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WO2002082066A1 - Concentration et identification de ligands potentiels a liaison de type moderee a forte dans des produits naturels par electrophorese capillaire - Google Patents

Concentration et identification de ligands potentiels a liaison de type moderee a forte dans des produits naturels par electrophorese capillaire Download PDF

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Publication number
WO2002082066A1
WO2002082066A1 PCT/US2002/009727 US0209727W WO02082066A1 WO 2002082066 A1 WO2002082066 A1 WO 2002082066A1 US 0209727 W US0209727 W US 0209727W WO 02082066 A1 WO02082066 A1 WO 02082066A1
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Prior art keywords
target
capillary
hit compound
concentration
capillary electrophoresis
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Alexei Belenky
Yuriy Dunayevskiy
Dallas E. Hughes
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Cetek Corp
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Cetek Corp
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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
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
    • G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules

Definitions

  • the present invention relates to a method of isolating, concentrating, and characterizing moderate-to-strong binding compounds present in complex biological material that bind to a target (e.g., protein, nucleic acid, protein/nucleic acid complexes, other cellular components) of interest using an initial capillary electrophoresis separation and affinity step followed by a detection analysis.
  • a target e.g., protein, nucleic acid, protein/nucleic acid complexes, other cellular components
  • the described method is particularly useful for identifying compounds having an equilibrium dissociation constant (K d ) of less than 100 nM (moderate-to-strong hit compounds) .
  • exemplary detection methods include, but are not limited to, mass spectroscopy (MS) , nuclear magnetic resonance (NMR) spectroscopy, photodiode array (PDA) detection, light scattering detection, infrared detection, liquid chromatography in association with a subsequent, different detection method (e.g., LC/MS, LC/PDA or LC/NMR) or ultra-violet spectroscopy.
  • Fig. 1 is a diagram representing movement of a target plug as it progresses through a capillary containing a mixture of potential ligands, or "hit compounds," and inactive mixture components in electrophoresis buffer during a capillary electrophoresis run according to the method of the invention;
  • Fig. 2 is a description of exemplary steps used to carry out the method of the invention.
  • Figs. 3A and 3B are histograms showing the results of carrying out the method of the invention to purify and concentrate a strong ligand Methazolamide (MZ) (3A) and a weak ligand Carzenide (CL) (3B) using the target carbonic anhydrase.
  • MZ Methazolamide
  • CL weak ligand Carzenide
  • the results show about a 100-fold increase in the concentration of MZ and about a 2.4-fold increase in the concentration of CL;
  • Figs. 4A-4C show HPLC chromatographic detection (4A, 4B) and MS/MS analysis (4C) of methotrexate (MTX) purified and concentrated from a crude natural extract according to the method of the invention using the target protein dihydrofolate reductase (DHFR) .
  • MZ Methazolamide
  • CL weak ligand Carzenide
  • Fig. 1 The principles of the method of the invention is directed to isolating, concentrating and characterizing moderate-to-strong hit compounds present in complex biological materials and/or natural samples (NS) according to the invention are illustrated diagrammatically in Fig. 1.
  • the capillary 10 of a capillary electrophoresis (CE) instrument is filled with running electrophoresis buffer containing a natural sample comprising a potential ligand 12 and inactive mixture components 14 dispersed throughout.
  • the natural sample can be included in the reservoir buffer chambers as well.
  • a plug of target protein sample 16 is injected into capillary 10, the capillary is inserted into the reservoir buffer chambers (not shown) , voltage is applied across the capillary, and the target migrates down the capillary (in a target zone) . As the target migrates, individual target molecules bind to any ligands (potential hit compounds) present in the electrophoresis buffer sample mixture.
  • the target zone 18 accumulates and concentrates ligands as they bind and, consequently, move with the target. Only a small portion of the target protein binds a ligand at any particular moment in time during migration, as the protein is present in excess of the ligand in the vicinity of the target zone (e.g., 5 ⁇ M protein and 1 nM ligand) .
  • a large excess local concentration of the protein target e.g., about 10 to 50 ⁇ M, over the concentration of possible hit compounds will drive the equilibrium of any binding reaction toward the formation of complex and, thus, complete association of the ligand, or "strong hit.”
  • the target zone migrates further, and the remaining free, or unbound, target protein is exposed to a new portion of strong hit in the buffer. Consequently, more protein/strong hit complex 20 can form.
  • the strong hit will be concentrated in the electrophoretic zone containing the target protein.
  • the strong hit will be spread in this zone between two extreme sub-zones: sub-zone containing the target/hit complex formed in the initial event of target-hit binding and sub-zone of free unbound target, i.e., the portion of the target that never associated with a hit compound or from which a hit compound has dissociated.
  • a weak-binding hit compound if present in the same sample, will not be concentrated, or will be concentrated much less, with the target protein in its electrophoretic zone as any weak hit/protein complex dissociates very rapidly due to the fast off-rate of the weak hit. Free or unbound target protein then moves away from the dissociated weak hit and interacts with a new portion of the sample, containing additional molecules of the strong hit, present in the running buffer.
  • the hit compound's electrophoretic mobility should be different from the mobility of the target protein.
  • the hit compound may have the same charge as the target so that its mobility is in the same direction as that of the target, but this mobility should then be slower or faster than that of the target.
  • the concentration of the strong hit accumulating in the target zone will be smaller than it would be in the case where the hit and target migrate in the same direction if there is no natural sample in the outlet end buffer reservoir to feed buffer containing additional sample into the capillary.
  • sample and any strong hit can migrate out of the outlet reservoir into the CE capillary toward the target protein, replacing any strong hit that has migrated past the target. This modification would require twice as much running buffer containing sample in each run (in terms of volume) and, therefore, will double the amount of natural sample required.
  • an intended target molecule is subjected to electrophoresis as described above and accumulates a potential hit compound in the natural sample as it migrates through the electrophoresis buffer in the capillary (Step 1) .
  • the run is stopped when the target (protein) passes a detector window
  • Step 2 This enables the operator to know where the target zone resides in the capillary.
  • the capillary is then carefully removed from the electrophoresis unit and cut into several small segments (Step 3) depending on the width and position of the target peak(s) for collection, keeping track of the segment containing the target zone with the target and any bound ligand.
  • the position of the target peak(s) may be preliminary determined by performing an exploratory run of the target.
  • other collection methods may be used, for example, by collecting the sample from the end of the capillary (retaining the capillary whole) for an off-line analysis.
  • the segment containing the target and any bound ligands is then flushed, e.g., with HPLC buffer into a fresh tube (Step 4) .
  • This material is then analyzed by, e.g., HPLC-UV to quantitate the amount of ligand present (Step 5) , although other detection methods may be used.
  • the HPLC can be performed with an organic mobile phase to denature the protein and release bound ligands, which are detected by UV absorbance .
  • the amount of ligand is quantified by comparing the HPLC peak area with known standards.
  • the method of the invention significantly improves the efficiency of isolating small quantities of pure compounds, or ligands, from natural extracts for further analysis.
  • dissociation of the ligand from the target can either occur on-line or off-line.
  • a liquid sheath at the capillary electrophoresis-mass spectrometer (CE-MS) interface for example, containing a small amount of organic (e.g., 50% methanol) and weak acid (e.g., 1% acetic acid).
  • organic e.g. 50% methanol
  • weak acid e.g., 1% acetic acid
  • Other combinations of organic chemicals and weak acid may be used, for example, TFA, acetonitrile, acetone or ammonium acetate.
  • CE-MS can be applied to a crude NS or a pre-fractionated sample.
  • a crude sample can be fractionated and the various fractions can be subjected to the method of the present invention.
  • detectors can be used to obtain physical and structural properties of the hit compound (dereplication) .
  • exemplary detectors include, but are not limited to, mass spectroscopy (MS) , nuclear magnetic resonance (NMR) spectroscopy, photodiode array (PDA) detection, light scattering detector, infrared detector, liquid chromatograph (LC) /MS, LC/PDA, LC/NMR or ultra-violet spectroscopy.
  • MS mass spectroscopy
  • NMR nuclear magnetic resonance
  • PDA photodiode array
  • LC liquid chromatograph
  • LC/PDA liquid chromatograph
  • LC/NMR ultra-violet spectroscopy
  • MS spectrometry will be used to get mass and/or mass substructure (MS n ) on the initial purified material.
  • MS (or MS n ) analysis can be performed using direct infusion, High Performance Liquid Chromatography/Mass Spectrometer (HPLC/MS), or a CE/MS interface.
  • HPLC analysis gives additional information of the hit compound such as polarity.
  • the mobile phase utilized in many HPLC protocols will usually be sufficient to dissociate the complex due to the presence of organic and acidic additives.
  • PDA detection is routinely used online with HPLC to obtain a UV/Vis spectrum on the hit compound, which is useful for identifying chromophores .
  • the detection limits of modern mass spectrometers are on the order of about 0.1 ⁇ M concentration.
  • ESD-TOF electrospray ionization-time-of-flight
  • NMR can be used to obtain additional structural information.
  • the NMR can be interfaced with HPLC if an initial separation step is required prior to NMR analysis.
  • the stability of the protein/hit complex in the capillary can be increased by lowering the capillary temperature during CE.
  • the temperature range can be from about 0°C to about 60 °C, preferably from about 5°C to about 37 °C.
  • more hits of lower affinities can be concentrated in the target protein's electrophoretic zone. For example, under the most stringent CE conditions (for example, temperature of about 37°C) , only a strong hit is detected and identified. Under less stringent CE conditions (for example, temperature of 20°C) , any hit of moderate-to-tight binding strength can be isolated and concentrated by the method.
  • concentration factor for example, using longer (e.g., 1 m length) and wider (e.g., 100 ⁇ m wide) capillaries can increase the concentration of a protein-ligand complex because more natural sample can be used and thus more hit accumulates in the target zone.
  • a larger volume of NS e.g., 7.85 ⁇ L
  • the target zone remains the same (e.g., lOnL) .
  • the concentration factor will be larger, as more of the strong hit will be migrating into the CE capillary from the buffer reservoirs.
  • capillary electrophoretic conditions Prior to the performance of the method of the invention, capillary electrophoretic conditions must be optimized to detect the protein and, possibly, a new protein peak representing protein/ligand complex. This can be done by preliminary exploratory runs with and without natural sample. The information from these runs is used to determine when to collect the protein peak(s), e.g., where to cut the capillaries, or when to collect fractions from the capillary.
  • the target protein and the target/ligand complex may generate different results since they may potentially have different mobility rates.
  • a preliminary run of the target is useful to determine the migration and mobility profile of the target.
  • the area where there is a shift in the mobility profile of the target indicates the presence of hit compounds where one can collect for further analysis. If a shift is not present, then the area where a target is detected is collected for further analysis.
  • the present method requires the use of at least one buffer, but may require two: (a) a sample buffer and (b) a running buffer. In most cases these buffers will be different but they may be the same buffer.
  • the "sample buffer” is a pH-balanced solution for preparing samples of the target used to isolate a hit compound.
  • the "running buffer” is a pH-balanced solution used in the capillary electrophoresis apparatus and contains the natural sample. Examples of sample buffers and running buffers include Good's biological buffers (e.g., TES, CAPSO, etc.) or Tris based buffers.
  • suitable pH for preserving the target's functional activity, e . g. , binding activity, and allowing binding of the target to its ligand.
  • the running buffer's pH value should produce suitable capillary electrophoretic profiles of the target alone and/or the target/ligand complex.
  • the running buffer should ideally allow the unbound target to produce a detectable peak when subjected to capillary electrophoresis, within a reasonable time period (e.g., preferably under 10 minutes).
  • Each buffer solution may include appropriate additives, as needed.
  • Suitable buffers are well-known in the art to one of ordinary skill.
  • Various capillary parameters may also be adjusted to allow optimal capillary electrophoresis conditions for a selected target molecule and its ligand. Some capillary dimensions or factors that may be optimized include, but are not limited to: capillary size or diameter; capillary temperature; capillary length; inner coatings for the capillary, if necessary; and any capillary pretreatment, if necessary.
  • a preferred capillary inner diameter is within the range of about 10—500 microns, preferably within about 25—100 microns.
  • the capillary length will depend on the amount of time needed for obtaining good capillary electrophoretic profiles of the selected target and/or the target/ligand complex. Longer or narrower total capillary lengths can be used to improve resolution. Longer capillaries will increase the total amount of ligand isolated by the method because there is more natural sample present.
  • a preferred capillary length is within a range of about 0.5 cm to about 1 meter, most preferably within about 0.5 cm to 40 cm.
  • the inner wall of the capillary may be coated with a polymer, a polymer blend, or other suitable material.
  • the inner capillary coating may serve to minimize any electrostatic charge on the capillary wall and to diminish adsorption of a selected target, a ligand, or the target/ligand complex to the capillary wall.
  • the coating may also be pre-treated as needed. For instance, it may be pre-treated with a non-specific protein, such as bovine serum albumin (BSA) , to help prevent target adsorption.
  • BSA bovine serum albumin
  • CE may also be carried out in capillaries in the form of open grooves or channels in a planar surface such as a fused silica or polymer microfabricated device or microchip.
  • the migration of the tracked target molecule is followed typically by the use of an on-column detector aligned with a small window etched into the capillary. Alternatively, it is possible to scan the entire capillary.
  • Preferred detection methods use UV absorbance, UV or laser-induced fluorescence, and visible light absorbance. Other on-column detection methods may also be used.
  • one may use on-line detection instruments coupled with the capillary electrophoresis apparatus, which use radionuclide, fluorescence polarization, NMR, mass spectrometry, electrochemical detection and other methods .
  • the detection variable for direct detection can be absorbance at 210 or 280 nm for most proteins and 260 nm for nucleic acids.
  • Indirect detection uses laser-induced (or other) emission of mainly visible wavelengths from dye-labeled target molecules which give high sensitivity.
  • Preferred are fluorescently labeled molecules.
  • fluorescent dyes include fluorescein, rhodamine, tetramethylrhodamine, Texas Red and ethidium bromide. It must be kept in mind, however, that these labels can influence the overall charge on the target molecule and may affect its binding capability.
  • UV sources and lasers include: deuterium, xenon and mercury lamps; argon, Ar/Kr, HeCd, HeNe, XeCl, KrF, nitrogen and solid state lasers.
  • Some target molecules such as carbohydrates and small molecules, may require pre-capillary derivatization to be detected.
  • the capillary electrophoresis process is adjusted to produce the optimal electrophoretic profiles for the unbound target and, possibly, a target/ligand complex.
  • the profiles, when superimposed, may display at least two distinct peaks corresponding to the selected target molecule alone and to the target when bound to the selected target/ligand complex.
  • Some electrophoretic parameters to be optimized include, but are not limited to: time, voltage, current and temperature.
  • the detection point may constitute at least one window in the capillary, at which is placed a detector.
  • a fluorescence detector there may be a fluorescence detector and an ultraviolet or laser light source to cause fluorescence.
  • the capillary electrophoresis procedure may be set to run for up to 2 hours or even longer, as needed.
  • ligands of a particular binding strength have certain similar characteristics.
  • MTBL Mode-to-tight binding ligands
  • K off off-rates
  • K D dissociation constants
  • tight-binding ligands have lower dissociation constants and slower off-rates, forming stable target/ligand complexes that remain bound to the target and accumulate in the target zone during electrophoresis as they migrate past a detector during capillary electrophoresis.
  • the characteristics of these ligand groupings are outlined in Table 1.
  • Natural samples including, but not limited to, any pure, partially pure, or impure sample that contains complex biological material is considered an appropriate sample to be analyzed by the method of the invention.
  • Complex biological material is intended to include any mixture of compounds that may contain compounds that are potentially useful in a biological system, e. g. , whether human, other mammalian, or agricultural.
  • large chemical libraries are frequently generated by combinatorial chemistry to enable investigators to screen extremely large numbers of chemical compounds for potential therapeutic or diagnostic purposes. These libraries can be, in essence, modified biological scaffolds and can be screened advantageously by the method of the invention.
  • Particularly suitable as exemplary natural samples are extracts of terrestrial and marine plants, cells from higher animals including humans, eubacteria, actinomycetes and other bacteria, extracts from non-recombinant or recombinant organisms, microbial fermentation broths, both filamentous and non-filamentous fungi, protozoa, algae, archaebacteria, worms, insects, marine organisms, sponges, corals, crustaceans, viruses, phages, tissues, organs, blood, soil, sea water, water from a fresh-water body (e.g., lake or river), humus, detritus, manure, mud, and sewage or partially pure fractions from isolation procedures performed on any of these samples (e.g., HPLC fractions) .
  • a fresh-water body e.g., lake or river
  • humus e.g., detritus, manure, mud, and sewage or partially pure fractions from isolation procedures performed on any of these samples (e
  • the natural sample may be one that is harvested from the environment and/or cultured under suitable environmental conditions (growth medium, temperature, humidity) .
  • the harvested sample is simply diluted to the extent necessary to practice the method of the invention.
  • the sample material can be treated by any combination of standard processes used by those skilled in the field to prepare the sample for analysis.
  • the crude sample may be subjected to a preliminary treatment such as freeze-thawing, homogenization, sonication, heating or microwave extraction to break down cell walls.
  • the sample could be heated at, e . g. , 50°C for 10 minutes to inactivate destructive enzymes.
  • Non-specific proteins may be added to prevent destruction of proteinaceous targets by heat-resistant proteases.
  • Extraction of cells or culture media with various solvents can be carried out, followed by filtration to remove particulate matter and/or high molecular- weight compounds .
  • the natural sample may also be fractionated by centrifugation, sequential extractions, high pressure—liquid chromatography, thin—layer chromatography, counter—current chromatography, and/or other chromatography techniques before use in the method of the invention.
  • Various fractions of a positive sample may be tested to help guide the detection and isolation of active compounds by the method of the invention.
  • the sample may be diluted in aqueous or non-aqueous solution, which may contain salts and buffers such as sodium chloride, sodium citrate or Good's biological buffers.
  • aqueous or non-aqueous solution which may contain salts and buffers such as sodium chloride, sodium citrate or Good's biological buffers.
  • the dilution step is required and preferably is the only treatment. However, dilution can also be performed as a final procedure after one or more of the preceding steps. A dilution of about 1:10 to about 1:200 (vol. /vol.) of the original complex biological material sample is usually preferred to achieve reproducible results in the method of the invention. Additional dilution factors may be desirable.
  • the target sample may be purified, partially purified, or even unpurified (e. g. , as in a bacterial extract), as long as the target and/or ligand/target complex give(s) a discernible CE peak.
  • Any molecule that is implicated in a disease process is a potential target.
  • the potential target may be any compound useful in diagnosing a specific condition.
  • other categories of target molecules can be contemplated.
  • the target could be a molecule representing an essential function of an insect pest.
  • target molecules that may be used in the method of the invention include: proteins, nucleic acids, carbohydrates, and other compounds .
  • Dihydrofolate reductase Cancer Other examples include DNA or RNA (used to search for nucleic acid—binding proteins, transcription factors, etc.) ribosomes, cell membrane proteins, growth factors, cell messengers, telomerases, elastin, virulence factors, antibodies, replicases, other protein kinases, transcription factors, repair enzymes, stress proteins, uncharacterized disease-related genes and/or their RNA and protein products, uncharacterized disease-related regulatory DNA or RNA sequences, lectins, hormones, metabolic enzymes, proteases and toxins.
  • This definition also includes any subcomponent of the listed molecules, such as protein subunits, active peptide domains of therapeutic proteins and active regions of small molecules.
  • the target molecule may be chemically, enzymatically, or recombinantly altered to improve its electrophoretic properties (e. g. , deglycosylated) or subjected to fluorophore or polyion addition to facilitate its separation and/or detection during CE.
  • the target should be detectable during capillary electrophoresis, as unbound target and/or as target complexed with a moderate- to tight-binding ligand. For instance, it may be detectable by observation of its ultraviolet (UV) or other light absorbance properties, or its fluorescence properties.
  • a detectable tag such as a tag of a fluorescent or other dye, a radio-label, a chemical tag or other marker.
  • a fluorescently labeled target may be detected by ultraviolet light absorption detection (typically having a micromolar detection limit) or, more preferably, by laser-induced fluorescence detection (typically having a picomolar to low nanomolar detection limit) .
  • An additional advantage of a fluorescent tag is the selectivity provided, particularly in complex samples that may have many UV-absorbing compounds present.
  • the need for a detectable tag, and the type used, will depend on the nature of the target molecule.
  • Proteins and peptides may be labeled by, e . g. , amino labeling of lysine residues or sulfhydryl labeling of cysteine residues.
  • Nucleic acid species and polynucleotides may be labeled by incorporating a labeled nucleotide in an in vitro synthesis reaction. Methods of labeling various targets are well-known in the art.
  • a modified target e . g. , a fluorescently labeled target
  • a fluorescently labeled target retains its functional activity. That is, one can confirm that the labeled target retains a functionally active site by using any available, well-established functional or binding assay whose result depends on a functionally active target.
  • the concentration of ligands was performed using the target protein carbonic anhydrase II, which is a target for glaucoma, a serious eye disease. Two ligands were tested to compare the extent of accumulation of a strong ligand and a weak ligand.
  • the target protein carbonic anhydrase II was at a concentration of 50 ⁇ M injected in a total volume of 10 nL. Therefore, the total amount of protein in this injection plug is
  • 14.5 ng. 14.5 ng of protein has the capacity to bind 118 pg of MZ
  • the capillary was cut into equal 0.80 cm segments that were individually flushed with HPLC buffer. The eluants were then analyzed by High Performance Liquid Chromatography using an ultraviolet detection (HPLC/UV) to quantitate the concentration of MZ . The histogram shows the concentration of MZ in several of the segments.
  • the negative value segments (-4, -3, -2, -1) represent the four capillary segments just before the target zone segment
  • the “0" segment is the target zone segment that contains the protein and any bound
  • M The positive value segments (+1, +2 , +3, +4) represent the four capillary segments just after the target zone (the protein has not migrated through these segments) . Other segments are not shown.
  • MZ migrates in the same direction as the protein and has a higher electrophoretic velocity than the protein, so MZ constantly migrates through the target zone and is replenished from the inlet buffer reservoir. This is the reason MZ was included in the inlet reservoir only.
  • the test sample can be placed in both the inlet and outlet reservoirs.
  • the capillary segment "0" containing the protein had an MZ concentration of 2.36 ⁇ g/mL or 10.0 ⁇ M, representing over a 100- fold increase in concentration relative to the starting concentration of 100 nM in the electrophoresis buffer.
  • the total mass of MZ in segment "0" is calculated to be about 330 pg. (If this were an unknown compound, this mass would probably not be enough to perform dereplication, so multiple, identical runs could be run and the samples pooled and concentrated for analysis.) All the "negative" segments contain approximately 100 nM MZ because the MZ is constantly replenished from the inlet reservoir buffer during the run.
  • the method of the invention comprises purifying and concentrating higher affinity ligands.
  • the method eliminates the need for extensive fractionation and purification procedures that are time- consuming and cost inefficient.
  • Fig. 4A shows an HPLC chromatogram (UV 2X4 ) of the electrophoresis buffer containing the NE spiked with MTX prior to the experiment. As expected, it is a very complicated profile due to the presence of multiple extract components. The MTX peak is not discernable because of its low concentration combined with the complex profile.
  • Fig. 4B shows an HPLC chromatogram run under the same conditions but on the material eluted from the capillary segment containing the protein and any bound ligands . The peak at 11.377 minutes represents MTX. The earlier peaks in the chromatogram are some contaminating components from the NE.
  • Exemplary starting conditions include the following: Concentration of original natural sample (NS) is about 25 mg/mL (the amount of NS is 1 mg in 40 ⁇ L) . The concentration of an exemplary strong hit present in the NS is at about 50nM or 25ng/mL (M.W. 500Da) . The capillary dimensions for capillary electrophoresis are 50 ⁇ m diameter and 60 cm length, leading to a total volume of 1.1 ⁇ L.
  • the CE capillary was filled with running buffer containing NS. An aliquot of the target protein sample was injected into the CE capillary as a narrow plug.
  • the target protein concentration is 5 ⁇ M, in a total sample volume of 3 ⁇ L.
  • the NS concentration in the running buffer is about 2% or a 50-fold dilution.
  • the concentration of a strong hit test compound in the running buffer is 1 nM hit.
  • the total volume of the running buffer with the NS is 200 ⁇ L (100 ⁇ L on each side of the capillary) .
  • the electrophoresis is performed with controlled electroosmosis.
  • manipulating the velocity of the electroosmotic flow e.g., by eliminating or modifying the polymeric coating of the capillary inner walls and by choosing appropriate ionic strength of the running buffer and/or pH of the running buffer in a manner well- known to an ordinary skilled artisan (see U.S. Patent No. 6,299,747, the whole of which is hereby incorporated by reference)
  • Any strong hit present in the running buffer will be concentrated with the target protein in its zone by two phenomena.
  • the first is when the migrating target zone continuously encounters " fresh" NS-containing buffer and any unbound, strong hit capable of binding to unbound target protein.
  • the second phenomenon is enrichment of the target zone during slow electrophoresis for any strong hit in NS-containing buffer that is pumped from the outlet buffer reservoir by electroosmotic flow.
  • the electroosmotic flow will carry neutral molecules and molecules that have the charge opposite to the protein into the capillary and through the target zone. The molecules of the same charge as the protein but lower mobility than the protein also will be carried with the electroosmotic flow.
  • the small molecules of the same charge as the protein but higher mobility than the protein will be coming from the inlet buffer reservoir and be enriched when reaching the target zone.
  • Example calculations are presented.
  • the amount of the strong hit concentrated in the target zone by the first phenomenon is 1.1 fmol (1 nM in 1.1 ⁇ L capillary volume).
  • the electroosmotic flow velocity is 100 nL/min.
  • the amount of the strong hit migrating with electroosmotic flow through the target zone is 1 fmol/ ⁇ L or 0.1 fmol/min.
  • the amount of the strong hit concentrated in the target zone during one hour of electrophoresis is 6 fmol (0.1 fmol/min x 60 min) .
  • Total amount of the strong hit accumulated in the target zone is about 7.1 fmol.
  • the concentration of the strong hit in the target zone is 0.71 ⁇ M.
  • the target zone can be collected for analysis by several means.
  • the capillary may be cut and the target and bound ligands eluted as in the previous examples.
  • the target zone can be pushed from the capillary into a collection chamber using pressure and then analyzed off-line.
  • the outlet end of the capillary is placed in a CE-MS interface, and the target zone and any hit contained therein are then allowed to migrate out of the CE capillary and enter into a mass spectrometer.
  • the advantage of this embodiment is that the protein has a very slow migration in the capillary as the sample migrates through it, so one should be able to pass much more sample across the target than would be possible in the embodiment of Example I. This would be advantageous for ligands present at very low concentrations.
  • Uncoated capillaries are usually used because an electroosmotic force (EOF) is required, so the protein must behave in an uncoated capillary environment (no protein adsorption to walls) . Also, the EOF and electrophoretic mobility of the target must be carefully controlled so that they nearly offset each other and this can be technically difficult and require significant optimization.
  • EEF electroosmotic force

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Abstract

La présente invention concerne un procédé qui permet d'accroître la concentration de n'importe quel composé (12) de ligand candidat dans un échantillon naturel qui se lie à une cible sélectionnée pendant une électrophorèse capillaire. Le procédé comprend les étapes suivantes: on remplit le capillaire (10) d'un appareil d'électrophorèse capillaire avec un tampon circulant contenant un échantillon naturel; on injecte dans le capillaire (10), un bouchon de la cible sélectionnée (16), dans lequel la concentration de ladite cible (16) dépasse la concentration de n'importe quel composé (12) de ligand candidat dans ledit échantillon naturel; on soumet le bouchon de la cible à l'électrophorèse capillaire; on recherche, on détecte et on suit la migration de n'importe quelle cible liée au composé (20) de ligand au niveau d'un point de détection; on récupère ladite cible liée au composé de ligand détecté au niveau du point de détection; et on isole ledit composé de ligand en le séparant de ladite cible.
PCT/US2002/009727 2001-03-28 2002-03-28 Concentration et identification de ligands potentiels a liaison de type moderee a forte dans des produits naturels par electrophorese capillaire Ceased WO2002082066A1 (fr)

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US60/283,988 2001-03-28

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7074334B2 (en) 2001-05-23 2006-07-11 Klaus Wanner Method for determining the binding behavior of ligands which specifically bind to target molecules
US7672786B2 (en) * 2003-07-02 2010-03-02 Sergey Krylov Non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM)—based methods for drug and diagnostic development
WO2021009669A3 (fr) * 2019-07-14 2021-03-18 Kashiv Biosciences, Llc Traitement de séparation et de quantification de protéines par électrophorèse capillaire
WO2023112000A3 (fr) * 2021-12-17 2023-08-03 Kashiv Biosciences, Llc Procédé amélioré de séparation de protéines de faible poids moléculaire
US12139510B2 (en) 2020-05-01 2024-11-12 Kashiv Biosciences, Llc Process of purification of protein

Citations (4)

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Publication number Priority date Publication date Assignee Title
US5145567A (en) * 1991-11-14 1992-09-08 Beckman Instruments, Inc. Capillary zone electrophoretic analysis of isoenzymes
US5630924A (en) * 1995-04-20 1997-05-20 Perseptive Biosystems, Inc. Compositions, methods and apparatus for ultrafast electroseparation analysis
EP0848251A2 (fr) * 1996-12-16 1998-06-17 Beckman Instruments, Inc. Dosages homogènes en ligne utilisant l'électrophorèse
US5810985A (en) * 1992-09-14 1998-09-22 Purdue Research Foundation Electrophoretically mediated chemical analysis

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5145567A (en) * 1991-11-14 1992-09-08 Beckman Instruments, Inc. Capillary zone electrophoretic analysis of isoenzymes
US5810985A (en) * 1992-09-14 1998-09-22 Purdue Research Foundation Electrophoretically mediated chemical analysis
US5630924A (en) * 1995-04-20 1997-05-20 Perseptive Biosystems, Inc. Compositions, methods and apparatus for ultrafast electroseparation analysis
EP0848251A2 (fr) * 1996-12-16 1998-06-17 Beckman Instruments, Inc. Dosages homogènes en ligne utilisant l'électrophorèse

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7074334B2 (en) 2001-05-23 2006-07-11 Klaus Wanner Method for determining the binding behavior of ligands which specifically bind to target molecules
US7672786B2 (en) * 2003-07-02 2010-03-02 Sergey Krylov Non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM)—based methods for drug and diagnostic development
WO2021009669A3 (fr) * 2019-07-14 2021-03-18 Kashiv Biosciences, Llc Traitement de séparation et de quantification de protéines par électrophorèse capillaire
US12139510B2 (en) 2020-05-01 2024-11-12 Kashiv Biosciences, Llc Process of purification of protein
US12378282B2 (en) 2020-05-01 2025-08-05 Kashiv Biosciences, Llc Process of purification of protein
US12435106B2 (en) 2020-05-01 2025-10-07 Kashiv Biosciences, Llc Process of purification of protein
WO2023112000A3 (fr) * 2021-12-17 2023-08-03 Kashiv Biosciences, Llc Procédé amélioré de séparation de protéines de faible poids moléculaire

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