EP1558925A1 - Dispositif de separation et procede de separation pour echantillon de matiere biomoleculaire - Google Patents

Dispositif de separation et procede de separation pour echantillon de matiere biomoleculaire

Info

Publication number
EP1558925A1
EP1558925A1 EP03810966A EP03810966A EP1558925A1 EP 1558925 A1 EP1558925 A1 EP 1558925A1 EP 03810966 A EP03810966 A EP 03810966A EP 03810966 A EP03810966 A EP 03810966A EP 1558925 A1 EP1558925 A1 EP 1558925A1
Authority
EP
European Patent Office
Prior art keywords
separating
separation
separating device
plane
sample components
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Withdrawn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
EP03810966A
Other languages
German (de)
English (en)
Inventor
Albert RÖDER
Alois Rainer
Hans-Joachim HÖLTKE
Carlo Effenhauser
Peter Berndt
Hanno Langen
Remo Hochstrasser
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.)
F Hoffmann La Roche AG
Roche Diagnostics GmbH
Original Assignee
F Hoffmann La Roche AG
Roche Diagnostics 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 F Hoffmann La Roche AG, Roche Diagnostics GmbH filed Critical F Hoffmann La Roche AG
Publication of EP1558925A1 publication Critical patent/EP1558925A1/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Withdrawn legal-status Critical Current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
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44773—Multi-stage electrophoresis, e.g. two-dimensional electrophoresis

Definitions

  • the invention relates to a separation device and a separation method for biomolecular sample material, in particular protein mixtures according to the preamble of patent claims 1 and 18, respectively.
  • proteomics should provide new insights into the function, regulation and interaction of the proteins.
  • quantifiable differences between normal cells and "degenerate”, cancer cells or differences in the protein repertoire should of "sick” and “healthy” are shown. This in turn allows a targeted search for therapeutically active substances.
  • the total amount of proteins is first separated into their individual components, i.e. the many thousands of individual proteins are physically separated from the mixture. This is usually done by so-called two-dimensional (2-D) electrophoresis.
  • the total proteins from a cell population or a tissue are isolated as quantitatively as possible. If necessary, certain fractionation steps are also carried out if one wishes to examine only a specific fraction of the total proteins, e.g. Proteins from certain cell organelles such as the mitochondria.
  • an isoelectric focusing takes place in the first dimension.
  • the proteins are separated in a special gel or on a solid support in a pH gradient on the basis of their isoelectric point p1.
  • the proteins migrate in the electric field and are focused on their pI, which is characteristic of each protein.
  • This first step of isoelectric focusing requires certain electrophoresis devices to hold membranes or "strips" with immobilized pH gradients or gels with ampholyte buffers.
  • the process of isoelectric focusing usually takes many hours (24 to 48 hours).
  • the gels or membranes are usually removed from the first apparatus and equilibrated in an electrophoresis buffer containing SDS (sodium dodecyl sulfate). Then each gel, each membrane is applied to a new, second gel in a second gel apparatus. These second gels are made of SDS / polyacrylamide. After applying an electrical voltage perpendicular to the IEF gel, the focused proteins migrate into the second gel and are separated there based on their molecular weight. This electrophoretic separation can also take many hours (> 16 h). In the next step, the proteins separated in this way are made visible.
  • SDS sodium dodecyl sulfate
  • the stained protein spots are isolated from the gel in the smallest possible volume. This is done manually or with automatic, software-controlled spot pickers.
  • the small protein-containing gel pieces (a few ⁇ l volume) are then incubated with a buffer, which ensures that SDS and the dye are removed from the gel.
  • the gel pieces are then dried, taken up in a buffer suitable for protease digestion and incubated with a protease (usually trypsin). This cleaves the proteins contained in the gel into defined fragments.
  • the protein fragments (peptides) are then washed out or eluted from the gel with ammonium bicarbonate.
  • the peptides isolated from the individual gel pieces are then applied separately to a support for mass spectrometry and, after drying and optionally recrystallization, subjected to the mass spectrometer
  • MALDI-TOF mass spectrometer
  • IEF electrophoresis A large number of devices are required (IEF electrophoresis, SDS-PAGE electrophoresis, stainer, imager, spot picker, gel piece incubator, dryer, automatic pipetting device, etc.). It only allows the separation of proteins that are accessible to the IEF. Many and particularly interesting protein classes, e.g. Membrane proteins / receptors, cell nucleus or DNA-associated proteins cannot or cannot be separated with the IEF. The overall process is not easily reproducible.
  • the invention is based on the object of avoiding the disadvantages encountered in the prior art and of a device and to improve a method of the type specified at the outset so that even very small sample quantities can be processed and analyzed reliably and with high accuracy with little handling effort in an automated process.
  • the invention is based on the idea of creating a three-dimensional transport structure for the sample material integrated in a component in order to enable not only the separation of sample components but also their targeted further processing. Accordingly, it is proposed according to the invention that the separating element has a channel structure for discharging separated sample components onto a carrier surface in a transport direction running transversely to the separating plane. In terms of the method, it is accordingly provided that the separated sample components are discharged from the separating element onto a carrier surface transversely to the parting plane.
  • the invention allows a rapid, easily automatable separation of the proteins as well as the direct, spatially resolved discharge onto a carrier surface or a substrate in a single three-dimensional basic element. This makes it possible to transfer the information content of a two-dimensional protein separation into the third dimension without loss of information.
  • the entire process chain which is otherwise carried out in separate devices, is combined in a single element.
  • the manual steps are reduced to the application of the sample.
  • the amount of biological material required can be reduced by several orders of magnitude, so that microgram amounts are sufficient for an analysis.
  • the overall process can be reduced to a few hours.
  • the substances deposited on a free surface of the separating element can be analyzed directly, for example by mass spectroscopy. If necessary, a separate target plate for mass spectroscopy is provided as the carrier substrate. In principle, other uses are also conceivable, for example functional assays for activity or binding tests of the separated proteins or peptides.
  • 1 and 2 a device for separating and further processing of proteins comprising a plate-shaped separating element in perspective and vertically broken representation;
  • FIG. 3 shows a vertical section through the separating element according to FIG. 1;
  • FIG. 4 is a perspective view of a central or separating plate of the separating element
  • FIG. 5a is a partial plan view of the partition plate of FIG.
  • FIG. 5b shows a section along the line bb of FIG. 5a with the carrier plate connected to the channel structure;
  • Fig. 5c an embodiment with a support surface directly on the
  • FIG. 8 shows a positioning frame for connecting a plurality of separating elements to a mass-spectroscopic carrier plate in a diagrammatic representation
  • Fig. 9 a recording unit for storage and processing of
  • the plate-shaped separating elements 10 shown in the drawing essentially consist of a separating plate 12 for the two-dimensional separation of protein material, a transport or channel structure 14 acting transversely thereto for the accurate removal of the separated sample components onto a support surface 16 suitable for mass spectroscopic examinations and a cover plate 18 and a base plate 20 for delimiting the partition plate 12 on both sides.
  • the cover plate 18 has a slit-like, continuous sample application opening 22, a cover chamber 24 which is open towards the separating plate 12, a lateral inlet opening 26 opening into the cover chamber 24, and a central valve opening 28.
  • the separating plate 12 forms two above one another lying areas a three-dimensional channel system, an upper two-dimensional separating area 30 lying in the plate or parting plane being delimited on the bottom side by the channel structure 14 running in the third dimension.
  • the base plate 20 serves to support and stabilize the plate arrangement and contains a collection chamber 32 which is open towards the channel structure 14.
  • the base material for the separating element 10 designed as a composite chip can consist of silicon, polypropylene, polycarbonate, other polymers (for example polymethyl methacrylate, polyethylene terephthalate, polystyrene, polydimethylsiloxane), synthetic resin, ceramic or a metal or various of these materials. It is important that the base material allows the shaping with the necessary precision and is compatible with the materials used for sample processing.
  • the base material should expediently be electrically insulating and thermally conductive in order to enable electrophoretic separations and, if appropriate, efficient cooling.
  • the separating element 10 has, for example, a base area of 6 ⁇ 4 cm (corresponding to 1/4 of a conventional microtiter plate).
  • the separating plate 12 shown separately in FIG. 4 has orthogonal separating paths 34, 36 running in the plane of the plate for sequential 2D electrophoresis.
  • two pairs of electrodes 38, 40 are provided, which can be supplied with a suitable direct voltage via electrical connection lugs.
  • the separation path 34 of the first dimension between the electrodes 38 is formed by an elongated recess 42, in which a gel or membrane strip (not shown) with a preformed pH gradient for isoelectric focusing (IEF) of the protein molecules is arranged.
  • An electrophoresis buffer can be placed in storage zones 44 in the area of the electrodes 38.
  • the separating paths of the second dimension are formed by a family of channels 36 which run parallel to one another between the electrodes 40 and are arranged distributed along the recess 42 and branch off at right angles therefrom.
  • the number of channels depends on the desired and achievable resolution or separation performance of the first dimension. It can be between 10 and several thousand, preferably 50 to 500.
  • the molecular These channels are separated by polyacrylamide gel electrophoresis (PAGE) in an electrophoresis buffer containing sodium dodecyl sulfate (SDS).
  • PAGE polyacrylamide gel electrophoresis
  • SDS sodium dodecyl sulfate
  • the cutouts 46, 48 in the region of the electrodes 40 can serve as a buffer reservoir.
  • the SDS-PAGE separating gel is first introduced into the channels 36 of the second dimension and into the buffer reservoirs 46, 48 and photopolymerized.
  • the recess 42 for the IEF gel strip can be mechanically separated (separator 50) in order to prevent mixing of the gel types. It is also advantageous if the channels 36 are filled in their initial part with a collecting gel which is connected upstream of the separating gel. In the later separation, the collection gel enables the proteins to be collected and compressed at the boundary zone to the separation gel, so that sharper zones of the individual protein bands are formed.
  • the channel structure 14 is formed by discharge channels 52 lined up at the bottom of the separation channels 36. These run perpendicular to the separating plane spanned by the separating channels 36 and form a channel matrix distributed over the separating area 30 in order to enable the 2D pattern obtained during the molecular separation to be discharged true to the image, as will be explained in more detail below.
  • the number of discharge channels 52 per separation channel 36 in turn depends on the desired and achievable resolution of the separation in the second dimension. It can be between 10 and 1000, preferably 30 to 400.
  • the discharge channels 52 are advantageously designed to taper conically, for example with an upper inlet diameter of 100 ⁇ m and a lower mouth cross section of 50 ⁇ m (FIG. 5b). However, other channel geometries are also conceivable in order to process the proteins in the third dimension as loss-free as possible after separation.
  • the base plate 20 can be removed and the underside 54 of the separating plate can be used for a mass spectrometric examination.
  • support plate 56 specifically a target plate designed for matrix-assisted laser desorption / ionization-time-of-flight mass spectrometry (MALDI-TOF).
  • the carrier plate can consist of different materials (metals, plastics, polymers). It is important that the material is compatible with the following mass spectrometer, especially for MALDI-TOF.
  • hydrophilic anchor zones 58 for the substances to be transferred can be arranged on an inherently hydrophobic surface of the carrier plate 56. The position and size of these anchor zones are adapted to the discharge channels, so that the eluted molecular fragments are collected directly and the geometric resolution achieved on the target plate during the separation process is essentially retained.
  • the carrier surface 16 is itself formed by a free surface of the separating plate 12. It is advantageous if the support surface 16 has annular hydrophilic anchor zones 58 for the sample material around the mouths 59 of the discharge channels 52, but is otherwise hydrophobic. This can be achieved by appropriate chemical derivatization or coating. Cup-shaped depressions around the outlet openings of the discharge channels 52 can also ensure better deposition of the discharged peptides on the surface 16 (not shown). The geometric resolution achieved during the separation process should also be retained here.
  • the separating plate 12 with the peptides deposited on its surface 16 is then inserted into a suitable receptacle for mass spectroscopy and subjected to the mass spectroscopic examination. Here, the peptides are desorbed directly from the surface 16 of the separating element by means of a laser.
  • the channels 34, 36, 52 of the three dimensions can be in direct contact all the time, or can be separated by barriers or valves, which are only opened when changing from one dimension to the other.
  • the contact can be made mechanically, by moving or rotating the channels, or mechanical barriers can be provided.
  • the channels can also be separated chemically, for example by polymers that are dissolved at the desired time. It is also conceivable to separate the channels of the different dimensions by means of semipermeable membranes, the permeability of which can be controlled.
  • the separation channels 36 of the second dimension, filled with SDS-PAGE separating gel 60 are bounded at the bottom by a porous bottom layer or frit 62, which forms a channel structure 14 through which the micropores communicate fluidly with one another.
  • a porous bottom layer or frit 62 which forms a channel structure 14 through which the micropores communicate fluidly with one another.
  • the many closely spaced pores of the bottom layer 62 are to be understood as microscopic discharge channels.
  • the exemplary embodiment shown in FIG. 7 additionally differs in that, instead of discrete separation channels for the separation in the second dimension, an ultra-thin polyacrylamide gel layer 64 is provided, which optionally adjoins the IEF gel strip laterally via an intermediate strip from a collecting gel and an SDS -PAGE electrophoresis enables.
  • four separating elements 10 can be jointly positioned and processed in a positioning frame 66 on a mass-spectroscopic carrier plate 56 the size of a microtiter plate.
  • the carrier plate 56 is positioned in parallel at a predetermined distance from the exit side of the respective channel structure 14, so that the molecules to be transferred can be transferred directly to the hydrophilic anchor zones 58.
  • the separating elements 10 can be inserted into a storage container 68 for storage and preparation and enclosed therein in an airtight manner by means of a detachable cover film 70 (FIG. 9).
  • the transfer into the positioning frame 66 with removal of the base plates 20 can be simplified or automated by a suitable sliding mechanism (not shown).
  • the entire separating device comprises further units for controlling the process sequence, which are known per se to the person skilled in the art and are not explained in detail here.
  • a suitable amount of the sample material is introduced into the first separating path 34 via the feed opening 22.
  • 1 ⁇ g total protein is added in a volume of approx. 50 nl to 1 ⁇ l carrier liquid.
  • the proteins migrate in the electrical field and pH gradients to a point corresponding to their isoelectric point.
  • the isoelectric focusing is ended by switching off the electrical voltage.
  • the buffer which was used for the separation in the first dimension may be removed from the proteins and for these to be re-equilibrated in a buffer which is suitable for the separation in the second dimension.
  • This can be done directly in the channel 42 by overlaying, the new buffer being fed in via the cover plate 18.
  • the SDS-PAGE buffer is then also filled into the two buffer reservoirs 46, 48.
  • voltage is applied to the two electrodes 40 and the IEF-focused proteins migrate from the IEF strip into a corresponding channel 36 of the second dimension in accordance with the position reached.
  • the proteins in the SDS-polyacrylamide-filled channels 36 of the second dimension are then separated electrophoretically, the various proteins being separated according to their molecular weight.
  • This SDS-PAGE gel electrophoresis which can be followed by adding suitable marker substances, is complete after about an hour. Then the electrical voltage is switched off.
  • the separation principles of the first dimension (IEF, hydrophobic interaction, affinity, ionic interaction) described above can of course also be used for the separation in the second dimension.
  • the further processing of the proteins until they are discharged onto the carrier surface 16 takes place in a liquid flow via the channel structure 14.
  • the required liquids are introduced via the cover chamber 24, while the valve opening 28 allows the displaced air to flow away.
  • the liquid flow is passed perpendicular to the parting plane and in a uniform distribution through the parting region 30, the floating pressure difference being able to be increased, for example, by applying a supporting vacuum to the collecting chamber 32 of the base plate 20.
  • Gege- if necessary, the homogeneous surface distribution can be improved by a perforated plate, not shown, which covers the channels 36.
  • SDS-free buffer solution is directed through the gel in the channels 36 of the second dimension. This removes SDS from the gel and from the separated proteins without changing the position of the proteins.
  • the buffer solution is discharged via the channel structure 14 into the lower collecting chamber 32 of the base plate 20.
  • a precisely metered amount of trypsin solution is then added in the same way.
  • the trypsin cleaves the proteins into defined polypeptides. The concentration and flow of trypsin are controlled so that the proteins are cleaved as completely as possible without significant amounts of the cleavage products being eluted from the gel.
  • the bottom plate 20 with the waste eluates is removed by a sliding mechanism.
  • the protein fragments generated by the protease cleavage are then eluted by adding a defined volume of elution buffer through the cover plate and targeted flow through the separating gel through the discharge channels 52.
  • the volume is controlled in such a way that the peptides generated from the separated proteins by the protease cleavage are deposited as completely as possible through the channel structure on the support surface 16, specifically localized in the region of the outlet openings of the discharge channels 52.
  • the invention relates to a separation device and a separation method for biomolecular sample material, in particular protein mixtures.
  • a separating element 10 is provided for the two-dimensional, preferably electrophoretic, separation of components of the sample material in the region 30 of a separating plane.
  • the separating element 10 has a channel or transfer structure 14 for the spatially resolved discharge of separated sample components in a transport direction running transversely to the separating plane onto a carrier surface 16 which is preferably suitable for mass-spectroscopic examinations.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Electrochemistry (AREA)
  • Pathology (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Peptides Or Proteins (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)

Abstract

La présente invention concerne un dispositif de séparation et un procédé de séparation pour un échantillon de matière biomoléculaire, notamment des mélanges protéiques. Ce dispositif comprend un élément de séparation (10) conçu pour effectuer une séparation bidimensionnelle de préférence électrophorétique de composants de l'échantillon de matière dans la zone (30) d'un plan de séparation. Selon cette invention, l'élément de séparation (10) présente une structure de canal ou de transfert (14) permettant de transférer de façon localement précise des composants d'échantillon séparés dans une direction de transport transversale au plan de séparation sur une surface de support (16) adaptée à des recherches par spectroscopie de masse.
EP03810966A 2002-11-09 2003-11-05 Dispositif de separation et procede de separation pour echantillon de matiere biomoleculaire Withdrawn EP1558925A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10252177 2002-11-09
DE10252177A DE10252177A1 (de) 2002-11-09 2002-11-09 Trennvorrichtung und Trennverfahren für biomolekulares Probenmaterial
PCT/EP2003/012320 WO2004044574A1 (fr) 2002-11-09 2003-11-05 Dispositif de separation et procede de separation pour echantillon de matiere biomoleculaire

Publications (1)

Publication Number Publication Date
EP1558925A1 true EP1558925A1 (fr) 2005-08-03

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ID=32308504

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Application Number Title Priority Date Filing Date
EP03810966A Withdrawn EP1558925A1 (fr) 2002-11-09 2003-11-05 Dispositif de separation et procede de separation pour echantillon de matiere biomoleculaire

Country Status (7)

Country Link
US (1) US7645369B2 (fr)
EP (1) EP1558925A1 (fr)
JP (1) JP4541894B2 (fr)
AU (1) AU2003301992A1 (fr)
CA (1) CA2505255C (fr)
DE (1) DE10252177A1 (fr)
WO (1) WO2004044574A1 (fr)

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US7105810B2 (en) 2001-12-21 2006-09-12 Cornell Research Foundation, Inc. Electrospray emitter for microfluidic channel
US7537807B2 (en) 2003-09-26 2009-05-26 Cornell University Scanned source oriented nanofiber formation
AU2006261309A1 (en) 2005-06-18 2006-12-28 Ge Healthcare Bio-Sciences Ab Methods and systems for adding a reagent to an analyte in a gel
EP1917522A1 (fr) * 2005-06-18 2008-05-07 GE Healthcare Bio-Sciences AB Méthode et dispositifs pour former une multitude de puits sur un gel
KR100792683B1 (ko) * 2006-05-09 2008-01-09 연세대학교 산학협력단 모세관등전집중-중공사흐름장흐름분획법을 이용한 단백질분리장치 및 분리방법
US20100108512A1 (en) * 2006-12-26 2010-05-06 Wataru Hattori Electrophoretic chip and method of using the same
DE102008020428B3 (de) * 2008-04-24 2009-07-16 Johannes-Gutenberg-Universität Mainz Vorrichtung und Verfahren und Gelsystem zur analytischen und präparativen Elektrophorese
AU2016353162B2 (en) 2015-11-10 2023-02-23 Woodham Biotechnology Holdings, LLC Gel electrophoresis and transfer combination using conductive polymers and method of use
US9702851B1 (en) 2016-06-17 2017-07-11 Woodham Biotechnology Holdings, LLC Gel electrophoresis and transfer combination using conductive polymers and method of use
WO2023248280A1 (fr) * 2022-06-20 2023-12-28 株式会社東陽テクニカ Micropuce, système de séparation bidimensionnelle d'échantillon et procédé de fabrication de micropuce

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Also Published As

Publication number Publication date
US20060000712A1 (en) 2006-01-05
CA2505255A1 (fr) 2004-05-27
WO2004044574A1 (fr) 2004-05-27
US7645369B2 (en) 2010-01-12
JP2006505786A (ja) 2006-02-16
WO2004044574A8 (fr) 2005-12-15
AU2003301992A1 (en) 2004-06-03
DE10252177A1 (de) 2004-06-09
CA2505255C (fr) 2010-10-26
JP4541894B2 (ja) 2010-09-08

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