WO2005088292A1 - Separation et identification d'analytes par une electrophorese sur gel - Google Patents

Separation et identification d'analytes par une electrophorese sur gel Download PDF

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WO2005088292A1
WO2005088292A1 PCT/IL2005/000305 IL2005000305W WO2005088292A1 WO 2005088292 A1 WO2005088292 A1 WO 2005088292A1 IL 2005000305 W IL2005000305 W IL 2005000305W WO 2005088292 A1 WO2005088292 A1 WO 2005088292A1
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gel
analytes
detector
mass spectrometer
group
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PCT/IL2005/000305
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English (en)
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Ovadia Lev
Shaul Mizrahi
Artem Melman
Jenny Gun
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Yissum Research Development Company Of The Hebrew University Of Jerusalem
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Publication of WO2005088292A1 publication Critical patent/WO2005088292A1/fr

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    • 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
    • G01N27/44747—Composition of gel or of carrier mixture

Definitions

  • the present invention generally relates to the separation of analytes by gel electrophoresis, and more specifically relates to a novel method and apparatus for the separation of analytes using non-polymeric small molecule organogels and hydrogels, and to the identification and/or quantification of the separated analytes by various detection methods such as mass spectrometry.
  • organic gelators or organogelling agents
  • hydrogels or small molecule hydrogels
  • organogels or small molecule organogels
  • the gels which are formed in a variety of organic or aqueous solvents, are thermo reversible. Upon heating they form liquid solutions, and upon cooling the gels reform. They are also thixotropic, becoming liquefied upon mechanical agitation.
  • Most of the known gelators contain one of a number of functional groups, the most common among them being sugars (Jung, J.H. el al. Chem. Eur. J. 2002, 8, 2684-2690; Kobayashi, H et al. J. Chem. Soc. Perkin Trans. 2 2002, 1930-1936), amino acids (Hanabusa, K. et al. Chem. Mater.
  • the aggregation occurs through non-covalent interactions, usually though not exclusively hydrogen bonding. Often one gelator combines several types of interactions such as ⁇ - stacking, van der Waals and hydrophobic interactions with hydrogen bonding. The resulting structures are twisted fibers which intertwine to trap solvent molecules, thus causing gelation. Bundles of fibers, rods and planar platelets or leaf-like supra-molecular structures are also encountered. While much of the focus has been on discovering new gelators and understanding the process of gelation, very little success has been recorded in finding applications for these materials. Several preliminary observations have been made regarding organogels. First, the ability to incorporate electrolytes into gels has been shown.
  • Organogels have also been used as templates for inorganic, sol-gel derived chiral materials (Placin, F. et al. Chem. Mater. 2001, 13, 117-121). They have been found to gelate liquid crystals which may make them suitable for electrooptical displays (Mizoshita, N. et al. J Mater. Chem. 2002, 12, 2197-2201). Gelators that can selectively gel one of a mixture of solvents have been found (Trivedi, D. R. et al. Chem. Mater. 2003, 15, 3971- 3973; Bhattacharya, S. et al. Chem. Commun. 2001, 185-186). This type of gelation may be used in handling oil spills.
  • gel electrophoresis in both its planar and capillary forms
  • methods such as UV-VIS, diode array, electrochemical detection, radiolabel detection, calorimetry, fluorescence and mass spectrometry.
  • matrices for gel electrophoresis include agarose, polyacrylamide, gelatin or another gel formed of cross linked polymers or long chain polymers.
  • polyacrylamide gels exist, that vary in the degree of cross-linking and the nature of the surfactant included in the gel; the surfactant having the most widespread use is sodium dodecyl sulfate (SDS).
  • SDS sodium dodecyl sulfate
  • the analytes are first separated on a gel plate (or in a capillary filled by a solid porous matrix). The gel is stained and/or fixated to view and quantify the resultant bands, using radioactive labels, fluorescent dyes or colorimetric reagents. Bands of interest can then be excised from the gel, and often must undergo de- staining of fluorescent or colored dyes, and removal of all traces of the gel, in order to proceed with analysis strategies.
  • the separated proteins and peptides In conventional electrophoresis, the separated proteins and peptides must be located by staining, cut out of the slab, extracted from the matrix, cleaned of the stain, and only then injected into the MS for identification.
  • the need to physically remove the proteins and peptides from the gels before MS is complicated, time-consuming and labor-intensive, and has prevented the development of a fully automated system for protein analysis. Moreover, these steps often necessitate the use of volatile solvents, which are potentially harmful to the researcher and to the environment.
  • a different method for separation of the analytes from the gel involves application of electric force to drive the analytes off the gel.
  • the gel can alternatively be blotted onto a membrane, and Edman degradation or Matrix-Assisted Laser Desorption-Ionization Mass Spectroscopy (MALDI-MS) can be performed directly on the membrane.
  • MALDI Matrix-Assisted Laser Desorption-Ionization Mass Spectroscopy
  • a laser pulse is used to desorb and ionize the species of interest from a matrix in which it is embedded.
  • the sequence or residues are read directly in the mass spectrometer.
  • the ionization and desorption are often followed by Time of Flight (TOF) mass spectroscopy (Pacolsky & Winograd, 1999, Chem. Rev. 99(10), 2977-3005; Ekstrom et al., 2000, Anal Chem. 72, 286).
  • TOF Time of Flight
  • the present invention relates to a method and apparatus for the separation of analytes by application of an electric field across a small molecule organogel or a small molecule hydrogel containing a mixture of the analytes.
  • Organogels and hydrogels are made of small organic molecules (referred to herein as organic gelators or organogelling agents) and a solvent that form a porous gel structure held by non-covalent interactions between the small molecules.
  • the molecules of the organogelling agent are capable of establishing, with each other, at least one physical non-covalent interaction (e.g., hydrogen bonding, ⁇ stacking, dipolar interactions and the like), leading to self-aggregation of these molecules so as to fonn a matrix which traps the solvent molecules, thereby forming the gel.
  • Organogels are formed from small molecule organogelling agents in organic solvents, and hydrogels are formed from small molecule organogelling agents in aqueous solvents.
  • the Applicants of the present invention have unexpectedly discovered that these organogels and hydrogels provide a new and versatile matrix for gel electrophoresis.
  • Application of an electric field across the organogel or hydrogel permits separation of analytes based on their different mobilities in the solution within the gel and on their interaction with the compounds that form the organogel or hydrogel itself.
  • the gels can be used for the separation of analytes such as amino acids, peptides, proteins, nucleic acids, oligonucleotides, porphyrins, polypyrrols, viruses, water pollutants, toxicants, pesticides, explosives, nitrocompounds, heavy metal ions, heavy metals complexes, pharmaceuticals and racemic mixtures of optically active compounds.
  • analytes such as amino acids, peptides, proteins, nucleic acids, oligonucleotides, porphyrins, polypyrrols, viruses, water pollutants, toxicants, pesticides, explosives, nitrocompounds, heavy metal ions, heavy metals complexes, pharmaceuticals and racemic mixtures of optically active compounds.
  • organogels and hydrogels i.e., their thermo reversibility and thixotropic nature
  • a detector such as a mass spectrometer
  • analytes such as amino acids, peptides and porphyrins
  • separation of analytes can be achieved by capillary and planar electrophoresis using an organogel comprising the organic molecule trans-(lS,2S)-l,2- bis(dodecylamido)cyclohexane in an acetate buffer of the organic solvent acetonitrile and in mixtures of buffered acetonitrile and methanol.
  • the applicants have also carried out electrophoresis of amino acids and proteins in hydrogels comprising the organic gelator N,N'-dibenzoyl-L-cystine-di(ethanolamide) in aqueous buffered solutions.
  • the applicants have also carried out electrophoresis of proteins in a hydrogel comprising N,N'- dibenzoyl-L-cystine-di(ethanolamide) in the presence of up to 0.1% (w/w) sodium dodecyl sulfate, which is commonly used in polyacrylamide gels. It was further shown that the gels of the present invention can easily be inserted into a capillary tube by simply heating the gel up, injecting it into the capillary and cooling. Modifying compounds such as polyethylene glycol (PEG) can also be added in order to enhance the separation. Moreover, importantly, it was discovered that after separation the gel can be injected directly into a mass spectrometer for identification of the separated analytes.
  • PEG polyethylene glycol
  • the gel was simply divided into small samples, which were liquefied by heating or by physically disturbing the gel with a syringe, and injected directly into a mass spectrometer. This type of direct transfer cannot be done with polymeric gels, because they cam ot easily be liquefied and because the polymer matrix interferes with the MS analysis. With the organogel and hydrogel sections however, the
  • the present invention relates to a method for the separation of analytes comprising the steps of contacting the analytes with a non- polymeric organogel or a non-polymeric hydrogel; and applying an electric field across the gel, thereby separating the analytes.
  • the gels of the present invention can be in the form of a thin or thick planar film (planar gel) or they can be filled in a capillary (capillary gel) or tube in-tube gel) or deposited in long cavities in a solid substrate (integrated microfluidic device). Any type of organic compound that is capable of gelation (i.e., formation of an organogel or a hydrogel upon contact with the appropriate solvent molecules), can be used for fonning the gels of the present invention.
  • Non-limiting examples of suitable organogelling agents include but are not limited to sugars, amino acids, cholesterols, short peptides (typically 1-11 amino acids) connected to an alkyl or aryl or another hydrophobic side chain, and compounds containing hydroxyl, carbonyl, amine, carboxylic acid, amide, benzyl, sulphonamide, carabmate, thiocarbamate, urea, thiourea, oxamido, guanidino and/or biguanidino functional groups.
  • a currently preferred organogelling agent is a disubstituted cyclohexane such as 1,2- bis(dodecylamido)cyclohexane.
  • organogelling agent is a cystine-based compound such as N,N'-dibenzoyl-L-cystine di(ethanolamide).
  • the organogelling agents can be mixed with any type of organic or aqueous solvent or solvent mixtures that will achieve adequate separation of the desired analytes.
  • a buffer is typically added to the solvent or solvent mixture.
  • any buffer system that is compatible with electrophoresis can be used.
  • An organogel is formed upon cooling of a homogeneous mixture that is formed by mixing an organogelling agent with an organic solvent or solvent mixture.
  • a currently preferred solvent for forming the organogels of the present invention is acetonitrile containing a buffer.
  • Polar co-solvents such as alcohols (e.g., methanol) can be added in various concentrations in order to alter the polarity and enhance the resolution.
  • a hydrogel is formed upon cooling of a homogeneous mixture that is formed by mixing an organogelling agent with an aqueous solvent or an organically-modified aqueous solvent (i.e., a mixture of a water-miscible organic solvent and water).
  • a currently preferred solvent for forming the hydrogels of the present invention is water containing a buffer or a mixture of a water-miscible organic solvent in buffered water. According to a currently preferred embodiment, the solvent is a mixture of ethanol in water, for example 15% ethanol in water. It is also possible to introduce into the gels modifying molecules that are not covalently bonded to the gelator molecules which fonn the organogel or hydrogel network.
  • modifier compounds include but are not limited to oligomeric compounds; water soluble polymers; anionic, cationic or nonionic surface active agents (surfactants); polysaccharides and their esters; polyethylene oxide; polyimine and block polymers; and chiral selector molecules.
  • a currently preferred such modifying compound is polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the methods of the present invention can further include one or more additional separation steps after the organogel or hydrogel electrophoretic separation.
  • additional separation steps include, but not limited to chromatography (for example high performance liquid chromatography (HPLC), gas chromatograph (GC), or thin layer chromatograph (TLC)); distillation, electrophoresis in a second dimension in columns, capillaries, or on a planar plate; and/or membrane filtration.
  • HPLC high performance liquid chromatography
  • GC gas chromatograph
  • TLC thin layer chromatograph
  • the separated analytes are removed from the gel via a syringe and injected into the inlet port of the separation device, or in the case of capillary organogel electrophoresis, the entire contents of the capillary are pumped into the inlet port of the second device.
  • the present invention further relates to an apparatus which allows separation of analytes by electrophoresis in the small molecule organogels or hydrogels.
  • the apparatus is comprised of a container comprising a non-polymeric organogel or hydrogel, which gel is connected at its two ends to a pair of electrodes that are com ected to a power supply. Separation is achieved by application of an electric field between the two electrodes.
  • the apparatus can be in the form of a box comprising a flat surface for holding a planar gel, or it can be in the form of a capillary or a tube for holding a capillary or an in-tube gel.
  • the apparatus can also be in the form of long cavities in a solid substrate (integrated microfluidic device).
  • the apparatus of the present invention can easily be incorporated into a system comprising means for detection of the separated analytes.
  • the system of the invention can be automated, either fully or partially.
  • One of the advantages of the organogels and hydrogels of the present invention is the ability to couple gel electrophoresis and a detector such as a mass spectrometer, thereby allowing the direct identification and/or quantification of the separated analytes.
  • the present invention thus provides a method for the identification and/or quantification of analytes, by contacting the analytes with a non-polymeric organogel or hydrogel; applying an electric field across the gel, thereby separating the analytes; and transferring a sample from the gel to a detector in order to identify and/or quantify the analytes.
  • prior art methods for the extraction of analytes from polyacrylamide and other planar electrophoretic gels prior to their analysis by mass spectrometry suffer from many disadvantages, including complicated and labor-intensive procedures in order to extract the separated analytes from the gels.
  • the methods described herein enable easy, fast and non-labor intensive coupling of gel electrophoresis to a detector in order to identify and/or quantify the separated analytes.
  • Using the small molecule organogels or hydrogels it is possible to introduce the gel and the analytes directly or indirectly into the detector without resorting to complex procedures for separation of the analytes from the gel.
  • MS detectors include but are not limited to an electrospray interfaced mass spectrometer (ESI-MS) and a matrix assisted laser desorption ionization mass spectrometer (MALDI-MS).
  • ESI-MS electrospray interfaced mass spectrometer
  • MALDI-MS matrix assisted laser desorption ionization mass spectrometer
  • FIGURE 1 Dependence of the mobilities of dansylated amino acids [serine (1), phenylalanine (2), tryptophan (3), proline (4) and histidine (5)] on solvent concentration. Dotted line and right axis represent the viscosity of acetonitrile- methanol mixtures.
  • FIGURE 2 Dependence of mobilities of dansylated amino acids [serine (1), phenylalanine (2), tryptophan (3), proline (4) and histidine (5)] on addition of PEG 200 to acetonitrile.
  • FIGURE 3 Dependence of mobilities of dansylated amino acids [serine (1), phenylalanine (2), tryptophan (3), proline (4) and histidine (5)] on gelator concentration. Smoothed lines were added to guide the eye, with no theoretical basis.
  • FIGURE 4 Electropherograms at 20 kV of 14 amino acids in (A) buffer filled and (B) gel filled capillaries. Peak numbers are identified in Table 3.
  • FIGURE 5 Electropherograms at l lkV of five peptides in (a) buffer-filled and (b) gel-filled capillaries. Peak numbers are identified in Table 3.
  • FIGURE 7 Relative abundances of histidine (1, m/z 389), proline (2, m/z 347), tryptophan (3, m/z 436), phenylalanine (4, m/z 397) and serine (5, m/z
  • FIGURE 8 Electropherograms at 15 kV of five metal porphyrins in (A) buffer and (B) gel filled capillaries. Peak numbers are identified in Table 4.
  • FIGURE 9 Electropherograms based on MALDI analysis of planar gel separation of (A) 100 ⁇ M and (B) 200 ⁇ M porphyrins (numbers according to Table 4) with matrix ⁇ -cyano-hydroxycinnamic acid.
  • FIGURE 10 Electropherogram based on MALDI analysis of planar gel separation of 100 ⁇ M porphyrins (numbers according to Table 4) with no matrix.
  • the present invention relates to a method for the separation of analytes by contacting the analytes with a non-polymeric organogel or hydrogel; and applying an electric field across the gel, thereby separating the analytes.
  • Oranogels and hydrogels do not suffer from most of the drawbacks of conventional gel systems such as polyacrylamide gels.
  • One of the advantages of these gel systems is their reversible nature.
  • organogels and hydrogels can be liquefied locally by mechanical agitation with the needle of a syringe, by heating, or by introduction of a chemical that cleaves or dismpts the non-covalent bonds between the gelator molecules.
  • the liquefied gel can then be injected directly into a detector system, for example a mass spectrometer. This simple technique allows the entire matrix or any part of the matrix to be analyzed.
  • a particular advantage of organogels is that they can be made in organic solvents in addition to water.
  • Conventional electrophoresis gels are aqueous polymeric gels.
  • Organogels and hydrogels are made of small organic molecules (organic gelators or organogelling agents) and a solvent that form a porous gel structure held by non- covalent interactions between the small molecules.
  • Organogels are formed from small molecule organogelling agents in organic solvents, and hydrogels are formed from small molecule organogelling agents in aqueous solvents.
  • the molecules of the organogelling agent are capable of establishing, with each other, at least one physical non-covalent interaction leading to self-aggregation of these molecules so as to form a matrix which traps the solvent molecules, thereby forming the gel.
  • the physical interactions are diverse and include hydrogen bonding interactions, ⁇ interactions between unsaturated nuclei, van-der-Waals hydrophobic interactions, dipole— dipole interactions, and coordination bonds with organometallic derivatives.
  • the establishment of these interactions can often be promoted by the architecture of the molecule, for example by the nature of the nuclei (e.g., aromatic nuclei), the presence of one or more unsaturated bonds, and the presence of asymmetric carbons.
  • each molecule of an organogelling agent can establish several types of physical interactions with a neighboring molecule.
  • the term '"small molecule” as used herein refers to a low molecular weight molecule, typically having a molecular weight of less than 1,000, for example a moleculai- weight of 500-1000 which is characteristic of oligopeptides and oligosaccharides; a molecular weight of 400-600, which is characteristic of cholesterol based gelators; or a molecular weight of less than 400 which is characteristic of amino acid based gelators and mono and disaccharide based gelators.
  • gelling means a thickening of the medium which can lead to a gelatinous consistency and even to a rigid, solid consistency which does not run under its own weight.
  • the capacity to form this network of long range superamolecular structures and thus the gelling depends on the nature (or the chemical category) of the organogelling agent, the nature of the substituents and the nature of the solvent and the external constraints (e.g., temperature, pressure etc.).
  • This gelling is reversible under the action of an external stimulus such as heat or physical force or stress such as by mechanical agitation, or by addition of a chemical which breaks or disrupts the non- covalent bonds established between the organogelling agent molecules.
  • non polymeric organogel or hydrogel refers to an organogel and hydrogel containing a non-polymeric organogelling agent. Any type of organic compound that is capable of gelation (i.e., formation of a gel upon contact with solvent molecules), can be used for forming the organogels and hydrogels of the present invention.
  • Non-limiting examples of suitable organogelling agents include but are not limited to sugars such as monosaccharides and disaccharides, amino acids, short peptides (typically 1-11 amino acids) connected to an alkyl or aryl or another hydrophobic side chain), cholesterols, and compounds containing hydroxyl, carbonyl, amine (in the deprotonated form), carboxylic acid (in the protonated form), amide, benzyl, sulphonamide, carabmate, thiocarbamate, urea, thiourea, oxamido, guanidino and/or biguanidino functional groups.
  • sugars such as monosaccharides and disaccharides, amino acids, short peptides (typically 1-11 amino acids) connected to an alkyl or aryl or another hydrophobic side chain), cholesterols, and compounds containing hydroxyl, carbonyl, amine (in the deprotonated form), carboxylic acid (in the protonated form),
  • a currently preferred organogelling agent is a disubstituted cyclohexane such as l,2-bis(dodecylamido)cyclohexane.
  • Another cun-ently preferred organogelling agent is a cystine-based compound such as N,N'- dibenzoyl-L-cystine-di(ethanolamide).
  • suitable gelling agents include but are not limited to: - amides of carboxylic acids such as tricarboxylic acids, for example cyclohexanetricarboxamides; - amides or esters of amino acids, for example esters of alanine and amides of valine; - amides of N-acylamino acids, for example the diamides resulting from the action of an N-acylamino acid with amines containing from 1 to 22 carbon atoms, such as those described in WO 93/23008, the contents of which are hereby incorporated by reference in their entirety, for example N-acylglutamides in which the acyl group is a C 8 to C 2 alkyl chain, and the dibutylamide of N-laurylglutamic; - diamides having hydrocarbon chains each containing from 1 to 22 carbon atoms, for example from 6 to 18 carbon atoms, these hydrocarbon chains
  • - bolaamphiphiles with al-glucosamide head such as N,N'-bis(ss-D- glucopyranosyl)-n-alkane-l- dicarboxamide, such as the compounds mentioned in the article by T. Shimizu, J. Am. Chem. Soc. , 119, pp. 2812-18,1997, the contents of which are hereby incoiporated by reference in their entirety
  • organogelling agents described in WO 03/105788, the contents of which are hereby incorporated by reference in their entirety. It is also possible to use mixtures of the various organogelling agents described above.
  • organogelling agents the following non-limiting examples are mentioned: - N, N'-bis (dodecanoyl)- 1 , 2-diaminocyclohexane, in particular in the trans fonn, also known as (2-dodecanoylamino- cyclohexyl) dodecanamide). This compound is described in particular by Hanabusa K.; Angew.
  • gelators that are especially suitable for forming hydrogels in aqueous solutions include but are not limited to compounds containing one or more of mono, di, and oligosaccharide moieties; compounds containing amino acids, di, tri and oligopeptides; fatty acids and amides; pyridinyl containing compounds; organic ammonium or phosphate salts; and gemini surfactants.
  • Hydrogels are formed by the same method as organogels. Any type of solvent system that will achieve adequate separation of the particular analytes can be used in the organogels and hydrogels of the present invention.
  • the polarity of the solvent can range from nonpolar solvents such as alkanes and toluene to polar solvents such as water and water/ethanol solutions.
  • Solvent mixtures are also possible.
  • the polarity of the solvent/solvent mixture can be adjusted to achieve optimal separation of the desired analytes.
  • a buffer is typically added to the solvent or solvent mixture, in order to ensure compatibility with electrophoretic systems. Suitable buffers include but are not limited to acetic acid/acetate or other carboxylic acid/carboxylate buffers, tris, phosphate, ortho- phosphate, borate and carbonate based buffers. Any other organic or inorganic weak acid - base pairs can also be used.
  • An organogel is formed upon cooling of a homogeneous mixture that is formed by mixing an organogelling agent with an organic solvent or solvent mixture.
  • Suitable organic solvents include, but are not limited to, acetonitrile; halogenated solvents such as chloroform and methylene chloride; ketones such as methyl ethyl ketone, methyl isobutyl ketone, diisobutyl ketone, cyclohexanone and acetone; alcohols such as methanol, ethanol, isopropanol and cyclohexanol; glycols such as ethylene glycol, propylene glycol and pentylene glycol; short-chain esters such as ethyl acetate, methyl acetate, propyl acetate, n-butyl acetate and isopentyl acetate; ethers such as diethyl ether, dimethyl ether and dichlorodiethyl ether; alkanes such as decane, heptane, dodecane, hexane and cyclohexane; cyclic aromatic compounds such as
  • Polar co-solvents such as alcohols (e.g., methanol, ethanol and the like) can be added in various concentrations in order to alter the polarity and enhance the resolution.
  • a currently preferred solvent system is a mixture of acetonitrile and buffer, with or without methanol.
  • a hydrogel is formed upon cooling of a homogeneous mixture that is formed by mixing an organogelling agent with an aqueous solvent or an organically-modified aqueous solvent (i.e., a mixture of a water-miscible organic solvent and water).
  • a preferred solvent for forming the hydrogels of the present invention is buffered water.
  • An organically-modified aqueous solvent i.e., a mixture of a water-miscible organic solvent and water, can also be used to fonn the hydrogels of the present invention.
  • a small amount of an organic solvent such as methanol, ethanol, DMSO etc. is added to help the gelator dissolve.
  • concentration of the water-miscible organic solvent in water can vary depending on the electrophoresis system being used, and the analytes being separated. Suitable concentrations include 0-50% water-miscible organic solvent in water.
  • the solvent is a mixture of water and an alcohol, for example ethanol.
  • a currently preferred aqueous system is 15% ethanol in water.
  • the gels of the present invention can be in the fonn of a thin or thick planar film, typically having a thickness ranging from 0.5 mm to 3 mm, and a length ranging between 3 and 30 cm for example 1 mm x 10 cm; 2 mm x 20 cm, or they can be filled in a capillary or tubes typically having a thickness of about 50-500 ⁇ m, for example 100 ⁇ m, 75 ⁇ m and 50 ⁇ m or deposited in long cavities in a solid substrate (integrated microfluidic device).
  • the cavity can be suitable for lab on a chip- separation as described for example in the article Integrated microfluidic devices Erickson D, Li DQ Analytica Chimica Acta 2004, 507 (1): 11-26.
  • the concentration of the gelator in the gel can vary. Typical concentration ranges are 0.1-10% by weight-gelator based on the weight of gelator plus solvent molecule, for example 0.3?/o, 1%, 3%, 5% and 10% by weight of the gelator molecule. It is also possible to introduce into the gels modifying molecules that are not covalently bonded to the gelator molecules which form the organogel or hydrogel network. These molecules can be used in order to enhance the separation of analytes by exploiting the different affinities between modifier and organogel. These molecules can also be used to alter the selectivity of the separation.
  • modifier compounds include but are not limited to oligomeric compounds (e.g., glycol ethers such as polyetheyle glycol and polypropylene glycol); water soluble polymers (e.g., polyvinyl alcohol (PVA), chitosan, polysaccharide and polyethyleneimine); anionic surface active agents or surfactants (e.g., sodium dodecyl sulfate), cationic surface active agents (e.g., cetylpyridinium bromide (CPB), and cetyltrimethylammonium bromide (CTAB)), nonionic surface active agents (e.g., poly(oxyethylene)ethers;) polysaccharides and their esters; polyethylene oxide; polyimine and block polymers such as PEO-PPO-PEO; and chiral selector molecules (e.g., cyclodextrins, crown ethers, amino acid containing compounds and polypeptides).
  • a currently preferred such modifying compound is polyethylene glycol (P
  • nanoparticulate dispersions that are not attached to the gels by covalent bonding and which alter the selectivity of the separation compared to the separation in a matrix containing only the gel or only the solid particles.
  • the nanoparticulate dispersions preferably have a particle size of less than 2000 nm, more preferably less than 1000 nm, and even more preferably less than 500 nm.
  • the nanoparticulate dispersions can be made of, e.g., metals such as gold, silica gels, organically modified silica gels (e.g.
  • octyl, cyanoalkyl, aryl, amine or amide-modified silica gels metal oxides, organically modified metal oxides (e.g., octyl, cyanoalkyl, aryl, amine or amide-modified metal oxides), polystyrene, latex and the like.
  • stationary gold nanoparticles interact with the analyte and alter their electrophoretic mobilities. The difference in the electrochromtographic mobilities results in improved separation (see for example Separation of long double-stranded DNA by nanoparticle-filled capillary electrophoresis Huang MF, et al. Analytical Chemistry, 2004, 76 (1): 192-196 2004).
  • Assymetric (chiral) organogels based on small assymetric molecules in solvents have already been reported, for example cholesterol based gelators such as 3-b- cholesteryl-4-(2-anthryloxy)butanoate (Y.-C. Lin, R. G. Weiss, Macromolecules 1987, 20, 414), or amino acid based gelators such as L-cystine derivatives (Menger FM, Caran KL 5 JACS 2000, 122, 11679). It is therefore also possible to carry out chiral separations of enantiomers by exploiting different non-covalent interactions between the gel forming molecules and the different enantiomers that are introduced in the sample.
  • cholesterol based gelators such as 3-b- cholesteryl-4-(2-anthryloxy)butanoate (Y.-C. Lin, R. G. Weiss, Macromolecules 1987, 20, 414)
  • amino acid based gelators such as L-cystine derivatives (Menger FM, Caran KL
  • Suitable chiral organogels include, but are not limited to, gelators containing cholesterol, amino acids, sugars, and disubstituted cyclohexanes.
  • the gels of the present invention can be used for the separation of analytes such as amino acids, peptides, proteins, nucleic acids, ohgonucleotides, porphyrins, polypyrrols, viruses, water pollutants, toxicants, pesticides, explosives, nitrocompounds, heavy metal ions, heavy metals complexes, pharmaceuticals and racemic mixtures of optically active compounds.
  • the analytes can be derivatized in order to allow for easier detection.
  • amino acids are commonly used model compounds which are derivatized to allow for easier detection by UV, calorimetry, fluorescence microscopy, radiolabel detection, etc.
  • One such derivatizing agent is Sanger's reagent (l-fluoro-2,4-dinitrobenzene (FDNB), which reacts with the N-terminal residue under alkaline conditions.
  • FDNB l-fluoro-2,4-dinitrobenzene
  • the derivatized amino acid can be hydrolyzed and will be labeled with a dinitrobenzene group that imparts a yellow color to the amino acid.
  • Separation of the modified amino acids (DNP-derivative) by electrophoresis and comparison with the migration of DNP-derivative standards allows for the identification of the N-terminal amino acid.
  • dansyl chloride which reacts with the N-terminal residue under alkaline conditions. Analysis of the modified amino acids is carried out similarly to the Sanger method except that the dansylated amino acids are detected by fluorescence. This imparts a higher sensitivity into this technique over that of the Sanger method.
  • Another example is the use of the reagent ninhydrin, which provides an intense blue color, except for proline, where a yellow color is obtained due to the presence of the secondary imino group. Furthennore, Edman degradation enables the identification of an entire peptide sequence, since it allows for additional amino acid sequence to be obtained from the N-terminus inward.
  • This method utilizes phenylisothiocyanate to react with the N-terminal residue under alkaline conditions.
  • the resultant phenylthiocarbamyl derivatized amino acid is hydrolyzed in anhydrous acid.
  • the hydrolysis reaction results in a rearrangement of the released N-tenninal residue to a phenylthiohydantoin derivative.
  • the N-terminal residue is tagged with an identifiable marker, however, the added advantage of the Edman process is that the remainder of the peptide is intact. The entire sequence of reactions can be repeated over and over to obtain the sequences of the peptide. This process has subsequently been automated to allow rapid and efficient sequencing of even extremely small quantities of peptide.
  • Another preferred derivatization involves the derivatization of proteins with 5-carboxyfluorescein, succinimidyl ester to form fluorescent spots in gel electrophoresis.
  • Another preferred derivatization involves the derivatization of ohgonucleotides with the cyanine dyes such as YO, YO-YO and YO-PRO to form fluorescent spots in gel electrophoresis.
  • the organogels and hydrogels of the present invention separate the analytes based on a particular characteristic of the analytes, for example electronic charge. If there are multiple analytes with the same charge, they will not separate.
  • the present invention also contemplates perfo ⁇ ning one or more additional separation step(s) after the organogel or hydrogel separation has taken place.
  • the additional separation is based on a different characteristic of the analytes, for example size.
  • additional separation steps include, but not limited to chromatography (for example high performance liquid chromatography (HPLC), gas chromatograph (GC), or thin layer chroniatograph (TLC)); distillation, electrophoresis in a second dimension in columns, capillaries, or on a planar plate; and/or membrane filtration, hi all these techniques the separated analytes are removed from the gel via a syringe and injected into the inlet port of one or more separation device(s), for example HPLC, GC or TLC, or a gel electrophoresis apparatus.
  • HPLC high performance liquid chromatography
  • GC gas chromatograph
  • TLC thin layer chroniatograph
  • the entire content of the capillary is pumped stepwise into the inlet port of the second device(s), thus achieving resolution of analytes that did not separate well in the capillary gel.
  • the additional separation step can also include one or more of the conventional unit operations described in the book (M. McCabe, JC Smith and P. Harriott, Unit operations in chemical engineering, 5 th ed. McGraw hill, Inc. NY, 1993), the contents of which are hereby incorporated by reference herein, such as distillation, extraction, adsoiption, absorption, stripping, drying, membrane separation processes, and crystallization.
  • the present invention further relates to an apparatus for use according to the methods of the present invention and which allows separation of analytes by electrophoresis in the small molecule organogels or hydrogels.
  • the apparatus is typically comprised of a container such as a mould box to cast the organogel film on a flat surface.
  • the flat surface is connected at its two ends to two electrodes (usually via a buffer solution) that are connected to a power supply. Separation is made by application of electric field between the two electrodes.
  • the choice of the appropriate electric field is known to a person skilled in the art.
  • the present invention further relates to an apparatus for use according to the methods of the present invention and which allows separation of analytes by electrophoresis in organogels or hydrogels that are filled in a capillary or a multiplicity of capillaries.
  • the apparatus is comprised of a capillary tubing filled with the gel (preferably the gel is introduced in a heated form above the gel formation temperature).
  • the capillary is connected at its two ends to two beakers in which are immersed two electrodes that are connected to a power supply.
  • the apparatus can further comprise one or more additional separation device(s) for further separating the analytes after the organogel or hydrogel electrophoretic separation.
  • the additional separation(s) can be conducted by methods such as chromatography (for example high performance liquid chromatography (HPLC), gas chiOmatograph (GC), or thin layer chromatograph (TLC)); distillation, electrophoresis in a second dimension in columns, capillaries, or on a planar plate; and/or membrane filtration.
  • chromatography for example high performance liquid chromatography (HPLC), gas chiOmatograph (GC), or thin layer chromatograph (TLC)
  • distillation electrophoresis in a second dimension in columns, capillaries, or on a planar plate
  • membrane filtration for example high performance liquid chromatography (HPLC), gas chiOmatograph (GC), or thin layer chromatograph (TLC)
  • distillation electrophoresis in a second dimension in columns, capillaries, or on a planar plate
  • electrophoresis in a second dimension in columns, capillaries, or on a planar plate
  • membrane filtration for example high performance liquid chromatography (HPLC
  • One of the objectives of the present invention is to provide a method that allows simple coupling of planar and capillary gel electrophoresis and a detector such as a mass spectrometer, thereby allowing the direct identification and/or quantification of the separated analytes.
  • a sample from the gel is liquefied, for example by heating, by mechanical agitation, or by introduction of a chemical that cleaves the non-covalent bonds between the gelator molecules, and the liquefied sample is simply transferred to a detector in order to identify and/or quantify the analytes.
  • the methods described herein enable easy, fast and non-labor intensive coupling of gel electrophoresis to a detector in order to identify and/or quantify the separated analytes.
  • a detector commonly known in the art can be used in the methods of the present invention. Suitable detectors include but are not limited to a mass spectrometer (MS), ultraviolet (LTV) detector, ultraviolet-visible (UV-VIS) detector, calorimeter, diodearray, electrochemical detector, fluorescence detector and radiolabel detector.
  • a particularly preferred detector for use in the methods of the present invention is a mass spectrometer.
  • MS detectors include but are not limited to an electrospray interfaced mass spectrometer (ESI-MS) and a matrix assisted laser desorption ionization mass spectrometer (MALDI-MS).
  • ESI-MS electrospray interfaced mass spectrometer
  • MALDI-MS matrix assisted laser desorption ionization mass spectrometer
  • MALDI-MS matrix assisted laser desorption ionization mass spectrometer
  • MALDI desorption-enhancing reagents include for example-2,4,6 - trihydroxy acetophenone; sinapinic acid; ⁇ -cyano-4-hydroxy cinnamic acid (CHCA); dihydroxybenzoic acid; hydroxy picolinic acid; anthranilic acid; nicotinic acid; salicylamide; succinic acid; ferulic acid; caffeic acid; poiphyrins; metal porphyrins; and
  • the present invention further provides a method and apparatus for the coupling of planar organogel or hydrogel electrophoresis and mass spectrometry by introduction of the separated analytes from the electrophoresis apparatus into a mass spectrometer such as MALDI - MS or ESI - MS.
  • the electrophoresis apparatus is comprised of a flat plate equipped at its two ends with electrodes that can be comiected to a DC power supply, which can be transferred easily to a detector such as a mass spectrometer, e.g. a MALDI-MS or ESI-MS apparatus.
  • the present invention further provides a method and apparatus for the coupling of capillary organogel or hydrogel electrophoresis and mass spectrometry by introduction of the separated analytes from the electrophoresis apparatus into a mass spectrometer such as MALDI - MS or ESI - MS. This is earned out by pumping a fluid that forces the gel and the analyte out of the capillary and into the mass spectrometry through an interconnected capillary. Detection of the analytes by mass spectrometry according to the methods of the present invention can be conducted in accordance with the literature procedure described in Michalski WP, et al.
  • thermo- reversibility and thixotropy which enables their simple and direct transfer to a detector for the identification and/or quantification.
  • the gel is normally solid, but it can easily be disturbed and liquefied by a number of methods such as by physically disturbing the gel, by heating, or by introduction of a chemical that cleaves the non-covalent bonds between the gelator molecules.
  • Physical disturbance can be achieved by mechanical agitation, such as by applying shear force or shear stress (e.g., by repeatedly moving the syringe).
  • Local heating of the gel can be achieved by using a heated needle of a syringe, a heating element, or an external infra red emitter.
  • Chemicals which can disrupt the non-covalent bonds of the organogels and hydrogels include but are not limited to urea, guanidine hydrochloride. dimethylamino benzaldehyde, sodium fluoride and salts of other small ions.
  • the liquefied gel can then be directly transferced to the detector with a syringe, with a pump, by applying a pressure gradient, by applying electrophoretic force, or by any other method known in the art.
  • the transfer can be manual or it can be automated (e.g., by using a robot arm).
  • the gel is first divided into several samples, and each sample is separately disturbed and transferred into the detector for identification/ quantification.
  • the gel is dried and laser inadiation in the
  • UV or visible or IR region is applied in order to desorb the analytes and transfer them to a mass spectrometer.
  • a mass spectrometer In order to facilitate the transfer of analytes to the mass spectrometer it is customary to add to the matrix compounds that assist the transfer.
  • Commonly used matrix compounds include but are not limited to2,4,6 -trihydroxy acetophenone; sinapinic acid; ⁇ -cyano-4-hydroxy cinnamic acid (CHCA); dihydroxybenzoic acid; hydroxy picolinic acid; anthranilic acid; nicotinic acid; salicylamide; succinic acid; ferulic acid; caffeic acid; porphyrins; metal porphyrins; and
  • the methods of the invention further include the addition of small molecules to the gel in-order to enhance matrix assisted laser desorption and thus allow matrix assisted laser desorption mass spectrometry.
  • small molecules include but are not limited to any of the matrix compounds listed above.
  • the small gelator molecules also enhance matrix assisted laser desorption and thus allow matrix assisted laser desorption mass spectrometry.
  • Example 1 Planar Electrophoresis Oganogels were formed by adding 6 mg of t/O77-.-(lS, 2S)-1,2- Bis(dodecylamido)cyclohexane (1) to 2 ml of buffer, heating until dissolution and casting into the electrophoresis cell.
  • the buffer used consisted of IM acetic acid and 25 mM ammonium acetate in acetonitrile or mixtures of acetonitrile with methanol.
  • the gel was deposited in a planar shallow box. Gel dimensions after deposition were 2 mm thick x 2.5 cm wide x 6 cm long. Separation was conducted by application of 300 V between two electrodes located at two cavities at the two ends of the organogel.
  • Figure 1 shows the mobilities of five dansylated amino acids in planar gels of different acetonitrile-methanol mixtures.
  • the following dansylated amino acids were used for separation: serine (1), phenylalanine (2), tryptophan (3), proline (4) and histidine
  • Different solvents can be used in order to control the separation of a group of analytes or a specific analyte pair.
  • Proline and tryptophan have the best resolution in pure acetonitrile, while phenylalanine and serine separate better in the 50% mixture. Analytes that are difficult to resolve can potentially be resolved with a change in the solvent.
  • Example 3 Effect of the Concentration of the Gelator Figure 3 shows the dependence of mobilities of dansylated amino acids [serine
  • Example 4 Capillary Electrophoresis The separation of a mixture of 14 dansylated amino acids was carried out in both gel-filled and buffer-filled capillaries. As stated above, the dansylated acids are all negatively charged in this system. Since in the gel there is no electroosmosis, and electrophoretic mobility is the only force acting on the analytes, separations were performed in negative polarity mode (i.e., with the detector located near the anode).
  • electroosmotic flow marker mesityl oxide was found to have a mobility of 3.4 x 10 "4 cm V "1 S "1 toward the cathode, which is greater than the electrophoretic mobilities of all the amino acids toward the anode, with the exception of aspartic acid. Therefore, positive polarity was used, and the compounds were detected in the reverse order of that in the gel-filled capillaries.
  • Figure 4 shows electropherograms of 14 amino acids in (A) buffer filled and (B) gel filled capillaries of different dansylated amino acids. The peaks were identified by individual injection of each acid. The retention times, mobilities, and plate counts for the gel separation are given in Table 3.
  • N 5.54(t t /WX where W is the half-height peak width
  • Electroosmosis in the acetonitrile- methanol gel-filled capillaries was negligible as in the acetonitrile-based gels, but the change of solvent rendered the electroosmosis in the conventional CE test (upper curve of Figure 5) negligible, and therefore the test was done in negative mode.
  • full baseline separation was achieved in the gel, which was not possible in the buffer-filled capillaries.
  • Example 6 Direct MS Interfacing
  • One of the important advantages of the organogels and hydrogels is the ability to inject the gel directly into the mass spectrometer, thus attaining by definition 100% recovery of the analytes. Full recovery is indeed common practice for capillary electrophoresis but not for planar gels. Theoretically, full recovery is possible also for other polymer-based gels, but in practice the polymeric matrix or its degradation products interfere with the mass spectrometric analysis.
  • the Applicants have compared the calibration curves of the different dansylated analytes in acetonitrile acetate buffer solution and organogels of 1. The sensitivities obtained for the different analytes that ⁇ vere injected from the gels ranged between 73% and
  • the m/z 436 peak is the molecular ion.
  • the /z 223 and 250 peaks appeal- in all the injections and are fragments of the dansyl group, oxidized demethylized dansyl and dansylamine, respectively.
  • the Applicants perfonned the separation of 5 dansylated amino acids as described above. After the separation was completed- the gel was divided into 13 compartments by placing a plastic grating on the gel. The 4-mm size of the grating provided a sample of about 10 mL which was necessary for injection into the MS.
  • FIG. 7 depicts the relative abundances of histidine (1, m/z 389), proline (2, m/z 347), tryptophan (3, m z 436), phenylalanine (4, m/z 397) and serine (5, m/z 337) in each of 13 different injections.
  • the different levels in each of the compartments are comiected by a line to guide the eye, forming a chromatogram presentation. This mode of planar electrophoresis offers full recovery of the analytes. Similar procedure was carried out with local liquefaction by local heating and gave similar results.
  • the reversible nature of organogels thus allows them to be injected directly into the MS for identification of the compounds separated on them and obviates the need for complicated and time-consuming extraction procedures.
  • Example 7 Porphyrin separation in capillaries
  • a further example of the use of trt7/7-.-(lS, 2S)-1,2- Bis(dodecylamido)cyclohexane is the separation metal porphyrins. Five porphyrins were separated in gel-filled and buffer-filled capillaries at 15 kN and detected by absorbance at
  • the buffer was IM acetic acid-25 mM ammonium acetate in acetonitrile.
  • the electropherograms are shown in Figure 8 with the data listed in Table 4.
  • porphyrins were separated on an acetonitrile slab gel of trans-( ⁇ S, 2S)-l,2-Bis(dodecylamido)cyclohexane, and the gel was divided into sections for MALDI-MS analysis. A sample from each section was placed onto a MALDI plate with matrix ⁇ -cyano-hydroxycinnamic acid. Calibration of the spectrometer with gels containing known concentrations of the porphyins allowed conversion of the signal intensities into concentrations. The resulting electropherograms are shown in Figure 9. Compound 5 is not detected because it is multiply charged, and 1 is not detected because it is too small.
  • Example 9 Porphyrin separation with MALDI-TOF-MS interfacing without added matrix
  • Example 10 Gel Electrophoresis Using Hydrogels
  • Hydrogels were formed by adding 24 mg of ⁇ , ⁇ '-dibenzoyl-L-cystine- di(ethanolamide) to 3 mL of buffer, heating until dissolution and casting into the electrophoresis cell.
  • the buffer consisted of 10 mM phosphate (pH 7) in a solution of 15% ethanol in water. A voltage of 500 V was applied for 20 minutes.
  • Amino acids were labeled with dansyl chloride, and non-colored proteins were labeled with 5- carboxyfluorescein, succinimidyl ester. Visualization was carried out by illumination with UV light.
  • the mobilities of the various amino acids and proteins are listed in Table 5. Table 5. Mobility Data for Planar Hydrogels
  • BDACH, trtf7M-(lS,2S)-l,2-bis(dodecylamido)cyclohexane (1) was synthesized according to the following procedure: Lauroyl chloride (0.768 g, 3.5 mmol) was added to a solution of (lS,2S)-l,2-diaminocyclohexane (0.2 g, 1.75 mmol) and triethylamine (1.77 g, 17.5 mmol) in 50 mL of tetrahydrofuran in a nitrogen atmosphere. The mixture was refluxed for 3 h and allowed to cool. The solvent was removed by rotoevaporation.
  • Samples for the MALDI-MS were taken by pushing a plastic grating (1-mm holes with 1- mm spacing) into the gel and withdrawing 2 ⁇ L with a pipet.
  • Capillary electrophoresis was perfonned in a Dionex CE instrument with a 42-cm long, 100-//m i.d. capillary (Biotaq, MD). Injection was done electrophoretically.
  • UV detection was carried out at 260 nm for dansyl amino acids and 465 nm for porphyrins.
  • Gel-filled capillaries were made by injecting hot organogel solution (60 °C) into the capillary and allowing it to cool. Relative viscosity measurements were made by flowing the solvents through an

Abstract

L'invention concerne un méthode et un appareil pour la séparation d'analytes, par l'application d'un champ électrique à travers un organogel présentant de petites molécules non polymères ou un hydrogel à petites molécules contenant un mélange de ces analytes. Les analytes séparées peuvent être subséquemment identifiées et/ou quantifiées par le transfert direct des échantillons du gel sur un détecteur, notamment un spectromètre de masse.
PCT/IL2005/000305 2004-03-18 2005-03-17 Separation et identification d'analytes par une electrophorese sur gel WO2005088292A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102006022569A1 (de) * 2006-05-15 2007-11-22 Johannes-Gutenberg-Universität Mainz Spezies-unabhängiges Nachweisverfahren für biologisches Material
DE102006022569B4 (de) * 2006-05-15 2011-05-05 Johannes-Gutenberg-Universität Mainz Spezies-unabhängiges Nachweisverfahren für biologisches Material
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WO2015148609A3 (fr) * 2014-03-26 2015-12-10 Li-Cor, Inc. Dosages immunologiques au moyen de cristaux colloïdaux
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US10793513B2 (en) * 2016-02-26 2020-10-06 Srm University Process for electrochemical separation of enantiomers of an amino acid from a racemic mixture
CN111171218A (zh) * 2020-01-17 2020-05-19 北京航空航天大学 一种具有多稳态力学和形状记忆性质的多相凝胶及其制备方法
CN111171218B (zh) * 2020-01-17 2021-10-26 北京航空航天大学 一种具有多稳态力学和形状记忆性质的多相凝胶及其制备方法

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