Classifications

• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
  • H01J49/00—Particle spectrometers or separator tubes
  • H01J49/02—Details
  • H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00277—Apparatus
  • B01J2219/00279—Features relating to reactor vessels
  • B01J2219/00306—Reactor vessels in a multiple arrangement
  • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
  • B01J2219/00315—Microtiter plates
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/00603—Making arrays on substantially continuous surfaces
  • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/00603—Making arrays on substantially continuous surfaces
  • B01J2219/00659—Two-dimensional arrays
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/0068—Means for controlling the apparatus of the process
  • B01J2219/00702—Processes involving means for analysing and characterising the products
  • B01J2219/00707—Processes involving means for analysing and characterising the products separated from the reactor apparatus
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00718—Type of compounds synthesised
  • B01J2219/0072—Organic compounds
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
  • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

• the present invention relates to an automated, high-throughput method of screening a plurality of chemical compounds to detect interaction of those compounds with a target using mass spectrometry ("MS”) or tandem mass spectrometry (“MS/MS”) and/or to determine the affinity of the binding interaction.
• MS mass spectrometry
• MS/MS tandem mass spectrometry
• Molecular recognition events are a hallmark of biological systems. For example, particular enzymes interact with specific substrates, histones specifically bind to DNA, major histocompatibility complexes are specifically recognized by T cell receptors, and immunoglobulins exhibit extremely specific binding to certain antigens.
• researchers in the life sciences seek to understand and exploit these molecular recognition events within a specific biological system to discover methods to manipulate the system to obtain a desired result.
• the paradigm approach is the use of a chemical compound (a drug) to inhibit binding of a naturally occurring ligand to a receptor in order to achieve a desired therapeutic effect.
• molecular recognition events to manipulate biological systems with chemical compounds is used by the agrochemical industry (e.g., herbicides, fungicides, insecticides) as well as the pharmaceutical and biotechnological industries.
• a specific molecular recognition event between a target and ligand as a means of separating the target from a complex mixture by attaching the ligand to a stationary phase, combining the mixture in a mobile phase (e.g., solvent or gas) with the ligand in a stationary phase under conditions in which the ligand and target will bind, eluting the undesired elements from the mixture, and then eluting the target from the ligand.
• a mobile phase e.g., solvent or gas
• the conventional process used to detect these molecular recognition events between a target and chemical compound relies on biological screens to detect the effect of the compound on the biological system, or subset of the biological system.
• biological screens For example, in order to detect the effect of a chemical compound on a receptor, one would ordinarily develop a screen involving the receptor, its natural ligand, and a reporter element (such as a fluorescent compound) that is activated in proportion to activation of the receptor. If the compound of interest inhibits the receptor-ligand interaction, the screen would quantitatively detect the inhibition effect.
• assay development and validation is usually an expensive and time-consuming process, even with high-throughput screening methods.
• Assay development usually requires extensive knowledge of the particular biological system.
• a new screening method must be developed and validated for each biological target, although similar methods may be used with similar targets.
• genomics technology has enabled researchers to link biological targets with disease states without knowledge of target function or the biological system in which the target participates.
• one In order to use conventional biological assays with such uncharacterized targets, one must identify the biological function of the target with little information to provide useful guidance. This problem exacerbates the time and expense required for assay development and validation.
• Electrospray and related atmospheric pressure ionization (API) techniques such as ionspray (pneumatically assisted electrospray) and atmospheric pressure chemical ionization (Apcl) form gas phase ions directly from solution at atmospheric pressure via protonation (or deprotonation in the negative ion mode) followed by ion evaporation.
• the atmospheric ionization techniques not only ionize small molecular weight compounds, but large macromolecules as well. In fact, these ionization techniques can also provide a multiple number of charges on macromolecules capable of stabilizing them.
• a continuous infusion electrospray MS interface is set up using a syringe infusion pump to continuously infuse solution mixtures containing a specific target, such as a macromolecule, and a molecule of interest, such as a potential ligand, into a mass spectrometer.
• the syringe is usually filled with the solution mixture and attached to the infusion pump.
• a small diameter capillary is attached from the syringe needle directly to the particular API interface of the mass spectrometer, and infusion of the solution begins.
• the normal infusion flow rate is slow, and typically ranges from about 2 ⁇ l /minute to about 20 ⁇ l/minute.
• the mixtures are sprayed, evaporated, and ionized with the aid of a pneumatically-assisted nebulizing gas while continually flowing into the mass spectrometer.
• the gas phase ions including the targets, the molecules of interest, and any complexes formed therefrom are detected and identified by the mass spectrometer.
• the continuous infusion method has been used with biologically relevant macromolecules because such targets require an aqueous solution in order to maintain the established solution equilibrium of the target-ligand complex under physiological conditions.
• the aqueous solution causes an unfavorable signal-to-noise ratio.
• continuous infusion of the mixture during the MS experiment (usually 10-15 minutes cycle time between different samples) is used so that the data may be acquired using the multiple channel average (MCA) mode of the mass spectrometer.
• MCA multiple channel average
• the MCA mode allows the individual scans to be continuously accumulated over a period of time. This accumulation of data enhances a relatively weak signal of a target-ligand complex above the random chemical background noise originating from the aqueous medium.
• the accumulated MCA scan provides better sensitivity, and a superior signal to noise ratio, than any single discrete scan or average number of scans. This method, however, requires a sample analysis time on the order of greater than ten minutes.
• the present invention is based on the finding that non-covalent binding interactions can be detected using a high-throughput, automated screening method that is more efficient than conventional high-throughput biological screening methods (including both assay development costs and screening costs).
• the method takes advantage of flow injection MS methodology. It has been discovered according to the invention that non-covalent interactions can be detected using flow injection analysis to reduce the sample analysis time significantly.
• the survival of non-covalent interactions with the electrospray MS technique allows one to directly transfer intact compound-macromolecule complexes from a solution into a mass spectrometer without chromatographic separation of the mixture of the compound, macromolecule, and complex. Once the molecules are transferred to the mass spectrometer and ionized, they can be identified by their mass-to-charge ratio.
• the methods of the invention not only confirm the hypothesis that non-covalent interactions can be detected using electrospray ionization MS techniques but enable a novel method for the high throughput screening of a plurality of compounds for a non-covalent interaction with a target.
• the ability to perform such a high throughput screening method was not known because of the conditions that researchers believed were required to maintain and detect a non-covalent interaction.
• the prior art methods which were used to examine the non-covalent binding interactions involved continuous infusion MS.
• an aqueous solution which is an adequate environment for maintaining a non-covalent binding interaction, causes an unfavorable signal-to-noise ratio.
• continuous infusion of the mixture is used so that the data may be acquired using the MCA mode of the mass spectrometer.
• the MCA mode which enhances a relatively weak signal, however, requires a sample analysis time on the order of greater than ten minutes. It was discovered according to the instant invention that non-covalent binding interactions can be screened using flow injection MS, under conditions in which the sample analysis time is on the order of five minutes or even as low as one minute or 30 seconds, as compared to the sample analysis time often minutes for continuous infusion MS.
• the methods of the invention also solve the problem that continuous infusion electrospray MS is too labor-intensive and time-consuming for practical use in screening the hundreds of thousands of compounds that currently exist in pharmaceutical company collections, as well as the millions of compounds being developed using combinatorial chemistry techniques. Rather than using the continuous infusion method, researchers in the life sciences would continue to use the more conventional high-throughput screening methods that are primarily based on biological function because these methods are less expensive on a per unit basis than continuous infusion electrospray MS. The methods of the invention, however, provide a rapid high throughput method for screening hundreds of thousands of molecules in a relatively short time.
• One aspect of the present invention is directed to a method for the rapid testing of a plurality of compounds for non-covalent interaction with a target.
• the method of the invention involves the steps of providing a continuous flow of a mobile phase into a mass spectrometer; providing settings for the mass spectrometer for detecting at least one target in the mobile phase; sequentially injecting individual samples of a plurality of mixtures, wherein each mixture comprises the at least one target and at least one compound of interest, into the mobile phase for delivery into the mass spectrometer; and obtaining a mass spectrum that indicates for each mixture the presence or absence of a complex of a target and a compound of interest.
• each sample is completely injected into the mobile phase within less than 5 minutes, and more preferably within less than 1 minute and even more preferably within less than 30 seconds.
• each sample is analyzed within less than 5 minutes, and more preferably within less than 1 minute and even more preferably within less than 30 seconds.
• the mass spectrometer is tuned to more accurately detect the target in the mobile phase. This is accomplished, in part, by adjusting the mobile phase to optimize overall mass spectrometric sensitivity while maintaining optimum solution equilibria for each mixture.
• the mass spectrometer may also be tuned by setting a minimum detection threshold based on the relative strength of interaction between the at least one target and the at least one compound of interest. This is preferably accomplished utilizing automated flow-injection analysis up-front collision induced dissociation mass spectrometry.
• the method of the invention may be multiplexed for higher throughput by using mixtures that contain one target and a plurality of compounds of interest, one compound of interest and a plurality of targets, or a plurality of compounds of interest and a plurality of targets.
• the target is a biological target.
• the biological target is a receptor protein.
• Each of the mixtures that are tested using the method of the invention are preferably formed in a plurality of addressable locations, such as the individual wells in a multiple-well plate.
• the samples are then sequentially obtained from the addressable locations, and sequentially injected into the mobile phase for delivery to the mass spectrometer, such as by obtaining the samples from a multiple-well plate using an autosampler.
• At least one complex of a target and compound of interest are dissociated to determine the relative strength of the interaction between the target and the compound of interest within each complex.
• a preferred method for dissociating the complex is through the use of automated selected reaction monitoring flow-injection analysis mass spectrometry.
• the identities of one or more of the targets and the identities of one or more of the compounds of interest that complex with the targets may be determined based on the molecular mass of the dissociated targets and compounds of interest.
• a method for rapid determination of relative non-covalent binding affinity of at least one compound of interest and at least one target includes the steps of providing a continuous flow of a mobile phase into a mass spectrometer; providing settings for the mass spectrometer for detecting at least one target in the mobile phase; sequentially injecting individual samples of a plurality of mixtures, wherein each mixture comprises the at least one target and at least one compound of interest, into the mobile phase for delivery into the mass spectrometer; dissociating at least one complex of a target and compound of interest; and obtaining a mass spectrum that indicates for each mixture the relative strength of the interaction between the target and the compound of interest within each complex based on ability of the complex to be dissociated.
• each sample is completely injected into the mobile phase within less than 5 minutes, and more preferably within less than 1 minute and even more preferably within less than 30 seconds. In another embodiment, each sample is analyzed within less than 5 minutes, and more preferably within less than 1 minute and even more preferably within less than 30 seconds.
• the step of disassociating at least one complex is performed by applying a series of potential energies of increasing strength to the mixtures as each mixture is transferred from a solution phase to a gas phase in the spectrometer.
• the mass spectrum indicates for each mixture the potential energy that was sufficient to dissociate the complex, the potential energy being indicative of the non-covalent binding affinity of a complex between the target and the compound of interest.
• the dissociation of the complex is conducted utilizing automated flow-injection analysis up-front collision induced dissociation mass spectrometry, and applying different up-front cone voltages to create different potential energies.
• the dissociation of the complex is conducted utilizing automated selected reaction monitoring flow- injection analysis mass spectrometry.
• the plurality of mixtures contains a single compound of interest and a single target and a complex thereof and wherein a series of potential energies of increasing strength is applied to the mixtures as each mixture is transferred from a solution phase to a gas phase, and obtaining a mass spectrum that indicates for each mixture which potential energy was sufficient to dissociate the complex, the potential energy being indicative of the non-covalent binding affinity of a complex between the target and the compound of interest.
• the plurality of mixtures each contain a single target and wherein the plurality of mixtures each contain a different compound of interest.
• the plurality of mixtures each contain a single compound of interest and wherein the plurality of mixtures each contain a different target.
• the method of the invention in another embodiment includes the step of forming the plurality of mixtures in a plurality of addressable locations. Prefereably each addressable location is an individual well in a multiple-well plate.
• the method may also include the steps of sequentially obtaining samples from the addressable locations, and sequentially injecting the samples into the mobile phase for delivery to the mass spectrometer. Preferably the samples are obtained from a multiple-well plate using an autosampler.
• the invention is a method for rapid analysis of structural binding properties of a compound that forms a non-covalent interaction with a target.
• the method includes the steps of generating a database of relative binding affinities by performing the method described above for determining relative non-covalent binding affinity and comparing the structures of the compounds of interest having the highest relative binding affinities to determine structural similarities amongst the compounds of interest having the highest relative binding affinities, wherein the structural similarities are indicative of structural binding properties of a compound that forms a non-covalent interaction with the target.
• Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each method.
• Fig. 1 is a chemical structure of insulin ligands.
• Fig. 2 is a mass spectra of insulin and insulin-ligand complexes.
• Fig. 3 is a summary of mass spectrometry results.
• Fig. 4 is a summary of mass spectrometry results.
• Fig. 5 is a graph depicting relative binding strength of 9 insulin-ligand complexes.
• Fig. 6 is a FIA chromatogram of FKBP/Rapamycin solution using a Micromass LCT.
• Fig. 7 is a mass spectra of FKBP/Rapamycin solution using a Micromass LCT.
• Fig. 8 is a mass spectra of Avidin/Biotin solution using a Micromass LCT.
• the present invention relates to an automated, high throughput method for screening a plurality of chemical compounds to identify interactions with one or more macromolecular targets.
• the method allows one of ordinary skill in the art to rapidly detect interactions between a given biological target and a compound of interest, and also provides a method to determine the relative strength of the interaction between the target and compound.
• An electrospray MS technique of detecting non-covalent complexes of a macromolecular target and a chemical compound can be applied to the screening of targets with chemical compounds for the discovery of new drugs.
• Drug molecules often form weak, non-covalent bonds with specific proteins or other biological molecules in a manner that is analogous to a lock and key, and thereby assert their influence on the body through these non-covalent bonds. The better a drug molecule can bind to a target, the stronger the efficacy (and often specificity) the drug will exhibit.
• the term "plurality of compounds of interest” refers to two or more compounds, and includes but it is not limited to, an array or library of compounds generated using combinatorial chemistry techniques, and also includes, but is not limited to, libraries of natural products, libraries of biological molecules (e.g., polypeptides, polynucleotides, oligosaccharides), libraries of compounds produced with combinatorial biology techniques, and any other collection of chemical compounds.
• a “target” as used herein is any structure which can form a non-covalent binding interaction with a compound such as a ligand.
• the target is a biological target.
• the preferred embodiments of the invention are described in terms of a biological target, but the description is not intended to limit the scope of the invention to biological targets. It is apparent to one of ordinary skill in the art that the appended claims cover all targets.
• a target can include multimeric structures as well as a monomeric structure.
• a “biological target” as used herein is a target which is ordinarily found in a living system or which interacts with a compound such as a ligand which is ordinarily found in a living system.
• Biological targets can be natural compounds or synthetic compounds and include for example but are not limited to receptors, polypeptides, proteins, polynucleotides, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), carbohydrates, polysaccharides, and lipids.
• Automated electrospray MS using flow injection analysis has been applied according to the invention to identify non-covalent complexes.
• the method of the invention uses an automated sampling device, such as an HPLC pump equipped with an autosampler, to transport a specific amount of sample in a continuous stream of solvent (mobile phase) to the mass spectrometer via a loop injection.
• an automated sampling device such as an HPLC pump equipped with an autosampler
• a continuous stream of mobile phase is established from the HPLC system to the mass spectrometer, in order to transport the aliquot of sample.
• the mobile phase is only the conduit to transport the aliquot of sample to the mass spectrometer, its composition can be adjusted to optimize the overall mass spectrometric sensitivity while maintaining optimum solution equilibria established for each target-ligand mixture.
• the mobile phase velocity and the amount of tubing connecting the HPLC to the MS determines the time that the injected aliquot will reach the mass spectrometer.
• the injected sample will reach the mass spectrometer faster, increasing the overall throughput.
• the best instrumental parameters can be identified by those of ordinary skill in the art in order to maximize both sensitivity and throughput.
• the overall cycle time from sample to sample will depend on the overall sensitivity of the instrument for the particular molecular target, the injection of the sample in the mobile phase is typically less than 1 minute, compared to about 10 minutes or more per samples for the continuous infusion process.
• the MS can be "tuned", such that the macromolecular target or targets can be readily detected. This is accomplished using the infusion pump method which is well known in the art. Once the MS is tuned to detect the target, the MS will also be able to detect the compound of interest and any complexes that are formed by the macromolecular target and a compound of interest.
• An HPLC/MS interface is set up by connecting the HPLC to an electrospray probe, and the flow rate is optimized for maximum sensitivity for the target macromolecular compound. Typically, a flow rate of about 0.01 to about 0.2 ml/min, preferably about 0.05 to about 0J5 ml/min, is selected as the optimal flow rate.
• Such a flow rate provides sufficient sensitivity to obtain mass spectra of macromolecules, such as receptor proteins or receptor-ligand complexes, within about 5 minutes (total cycle from sample to sample injection), preferably about 2.5 minutes per sample.
• the mass spectrometric parameters can be optimized to eliminate fragmentation of any complexes formed, due to the application of cone voltages, source temperature, etc.
• an HPLC system is set up to provide a flow rate of less than about 0.2 ml/minute, preferably less than about 0J ml/minute, in order to provide an overall analysis cycle time from sample to sample in less than about 5 minutes, preferably less than about 1 minute or 30 seconds, without manual intervention.
• This is a great improvement over the methods of the prior art, which require more than 10 to 15 minutes to analyze a single sample with manual intervention by the operator for each sample. Assuming each sample contains only molecules of a single compound of interest and a single target, plus solvent, the MS will produce, at most, 3 peaks or sets of peaks, depending on the amount of charge distribution that the macromolecule can stabilize.
• the peaks observed include a peak for the macromolecular target, a peak for the small molecular weight compound of interest in the sample, and, if the compound of interest has an interaction with the target, a peak or sets of peaks attributed to the non-covalent complex formed by the two molecules.
• the size of the peak for the complex indicates the relative strength of the interaction between the chemical compound and the macromolecular target. If the interaction is strong, a large peak will result; if the interaction is weak, a smaller peak will result.
• the automated MS method of the invention is particularly useful for enabling high- throughput screening of compounds in combinatorial chemistry libraries to detect interaction between the compounds and a particular protein target by the rapid and direct detection of non- covalent receptor-ligand complexes in the gas phase in a manner that approximates solution phase equilibria.
• Another aspect of the invention provides a method for determining the relative strength of the interaction between a biological target and various compounds of interest by comparing the binding strength of each ligand in relation to the other members of a particular set using automated FIA up-front collision induced dissociation (CID) MS.
• CID collision induced dissociation
• different up- front cone voltages are applied so that different potential energies are created. The greater the potential energy in the instrument, the more energy imparted to the complex being transferred from the solution phase into the gas phase. If the imparted energy is greater than the binding energy involved in the non-covalent interaction, the complex will dissociate prior to entering the first MS and the complex will not be detected. Thus, by changing the cone voltage, only- complexes of a certain binding energy will be seen.
• FIA MS and up-front CID MS can be used to screen large number of compounds (in the hundreds of thousands or more) for interaction with specific biological targets. These compounds can be further studied by an automated selected reaction monitoring (SRM) FIA MS method. Samples are introduced into the mass spectrometer using FIA and maintaining the cone voltage constant. The first MS analyzer (MSI) is set to pass only masses equal to that of the complex.
• SRM selected reaction monitoring
• the selected parent ion such as a receptor-ligand complex
• CID collision induced dissociation
• MS2 second mass analyzer
• This FIA-SRM-MS technique is monitored as a function of increasing collision energy under a constant cone voltage and collision gas thickness, allowing rapid monitoring of the relative binding strengths by dissociating the complex in the gas phase. Because only one ion is selected by MS 1 at any given time for CID, this approach provides increased selectivity and specificity to monitor the relative strength of binding interactions.
• the screening process with the method of the invention. Multiplexing is performed by mixing one compound of interest with a plurality of targets, by mixing a plurality of compounds of interest with one target, or by mixing a plurality of targets with a plurality of compounds of interest.
• the composition of any complex can be determined from the molecular mass of the complex, i.e., the molecular mass of the complex is the sum of the compound and the target that comprise the complex. In a typical random screening against a single target, only one compound in 10,000 will interact with a target and thereby form a complex with the target. Therefore, when the method of the invention is multiplexed, the odds for the formation of multiple complexes in a single sample are very low.
• the multiple MS peaks observed can be differentiated and identified based on their different molecular masses.
• this approach will not address the rare situation where multiple complexes with the same molecular mass are observed. This rare situation may be eliminated with careful selection of targets and compounds in a multiplexed screen to ensure that either the targets or compounds have distinct molecular masses such that isobaric complexes of different molecular structures will not be observed.
• the few isobaric complexes that are likely to occur may be re-analyzed with LC, LC/MS, or MS/MS to identify the components.
• the method of the invention allows one of ordinary skill in the art to rapidly detect interactions between a given biological target and a compound of interest, and also provides a method to determine the relative strength of the interaction between the target and compound.
• the biological function of the compound of interest on the target can be assessed. For instance, if the target compound is a receptor, a biological assay can be performed to determine whether the binding compound functions as an agonist or an antagonist. An assay based on biological function can be performed to confirm that the compound has a desired activity.
• the method of the invention dramatically reduces the number of compounds that require biological testing, such that even the most expensive in vivo animal tests become practical in many cases, based on the assumption that only about one compound in 10,000 will exhibit sufficiently strong interaction with a biological target to warrant confirmatory biological testing.
• the invention therefore provides a rapid, cost-effective method to screen large numbers of compounds to detect a specific interaction with a biological target without knowledge of the biological function of the target or knowledge of the biological system with which that target interacts.
• the method avoids the need for biological assay development and validation in a high- throughput format, although some assay development is useful unless whole animal testing is used.
• the expense of biological screening methods is reduced by decreasing the numbers of compounds that must be tested for a particular target.
• the per unit cost of screening by the method of the invention is less expensive than most screens based on biological function.
• affinity chromatography a mixture of molecules is passed through an affinity column that contains immobilized ligands with an affinity for one of the molecules in the mixture. That molecule will be retained in the column as the other components in the mixture pass through the column. A solvent that causes the molecule to be released from the ligands is then passed through the column, and a solution of the substantially pure molecule is obtained.
• affinity chromatography a mixture of molecules is passed through an affinity column that contains immobilized ligands with an affinity for one of the molecules in the mixture. That molecule will be retained in the column as the other components in the mixture pass through the column.
• a solvent that causes the molecule to be released from the ligands is then passed through the column, and a solution of the substantially pure molecule is obtained.
• large numbers of potential affinity ligands may be screened in a short period of time.
• the present invention provides an automated, high throughput MS and MS/MS method for rapid sequential screening of a plurality of compounds of interest to determine which form non-covalent complexes with macromolecular targets.
• the analysis cycle time from sample to sample, each containing a mixture of at least one compound of interest and at least one macromolecular target compound takes less than about 5 minutes and, preferably, less than about 1 minute and more preferably less than about 30 seconds.
• the invention also encompasses a method for analyzing the structural binding properties of a compound. The method can be accomplished by generating a database of relative binding affinities of a plurality of similar compounds with a target.
• the compounds which have the highest binding affinities for the target reveal the structural binding properties of the compound.
• the only limitation of the methods described herein is based on the state of the art of the equipment. Using the MS equipment available today it may be difficult to detect large, high molecular mass targets. As will be readily understood by one of ordinary skill in the art, as mass spectrometers are developed that allow the detection of molecules of higher molecular mass, the method of the invention can be extended to larger targets.
• a stock solution of 100 ⁇ M hr insulin was prepared in 3% acetic acid.
• a working solution of 25 ⁇ M hr insulin was freshly prepared before use.
• Eighty AA-ADA compounds, individually synthesized and disposed in individual wells of a 96-well microtitre plate, were originally dissolved in 100% DMSO to give a concentration of 5 mM of each. These compounds were respectively diluted with 100% acetonitrile (1 to 5 dilution) to obtain a concentration of 1 mM.
• hr insulin 25 ⁇ M
• 100 ⁇ l of each AA-ADA compound 1 mM
• an approximate molar ratio of 1 to 5 hr insulin to AA-ADA/dimer
• L-Lysine-ADA/dimer known to bind with hr insulin
• L-lysine- AD A/monomer known to disassociate from hr insulin
• Eight QC samples were placed and analyzed in a 96-well microtitre plate between every other 10 samples. These QC samples included hr insulin, AQ41440/monomer, AQ41440/dimer, a mixture of insulin + AQ41440/monomer, and a mixture of insulin + Al 41440/dimer. 10 ⁇ l of each sample was injected through a loop injection into a mass spectrometer.
• Figure 4D shows that there were only two insulin-ligand complexes in wells E4 and F4 observed at a cone voltage of 30 V.
• Figure 4A-4D show that these insulin-ligand complexes were found mostly in row 4 and column E, implying that these ligands made from particular chemical components X and Y used in row 4 and column E tended to associate with insulin.
• a second example of non-covalent interaction that was studied was the well known complex of FKBP (MW 11,822 Daltons) with Rapamycin.
• An 8.5 micromolar solution of FKBP was prepared in 10 millimolar ammonium acetate (pH ⁇ 7).
• a 109 micromolar solution of Rapamycin was prepared in methanol.
• Nine parts of the FKBP were combined with 1 part of the Rapamycin solution to prepare a 7.7:1 micromolar ratio, respectively.
• Two microliters of the resulting solution were injected into a Micromass electrospray time-of- flight instrument equipped with a Shimadzu SCL 10 Avp HPLC system pumping 10 millimolar ammonium acetate (pH ⁇ 7) at 0J mL/min.
• the source temperature of the mass spectrometer was 30C, the cone voltage was 40V and the mass range of 200-3000 amu was scanned in 0J seconds.
• the FIA chromatogram obtained from this analysis is given in Figure 6. Note that because of the faster scan rate possible with a time-of-flight instrument, there are more scans per unit time available, and therefore an enhancement in total sensitivity.
• the mass spectrum of the flow injection analysis of FKBP with Rapamycin is shown in Figure 7.
• the quality of the given spectrum, obtained by FIA/MS is better than typically obtained by continuous infusion compared to quadrupole instruments. Note that the free protein, the free Rapamycin and the protein/Rapamycin complex are easily distinguished in the spectrum with excellent signal-to- noise ratio.
• This method was further investigated using another well-known non-covalent complex that includes a higher molecular weight protein, avidin (MW 63,872 Daltons) complexing with four biotin molecules.
• avidin MW 63,872 Daltons
• a 15.6 micromolar solution of avidin was prepared in 10 millimolar ammonium acetate (pH ⁇ 7).
• a 4J millimolar solution of biotin was also prepared in 10 millimolar ammonium acetate (pH ⁇ 7).
• Nine parts of the avidin were combined with 1 part of the biotin solution to prepare a 14:410 micromolar ratio, respectively.
• a 10 microliter aliquot of the avidin/biotin solution was injected into the electrospray time-of-flight instrument system identified above.

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