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
  • G01N27/44795—Isoelectric focusing
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D57/00—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C
  • B01D57/02—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C by electrophoresis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/24—Extraction; Separation; Purification by electrochemical means
  • C07K1/26—Electrophoresis
• • 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
  • G01N27/44708—Cooling

Definitions

• the present invention relates to an apparatus and a method utilising that apparatus for sub-fractionation and subsequent separation of the fractions from highly complex protein/peptide mixtures, such as those found in total cell lysates.
• body fluids e.g.. plasma, sera, cerebrospinal fluid, urine
• tissue extracts in general.
• the present invention seeks to provide a new instrument which is capable of being operated to provide improvements in resolving power and preferably improvements in detection sensitivity.
• a novel multi-compartment electrolyser design to pre-fractionate complex protein mixtures, for use prior to the implementation of 2-D maps.
• Such a sub-fractionation processes can effectively remove, via suitable narrow range isoelectric membranes, proteins present in large excess in a cell lysate or in body fluids.
• the remaining protein mixture devoid of such major components, can be loaded in a narrow pH range 2-D map at much higher levels, thus ensuring a greater sensitivity and detection capability of low-abundance proteins.
• the apparatus embodying the present invention produces protein fractions which are fully compatible with the subsequent 2-D protocols, since it is based on a focusing technique, which yields samples highly concentrated and devoid of salts and buffers.
• the process of the present invention may be carried out using a multi- compartment electrolyser. which is characterised in that recirculation of the samples is not required.
• the present invention provides a process for electrophoretic separation of a mixture of macromolecules into groups comprising the steps of placing a sample mixture of macromolecules in a separation apparatus comprising a series of chambers separated by isoelectric membranes of known pi.
• the apparatus also having chambers located at each end of the series of chambers containing means for applying an electric field across the series of chambers: and applying an electric field across the chambers to separate the macromolecules on the basis of their isoelectric point characterised in that the sample mixture is agitated within the chambers and is not recirculated
• Each chamber preferably defines a well or recess adapted to receive a magnetic stirrer bar.
• Electrodecantation may be prevented by placing the device in a multi- place magnetic stirring platform and mixing the contents of each chamber of the electrolyser with the magnetic stirrer bar.
• the separator apparatus of the present invention may utilise an associated platform which provides power, cooling and a magnetic stirrer platform.
• the location of the magnets in the magnet array of the magnetic stirrer platform have to coincide with the location of the stirrer magnets in the electrolyser.
• Heat dissipation is implemented by the use of embedded Peltier elements in the magnetic stirrer platform which can be regulated to maintain the correct temperature.
• the platform may also be configured to accommodate a plurality of gel trays for isoelectric focusing in carrier ampholyte pH gradients or immobilised pH gradients.
• the multi- compartment electrolysers are assembled from a plurality of separate chambers, operating in an electric field, with a set of isoelectric membranes (having pi values increasing monotonically from anode to cathode) able to trap a desired protein population within a given chamber.
• the pre- fractionation mode using one or more multi-compartment electrolysers.
• the device can be operated under denaturing conditions (with mixtures of chaotropes.
• the temperature will be set to about 20°C.
• the device can be operated under native conditions, in the absence of denaturants. when native proteins are required for further analysis exploiting biological activity and this it typically done at about 4°C.
• the isoelectric membranes may be made either with standard acrylamide monomers or with stable acrylamide derivatives, such as N- acryloyl amino ethoxy ethanol or N-acryloyl amino propanol. or mixtures thereof.
• Membrane pH is defined by a suitable choice of buffering and counter-ion species, which are acrylamido-derivatives able to be copolymerised with polyacrylamide, or its derivatives, and possessing good buffering power.
• the isoelectric membranes may be made macroporous either by high levels of cross linkers, or by lateral aggregation of the acrylamide chains or by a combination of both means.
• the sub-fractionation of complex protein/samples may be obtained under native conditions in presence of suitable solubilizers compatible with maintenance of protein integrity, such as sugars, non-detergent sulfobetaines. glycerol, ethylene and propylene glycols and mixtures thereof.
• the sub-fractionation of complex protein/peptide mixtures may be obtained under denaturing conditions in presence of appropriate solubilizing cocktails, such as organic solvents, chaotropes. neutral and zwitterionic surfactants and amido-sulfobetaines and, when required, in presence of suitable disulfide bridge reducing and/or alkylating agents.
• solubilizing cocktails such as organic solvents, chaotropes.
• neutral and zwitterionic surfactants and amido-sulfobetaines and, when required, in presence of suitable disulfide bridge reducing and/or alkylating agents.
• the preferred embodiment for sample application may be by pulse- loading in a single chamber of the electrolyser, said chamber being preferably in the neutral or basic region of the pH scale.
• the sub-fractionation process may be performed in a cascade fashion, such that a wider pH fraction obtained in a first run in the electrolyser. and may subsequently be further fractionated in a very narrow pH window, as required by the sample complexity.
• the device described in the present invention also comprises a second operational mode for simultaneous separation of certain protein fractions, using isoelectric focusing in carrier ampholyte pH gradients or immobilised pH gradients.
• a second operational mode for simultaneous separation of certain protein fractions, using isoelectric focusing in carrier ampholyte pH gradients or immobilised pH gradients.
• Figure 1 is a schematic exploded view of a multi-compartment electrolyser apparatus embodying the present invention
• Figure 2 is a schematic view of two multi-compartment electrolysers set up accommodated on an electrophoresis platform containing a power supply, Peltier cooling and a multi-place magnetic stirrer;
• Figure 3 is a schematic view of two trays for isoelectric focusing accommodated on an electrophoresis platform containing a power supply; Peltier cooling and a multi-place magnetic stirrer:
• Figure 4 is a silver stained 2-D map of a pi 4-5 fraction of E. coli. as purified in the multi-compartment electrolyser of Fig. 1, run in a 7cm pH 3- 10 IPG strip in the first dimension:
• Figure 1 shows a disassembled separation apparatus in the from of a multi-compartment electrolyser apparatus 10.
• the apparatus includes five chamber blocks, defining three inner fractionation chambers 12 and two. outer, electrode chambers 14.
• Each chamber block is generally square in transverse cross-section.
• a cylindrical through bore 16 extends through the centre of each of the fractionation chamber blocks and part way into the outer electrode chambers 14.
• Four narrower through bores 18 located at the corners of the square cross-section of the blocks extend through all five chambers.
• the bores 18 receive threaded tie rods (not shown) which can be used to align the bores together and join the chambers together.
• Each chamber also includes a sample inlet 26 in the upper face of the chamber block which extends from the upper face of the block down to the bore 18.
• each chamber block 12 defines an annular spigot 22 extending around the bore 16.
• the opposite side defines a corresponding recess 24.
• the open side of one electrode chamber 14 defines a recess.
• the open side of the other electrode chamber 14 defines a spigot.
• the multi-compartment electrolyser apparatus is assembled by placing septa or dividing walls (not shown) between adjacent chambers and inserting tie rods through the holes in each chamber and tightening until the unit is sealed.
• the septa between the various chambers are isoelectric, buffering membranes, cast onto a support of glass fibres or other suitable material.
• the membranes are sandwiched between two washers and an O-ring is disposed outside the membrane to provide additional sealing. Such membranes are flow-tight and ensure proper pH control.
• novel apparatus of the present invention may be used for sub- fractionation of entire cell lysates, tissue extracts, body fluids and of any complex protein/peptide mixture, as a sample treatment, prior to a subsequent two-dimensional analysis step.
• the membranes at each end of a chambers have pH values encompassing the pis of the proteins to be confined within said chamber.
• the electrode solutions and sample solutions are added and removed via the sample inlets 26 in the top of each chamber.
• the sample inlets also allow excess fluid in a particular chamber to escape.
• the manner of mounting the magnetic stirrers in the chamber suspended from the cap means that the stirrers can be easily removed from the chamber for re-use. whilst the chamber can be disposed of.
• Figure 2 illustrates the use of a plurality of multi-compartment electrolysers 10. as shown in Fig.l, on an electrophoresis platform 50 with an integral power supply, multi-position magnetic stirrer and Peltier cooling. (The floor 52 of the platform is cooled by the Peltier elements)
• Figure 2 shows the use of two multi-compartment electrolysers 10 on a Peltier cooled multi-position magnetic stirrer platform, it will be clear to those skilled in the art that an electrophoresis platform of this design could be made to contain more or less than 2 multi-compartment electrolysers.
• Figure 3 illustrates using the same electrophoresis platform 50 as shown in Fig.
• Figure 4 shows a pi 4.0-5.0 fraction fractionated in the multi-compartment electrolyser shown in Fig. 1.
• a IPG 3- 10 strip was utilised for the first. IEF dimension, thus demonstrating that indeed, in the 2-D map, only this acidic fraction could be displayed, all other proteins in the remainder of the pH scale having been efficiently removed.
• Figure 5 shows 2 silver stained pH 3-6 IPGs with whole plasma in the left panel and fractionated plasma in the right panel. The 3 black arrows show matching areas on the 2 gels. The vertical arrow on the right panel indicates pH 5.6 which was the pH of the delimiting membrane between the acidic and albumin chambers.

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• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Molecular Biology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Electrochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• Pathology (AREA)
• General Physics & Mathematics (AREA)
• Immunology (AREA)
• Physics & Mathematics (AREA)
• Organic Chemistry (AREA)
• Medicinal Chemistry (AREA)
• Genetics & Genomics (AREA)
• Biophysics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• General Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Environmental & Geological Engineering (AREA)
• Peptides Or Proteins (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Sampling And Sample Adjustment (AREA)

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• 2000
  • 2000-11-14 JP JP2001538938A patent/JP2003514829A/ja active Pending
  • 2000-11-14 WO PCT/AU2000/001391 patent/WO2001036449A1/en not_active Application Discontinuation
  • 2000-11-14 EP EP00974180A patent/EP1230258A4/de not_active Withdrawn
• 2003
  • 2003-01-28 HK HK03100703.5A patent/HK1049343A1/zh unknown

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