EP1399734A2

EP1399734A2 - Method for electrophoretic sample separation

Info

Publication number: EP1399734A2
Application number: EP02738133A
Authority: EP
Inventors: Kai Hendrik Te Kaat
Original assignee: Xzillion Proteomics & Cokg GmbH
Current assignee: Xzillion Proteomics & Cokg GmbH
Priority date: 2001-06-06
Filing date: 2002-05-31
Publication date: 2004-03-24
Also published as: AU2002312963A1; US20040154921A1; WO2002099404A2; DE10127406A1; WO2002099404A3

Abstract

The invention relates to a method for the electrophoretic separation of peptides and proteins, through isoelectric focusing in particular, using a mixture containing a polar liquid and at least one amphiphilic substance which forms a liquid crystal phase, as a separation matrix.

Description

Methods for the electrophoretic separation of samples.

Description:

The present invention relates to a method for the electrophoretic separation of samples, in particular by isoelectric focusing, by means of a suitable separation matrix. The invention is particularly suitable for the separation of lipophilic peptide and protein components of a sample.

Electrophoresis has long been a standard technology for the separation of charged molecules in the life sciences. Charged molecules migrate in an electrical field depending on their surface charge, their molecular weight, their spatial dimensions and in

Dependence on the viscosity of the separation medium. The separation is due to. different migration speeds of individual charged molecules, which are caused by the above-mentioned dependencies. The electrophoresis of biomolecules is typically carried out in aqueous solutions. To prevent mixing of the sample due to convection, the

Electrophoresis usually in highly porous matrices, such as. B. in polyacrylamide or agarose gels. Here, the sample components are separated in a first approximation via the average pore size of the gel and the molecular weight of the sample component. This principle is used e.g. B. in SDS-PAGE electrophoresis for the separation of proteins in a sample. a

An overview of the existing processes and the separation media used for this is given, for. B. in Righetti, PG et al., J. Chromatogr. B 699 (1996) 63-75; Righetti, PG et al., J. Chromatogr. A 698 (1995) 3-17; Righetti, PG et al., Elektrophoresis 1995, 16, 1815-1829; Manabe, T., Electrophoresis, 21, 1 116-1 122. An alternative method is the separation using a pH gradient (see, for example, Molloy, MP Analytical Biochemistry 280, 1-10 (2000); Righetti, PG et al., J. Chromatogr. B 699 (1997) 77- 89), which can be set up between the two electrodes to separate molecules based on their isoelectric point. Amphoteric analytes carry a net

Surface charge in a pH gradient until the analyte has migrated to a region of the gel in which the pH corresponds to the isoelectric point of the analyte. The migration of the analyte in the electric field at the isoelectric point comes to a standstill, the analyte is focused in this region. Typically, anticonvective media with a high average pore size are used for isoelectric focusing, which hinder the migration of the analyte as little as possible. Agarose gels or polyacrylamide gels under low salt conditions are also preferably used for the isoelectric separation of biomolecules.

The protein components of an electrophoretically separated sample can be divided into easily separable water-soluble proteins and fat-soluble proteins. Proteins spanning the cell membranes in particular have extensive hydrophobic areas within the transmembrane domains and hydrophilic intracellular and extracellular head domains. Such membrane proteins are only very poorly soluble in aqueous phases and thus elude electrophoretic separation using conventional means. In order to solubilize membrane proteins in a suitable manner for electrophoresis, detergents are generally used (Rabilloud, T., Electrophoresis 1996, 17, 813-829; Herbert, B. Electrophoresis 1999, 20, 660-663, Molloy, MP Analytical Biochemistry 280 , 1-

10 (2000)). For isoelectric focusing in particular, either uncharged or zwitterionic detergents, which have only a limited capacity to dissolve lipophilic proteins, must be used. Thus, very efficient methods are available for the electrophoretic separation of water-soluble proteins, while the separation of protein mixtures which contain lipophilic proteins still has problems. The object of the present invention was therefore to provide a method which enables the separation of chemical and biological samples, in particular with the inclusion of the hydrophobic sample components.

The task could surprisingly be done by using a

Separating medium, comprising a mixture of a polar liquid and a liquid-crystalline phase of at least one amphiphilic substance, can be dissolved, the liquid-crystalline phase serving as a separation matrix.

The amphiphilic substances in polar liquids form a three-dimensional, double-membrane-like liquid-crystalline network that is traversed by channels of the liquid phase.

The electrophoretic separation is carried out after application of the dissolved sample to this separation medium by applying an electrical field. In the case of isoelectric focusing, the sample can also be mixed with the separation medium or with the amphiphilic substances.

Due to the amphiphilic liquid-crystalline separation matrix lipophilic substances, such as. B. lipophilic peptides, proteins or glycoproteins surprisingly migrate directly by applying an electric field without diffusing into the polar liquid. The liquid-crystalline phases of amphiphilic substances are also able to solubilize hydrophobic peptides and proteins without the use of detergents and thus bring them into an electrophoretically separable state. In addition, such phases in polar liquids form a porous separation matrix, which additionally ensures separation of substances soluble in polar solvents, so that a mixture of hydrophobic and hydrophilic peptides and proteins can also be separated efficiently. The separation using the methods according to the invention is consequently particularly suitable for mixtures which, in addition to peptides and proteins, also contain glycolipids, ionic lipids, carbohydrates, sugars, amino acids, nucleic acids or secondary metabolites. Furthermore, the separation matrix prevents convection currents and the associated undesired mixing of the Separation medium. Surprisingly, the separation matrix was stable under the electrophoresis conditions.

To generate the separation matrix, all uncharged amphiphilic substances with a hydrophobic molecular unit (tail) and a hydrophilic one

Molecular unit (head) are used, which are able to form a liquid-crystalline phase in polar liquids. These include above all alcohol fatty acid esters, in particular from short-chain C ₂ -C ₄ alcohols with up to 4 hydroxyl groups and C ₈ -C ₃ o-fatty acids, some of the hydroxyl groups being able to be in free form, or with other chemical groups, such as B.

Phosphoric acid derivatives, amino alcohols or saccharides with the formation of phosphatides, glycolipids or aminolipids can be present.

Preferably, the amphiphilic esters used contain as acyl groups mono- to pentasaturated C ₂ -C ₂₄ fatty acids or saturated C ₈ -C ₂₀ fatty acids, particularly preferably the esters contain monounsaturated ice ₄ -C ₂₂ acyl groups, such as. B. 1-monooleoyl-rac-glycerol, 1-monomyristoyl-rac-glycerol or 1-monopalmitoyl-rac-glycerol.

Particularly suitable amphiphilic substances are mono- or diacylglycerides,

Acylglycerol phosphatide, glycoacylglyceride, aminoacylglyceride or a mixture containing such substances.

The preparation and the properties of liquid-crystalline phases, starting from the amphiphilic substances described, is described, for. B. in WO 84/02076, WO

98/10281. A detailed description of liquid-crystalline cubic phases can also be found in Landau, E.M. et al., Proc. Natl. Acad. Be. Vol. 93, pp. 14532-14535 Dec. (1996); Nollert, P. et al., FEBS Letters 457 (1999) 205-208; Caffrey M., Current Op. Structural Biol. 10: 486-497 (2000); Hong, Q. et al., Biomaterials 21, 223-234 (2000), however, these phases exclusively for

Use protein crystallization. Different liquid-crystalline phases can be used for the purpose of electrophoretic separation of chemical and biological samples. However, preference is given to cubic phases which form a single coherent network with the polar liquid, through which channels are connected to one another. In such phases, e.g. B. the hydrophobic peptides and

Proteins migrate in the double membrane-like separation matrix without having to pass through the liquid channels. However, liquid-crystalline phases with a lamellar, hexagonal or inverted hexagonal structure are also preferably suitable for electrophoretic separation. It is advantageous here if the membrane-like lamellae are aligned in the longitudinal direction of the electrical field in order to ensure a migration within the separation matrix that is as trouble-free as possible.

Cubic phases are also particularly suitable for the electrophoretic separation of peptides and proteins for the following reasons:

The thickness of the membrane-like cubic phase is almost homogeneous, other phases, such as lamellar or hexagonal or inverted hexagonal phases, have hydrophobic membrane areas of different thickness. The liquid crystalline cubic phase is an isotropic phase. The physical and chemical properties of this phase are identical in all three dimensions, so that an alignment of the phase relative to the applied electric field is not necessary. In the case of anisotropic phases, it is advantageous to align the membrane planes parallel to the electrical field in order to enable mobility of the proteins in the membrane plane. - The liquid crystalline cubic phase is on in the presence of an excess

Water stable. Various buffers can be used in electrophoresis to allow current conduction between the electrodes and the ends of the matrix. It is therefore not desirable for the matrix to slowly disintegrate in excess buffer.

According to the phase diagram mono-oleic water, cubic phases are formed over a wide range from approx. 10 to max. 50% water content at temperatures from 5 to approx. 95 degrees Celsius at atmospheric pressure. One is preferred Composition aqueous phase: MO from 25: 75 to 40: 60 (v / v) at a temperature of 20 to 40 degrees Celsius.

Various auxiliaries can be added to the liquid-crystalline phases at a concentration of 0.1% to 15% (w / w), preferably between 1% to 5% (w / w). Lipids and detergents in particular must be taken into account here, as admixtures of these substances may be important when solubilizing difficult membrane proteins. There are also a number of membrane proteins that have specifically bound a particular lipid, e.g. B. as a biological cofactor.

The amphiphilic phase can e.g. B. synthetic or naturally occurring lipids, such as phosphatidylcholine, phosphatidylethanolamine, charged or uncharged phospholipids, sphingolipids or glycolipids, fat-soluble biological cofactors, chlorophylls, retinol, steroids, carotenoids, quinones or C ₈ -C ₃₀ fatty acids,

-Alkanes, -alkenes, -alkynes or -alcohols can be added. Lipids or detergents with ionic or zwitterionic head groups are preferably added auxiliaries.

Small polar amphiphiles can be added to the polar liquid as auxiliary substances

Substances, polar glycols, sugar monomers or multimers, amino acids or zwitterionic substances are added. Depending on the electrophoretic method z. B. as auxiliaries glycerol, ethylene glycol, propylene glycol, polyethylene glycol, glycine, urea or guanidine in question.

Carrier ampholytes can also be added to the polar liquid, with the aid of which a pH gradient can be built up in the separation medium. As a carrier ampholyte z. B. Ampholine ® in a concentration range of 0.1 to 40% by volume, preferably 2 to 8% by volume. Electrophoresis in a pH gradient enables the separation of proteins based on their isoelectric

Point by means of isoelectric focusing. As a rule, the separation medium is buffered; Tris buffers are preferably used. In isoelectric focusing, an acid, preferably dilute phosphoric acid, is used as the anolyte. The electrophoretic separations are preferably carried out in a capillary at a voltage below 8000V, preferably at a voltage between 100V and 600V. The

Runtimes are usually between 0.5 h to 10 h. However, voltages and running times are preferred which result in a Vh value of less than 4000 in order to prevent any change in the separation medium.

Another advantage of the method according to the invention lies in the ease

Sample preparation. The addition of detergents for the solubilization of lipophilic peptides and proteins can thus be dispensed with. In contrast, z. B. a centrifuged vesicle fraction or membrane fraction can be mixed directly with the amphiphilic substances forming the liquid-crystalline phase. The peptides and proteins contained in the membranes dissolve easily in the amphiphilic substance, the solution can be applied directly to a finished separation medium containing an amphiphilic liquid-crystalline phase or alternatively used to prepare the separation medium by mixing with a polar liquid become. The latter is suitable for. B. to prepare the separation medium for isoelectric focusing.

The method according to the invention can be used to separate ionic analytes or analytes which can be ionized in the electrical field. But also non-ionic substances can be modified with ionic groups and thus made accessible to an electrophoretic process. The methods described are particularly for

Separation of samples containing ionic lipids, hydrophobic secondary metabolites, glycolipids, glycoproteins, peptides and proteins is suitable. But the simultaneous separation of sugars, carbohydrates, nucleic acids, amino acids etc. in the polar liquid phase of the same separation medium is also possible. For this reason, the described method is also suitable for the investigation of

Total extracts as they are examined in proteome analysis. A combination with a second separation method in a 2D process seems advantageous, wherein a separation according to the method according to the invention is preferably carried out as the first separation step.

Analytes and auxiliary substances are preferably added to the polar liquid or the amphiphilic substance in a concentration which does not prevent the formation of a cubic liquid-crystalline amphiphilic phase.

Another advantage of the use of liquid-crystalline amphiphilic phases for the electropheretic separation according to the described method lies in the adjustability of the fluidity of the phase through its composition. So z. B. the increased use of glycides or phosphatides with saturated acyl groups, the fluidity of the liquid-crystalline phase can be reduced. Unsaturated acyl groups, especially in the cis configuration, increase fluidity. In this way, the migration rate of the sample components to be separated, in addition to the temperature or the applied voltage, can also be set via the composition of the cubic-crystalline phase.

Brief description of the figures:

Fig. 1 shows the electrophoresis of polar molecules in a separation medium from a cubic liquid-crystalline phase of separation matrix and an aqueous

Phase.

Fig. 2 shows the separation of two dyes bromophenol blue and orange G with a tris-glycine-SDS buffer system. 3 show the establishment of a pH gradient in the separation medium using

Bromophenol blue as an indicator, Fig. 3a shows a uniform pH in

Separation medium at a slightly basic pH, FIG. 3b shows a pH gradient built up by carrier ampholytes from a slightly basic pH to a pH of significantly below 3.0. 4 shows the separation of a heterogeneous mixture of a fluorescence-labeled protein by isoelectric focusing with variation of the separation conditions. 5 shows the electrophoretic migration of a fluorescence-labeled phosphatidyl ethanolamine under variation of the electrophoresis conditions. 6 shows the electrophoretic separation of a fluorescein isothiocyanate (FITC) -labeled peptide on an LCP separation matrix with different transit times.

FIG. 7 shows the electrophoretic mobility of a FITC-labeled peptide on the basis of the test results shown in FIG. 6 in a graphical representation.

To illustrate the method according to the invention, a few exemplary embodiments are given below.

General:

The liquid-crystalline cubic phase (LCP) is produced by mixing an aqueous phase with molten mono-olein (MO, 1 -monooleoyl-rac-glycerol) in a volume ratio of 30:70.

100-150 μg solid MO is weighed into an Eppendorf tube and covered with N ₂ . The weighed-in MO is melted in the oven at approx. 60 degrees C for 5 minutes. The molten MO is then degassed in the ultrasonic bath for about 5 minutes. 70 μl of liquid MO are then bubble-free into a preheated one

Hamilton syringe sucked up.

For isoelectric focusing, an aqueous phase with the following is used

Composition used: 10 μl of the carrier ampholyte Ampholine ® (Amersham-Pharmacia)

(Final concentration up to approx. 8% in the aqueous phase) and H ₂ 0 qsp 50 μl.

The aqueous phase is mixed and degassed by ultrasound, then

30 μl of the aqueous phase are filled into a second Hamilton syringe.

The syringes of the two syringes are coupled head-to-head, and the two phases are mixed from one syringe to the other by pressing about 100-200 times. The LCP is formed spontaneously when the aqueous phase is mixed homogeneously with the melted MO and is recognizable by the fact that the phase appears completely clear and transparent.

As a sample z. B. purified protein, a protein mixture, a vesicle

Preparation or dyes used.

The separation takes place in the following exemplary embodiments at a temperature of 30 degrees Celsius.

General electrophoresis conditions, unless otherwise stated in the explicit exemplary embodiments: - Cathode buffer: 0.1 M TRIS-SO pH 9.3 - Anode buffer: 0.08 MH ₃ PO - Field strengths (Sl is V / m, here V / column length) and electrophoresis duration:

1 h at approx. 100V / 60 mm

1 h at approx. 240 V / 60 mm

4 - 24 h at 600 V / 60 mm

BeispieM:

Electrophoresis of polar molecules in a liquid crystalline cubic phase

(LCP):

35 μl of an LCP are mixed with 100 mM Tris-HCl pH 7.4; Tris: Mono-olein 30:70 (v / v) mixed so that an LCP is formed. A 100 mM Tris HCl pH 7.4 buffer is used as the electrophoresis buffer on the anode and cathode. An anionic dye Orange G (1 mg / ml) is used as analyte. 5 μl of the analyte were deposited on the cathode on the surface of the LCP on the threaded side of the syringe and exposed to a voltage of 300 V for about 15 min. Orange G has migrated into the clear LCP. A sharp migration front can be seen (see Fig. 1). Electrophoresis of charged molecules in LCP is therefore possible. Example 2:

Separation of two dyes with a Tris-Glycine-SDS buffer system.

In 100 μl of an LCP consisting of electrophoresis buffer: mono-olein in one

Ratio of 30:70 (v / v), the electrophoresis buffer consisting of 25 mM Tris, 192 mM glycine and 0.1% (w / v) sodium dodecyl sulfate (SDS), 5 μl of one

Analytes from a mixture of bromophenol blue and orange G, both anionic

Dyes, in the concentration of approx. 1 mg / ml on the surface of the LCP at the

Cathode deposited. The electrophoresis is at 1 h at 100 V, 1 h at 240 V and 6 h at

600V performed. The electrophoretic mobility of the two dyes is different, which leads to their separation in the LCP (FIG. 2).

Longer electrophoresis can lead to a change in the matrix (milky white

Areas in the matrix at the right end near the anode).

Example 3:

Establishment of a pH gradient.

For this purpose, the LCP consists of an aqueous phase and mono-olein in

Ratio of 30:70 (v / v) used. The aqueous phase is composed of 5% Ampholine ® 3.5-9.5 (Amersham-Pharmacia) in water with bromophenol blue as a pH indicator. A 0.08M H3PO4 solution is used as the anolyte, and a 0.1 M Tris-SO4 pH 9.3 solution is used as the catholyte. The electrophoresis is carried out for 1 h at 100V, 1 h at 240 V, 4 * 1 h at 600V. A pH gradient, recognizable by the different color of the pH indicator, could be established.

The pH gradient is established by carrier ampholytes. The LCP is colored with bromophenol blue. In the basic bromophenol blue is colored blue, the pH change range is from pH 4.6 - 3.0 over green to yellow from pH 3.0. 3a shows the homogeneously mixed LCP before electrophoresis. 3b shows the built-up pH gradient after electrophoresis.

Example 4:

Separation of a heterogeneous mixture of fluorescence-labeled protein. Using a Cy3 fluorescent dye-labeled heterogeneously labeled lysozyme, it was possible to detect in the LCP using a fluorescence scan and to focus in different bands. The electrophoresis conditions were chosen as in Example 3.

4 shows the results of the electrophoretic separation of Cy3-labeled lysozyme in an LCP. Fig. 4 shows from top to bottom:

1. Homogeneously mixed LCP with Cy3 lysozyme,

2. after 1.5 h isoelectric focusing at 100V (150 Vh) 3. after 1 h isoelectric focusing at 600 V (750 Vh)

4. after 2.5 h isoelectric focusing at 600 V (1650 Vh)

5. after 3.5 h isoelectrical focusing at 600V (2250 Vh)

6. after 4.7 h isoelectric focusing at 600V (2970 Vh)

7. After 6.5 h isoelectric focusing at 600 V (4050Vh) 8. After 8 h isoelectric focusing at 600 V (4950 Vh)

It can be seen that the different proteins are focused in bands and then drift to the cathode.

Example 5:

Direct solubilization of membrane proteins, solubilization of membrane proteins in an LCP.

Chromatophores are used as a model system for this. The chromatophores are cytoplasmic membrane vesicles of a prokaryote with photosynthetic membrane proteins, the color pigments, such as. B. chlorophylls, cytochromes, etc. bound. After mixing the vesicles with MO in a ratio of 30:70 (v / v) to a homogeneously greenish-colored LCP, which shows no visible pellet even after 10 h centrifugation at 55,000 g, a total of 150 μg total protein is dissolved in 100 μg LCP , A detergent-free, direct solubilization of a membrane protein mixture is therefore possible.

Example 6: Electrophoretic separation of a fluorescence-labeled phosphatidyl

Ethanolamines on an LCP separation matrix.

A monofunctional fluorescent dye Cy5-Dye (Amersham, No .: PA25001) became one with phosphatidyl ethanolamine (PE) according to the manufacturer

Cy5-PE conjugate coupled (solvent: 50 mM HEPES pH 9.0 in 90% MeOH) and purified by TLC (silica 60 HPTLC plates, Merck 1.05631; mobile solvent

60: 35: 8 v: v: v CHCI3: MeOH: H2O).

The reaction product showed the expected mass distribution in MALDI-TOF-MS.

For isoelectric focusing, an LCP with a composition of 70 μl liquid, degassed mono-olein (MO) 30 μl aqueous phase from 1: 5 diluted Ampholine ® 9.5-3.5 (Amersham-Pharmacia) in H ₂ 0 with Cy5-PE as analyte is used used. A 0.08 MH ₃ PO solution is used as the anolyte, and a 0.1 M TRIS-SO pH 9.3 solution is used as the catholyte.

The results of the isoelectric focusing under variation of the focusing conditions are shown in FIG. 5. 5 shows the results of the following experiments from top to bottom:

1. On the left in the aqueous phase with Ampholine ® 9.5-3.5 the fluorescent Cy5-PE conjugate is recognizable as a black precipitate, the syringe on the right contains molten MO.

2. shows a still inhomogeneous distribution of the analyte in the separation medium and thus a still inhomogeneous distribution of the LCP in the aqueous phase after about 100 mixing steps, 3. shows a homogeneous distribution of the analyte in the separation medium after about 200

mixing

4. shows the result of electrophoresis at 100V after 1 h (100 Vh)

5. shows the result of electrophoresis at 100V after 3 h (300 Vh),

6. shows the result of electrophoresis at 600V after 1.5 h (900 Vh), 7. shows the result of electrophoresis at 600V after 2.5 h (1500 Vh),

8. shows the result of electrophoresis at 600V after 3.75 h (2250 Vh),

9. shows the result of electrophoresis at 600V after 4.85 h (2910 Vh). Cy5-PE is not detectable in the anolyte (100 μl end of the syringe). Example 7:

Electrophoresis of a fluorescein isothiocyanate (FΙTC) -labeled peptide on one

LCP Seperationsmatrix.

A fluorescein isothiocyanate (FΙTC) -labeled peptide with the

Sequence YVAD used.

A mixture of an LCP consisting of 70 μl of liquid, degassed mono-olein (MO) and 30 μl of an aqueous phase of 50 mM HEPES pH is used as the separation medium

8.0 containing the analyte with a concentration of 0.125 mg / ml FITC-YVAD.

The electrophoresis is carried out with 50 mM HEPES as anolyte and catholyte at a constant 150V. Due to the highly hydrophobic amino acids YVA, the peptide used is only partially soluble in water and carries negative charges on the fluorescein and in the aspartate side chain. The migration of the labeled protein to the anode can be observed during electrophoresis (FIG. 6, left 100 μl end of the syringe). In accordance with the limited water solubility, the peptide can also be found in the anolyte, which shows that the aqueous domain of the LCP is continuously bonded to the surrounding buffer (see also Example 6).

6 shows the result of the electrophoretic migration (anode left, cathode right) of the peptidic analyte at an electrophoresis voltage of constant 150V:

1. after OVh 2. after 182.5 Vh

3. after 402.5 Vh

4. after 535 Vh

5. after 702 Vh

Since a uniform buffer system was used, the electrophoretic

Mobility of the analyte can be derived: The migration distance of the front is at the evaluation measured in pixels, with a resolution of the fluorescence scanner of 100 μm / pixel, one pixel corresponds to a migration distance of 100 μm:

Table 1 shows the electrophoretic mobility of FITC-YVAD determined according to Example 7.

Table 1

The graphical representation of the electrophoretic mobility is shown in FIG. 7.

Claims

claims

1. A method for electrophoretic separation of a sample, characterized in that a mixture of a polar is used as the separation medium

Liquid and a separation matrix of at least one amphiphilic substance, which forms a liquid-crystalline phase, is used.

2. The method according to claim 1, characterized in that the separation medium used a ratio of polar liquid to liquid-crystalline

Phase between 10:90 and 50:50 (v / v).

3. The method according to any one of the preceding claims, characterized in that water is used as the polar liquid.

4. The method according to any one of the preceding claims, characterized in that the amphiphilic substances used form a lamellar, hexagonal, inverted hexagonal or cubic crystalline phase.

5. The method according to any one of the preceding claims, characterized in that the amphiphilic substances used are uncharged.

6. The method according to any one of the preceding claims, characterized in that the amphiphilic substances used are alcohol fatty acid esters.

7. The method according to claim 6, characterized in that the amphiphilic substances used contain a C ₂ -C ₄ alcohol with 1 to 4 partially or completely esterified hydroxy groups.

8. The method according to any one of the preceding claims, characterized in that the amphiphilic substance used from the group of the mono- or diacylglycerides, the acylglycerol phosphatides, the glycoacylglycerides, the Aminoacylglyceride or from a mixture containing such substances is selected.

9. The method according to claim 8, characterized in that the amphiphilic substances used contain C ₈ -C ₃ o-acyl groups.

10. The method according to claim 8, characterized in that the amphiphilic substances used contain as acyl groups mono- to pentasaturated unsaturated esterified C ₂ -C ₂₄ fatty acids or saturated esterified C ₈ -C ₂₀ fatty acids.

11. The method according to claim 8, characterized in that the amphiphilic substances used contain as unsaturated acyl groups monounsaturated C ₁₄ -C ₂₂ fatty acids.

12. The method according to claim 8, characterized in that 1-monooleoyl-rac-glycerol, 1-monomyristoyl-rac-glycerol or 1-monopalmitoyl-rac-glycerol is used as the amphiphilic substance.

13. The method according to any one of the preceding claims, characterized in that the liquid crystalline phase further auxiliaries from the group of detergents, the synthetic or naturally occurring lipids, the fat-soluble biological cofactors, fatty acids or the C ₈ -C ₃ o-alkanes, Alkenes, alkynes or alcohols can be added.

14. The method according to claim 13, characterized in that phosphatidylcholine, phosphatidylethanolamine, charged or uncharged phospholipids, sphingolipids or glycolipids, chlorophylls, retinol, steroids, carotenoids or quinones are added as auxiliary substances to the amphiphilic phase.

15. The method according to claim 13, characterized in that lipids or detergents with ionic or zwitterionic head groups are added.

16. The method according to any one of the preceding claims, characterized in that carrier ampholytes are added to the polar liquid.

17. The method according to claim 16, characterized in that as a carrier

Ampholyt Ampholine ® is used in a concentration range of 0.1 to 40 vol%.

18. The method according to claim 16, characterized in that Ampholine ® is used as a carrier ampholyte in a concentration range of 2 to 8% by volume.

19. The method according to any one of the preceding claims, characterized in that small polar amphiphilic substances, polar glycols, sugar monomers or multimers, amino acids or zwitterionic substances are added as auxiliary substances to the polar liquid.

20. The method according to claim 19, characterized in that the added auxiliaries are selected from the group consisting of glycerol, ethylene glycol, propylene glycol, polyethylene glycol, glycine, urea and guanidine.

21. The method according to any one of the preceding claims, characterized in that the polar liquid used is buffered.

22. The method according to any one of the preceding claims, characterized in that a pH gradient is built up in the separation medium.

23. The method according to claim 22, characterized in that the electrophoretic separation process is an isoelectric focusing.

24. The method according to claim 23, characterized in that the analytes are dissolved in the separation medium before the start of the separation.

25. The method according to claim 23, characterized in that the hydrophobic analytes are dissolved in the liquid-crystalline phase before the start of the separation.

26. The method according to any one of the preceding claims, characterized in that the electrophoretic separation is carried out at a temperature of 20 to 40 ° C.

27. The method according to any one of the preceding claims, characterized in that the electrophoretic separation is carried out at a voltage between 100V and 8000V.

28. Use of amphiphilic substances which form liquid crystalline phases in polar liquids for the electrophoretic separation of ionic or ionized lipids, metabolites, carbohydrates, sugars, glycolipids, nucleic acids, amino acids, glycoproteins, peptides and proteins.

29. Use of amphiphilic substances which form liquid crystalline phases in polar liquids for the electrophoretic separation of ionic or ionized lipids, metabolites, carbohydrates, sugars, glycolipids, nucleic acids, amino acids, glycoproteins, peptides and proteins by means of isoelectric focusing.

30. Use of amphiphilic substances which form liquid crystalline phases in polar liquids for the electrophoretic separation of peptide- and protein-containing samples containing lipophilic and hydrophilic constituents.

1. Use of amphiphilic substances which form liquid crystalline phases in polar liquids for the electrophoretic separation of lipophilic constituents of peptide and protein-containing samples.