEF) ou par isotachophorèse, et liés avec une substance de marquage, en ce que, dans une seconde étape, les fractions de protéines sont séparées, par électrophorèse capillaire, dans un ou plusieurs capillaires, et en ce que dans l'un ou plusieurs de ces capillaires, au moins une substance de marquage liée aux fractions de protéines est détectée.
EP01996736A 2000-11-16 2001-11-14 Procede de separation et de detection de proteines par electrophorese Withdrawn EP1334353A1 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
DE2000156838 DE10056838A1 (de) 2000-11-16 2000-11-16 Verfahren zur Trennung und Detektion von Proteinen
DE10056838 2000-11-16
DE2001120803 DE10120803A1 (de) 2001-04-27 2001-04-27 Verfahren zur Trennung und Detektion von Proteinen
DE10120803 2001-04-27
DE10135497 2001-07-20
DE2001135497 DE10135497A1 (de) 2001-07-20 2001-07-20 Verfahren zur Trennung und Detektion von Proteinen
PCT/EP2001/013195 WO2002040983A1 (fr) 2000-11-16 2001-11-14 Procede de separation et de detection de proteines par electrophorese

Publications (1)

Publication Number Publication Date
EP1334353A1 true EP1334353A1 (fr) 2003-08-13

Family

ID=27214156

Family Applications (1)

Application Number Title Priority Date Filing Date
EP01996736A Withdrawn EP1334353A1 (fr) 2000-11-16 2001-11-14 Procede de separation et de detection de proteines par electrophorese

Country Status (7)

Country Link
US (1) US20040031683A1 (fr)
EP (1) EP1334353A1 (fr)
JP (1) JP2004514136A (fr)
AU (1) AU1702902A (fr)
CA (1) CA2428372A1 (fr)
NO (1) NO20032209L (fr)
WO (1) WO2002040983A1 (fr)

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DE10047088C2 (de) * 2000-09-21 2002-10-17 Gerhard Weber Medium für analytische und präparative Elektrophorese
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DE10063096C1 (de) * 2000-12-18 2002-09-12 Gerhard Weber Elektrophoresevorrichtung, Elektrophoreseverfahren unter Verwendung einer Elektrophoresevorrichtung und Verwendung der Elektrophoresevorrichtung
SE0202401D0 (sv) * 2001-12-11 2002-08-13 Thomas Laurell Biomolecule handling method and machine using an arry dispenser
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Also Published As

Publication number Publication date
CA2428372A1 (fr) 2002-05-23
JP2004514136A (ja) 2004-05-13
US20040031683A1 (en) 2004-02-19
NO20032209D0 (no) 2003-05-15
NO20032209L (no) 2003-07-15
WO2002040983A1 (fr) 2002-05-23
AU1702902A (en) 2002-05-27

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