WO2015156730A1 - Procédé et appareil pour criblage de médicaments par site de liaison - Google Patents

Procédé et appareil pour criblage de médicaments par site de liaison Download PDF

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WO2015156730A1
WO2015156730A1 PCT/SE2015/050416 SE2015050416W WO2015156730A1 WO 2015156730 A1 WO2015156730 A1 WO 2015156730A1 SE 2015050416 W SE2015050416 W SE 2015050416W WO 2015156730 A1 WO2015156730 A1 WO 2015156730A1
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ligand
pooled
sample
samples
wells
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PCT/SE2015/050416
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Roman Zubarev
Juan Astorga Wells
Thorleif Lavold
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Biomotif Ab
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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/26—Mass spectrometers or separator tubes

Definitions

  • This invention relates to the field of drug screening where libraries of thousands of potential drug candidates are screened against a protein target, and utilizes hydrogen deuterium exchange mass spectrometry as a site-specific binding assay.
  • a disease condition can be defined as an abnormal interaction between groups of biomolecules, which - at the end - will be reflected into a dysfunctional physiological state.
  • the end - will be reflected into a dysfunctional physiological state.
  • the present leading screening technologies are based on the immobilization of the target protein (e.g. surface plasmon resonance, SPR or quartz crystal microbalance, QCM), or by chemical modification in order to produce a detectable signal upon binding (fluorescence). All these technologies present some drawbacks.
  • the immobilization procedure does not always produce a functional protein target.
  • fluorescent labeling is particularly more susceptible for interfering with the binding assay since the drug or the protein needs to be chemically modified to produce a proper signal. In this context, there is an increased interest in developing label-free binding assays.
  • the most commonly used label-free binding assays are isothermal titration calorimetry (ITC), x-ray crystallography and nuclear magnetic resonance (NMR). Although informative, these methods have a number of drawbacks, such as, low sample throughput, high protein consumption, and serious buffer incompatibilities issues. In addition, NMR and X-ray crystallography require extensive sample preparation prior to analysis (crystallization optimization in X-ray crystallography and proper protein expression for NMR).
  • HDX MS Hydrogen/deuterium exchange mass spectrometry
  • the HDX system usually combines on-line pepsin digestion (or uses alternative/complementary proteolytic enzymes) with desalting and LC separation of the resultant peptides by reverse phase chromatography. This is directly followed by electrospray ionization (ESI) MS analysis.
  • ESI electrospray ionization
  • the operations following the quenching such as digestion, sample cleanup and LC separation, must be run in an acidified aqueous solutions kept at low temperature (0 - 4 °C ). These steps must be performed in less than 15-20 min since the incorporated deuterium will slowly be exchanged back by the hydrogen present in the solutions.
  • the HDX MS analysis can be done using one of the two generic approaches.
  • the intensity weighted centroid mass of each peptide is calculated at every incubation time, and the amount of deuterium incorporated is correlated with the corresponding sequence of the protein [Reference 5] .
  • the deuteration kinetics of each particular peptide is compared between the experiments performed with and without the ligand.
  • a single HDX MS analysis including sample digestion, cleanup, and LC MS of a medium size protein requires on average 20-25 min of analysis time.
  • the multifunctional nature of the HDX MS instrumentation which includes two parallel and interconnected fluidic systems (for digestion, cleanup and LC) and the inherent difficulties of working with protein samples result in relatively long regeneration times prior to the injection of the next sample.
  • the regeneration process can take up to 20 min or more.
  • the sample throughput is normally restricted to an average of 45 min per sample cycle for a medium size protein. Therefore, for example, with a sample throughput of 1 sample per 45 min, the screening of a library containing 10,000 compounds will take about 312 days (10 months) to be completed. Such a sample throughput is unacceptably long for most screening project, which limits the use of HDX MS as a screening technique.
  • DeArmond et al. [Reference 6] describes a hydrogen/deuterium exchange- and MALDI mass spectrometry-based assay for screening chemical libraries, with the object to identify ligands to a target protein.
  • Ligands are pooled to obtain a faster, first round of screening, followed by a separate analysis of each ligand from each pool that bound to the target protein.
  • a library of compounds is pooled into groups of 10, without any ligand overlap. For example, a library of 100 compounds is pooled into 10 samples, each one containing a unique set of ligands. Each compound is analyzed only once.
  • the present disclosure provides a method and an apparatus that improve sample throughput, converting HDX MS into a high-throughput technique suitable for drug screening.
  • HDX MS can be used with practically any protein/ligand models without major limitations due to the molecular weight or buffer interferences.
  • HDX MS can be considered "label free", since at the protein level, deuterium (D) behaves very similarly to hydrogen (H).
  • HDX MS is a particularly valuable, and highly complementary to conventional, approach to site-specific drug screening.
  • HDX-MS has been extensively used in binding site characterization
  • its use as a screening tool has not been extensively exploited so far since its throughput has been limited to a few compounds per hour at best, and most commonly only one compound per 45-60 min, as mentioned above.
  • the present disclosure provides a method for screening a plurality of ligands against a target polypeptide by using hydrogen deuterium exchange mass spectrometry, comprising:
  • the expression "the pooled ligand samples from each pair of intersecting rows and columns” means “the pooled ligand samples from each pair of row and column that contain the same unique ligand sample”.
  • Step vi) of the method of the present disclosure may also be described as calculating a deconvoluted signal response of each unique ligand sample, wherein the value of the deconvoluted signal response of each unique ligand sample is the result of a mathematical analysis that combines the hydrogen deuterium exchange signals of the pooled ligand samples from each pair of intersecting rows and columns (i.e. the pooled ligand samples from each row and column that contain the same unique ligand sample.
  • the hydrogen deuterium exchange mass spectrometry may be performed by a membrane-based deuteration device.
  • the solid support used in the method may, for example, be a 96 or 384 or 1536 multiwell or microwell or microtiter well plate or similar wells arrangements.
  • the hydrogen deuterium exchange mass spectrometry signal comprises a selective group of reporting peptides that are either related to the binding site or allosteric regions of the target polypeptide, thereby facilitating and simplifying data acquisition and analysis.
  • the array of sample wells or spots arranged in rows and columns may have more than two dimensions, such as three dimensions.
  • each pooled ligand sample may be mixed with one single target polypeptide or a plurality of different target polypeptides, such as at least two different target polypeptides and up to several hundreds of different target polypeptides, for example 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, 150, 200, 300, 400 or 500 different target polypeptides.
  • each pooled ligand sample is mixed with whole cells or whole cell lysates comprising a plurality of different target polypeptides.
  • the present disclosure further relates to an apparatus for screening a plurality of ligands against a target polypeptide, comprising:
  • a solid support having an array of wells or spots arranged in a plurality of rows and a plurality of columns, possibly wherein each well or spot is configured to contain a unique ligand sample comprising a unique subset of said plurality of ligands;
  • a mixing device configured to mix aliquots of the unique ligand samples of the wells or spots located in the same row to generate a pooled ligand sample from each row, and configured to mix aliquots of the unique ligand samples of the wells or spots located in each column to generate a pooled ligand sample from each column;
  • a mixing device configured to mix each of said pooled ligand samples with a target polypeptide; iv) a mixing device configured to add deuterated water to said pooled ligand samples;
  • a liquid chromatography apparatus configured to be cooled to below room temperature, comprising online digestion, precolumn and analytical column, and a control unit configured to control the injection, digestion, cleanup and/or separation of each pooled ligand sample;
  • a mass spectrometer configured to record a mass spectrum of each pooled ligand sample after elution of each pooled ligand sample from the liquid chromatography apparatus;
  • a computing device comprising a processor and a memory, where the memory is adapted to comprise a computer program which when loaded in the processor is adapted to extract data from each recorded mass spectrum, and to calculate a deconvoluted signal response of each unique ligand sample by applying an algorithm that mathematically combines the hydrogen deuterium exchange signals of the pooled ligand samples from each pair of intersecting rows and columns.
  • the above-mentioned liquid chromatography apparatus is preferably cooled to below room temperature, such as to a temperature between -10°C to 15°C.
  • the apparatus may comprise a membrane-based deuteration device or any other deuteration device compatible with performing hydrogen deuterium exchange mass spectrometry.
  • the array of sample wells or spots arranged in rows and columns may have more than two dimensions, such as three dimensions.
  • FIG. Schematic view of the generation of multiplexed pooled samples, showing a standard 96 well plate (1) arranged into 8 rows (A to H) and columns (1-12). Arrows represent the generation of pooled ligand samples (on each row and column), in accordance with the present invention.
  • FIG. Schematic view of the generation of convoluted ligands (Pooled Ligands).
  • FIG 3. Theoretical Modeling of the Signal Deconvolution Process, showing the data generated from a model HDX MS analysis of a set of Pooled Ligands originating from a 96 well plate (as those shown in FIG 2).
  • Table A shows the mass increase of a given positive reporting polypeptide (compared to the mass of its undeuterated form) in the presence of each pooled ligand (Pooled Ligands A to H and 1 to 12).
  • the deconvolution signal is obtained by calculating the mathematical average of the signal from each pair of intersecting row and columns. Signal deconvolution on a 8 rows/ 12 columns array (B), and the corresponding 3-dimensional surface plot (C) are depicted.
  • FIG 4. A 4-column apparatus for HDX MS Hardware Multiplexing.
  • the apparatus comprises of: autosampler (1), Isocratic Pump for the autosampler (2), pepsin-column washing pump (3), binary LC MS pump, precolumn washing pump (5), a 6-port valves (6, 7), 10-port valves (8, 9), 4-port valves (10, 11), Pepsin Columns (12, 13, 14, 15), Reverse Phase Columns (16, 17, 18, 19), T-connector (20).
  • Letters "W” and “MS” describes the "liquid waste reservoir” and a "mass spectrometer", respectively.
  • FIG. A 6-column apparatus for HDX MS Hardware Multiplexing.
  • the apparatus comprises of: autosampler (1), Isocratic Pump for the autosampler (2), pepsin-column washing pump (3), binary LC MS pump, pre-column washing pump (5), a 6-port valves (6, 7), 10-port valves (8, 9), 4-port valves (10, 11), Pepsin Columns (12, 13, 14, 15), Reverse Phase Columns (16, 17, 18, 19), T-connector (20, 27), analytical columns (23, 24, 25, 26), analytical-column washing pump (27).
  • Letters "W” and “MS” describe the "liquid waste reservoir” and a "mass spectrometer", respectively. This configuration allows the presence of 4 analytical columns.
  • ligand refers to a molecule that binds to a receptor molecule.
  • the receptor molecule may be a protein, a polypeptide, or a peptide.
  • the ligand may be an atom, an ion, a small molecule, RNA, DNA, carbohydrate, polypeptide or lipid.
  • screening refers to the use of a binding assay or an activity assay, in which a plurality of potential ligands are tested against a particular target or a group of targets.
  • the term "chemical library” or “ligand library” refers to a plurality of different chemical compounds originating from chemical synthesis or natural products, which are available to be screened against a given target.
  • the term “convolution” refers to a process by which at least two components are converted or merged into a single component derived by a function, process or operation, with the aim to simplify or improve a given process.
  • deconvolution refers to a process by which the convolution is reversed, and by which the information converted or merged in the convolution process is recovered.
  • reporting polypeptide or “reporting peptide” or “reporting peptide” refer to a single polypeptide/peptide, and a group of polypeptides/peptides, respectively, that are selectively chosen to measure their corresponding mass-to-charge ratio during the HDX MS analysis.
  • peptides from the expected or known binding site are used as positive reporting peptides, and peptides from non-binding sites are used as negative reporting peptides.
  • the term “a plurality” means “more than 1", or “at least 2”.
  • the term “a subset of a [plurality]” is taken to mean “at least 1 and less than the plurality”; more particularly “least 1 and at most one less than the plurality", i.e. 1 ⁇ subset ⁇ ([plurality]-l).
  • the term “hydrogen deuterium exchange signal” refers to the intensity weighted centroid mass of a peptide or it can also refers to the mass increase (in values of intensity weighted centroid mass) of a deuterated peptide compared to its undeuterated mass.
  • HDX MS In ligand screening, a library of chemical compounds is tested against a target protein with the aim to identify those compounds that bind to a particular target.
  • HDX MS is subjecting each single compound to a HDX MS assay against the target polypeptide (one binding assay per each HDX MS analysis). This results in a long time of analysis per ligand, and thus leads to low throughput, which presents a problem when HDX MS is employed in ligand screening.
  • the present invention circumvents this problem by providing the means to perform several binding assays per each HDX MS analysis. It is based on sample convolution, binding assay and subsequent deconvolution via signal processing.
  • Ligands Convolution uses a plurality of wells (an array) arranged into rows and columns (e.g. a microtiter plate), where each well contains a unique set of ligands (FIG 1).
  • the convolution is performed by generating a series of pooled samples from each and every individual column and row. In this manner, a unique pooled sample is generated from every single row and column of the array, converting the array of a plurality of individual ligands into discrete pools of ligands.
  • the generation of pooled samples can be done manually or by a pipetting robot.
  • HDX MS-based Binding Assay Each pooled sample of ligands is mixed with the target protein, and later mixed with deuterium oxide. After an appropriate incubation time, the sample is quenched and subsequently digested, cleaned and concentrated, with peptides separated by liquid chromatography and detected by mass spectrometry. The latter technique measures the number of incorporated deuterium atoms in each positive and negative reporting peptide.
  • a positive binding event will involve a change in the deuterium incorporation into positive reporting peptides in comparison to an assay performed with a non-binding ligand or performed with the target protein alone. Simultaneously, no change should occur in deuterium incorporation in negative reporting peptides.
  • the numerical output signal generated from the binding assay might be expressed in different manners:
  • the numerical value of the output signal may be represented by the intensity weighted centroid mass of at least one positive reporting peptide.
  • the numerical value of the output signal may be represented by the mass increase (intensity weighted centroid mass) of at least one positive reporting peptide compared to the undeuterated mass of the same reporting ion.
  • the present invention can be used with both generic HDX MS approaches (with or without the digestion step).
  • the digestion step is desirable when the binding site characterization is needed.
  • Global Exchange is desirable when the binding site characterization is not needed, or when Top-Down fragmentation is available or possible to apply to a given protein/ligand complex (e.g. ETD or ECD or any other fragmentation technique that does not result in hydrogen scrambling).
  • the present invention may use global HDX with mass spectrometrical techniques - or fragmentation conditions - that produces positional hydrogen/deuterium scrambling during fragmentation.
  • the signal will not provide the localization of the binding site within the protein, but rather it may only provide information as to whether the binding occurred or not.
  • This approach may be useful when the digestion step is difficult to implement or when the digestion is detrimental for the overall sample throughput.
  • the mass spectra of undigested protein sample contain significantly fewer molecular components to analyze, which may result in shorter LC MS gradients and shorter system cleanup.
  • the main problem associated with the HDX MS analysis of intact proteins is related to the increased difficulties to measure the intensity-weighted centroid mass of high mass and multiply charged deuterated species produced by HDX MS.
  • the latter problems may be alleviated by gas-phase fragmentation of the intact protein in order to provide fragments having a lower mass and being less charged.
  • the fragmentation may be done by using fragmentations techniques that do not produce hydrogen scrambling, such as electron transfer dissociation, or those fragmentations techniques (or conditions) that produce hydrogen scrambling, such as collisional dissociation.
  • Ligands Deconvolution This step allows the generation of a numerical (quantitative) binding response for each individual well.
  • the numerical binding value for each ligand is obtained by the mathematical analysis of the signals of two pooled ligand samples where the given ligand is present, using at least one of the given algorithms.
  • the signal deconvolution of a ligand located at the first column and first row is performed by applying a particular algorithm to the signal obtained from the pooled ligands from the first column and the first row. Therefore, the deconvoluted signal from the ligand located on Al position on the 96 well plate in FIG 2, corresponds to the result of the mathematical analysis of Row A and Column 1.
  • the process is repeated for each and every pooled sample from each pair of intersecting rows and columns (Row A / Column 1, Row A / Column 2, Row A / Column 3, et cetera), resulting in a single numerical binding response for each and every single ligand. In the case where only one ligand is present in each well, the result is thus a single numerical binding response for each and every single well.
  • the method is capable of obtaining a quantitative signal response for each individual ligand located on the 96-well plate.
  • each well on the microtiter plate contains a single ligand.
  • the wells with ligands are organized in rows and columns (FIG 2), labeled from A to H and 1-12, respectively.
  • the well contents are then pooled according to rows and columns, mixed with the target polypeptide and analyzed by HDX MS.
  • the signal is related to the site-specific binding of at least one ligand to the target. It is assumed that most ligands do not bind, so only one or a few ligands in the 96-well plate will bind and produce a signal.
  • the generated data on each pooled sample (FIG 3A) is deconvoluted using at least one mathematical function or algorithm.
  • the signal deconvolution shown in FIG 3B is performed by the calculation of the mathematical average of the signal generated by each pair of intersecting rows and columns.
  • the deconvoluted binding signal from each individual ligand is shown on FIG 3B, where a two-colored heat map depicts the signal intensity distribution on a 12x8 sample matrix. In this case, a positive binding signal (decrease in deuterium incorporation) is observed on Row D and Column 7 (FIG 3A).
  • Example 1 Analysis of 96 ligands using ligand multiplexing.
  • the group of 96 ligands is arranged into a 12 x 8 array (12 columns and 8 rows) in such a manner that each well contains a single ligand.
  • the second step involves the generation of pooled samples which are prepared by mixing aliquots of all the components present in the same column as well as all the components present in the same row, thus resulting into 20 pooled samples (12 pooled sample coming from each column and 8 pooled samples coming from each row).
  • the third step involves the HDX MS analysis of each of the 20 pooled samples, followed by sample deconvolution. In this manner, the screening of 96 compounds will only reduce to the analysis of 20 multiplexed samples.
  • the present invention will reduce the analysis time from 67 hrs to only 15 hrs.
  • Each candidate predicted by deconvolution will need to be tested individually, which will add a few more analyses, but the gain in the analysis time will still be very significant.
  • Example 2 Analysis of 10,000 ligands using ligand multiplexing. Taking as a base the previous example: a 96 well plate, providing 20 multiplexed samples, at 45 min analysis per sample, resulting in the screening of 96 compounds per 15 hrs, the screening of 10,000 compounds using the present invention will take 1,562 hrs (or 65 days) in contrast to 7,500 hrs or 312 days of analysis without using the present invention.
  • Example 3 Analysis of 96 ligands using a Multiplexed HDX MS Apparatus.
  • the first independent assembly starts the sample cycle at time zero
  • the second independent assembly starts the sample cycle at time 6 min
  • the third independent assembly starts the sample cycle at 12 min.
  • the effective speed is 6 min per sample, and the total throughput is 10 samples per 1 hr or 240 per day or 7,200 per 30 days. Therefore, under these conditions the analysis of 96 ligands without ligand multiplexing can be performed in less than 10 hrs as opposed to ca. 29 h without the instrumental multiplexing.
  • Example 4 Analysis of 96 ligands using ligand multiplexing and a multiplexed HDX MS apparatus.
  • the screening of 96 ligands by ligand multiplexing (20 samples) is performed in 2 hrs, as opposed to ca. 10 h without ligand multiplexing or 29 h with neither ligand nor instrument multiplexing.
  • Example 5 Analysis of 10,000 ligands using ligand multiplexing and a multiplexed HDX MS apparatus.
  • the present invention is utilized to screen a library of compounds against a particular protein.
  • the steps involving ligand convolution, binding assay by HDX MS and ligand deconvolution are performed to screen a ligand library towards a given protein.
  • the present invention is utilized to screen a library of compounds against two or more proteins.
  • the steps involving ligands convolution, binding assay by HDX MS and ligand deconvolution are performed to screen a ligand library towards two or more proteins.
  • the present invention involves the ligand multiplexing in combination with HDX MS, and it is used against one or more proteins, wherein the HDX MS step is performed by measuring the mass of one or more peptides related to the binding site - and neglecting most other peptides -, and by this means facilitating the site-specific HDX MS analysis.
  • the advantages of this embodiment are three-fold. First, this embodiment is particularly useful when shorter analysis time is desirable, since the washing and/or LC MS separation conditions are adjusted to separate and detect only a few peptides rather than focusing on obtaining higher sequence coverage values. Second, smaller sample size is needed because targeted MS analysis is more sensitive than broad-mass analysis. Third, this embodiment greatly simplifies signal detection and analysis, since instead of measuring the mass of several peptides (wide m/z range) the detection and analysis is done on a limited m/z range.
  • the present invention involves the ligand multiplexing in combination with HDX MS, and it is used against whole cells or whole cell lysates, wherein the HDX MS step is performed by measuring the mass of one or more peptides related to the binding site , or as well as related to conformational changes upon binding, or as well as related to peptides coming from sequences from protein-protein interactions that are linked to given protein pathway - and neglecting most other peptides -, and by this means facilitating the HDX MS analysis.
  • the advantage of this embodiment is that the screening is made using the full collection of proteins of a given proteome, which greatly expands the possibility to find novel biomolecular targets. 5.
  • the multiplexed HDX MS system comprises a plurality of cooling systems, pumps, injection ports, valves, digestion columns, precolumns and HPLC columns, as well as means to automate the analytical procedures necessary to perform the method.
  • An example of such apparatus can be seen in FIG 4, where a 4 column system allows sequential and parallelized sample processing.
  • such apparatus allows the performance of 3 processes in parallel: 1) LC-MS analysis of a given sample, 2) the digestion/clean-up of a subsequent sample, and 3) the system cleanup and equilibration of two other column systems that are waiting for subsequent samples.
  • Valve 6 is in B position when the flow of the Pump 3 passes by the Pepsin column 13 (when the following pair of ports 1-10, 9-8, 7-6, 5-4 and 3-2 are connected by the rotor).
  • Valve 7 is in position A when the flow of the Pump 3 pass by the Pepsin Column 14 (when the following pair of ports 1-2, 3-4, 6-5, 7-8 and 9- 10 are connected by the rotor).
  • Valve 7 is in B position when the flow of the Pump 3 passes by the Pepsin column 15 (when the following pair of ports 1-10, 9-8, 7-6, 5-4 and 3-2 are connected by the rotor).
  • Valve 8 is in position A when the flow entering from the port 6 (Valve 8) is in contact with the Reverse Phase Column 16 (when the following pair of ports 1-2, 3-4, 6-5, 7-8 and 9-10 are connected by the rotor). Valve 8 is in position B when the flow entering from the port 6 (Valve 8) is in contact with the Reverse Phase Column 17 (when the following pair of ports 1-10, 9-8, 7-6, 5-4 and 3-2 are connected by the rotor). Valve 9 is in position A when the flow entering from the port 6 (Valve 9) is in contact with the Reverse Phase Column 18 (when the following pair of ports 1-2, 3-4, 6-5, 7-8 and 9-
  • Valve 9 is in position B when the flow entering from the port 6 (Valve 9) is in contact with the Reverse Phase Column 19 (when the following pair of ports 1-10, 9-8, 7-6, 5- 4 and 3-2 are connected by the rotor).
  • Valve 11 is in position A when the flow delivered by Pump 4 flows into the Valve 9 (when the following pair of ports 1-2 and 4-3 are connected by the rotor).
  • Valve 11 is in position B when the flow delivered by Pump 4 flows into the Valve 8. (when the following pair of ports 1-4 and 4-3 are connected by the rotor).
  • Valve 10 is in position A when the flow delivered by Valve 9 is delivered to the mass spectrometer (when the following pair of ports 1-2 and 4-3 are connected by the rotor).
  • Valve 10 is in position B when the flow delivered by Valve 9 is delivered to the mass spectrometer (when the following pair of ports 1-2 and 4-3 are connected by the rotor).
  • a potential cycle of screening can be described as following (non-restricted example):
  • Step 1 Sample 1 is injected into Valve 6, Pepsin Column 12 and Reverse Phase Column 17 when Valve 6 is in position B and Valve 8 is in position A; in parallel Valve 7 is in position B, Valve 9 in position A, Valve 10 in position A and Valve 11 in position B.
  • Step 2 Sample 1 is analyzed by MS by means of switching Valve 8 to position B. In addition, Sample 2 is injected into Valve 7 (in position B), Pepsin Column 14 and Reverse Phase Column 19 (Valve 9 in position A). During this step Valve 11 is in position B and Valve 10 is in position A.
  • Step 3 Sample 2 is analysed by MS by means of switching Valves 9 and 10 to position B and Valve
  • Step 4 Sample 3 is analysed by MS by means of switching Valves 8 and 10 to position A and Valve 11 to position B. In addition, sample 4 is injected into Valve 7 (in position A), Pepsin Column 15 and Reverse Phase Column 19 (Valve 9 in position A).
  • Step 5 Analysis cycle is repeated by performing Step 1 to 4 for the subsequent samples.

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Abstract

La présente invention concerne des procédés et un appareil qui facilitent le criblage de médicaments à l'aide d'un multiplexage multidimensionnel de ligand en association avec une spectrométrie de masse par échange d'hydrogène/deutérium (HDX MS). La présente invention est basée sur la convolution de ligand, le biotest de détection de liaison et une déconvolution subséquente au moyen de traitement de signal. Le procédé utilise une pluralité de puits disposés en rangées et en colonnes, où chaque puits contient au moins un seul ligand. La convolution de ligand est effectuée par la génération d'échantillons groupés à partir de chaque colonne et rangée. Chaque échantillon groupé est mélangé à la protéine cible et soumis à la spectrométrie de masse par échange d'hydrogène/deutérium. La présente invention concerne les moyens pour déconvoluer les signaux afin d'individualiser un signal de liaison positive. L'appareil fournit le moyen de mettre en œuvre le procédé et d'utiliser une pluralité de valves, de colonnes et de pompes parallélisées pour améliorer la production d'échantillon. Par conséquent, La présente invention concerne une augmentation significative du débit de la spectrométrie de masse par échange d'hydrogène/deutérium.
PCT/SE2015/050416 2014-04-08 2015-04-07 Procédé et appareil pour criblage de médicaments par site de liaison WO2015156730A1 (fr)

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SE1450437 2014-04-08
SE1450437-7 2014-04-08

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