WO2000003240A1 - Method to detect and analyze tight-binding ligands in complex biological samples - Google Patents
Method to detect and analyze tight-binding ligands in complex biological samples Download PDFInfo
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- WO2000003240A1 WO2000003240A1 PCT/US1999/015458 US9915458W WO0003240A1 WO 2000003240 A1 WO2000003240 A1 WO 2000003240A1 US 9915458 W US9915458 W US 9915458W WO 0003240 A1 WO0003240 A1 WO 0003240A1
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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
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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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
- G01N33/561—Immunoelectrophoresis
Definitions
- the invention relates generally to methods of screening complex biological materials for effective regulatory, therapeutic, or diagnostic compounds.
- the invention encompasses using capillary electrophoresis and mass spectrometry together in a method particularly advantageous for detecting and characterizing tight-binding ligands in mixtures that may also include much higher concentrations of competing, weaker-binding ligands.
- the method also allows ranking of ligands according to their relative binding strengths .
- BACKGROUND OF THE INVENTION Developing screening protocols to identify new, biologically active compounds can present unique and difficult challenges, especially when screening complex materials, particularly a "complex biological sample" (CBS) : any sample of material that may have an effect in a biological system.
- Examples of CBS include but are not limited to: a natural product; a natural extract; a biological preparation; a chemical mixture; a pure compound library; and a combinatorial library.
- capillary electrophoresis has previously been used to detect and/or to analyze known compounds and materials of known composition, this technology has not been widely used, until recently, to screen complex biological samples for target-binding compounds that were previously unknown or unidentified as being ligands to a selected target molecule.
- a major obstacle to successful and cost-effective drug screening has been the presence of high concentrations of one or several weak, target-binding ligand compound (s) in a screened sample, which can mask the presence of more valuable, moderate-to-tight-binding or tight-binding ligands occurring at a lower concentration within the same sample.
- Another major obstacle is obtaining structural information about the high affinity ligands, especially when they are present in very complex mixtures.
- capillary electrophoresis specifically capillary zone electrophoresis
- capillary electrophoresis enables selective identification of particular candidate ligand(s) that bind(s) tightly to a target of interest.
- the CE steps are optimized to screen out all but those ligands that bind to the target molecule of interest at a selected binding strength, as taught in International Application No. PCT/US98/27463, herein incorporated by reference.
- MS Mass spectrometry
- Suitable MS ionization techniques include, but are not limited to electron impact ionization (El), electrospray ionization (ESI), chemical ionization (CI), atmospheric- pressure chemical ionization (APCI), matrix-assisted-laser- desorption ionization (MALDI), thermospray (TSP) , and fast atom bombardment (FAB) ionization.
- El electron impact ionization
- ESI electrospray ionization
- CI chemical ionization
- APCI atmospheric- pressure chemical ionization
- MALDI matrix-assisted-laser- desorption ionization
- TSP thermospray
- FAB fast atom bombardment
- MALDI-TOF mass spectrometry is valued for ease of sample preparation, predominance of singly charged ions in mass spectra, sensitivity, and high speed.
- Ion trap and Fourier transform MS allow re-analysis of the ions, as needed.
- the ions may be subjected to fragmentation, such as collision-induced dissociation (CID) , during mass spectrometry to provide additional structural or substructural data.
- CID collision-induced dissociation
- MS allows compounds to be detected and differentiated by their molecular weight and/or size, it may be used to observe selectively the target's migration pattern after undergoing CE either alone or in the presence of a complex biological sample. Therefore, MS allows one to eliminate any separate detector and/or any derivatization of the target, if one so desires.
- UV ultraviolet absorbance
- fluorescence detector e.g., light-induced fluorescence (LIF)
- LIF light-induced fluorescence
- Identifying and immediately characterizing those candidate ligands that form the most stable complexes with the selected target minimizes the time and resources needed for isolating and characterizing these compounds.
- the invention provides a more cost-effective screening protocol since the most stable target-binding ligands are those most likely to be effective therapeutic, regulatory, and/or diagnostic compounds and drugs.
- the method generally combines a capillary electrophoresis (CE) technique for screening complex biological samples with a mass spectrometry (MS) analysis step to provide a streamlined procedure for selectively identifying and characterizing any candidate ligand(s) in a complex biological sample that binds at a selected binding strength to a selected target molecule.
- CE/MS method can selectively identify and characterizes moderate-to-tight binding ligands (MTLs) , especially tight- binding ligands (TLs) , even in the presence of high concentrations of weak ligands (WLs) that often mask lower concentrations of tight-binding ligands in the same sample.
- MTLs moderate-to-tight binding ligands
- WLs tight- binding ligands
- the present method improves over prior CE screening methods, by selectively detecting TLs over WLs, and by providing valuable structural data about candidate tight-binding ligands, all in one procedure.
- the present method also allows the ranking of target-binding ligands detected in a complex biological sample according to their relative binding strengths.
- Mass spectrometric analysis of compounds screened and separated by capillary electrophoresis offers the advantages of rapidly obtaining structural characterization of high- affinity ligands identified by CE.
- the mass spectrometer itself can be used to track the CE migration of the target, by selectively monitoring the mass/net charge (m/z) ratio of the ionized target, and thus to selectively detect any candidate ligand of a desired binding strength.
- m/z mass/net charge
- Using the mass spectrometer as a detector eliminates the need to use any additional detector and the need to label or otherwise derivatize the target to make it detectable during CE.
- the method involves interfacing a capillary or microfabricated chip of a CE instrument with a post-capillary mass spectrometer.
- a suitable interface is provided by, e.g, the microscale fluid handling system disclosed in Karger et al., U.S. Patent 5,872,010.
- the present method provides direct mass and structural analysis of candidate ligands that form target/ligand complexes that migrate stably through the CE instrument, a detectable amount (e.g., at least 50% or at least 80%) of which remains tightly bound, i.e., for a substantial portion of the CE run time, preferably at least 50% or, even better, at least 80%, most preferably until they reach the outlet end of the CE capillary.
- the present method can preferentially identify and structurally characterize tight- binding ligands in complex biological samples.
- the present method screens complex biological material for and characterizes any candidate ligand that binds to a selected target at or above a selected binding strength, by the steps disclosed herein.
- a complex biological sample is combined with a selected target to form a sample/target mixture.
- a plug of the sample/target mixture is then injected into an inlet end of a conduit of a capillary electrophoresis instrument (e.g., a capillary or a channel of a microchip) .
- the compounds within the sample/target plug are subjected to capillary electrophoresis (CE) under predetermined conditions.
- CE capillary electrophoresis
- the predetermined CE conditions have been optimized so that any first complex, formed between the target and any candidate ligand binding to the target at or above a selected binding strength (e.g., a tight binder) , remains bound for a substantial part the capillary electrophoresis run time, and so that any additional complex (es), formed between the target and any additional ligand (s) binding to the target below the selected binding strength (e.g. a weak binder), dissociates prior to reaching the CE/MS interface at the outlet end of the CE conduit.
- a selected binding strength e.g., a tight binder
- the migration of the target during capillary electrophoresis is tracked, enabling one to obtain at least one capillary electrophoretic profile of the sample/target plug.
- the target's migration may be tracked by, e.g., an ultraviolet light absorbance or fluorescence detector coupled to the CE capillary.
- on-line MS detection can be used exclusively, obviating the need for absorbance or fluorescence detection.
- the compounds from the electrophoresed sample/target plug are introduced from the capillary electrophoresis instrument into an on-line mass spectrometer interfaced with the CE instrument.
- the compounds from the electrophoresed sample/target plug are subjected to ionization and then mass spectrometry analysis (i.e., the mass spectra of the ions are analyzed).
- the mass spectrometry data are gathered.
- the mass spectrometry data of the ionized sample/target compounds are analyzed to determine the mass and other structural data of any detected target-binding ligand, including a candidate ligand having the desired binding strength.
- Structural data may be provided by performing collision-induced dissociation (CID) mass spectrometry or other appropriate fragmentation process, e.g., post-source decay, in-source fragmentation, etc.
- CID collision-induced dissociation
- Figure 2 is a schematic representation of the capillary electrophoretic assay screening method of the invention.
- the method of the invention allows on-line identification and characterization of candidate ligand (s) present in a complex biological sample that can bind to a selected target at a selected binding strength. For instance, tight-binding ligands can be detected preferentially even in the presence of high concentrations of weaker-binding ligands.
- the candidate ligand is often one that was previously unidentified as a ligand to the target. Characterization of any candidate ligand detected includes determination of its mass data.
- Fig. 1 summarizes the method generally.
- Fig. 2 illustrates pictorially the method of the invention in general.
- the target (TG) is first mixed with the complex biological sample (CBS) to be screened.
- a target molecule (TG) used in the present method may be, for example, a protein or nucleic acid involved in a disease process or a protein or gene to be regulated in a physiological setting.
- Examples of "complex biological samples” (CBS) include but are not limited to: naturally occurring samples, products or extracts; various biological preparations; chemical mixtures; libraries of pure compounds; and combinatorial libraries of synthetic compounds.
- natural extracts include extracts of terrestrial plants; extracts of marine plants; extracts of marine organisms; microbial broths; and microbial extracts.
- the TG will form complexes with any binding ligand (s) present in the sample, including weak-binding ligands (WLs) , moderate-to-tight binding ligands (MTL) , and tight-binding ligands (TLs) .
- WLs weak-binding ligands
- MTL moderate-to-tight binding ligands
- TLs tight-binding ligands
- the electrophoretic conditions affect the stability of weak and strong ligand/target complexes to different degrees. Therefore, one can set the CE conditions so that weak ligand/target complexes will substantially dissociate during the CE so that any weak ligand/target complexes remaining will be below the particular detector's detection limits. At the same time, the CE conditions should allow tight-binding ligand/target complexes to remain present at a level above the detection limits of the detector.
- WLs weaker-binding ligands
- TLs tight-binding ligands
- Factors governing the relative binding strength of a ligand to a target include, but are not limited to: capillary length; the distance between the CE starting point and the detector; CE run time; voltage and temperature during CE; and buffer composition, such as its pH and/or salt concentration.
- buffer pH values may be within the range of about pH 3 to about pH 10; CE voltages may be about 5-30kV; and salt concentrations may be within the range of about 0-100 mM.
- the CE conditions are experimentally predetermined prior to carrying out the CE/MS screening method. That is, one can establish the exact running buffer(s), temperature, voltage, and other CE conditions by using known WLs and TLs, and/or by generally usually known guidelines regarding interactions between particular types of molecules (e.g., protein-protein, protein-DNA. etc.).
- the conditions are set so that WLs have high off-rates and substantially dissociate from the target (TG) before reaching the outlet end of the CE conduit and entering the mass spectrometer, preferably early in the CE run.
- TLs typically have low off-rates and stay bound to the target for sufficient time during CE and in an adequate amount so as to shift the target's CE profile or pattern detectably (whether in terms of a time shift or change in peak shape or size) , whether detection is by post- CE MS or by an absorbance or fluorescence detector during CE.
- the CE conditions are preferably such that a detectable amount of TL remains bound to the target for a substantial part of the CE run time, preferably at least 50%, preferably at least 80%, most preferably for substantially the entire CE run so that the TL/TG complex reaches the outlet end of the CE conduit.
- the method of the invention is particularly advantageous in identifying and characterizing, in a screened sample, candidate ligand compound (s) having a binding strength higher than a selected threshold and for determining their relative binding strengths.
- “Weak- binding” ligands typically have faster off-rates (K off ) and higher dissociaton constants (K d ) , and form target/ligand complexes that are unstable and fall apart relatively quickly before reaching the outlet end of a CE conduit such as a capillary or microchip channel.
- "tight- binding" ligands have lower K d and slow off-rates (K Gff ) , forming target/ligand complexes, a substantial amount of which remains bound for a substantial portion of the CE run, preferably at least until the outlet end of the capillary.
- “Moderate-binding" ligands will have intermediate K d and K off values. Exemplary criteria for defining ligands of a relative binding strength are shown in Table 1.
- tight versus moderate- to-weak binding ligands may be determined by how they respectively affect or modulate the shape of the target's migration peak, e.g., by reducing the area of the unbound target peak and/or by producing a new bound target/ligand complex peak.
- a functional definition of a tight-binding ligand may be one that forms a target/ligand complex of which a detectable amount holds together during CE so that the complex's dissociates by no more than 50% after about 1.5-5.0 minutes into a CE run.
- a weak-binding ligand would then be one that forms a target/ligand complex of which a substantial amount, at least 50% and preferably at least 80%, dissociates during CE so that the complex's migration peak is reduced by at least 90% after about 1.5-5.0 minutes into a CE run.
- the screening method itself uses the fact that the rate of ligand/target complex dissociation is different for weak-binding ligands and tight-binding ligands. Therefore, using optimized CE conditions, one can achieve complete dissociation of weak ligand/target complexes, or at least enough dissociation so that any remaining weak ligand/target complex falls below the threshold for detection during CE.
- the tight-binding ligand/target complex may dissociate to some degree, but sufficient TL/TG complex must remain to be at a concentration above the detection limits of the particular detector. For instance, a mass spectrometer's detection limit is typically within the range of about lOOnM-lOO ⁇ M of a compound.
- the only other distinctive signal (s) that would be detected would be any signal due to the shifted CE migration or mobility of the target when bound to a tight-binding ligand.
- One of ordinary skill in capillary electrophoresis will be able to determine, in light of this disclosure, how to distinguish between weak and tight-binding ligands to a selected target, depending on the particular CE conditions and the particular detection limits.
- This method allows one to determine whether one or more tight-binding ligand (s) is/are present in the biological sample even in the presence of high concentrations of weak ligands. This fact will hold true as long as the tight- binding ligand/target complex is formed in the pre-capillary incubation step of a complex biological sample with a target in a concentration above the detection limits of the detector used to track the migration of the target.
- the detector may be, for example, a UV absorbance detector or a laser-induced fluorescence (LIF) located along the length of the CE conduit, preferably near the outlet end of the conduit, or may be the mass spectrometer itself.
- any weak-binding ligand/target (WL/TG) complex should rapidly dissociate and the TG should migrate essentially as unbound TG.
- Any candidate tight-binding ligand/target (TL/TG) complex that forms must remain largely intact, with a substantial amount of the complex staying bound together for a substantial portion of the CE run time, at least approximately 50% and more preferably, approximately at least 80%. That is, enough of the TL/TG complex must remain together for a sufficient time during CE so as to be detectable during MS.
- the method of the invention may be practiced with or without a known, charged, preferably weak- binding, competitive ligand (CL) in the CE running buffer (RB) , the CL serving to change the CE migration time of the target as it moves through this buffer.
- CL competitive ligand
- An exemplary known charged competitive ligand is one that binds weakly to the target and has a dissociation constant (K d ) greater than 1.0 ⁇ M and an off-rate (K C ff) greater than 1.0 (s _1 ) .
- a candidate TL happens to be neutrally charged, a new TL/TG complex peak may not be detected in running buffer free of CL as the complex's migration may not shift from that of unbound target.
- the neutral TL's presence may be detected only by including a known, charged, preferably weak-binding, CL in the running buffer.
- the target bound by the neutral, tight-binding candidate ligand would not be able to interact with the CL, so that the target's migration rate through the CL-containing running buffer would no longer be shifted by the CL but would revert back towards a migration rate of the target alone when in running buffer free of CL.
- a new TL/TG peak should be detected whether or not the CL is present in the running buffer, unless the TL has the same charge as the CL so that the TL shifts the target's migration to the same extent as the CL.
- two CE runs of the sample/target plug may be desired, one with and one without CL in the running buffer, to establish unequivocally the presence in the CBS of a candidate ligand having the desired binding strength.
- determining the presence of a candidate ligand comprises comparing the sample/target plug' s capillary electrophoretic profile in that buffer with a reference standard comprising a capillary electrophoretic profile of the target in running buffer free of any target-binding ligand and a capillary electrophoretic profile of the target in running buffer that includes the known, charged competitive ligand. Determining the mass and/or structure of the candidate ligand will be simplified if the mass spectra of the known competitive ligand is known and can be used as a reference standard in addition to the mass spectra of the target alone.
- Detection of the target and stable ligand/target complexes can be performed by several well-established detection methods, including but not limited to UV light absorbance, laser-induced fluorescence (LIF) , or on-line MS. Tracking the migration of the target produces a capillary electrophoretic profile or migration pattern, which can be analyzed to determine whether the screened CBS contains any ligand capable of binding to the target at a selected binding strength.
- LIF laser-induced fluorescence
- MS ionization technique including: electron ionization (El); electrospray ionization (ESI); matrix-assisted laser desorption ionization (MALDI) ; chemical ionization (CI) ; atmospheric pressure chemical ionization (APCI); and thermospray ionization (TSI) .
- El electron ionization
- ESI electrospray ionization
- MALDI matrix-assisted laser desorption ionization
- CI chemical ionization
- APCI atmospheric pressure chemical ionization
- TSI thermospray ionization
- the mass spectrometer may be configured for time-of- flight (TOF) mass spectrometry, single-quadrupole mass spectrometry, triple-quadrupole mass spectrometry, Fourier transform mass spectrometry, or fast atom bombardment (fab) mass spectrometry.
- TOF time-of- flight
- single-quadrupole mass spectrometry single-quadrupole mass spectrometry
- triple-quadrupole mass spectrometry triple-quadrupole mass spectrometry
- Fourier transform mass spectrometry or fast atom bombardment (fab) mass spectrometry.
- fab fast atom bombardment
- MS techniques enabling high through-put of samples.
- the on-line CE/MS set-up allows one to detect the target's CE migration pattern by MS data alone, if so desired, as can be appreciated by one of ordinary skill in the art in view of the teachings of this disclosure. For instance, one can use single-ion monitoring to track the target's CE mobility through its m/z (mass
- the CE profile of the target can be provided by mass spectrometry data because a plug of unbound target has a different CE mobility from that of a target tightly bound to a charged ligand. Therefore, after CE, unbound target and target that has been bound tightly to a ligand will arrive at and enter the mass spectrometer at respectively different times. As a result, there will be different peaks or distributions in the mass data occurring at different times, which will correspond to unbound TG, to TG that has been complexed to TL, and to the TL. As well, the size or area of each peak may differ and changes can be monitored. One can monitor the target alone, observe its location and compare its mass spectra in relation to its time of arrival, and then compare these data to the MS data for a mixture of target and ligand.
- the mass spectrometer as the sole detector is that it eliminates the need for labelling or derivatizing the target molecule to make it detectable. As well, the mass spectrometer allows a certain selectivity in looking for candidate ligands that bind at or above a desired binding strength.
- a mass spectrometer is a particularly useful detector when using a known, charged competitive ligand (CL) in the CE running buffer, since the mass spectra of the target and any other target-binding ligand will not be affected by the CL. Moreover, if one detects a tight-binding ligand/target
- the complex may break apart or stay together. In either case, the individual ion masses are determined in the mass spectrometer. If detection is by another method, such as light absorbance detection (e.g., ultraviolet (UV) absorbance) or fluorescence detection
- LIF laser-induced fluorescence
- CE/MS control runs must be performed. The first is performed with complex biological sample alone, to determine those components in the CBS that co-migrate with the TL/TG complex. The second is performed with TG alone to determine any masses that are associated with the TG. By subtractive analysis of the data from the one experimental CE/MS run of a plug of the sample/target mixture, and from the two control runs, one then determines the mass(es) of the TL(s).
- the complex biological sample is composed of a mixture of compounds of known masses, such as mixtures of synthesized compounds, a single MS analysis may be enough to completely identify the compound. Sometimes it may be necessary to re-analyze the ions in the mass spectrometer to obtain additional structural information about the TL. Such re-analysis is possible if one uses, e.g. an ion trap mass or Fourier Transform mass spectrometer.
- the method may further comprise subjecting the electrophoresed sample/target compounds to collision-induced dissociation (CID) during mass spectrometry to generate CID data about the target, any detected candidate ligand, or both.
- CID collision-induced dissociation
- This method is useful to detect the structure and molecular weight of unknowns, such as may be found in natural products. This step is particularly useful for differentiating compounds having the same molecular weight, by the fragmentation behaviors of the ions upon CID. For instance, CID analysis is helpful when screening for a candidate target-binding ligand among a library of known compounds having known molecular weights. Analyzing CID data of an electrophoresed sample/target plug allows one to identify and to determine the structure of any candidate ligand detected.
- the invention provides a method of screening complex biological material for and characterizing any candidate ligand that binds to a selected target at or above a selected binding strength.
- one embodiment of the method comprises the steps of:
- step (3) injecting a plug of the sample/target mixture from step (2) into an inlet end of a conduit of a capillary electrophoresis instrument;
- a "capillary electrophoretic profile” is the migration pattern obtained by tracking the target upon capillary electrophoresis of, e.g., a plug of target alone or a sample/target plug.
- the target's migration may be tracked by various detection means, including a fluorescence or absorbance detector or a mass spectrometer.
- a "candidate ligand” is one that binds tightly to the selected target at or above a selected binding strength, preferably a tight-binding ligand.
- a “detectable amount of the first complex” is that amount of candidate ligand/target complex that is above the lower detection limit of the particular detector used, especially the mass spectrometer. Generally at least approximately 50%, more preferably at least approximately 80%, of the first complex remains bound for a substantial part of the capillary electrophoresis run time. The "substantial portion of the capillary electrophoresis run time” is at least approximately 50%, and more preferably at least approximately 80% of the entire duration of the CE run. The "substantial amount of any additional complex" of any additional, weak-binding ligand and target that dissociates, is such that the amount of any remaining additional complex that reaches the mass spectrometer is below the mass spectrometer's lower detection limit.
- CE conditions are those that enable a detectable amount of the first complex to remain substantially intact for the entire capillary electrophoresis run while at least 50% of any additional complex (es) of a weaker-binding ligand (s) and target dissociate (s) within the first 20% of the capillary electrophoresis run time.
- the sample/target plug's capillary electrophoretic profile may be obtained by tracking the target's migration at a detection point along a length of the conduit, by means of, e.g., a light absorbance detector or a fluorescence detector.
- the sample/target plug's CE profile may be provided by the post-capillary mass spectrometry data.
- Determining the presence, in the complex biological sample, of a candidate target-binding ligand having the requisite binding strength comprises comparing the sample/target plug's capillary electrophoretic profile with a reference standard comprising a capillary electrophoretic profile of a plug of the target in the absence of any target-binding ligand.
- Determining the mass of any detected candidate ligand comprises comparing mass spectrometry data
- any detected candidate ligand may entail comparing not only the mass spectra of various compounds, including the target and various ligands, but also comparing the fragmentation data (e.g., collision- induced dissociation data) of the electrophoresed sample/target compounds to a reference standard comprising fragmentation data of the target alone in the absence of any target-binding ligand and/or fragmentation data of the target together with a known target-binding ligand.
- CE conditions e.g., temperature, buffer composition, voltage, etc.
- PCT/US98//27463 International Application No. PCT/US98//27463
- the present method can be used to rank detected candidate ligands according to their relative affinities for a selected target and thus, their potential value as therapeutic or diagnostic compounds.
- small molecules e.g., many drug-like compounds
- their CE off-rates correlate to their relative binding strengths. Small molecules are approximately ⁇ 1000 daltons in size. Therefore, the present method can be used to rank candidate ligands according to their relative affinities for a selected target and thus, their potential value as therapeutic or diagnostic compounds.
- CE/MS screening method of the invention may be practiced to detect and to characterize moderate-binding ligands that may not be as easily detected in the previously discussed embodiments.
- a CE running buffer is made up that includes the complex biological sample (e.g., a mixture of synthetic compounds that are potential ligands to a selected target) .
- a plug of the target is injected into and electrophoresed in a CE conduit filled with this CBS-containing running buffer. As the target migrates through this buffer, the target is able to bind to any target-binding ligands present in the CBS.
- any potential target-binding ligand (s) tend(s) to be present in higher concentrations, as compared to the previous embodiment in which the target and CBS are first mixed together and then a plug of that mixture is injected into the CE conduit (i . e. , where the running buffer does not include CBS) . Therefore, moderate-to-weak ligands are then better able to stay bound to the target and thus to be detected.
- the steps of this embodiment, having CBS in the running buffer comprise:
- step (6) using mass spectrometry data from step (4) to determine a mass of any candidate target-binding ligand detected.
- the data collected from this screening method will be compared to reference standards comprising the CE profile and mass spectrometry data from a target plug alone electrophoresed in buffer free of CBS, and/or mass spectrometry data from CBS alone.
- the method of the invention is particularly advantageous for screening a split-and-pool combinatorial library.
- This library is a mixture of synthetic compounds containing anywhere from several tens to several thousands of compounds of known masses. Often, many of these compounds have weak binding activity, i.e., are weak-binding ligands (WLs) . They are also frequently present at very high concentrations, in total. It is desirable, but difficult, to find the rare combinatorial derivative compounds in the library that are tight-binding ligands
- TLs TLs
- MS in conjunction with optimized CE conditions, not only detects such a rare TL, but also identifies its mass and sometimes its substructure as well (through ion fragmentation, such as by collision-induced dissociation during MS) . Using this information, one may be able to directly determine, without further steps, which component of the original combinatorial library is the TL.
- an active split-and-pool library i.e., one that contains a potential target-binding ligand
- the present on-line CE/MS screening method eliminates or substantially reduces the need to perform such complex procedures and analyses.
Abstract
Description
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
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JP2000559424A JP2004500541A (en) | 1998-07-10 | 1999-07-09 | Methods for the detection and analysis of strong binding ligands in complex biochemical samples |
EP99933790A EP1095267A1 (en) | 1998-07-10 | 1999-07-09 | Method to detect and analyze tight-binding ligands in complex biological samples |
CA002336746A CA2336746A1 (en) | 1998-07-10 | 1999-07-09 | Method to detect and analyze tight-binding ligands in complex biological samples |
US09/743,499 US6432651B1 (en) | 1998-07-10 | 1999-07-09 | Method to detect and analyze tight-binding ligands in complex biological samples using capillary electrophoresis and mass spectrometry |
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US9240398P | 1998-07-10 | 1998-07-10 | |
US60/092,403 | 1998-07-10 | ||
US9429798P | 1998-07-27 | 1998-07-27 | |
US60/094,297 | 1998-07-27 | ||
USPCT/US98/27463 | 1998-12-23 | ||
PCT/US1998/027463 WO1999034203A1 (en) | 1997-12-24 | 1998-12-23 | Capillary electrophoretic method to detect target-binding ligands and to determine their relative affinities |
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Cited By (14)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2003054549A2 (en) * | 2001-12-08 | 2003-07-03 | Micromass Uk Limited | Method of mass spectrometry |
US6680203B2 (en) | 2000-07-10 | 2004-01-20 | Esperion Therapeutics, Inc. | Fourier transform mass spectrometry of complex biological samples |
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US6919178B2 (en) | 2000-11-21 | 2005-07-19 | Sunesis Pharmaceuticals, Inc. | Extended tethering approach for rapid identification of ligands |
WO2005080960A1 (en) * | 2004-02-23 | 2005-09-01 | Toyo Kohan Co., Ltd. | Solid support and method of mass spectrometry through desorption/ionization of multiple substances or composites on solid support |
US6965832B2 (en) | 2000-04-07 | 2005-11-15 | Millennium Pharmaceuticals, Inc. | Investigating different physical and/or chemical forms of materials |
US6998233B2 (en) | 1998-06-26 | 2006-02-14 | Sunesis Pharmaceuticals, Inc. | Methods for ligand discovery |
US7061605B2 (en) | 2000-01-07 | 2006-06-13 | Transform Pharmaceuticals, Inc. | Apparatus and method for high-throughput preparation and spectroscopic classification and characterization of compositions |
US7108970B2 (en) | 2000-01-07 | 2006-09-19 | Transform Pharmaceuticals, Inc. | Rapid identification of conditions, compounds, or compositions that inhibit, prevent, induce, modify, or reverse transitions of physical state |
JP2008180728A (en) * | 2008-03-07 | 2008-08-07 | Toyo Kohan Co Ltd | Solid support, and method of analyzing mass-spectrometrically a plurality of substances or complex on solid support by eliminating/ionizing those |
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 |
US7927791B2 (en) | 2002-07-24 | 2011-04-19 | Ptc Therapeutics, Inc. | Methods for identifying small molecules that modulate premature translation termination and nonsense mediated mRNA decay |
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US6998233B2 (en) | 1998-06-26 | 2006-02-14 | Sunesis Pharmaceuticals, Inc. | Methods for ligand discovery |
US7108970B2 (en) | 2000-01-07 | 2006-09-19 | Transform Pharmaceuticals, Inc. | Rapid identification of conditions, compounds, or compositions that inhibit, prevent, induce, modify, or reverse transitions of physical state |
US7061605B2 (en) | 2000-01-07 | 2006-06-13 | Transform Pharmaceuticals, Inc. | Apparatus and method for high-throughput preparation and spectroscopic classification and characterization of compositions |
US6965832B2 (en) | 2000-04-07 | 2005-11-15 | Millennium Pharmaceuticals, Inc. | Investigating different physical and/or chemical forms of materials |
US6680203B2 (en) | 2000-07-10 | 2004-01-20 | Esperion Therapeutics, Inc. | Fourier transform mass spectrometry of complex biological samples |
US6919178B2 (en) | 2000-11-21 | 2005-07-19 | Sunesis Pharmaceuticals, Inc. | Extended tethering approach for rapid identification of ligands |
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US7927791B2 (en) | 2002-07-24 | 2011-04-19 | Ptc Therapeutics, Inc. | Methods for identifying small molecules that modulate premature translation termination and nonsense mediated mRNA decay |
US8224582B2 (en) | 2003-07-02 | 2012-07-17 | Sergery Krylov | Non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM)-based methods for drug and diagnostic development |
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 |
WO2005080960A1 (en) * | 2004-02-23 | 2005-09-01 | Toyo Kohan Co., Ltd. | Solid support and method of mass spectrometry through desorption/ionization of multiple substances or composites on solid support |
JP4644263B2 (en) * | 2008-03-07 | 2011-03-02 | 東洋鋼鈑株式会社 | Solid support and method for mass spectrometry by desorption / ionization of a plurality of substances or complexes on the solid support |
JP2008180728A (en) * | 2008-03-07 | 2008-08-07 | Toyo Kohan Co Ltd | Solid support, and method of analyzing mass-spectrometrically a plurality of substances or complex on solid support by eliminating/ionizing those |
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