Method for analysing analytes of interest

Abstract

The present disclosure may broadly provide a method for analysing analytes of interest comprising: a) mixing a biomaterial containing a protein target, with one or more candidate compounds for binding to the biomaterials to obtain a sample comprising one or more biomaterial-candidate complexes, b) introducing the sample to a mass spectrometer by ionising the sample under conditions that retain the one or more biomaterial-candidate complex in their native state, c) separating the one or more biomaterial-candidate complex from unbound candidate compounds by selecting for ions having a high mass range using a mass analyser configured to capture the one or more biomaterial-candidate complexes and excluding unbound candidate compounds based on a difference in oscillation frequencies of their respective ions, d) dissociating the biomaterial-candidate complex using collisional dissociation to obtain one or more dissociated candidate compounds, and e) determining the dissociated candidate compound based on mass-to-charge ratio (m / z).

Description

METHOD FOR ANALYSING ANALYTES OF INTERESTFIELD OF THE INVENTION

[0001] The present invention relates to methods for analysing analytes of interest. More particularly, the present invention relates to mass spectrometry (MS)-based methods for identifying binding of ligands to protein targets in drug discovery applications.BACKGROUND

[0002] Mass spectrometry (MS)-based methods are known and have been used to analyse analytes of interest.

[0003] One such method is affinity selection mass spectrometry (AS-MS). Conventional AS-MS methods typically require an initial labour intensive sample preparation phase before the analyte of interest maybe analysed in the mass spectrometer. For example, target proteins may be incubated with a mixture of compounds, followed by an initial separation step using techniques such as such as pulsed ultrafiltration and size exclusion chromatography (SEC) to separate unbound molecules from protein-ligand complexes. A secondary separation step may be required to dissociate the ligand from the protein prior to MS analysis.

[0004] AS-MS involves labour intensive preparatory steps that may limit the efficiency of such methods in screening applications. Additionally, such methods may require a separate process to dissociate the ligand from the protein-ligand complex prior to introduction into the mass spectrometer for analysis that may reduce the sensitivity of the methods resulting in false-negative results.

[0005] Collision induced dissociation mass spectrometry (CIAS-MS) integrates the separation and dissociation steps within a direct injection Fourier-transform ion cyclotron resonance mass spectrometer. However, CIAS-MS uses collision induced dissociation (CID) which may have limitations when it comes to attaining broader distribution of ions.Additionally, the instrument used to perform CIAS-MS typically involve high operational costs.

[0006] It is desired to address or ameliorate one or more disadvantages or limitations associated with existing methods, provide a drug discovery method or to at least provide the public with a useful alternative.SUMMARY

[0007] According to a first aspect, the present disclosure may broadly provide a method for analysing analytes of interest comprising :• mixing a biomaterial containing a protein target, with one or more candidate compounds for binding to the biomaterials to obtain a sample comprising one or more biomaterial-candidate complexes,• introducing the sample to a mass spectrometer by ionising the sample under conditions that retain the one or more biomaterial-candidate complex in their native state,• separating the one or more biomaterial-candidate complex from unbound candidate compounds by selecting for ions having a high mass range using a mass analyser configured to capture the one or more biomaterial-candidate complexes and excluding unbound candidate compounds based on a difference in oscillation frequencies of their respective ions,• dissociating the biomaterial-candidate complex using collisional dissociation to obtain one or more dissociated candidate compounds, and• determining the dissociated candidate compound based on mass-to-charge ratio (m / z).

[0008] According to another aspect, the present disclosure may broadly provide a method for analysing analytes of interest comprising:• introducing a sample comprising one or more biomaterial-candidate complexes to a mass spectrometer,• separating the one or more biomaterial-candidate complex from unbound candidate compounds by selecting for ions having a high mass range using an ion filter, such as a quadrupole,• dissociating the biomaterial-candidate complex using higher energy collisional dissociation (HCD) to obtain one or more dissociated candidate compounds,• capturing the dissociated candidate compound by selecting for ions having a low mass range, and• determining the dissociated candidate compound based on mass-to-charge ratio (m / z) using a mass analyser such as a time-of-flight (TOF) mass analyser, an Orbitrap mass analyser, a linear ion trap (LIT) mass analyser, or quadrupole mass analyser.

[0009] According to one aspect, the present disclosure may broadly provide a method for analysing analytes of interest comprising:• mixing a biomaterial containing a protein target, with one or more candidate compounds for binding to the biomaterials to obtain a sample comprising one or more biomaterial-candidate complexes,• introducing the sample to a mass spectrometer by ionising the sample under conditions that retain the one or more biomaterial-candidate complexes in their native state,• separating the one or more biomaterial-candidate complex from unbound candidate compounds by selecting for ions having a high mass range using an ion filter configured to capture the one or more biomaterial-candidate complexes and excluding unbound candidate compounds,• dissociating the biomaterial-candidate complex using higher energy collisional dissociation (HCD) to obtain one or more dissociated candidate compounds,• capturing the dissociated candidate compound by selecting for ions having a low mass range, and• determining the dissociated candidate compound based on mass-to-charge ratio (m / z).

[0010] According to another aspect, the present disclosure may broadly provide a method for analysing analytes of interest comprising:• mixing a biomaterial containing a protein target, with one or more candidate compounds for binding to the biomaterials to obtain a sample comprising one or more biomaterial-candidate complexes,• introducing the sample to a mass spectrometer by ionising the sample under conditions that retain the one or more biomaterial-candidate complexes in their native state,• separating the one or more biomaterial-candidate complexes from unbound candidate compounds by selecting for ions having a high mass range using an ion filter configured to capture the one or more biomaterial-candidate complexes and excluding unbound candidate compounds based on a difference in oscillation frequencies of their respective ions,• dissociating the biomaterial-candidate complex using higher energy collisional dissociation (HCD) to obtain one or more dissociated candidate compounds,• capturing the dissociated candidate compound by selecting for ions having a low mass range, and• determining the dissociated candidate compound based on mass-to-charge ratio (m / z).

[0011] According to another aspect, the present disclosure may broadly provide a method for screening a phenotypic panel comprising:• preparing a first sample by incubating a first cell lysate derived from diseased cells overexpressing one or more protein targets with a candidate compound such that the protein target or protein targets within the first cell lysate forms a complex with the candidate compound,• preparing a second sample by incubating a second cell lysate derived from nondiseased cells with the candidate compound, wherein for each sample,• introducing the sample to a mass spectrometer under conditions that retain the protein-candidate complex, when present, in their native state,• separating the protein-candidate complex, when present, from unbound candidate compounds by selecting for ions having a high mass range corresponding to the protein target using an ion filter configured to capture the one or more biomaterial-candidate complexes and excluding unbound candidate compounds based on a difference in oscillation frequencies of their respective ions,• dissociating the protein-candidate complex using higher energy collisional dissociation (HCD) to obtain one or more dissociated candidate compounds,• capturing the dissociated candidate compound by selecting for ions having a low mass range, and• comparing a mass spectrum of the first sample, and a mass spectrum of the second sample to determine the difference in binding of the candidate compound to the first and second cell lysates.

[0012] Any one or more of the following embodiments may relate to any of the above aspects.

[0013] The HCD may be configured to fragment the one or more dissociated candidate compounds into smaller fragments during dissociation from the biomaterial-candidate complex.

[0014] The smaller fragments may be less than 1,500, 1,400, 1,300, 1 ,200, 1,100, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 m / z, and suitable ranges may be selected from between any of these values.

[0015] The HCD may be performed with a plurality of collision energies to obtain a plurality of fragmentation patterns of the one or more dissociated candidate compounds.

[0016] The plurality of collision energies may comprise at least one low collision energy, and at least one high collision energy.

[0017] The low collision energy may correspond to about 1 to about 49% of a total available voltage of the HCD, and the high collision energy may correspond to about 50 to 100% of the total available voltage of the HCD.

[0018] The HCD collision energy may be increased at a predetermined rate to determine dissociation to obtain a plurality of fragmentation patterns of the dissociated biomaterial and / or dissociated candidate compound.

[0019] The predetermined rate may be about 5% to about 10% of the total available voltage.

[0020] A voltage of the HCD may be incrementally increased to determine dissociation behaviour of the biomaterial and candidate compound.

[0021] The voltage of HCD is increased incrementally from about 10, 20, 30, 40, 50, 60, 70, 80, 90 to 100 volts, and suitable ranges may be selected from between any of these values.

[0022] The method may further comprise comparing the dissociation behaviour of two or more candidate compounds to the biomaterial containing the target proteins.

[0023] The high mass range may be about 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 m / z, and suitable ranges may be selected from between any of these values.

[0024] The low mass range may be about 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000, 1,100, 1,200, 1,300, 1 ,400, or 1,500 m / z, and suitable ranges may be selected from between any of these values.

[0025] The mass analyser may comprise a central electrode and two outer electrodes, wherein ions of the biomaterial-candidate complex and unbound candidate compounds oscillate around the central electrode between the outer electrodes such that different ions oscillate at different frequencies that used to separate the ions.

[0026] The mass analyser may be selected from the group comprising an Orbitrap, a quadrupole, linear ion trap, and time of flight.

[0027] When the mass analyser is an Orbitrap, the mass analyser may be configured to have an automatic gain control (AGO) target of about 100 to about 500%.

[0028] The AGO may be configured to have a maximum injection time of about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or about 200 ms, and suitable ranges may be selected from between any of these values.

[0029] The biomaterial may be derived from a cell lysate.

[0030] The cell lysate may contain an overexpressed protein target.

[0031] The cell lysate may be prepared by• lysing cells by mechanical disruption,• separating lysed cells from unlysed cells and cellular debris by centrifugation at predetermined speed and time, or filtering the cell lysate through a device of appropriate mass cut-off, and• retaining the supernatant containing the cell lysate.

[0032] The mechanical disruption may comprise sonication.

[0033] The candidate compounds may comprise a library of compounds.

[0034] The candidate compounds may be 1500 Daltons or less.

[0035] The one or more candidate compounds may be soluble in an aqueous solution.

[0036] The sample may be introduced into the mass spectrometer via electrospray ionisation (ESI).

[0037] The sample may be introduced into an electrospray ionisation source by direction injection.

[0038] The sample may be introduced by a high-performance liquid chromatograph autosampler.

[0039] The liquid chromatograph may be operated with or without a chromatography column.

[0040] The liquid chromatograph may be operated at flow rates of about 0.1 , 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, or about 1000 pL / min, and suitable ranges may be selected from between any of these values.

[0041] Determination of the dissociated compound may be based on comparing the mass-to-charge ratio of the dissociated compound with a reference or database of compounds.

[0042] The method may further comprise determining one or more characteristics of the dissociated compound based on the plurality of fragmentation patterns.

[0043] The one or more characteristics of the dissociated compound may comprise compound structure, and site of attachment.

[0044] The candidate compound may be known to bind the overexpressed protein in the diseased cell.

[0045] The method may further comprise:• incubating the cell lysate derived from cells overexpressing the protein target with a plurality of candidate compounds, and• incubating the cell lysate derived from non-diseased cells with a candidate compound from the plurality of candidate compound which binds to a target in cell lysate of the diseased cell.

[0046] The method may further comprise:• dissociating biomaterial-candidate compound complex by applying a plurality of collision energies,• capturing the dissociated candidate compounds at each of the plurality of collision energies, and• determining dissociation constant of the dissociated candidate compounds.

[0047] The dissociation constant may be determined by comparing the relative amounts of a dissociated candidate at a plurality of dilution factors of the biomaterial and plurality of collision energies.

[0048] The method may further comprise ranking at least two dissociated candidate compounds based on their respective dissociation constants.

[0049] The term “comprising” as used in this specification means “consisting at least in part of”. When interpreting statements in this specification which include that term, the features, prefaced by that term in each statement, all need to be present but other features can also be present. Related terms such as “comprise” and “comprised” are to be interpreted in the same manner.

[0050] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7).

[0051] This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.)

[0052] To those skilled in the art to which the invention relates, many changes in construction and widely differing embodiments and applications of the invention will suggest themselves without departing from the scope of the invention as defined in the appended claims. The disclosures and the descriptions herein are purely illustrative and are not intended to be in any sense limiting.BRIEF DESCRIPTION OF THE FIGURES

[0053] The invention will now be described by way of example only and with reference to the following drawings.

[0054] Figure 1 illustrates a method of analysing analytes of interest according to the present disclosure.

[0055] Figure 2 illustrates a method for screening a phenotypic panel according to the present disclosure.

[0056] Figure 3A-F are mass spectra illustrating identification of hCAI-furosemide interactions according to a method of the present disclosure.

[0057] Figures 4A-D are mass spectra illustrating identification of hCAI-furosemide interactions within pooled compounds according to a method of the present disclosure.

[0058] Figures 5A-F are mass spectra illustrating identification of hCAI- diclofemanide interactions according to a method of the present disclosure.

[0059] Figures 6A-D are mass spectra illustrating identification of hCAI- diclofemanide interactions within pooled compounds according to a method of the present disclosure.

[0060] Figures 7A-F are mass spectra illustrating identification of hCAI interactions with furosemide within a bacterial lysate according to a method of the present disclosure.

[0061] Figures 8A-D are mass spectra illustrating identification of hCAI interactions with furosemide in a pooled compound within a bacterial lysate according to a method of the present disclosure.

[0062] Figures 9A-F are mass spectra illustrating identification of hCAI interactions with diclofenamide within a bacterial lysate according to a method of the present disclosure.

[0063] Figures 10A-D are mass spectra illustrating identification of hCAI interactions with diclofenamide in a pooled compound within a bacterial lysate according to a method of the present disclosure.

[0064] Figures 11A-F are mass spectra illustrating identification of hCAI-furosemide interactions in mammalian cell lysate overexpressing hCAI according to a method of the present disclosure.

[0065] Figures 12A-D are mass spectra illustrating identification of hCAI-furosemide interactions in a complex mixture of overexpression cell lysate and pooled small molecules according to a method of the present disclosure.

[0066] Figures 13A-F are mass spectra illustrating identification of hCAI-diclofemanide interactions in mammalian cell lysate overexpressing hCAI according to a method of the present disclosure.

[0067] Figures 14A-D are mass spectra illustrating identification of hCAI-diclofemanide interactions in a complex mixture of overexpression cell lysate and pooled small molecules according to a method of the present disclosure.

[0068] Figures 15A-F are mass spectra illustrating identification of bCAII-furosemide interactions in mammalian cell lysate overexpressing bCAII according to a method of the present disclosure.

[0069] Figures 16A-D are mass spectra illustrating identification of bCAII-furosemide interactions in a complex mixture of overexpression cell lysate and pooled small molecules according to a method of the present disclosure.

[0070] Figures 17A-F are mass spectra illustrating identification of bCAII-diclofemanide interactions in mammalian cell lysate overexpressing bCAII according to a method of the present disclosure.

[0071] Figures 18A-D are mass spectra illustrating identification of bCAII- diclofemanide interactions in a complex mixture of overexpression cell lysate and pooled small molecules according to a method of the present disclosure.

[0072] Figures 19A-F are mass spectra illustrating detection protein-ligands in various buffering salts and agents according to a method of the present disclosure.

[0073] Figures 20A-B are mass spectra illustrating sensitivity of the method of the present disclosure compared to CIAS-MS.

[0074] Figures 21A-C are mass spectra illustrating narrow mass filter isolation range according to a method of the present disclosure.

[0075] Figures 22A-F are mass spectra illustrating detection of dissociation of ertapenem from the OmpF-detergent-ertapenem complex according to a method of the present disclosure.

[0076] Figures 23A-D are mass spectra illustrating detection of membrane proteinligand interactions in complex mixtures of pooled compounds according to a method of the present disclosure.

[0077] Figures 24A-D are graphs illustrating the modulation of energy during collisional activation according to a method of the present disclosure.

[0078] Figures 25A-C are graphs illustrating ranking of dissociation constants according to a method of the present disclosure.DETAILED DESCRIPTION

[0079] Mass spectrometry is an analytical tool which uses the mass-to-charge ratio (m / z) of one or more molecules to calculate their exact molecular weight. The exact molecular weight can be used to help determine the chemical structure of unknown compounds or to validate the structure of a known compound. A mass spectrometer may consist of an ionisation source, a mass analyser and an ion detection system.

[0080] Mass spectrometry-based methods have been used to analyse biomaterialcandidate compound binding kinetics as part of a target-based approach to drug discovery. One such method is hyphenated mass spectrometry that identifies a bound ligand using affinity capture and subsequent dissociation.

[0081] Hyphenated mass spectrometry may be performed using pulsed ultrafiltration affinity selection mass spectrometry (PUF AS-MS), size exclusion AS-MS, and magnetic microbead affinity selection screening (MagMASS). However, these methods may require additional steps such that the methods are more time consuming to perform. Additionally, some filtration-based methods may be subject to inaccuracies due to limitations of the filtration process.

[0082] In another AS- MS- based method, a quadrupole is used to capture proteinligand complex and collision induced dissociation (CID) is then used to dissociate protein and ligand from the protein-ligand complex. However, CIAS-MS may be limited by its ability to achieve a broader distribution of ions. The narrower distribution of ions may also reduce fragmentation patterns resulting a reduced scope of information derivable from CIAS-MS.

[0083] Other limitations of CIAS-MS include:1. Requires a greater sample volume of at least 100-500 pL and direct injection only.2. The sample is prepared strictly in 100-200 mM ammonium acetate - this is not as versatile since some proteins may not do well in ammonium acetate.3. Enables mass filtering with cut off only, which is a very broad range.4. CID on and CID off need to be performed in two individual runs.

[0084] Described is a method for analysing analytes of interest. A sample is introduced to a mass spectrometer. The sample may comprise one or more biomaterialcandidate complexes. The one or more biomaterial-candidate complexes, if present, are separated from unbound candidate compounds by selecting for ions having a high mass range using an ion filter. The biomaterial-candidate complex is dissociated using higher energy collisional dissociation (HCD) to obtain one or more dissociated candidate compounds. The dissociated candidate is determined based on its mass-to-charge ratio (m / z).

[0085] Further described is a method for analysing analytes of interest. A biomaterial containing a protein target is mixed with one or more candidate compounds for binding to the biomaterials, to obtain a sample comprising one or more biomaterial-candidate complexes. The sample is introduced to a mass spectrometer by ionising the sample under conditions that retain the one or more biomaterial-candidate complex in their native state. The one or more biomaterial-candidate complex(es) are separated from unbound candidate compounds by selecting for ions having a high mass range using an ion filter configured to capture theone or more biomaterial-candidate complexes and excluding unbound candidate compounds based on a difference in oscillation frequencies of their respective ions. The biomaterialcandidate complex is dissociated using higher energy collisional dissociation (HCD) to obtain one or more dissociated candidate compounds. The dissociated candidate compound is captured by selecting for ions having a low mass range. The dissociated candidate compound is determined based on mass-to-charge ratio (m / z).

[0086] The present disclosure may provide a rapid, sensitive, and cost-effective method for analysing analytes of interest. For example, the method may determine binding of small molecule drug candidates for binding to a protein target relatively quickly, with a high degree of sensitivity while minimising the cost when compared to existing method.

[0087] The method according to the present disclosure may enable direct sampling of biomaterials and candidate compounds into a mass spectrometer without prior separation and dissociation steps.

[0088] The method according to the present disclosure may enable broader ion detection, increased ion dissociation and / or fragmentation for higher quality MS spectra, and a wider range of ion dissociation and / or fragmentation pathways.

[0089] The method according to the present disclosure may be used to analyse any analytes of interest. For example, the method may be used for identifying candidate compounds or molecules that bind with a selected affinity to a target from a plurality of compounds or molecules.

[0090] Figure 1 illustrates a method for analysing analytes of interest according to the present disclosure. The method may broadly comprise the steps of preparing a sample (or assay mixture) comprising one or more candidate compounds and a biomaterial containing a target to which the one or more candidate compounds may bind, transferring the sample into a mass spectrometer, separating the unbound materials (such as candidate compounds) using an ion filter, dissociating the bound compound from the biomaterial-candidate complex, and measuring the dissociated candidate compound using the mass analyser for mass spectrometry analysis.(a) Sample preparation step

[0091] The sample preparation step may comprise mixing the biomaterial containing the protein target, with one or more candidate compounds (or ligand). When mixed, one or more candidate compounds may bind to a target or targets in the biomaterial to form biomaterial-candidate compound complexes (or protein-ligand complexes).

[0092] The sample (or assay mixture) may comprise one or more candidate compounds and a biomaterial containing a target.

[0093] The candidate compound and biomaterial may be mixed with assay components for conducting affinity selection assay. The assay components may comprise any components for conducting an affinity selection including but are not limited to solvents, and buffer salts.

[0094] Conventional mass spectrometry techniques, such as native condition MS screening may be limited to using a buffer system comprising ammonium acetate. However, buffer systems comprising only ammonium acetate may not be suitable for suitable certain protein targets, therefore, limiting the range of protein targets that may be analysed.

[0095] The method according to the present disclosure may comprise a buffer system comprising a combination of salts and / or buffering agents. The buffer system may comprise one or more of ammonium acetate, NaCI, Tris, Na2SO4, and HEPES.

[0096] The biomaterial and candidate compound(s) may be incubated under suitable assay conditions to enhance biomaterial-candidate compound complex formation. The biomaterial and candidate compound(s) may be incubated for 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 minutes, and useful ranges may be selected between any of these values (for example, about 5 to about 90, about 10 to about 80, about, 20 to about 70, about 30 to about 60, about 40 to about 50 minutes).

[0097] The biomaterial and candidate compound(s) may be incubated at a temperature of 15°, 16°, 17°, 18°, 19°, 20°, 21°, 22°, 23°, 24°, 25°, 26°, 27°, 28°, 29° or 30° Celsius, and useful ranges may be selected between any of these values (for example, about 15° to about 30°, about 18° to about 28°, about 20° to about 26°, about 22° to about 24° Celsius)

[0098] It will be appreciated by the person skilled in the art that assay conditions including concentration of compounds and biomaterial, duration of incubation to achieve equilibrium, temperature, and buffer salt concentration may vary depending on assay requirements.

[0099] The molecule or compound may be a candidate drug molecule or compound for screening against a drug target. In some examples, the candidate compound may comprise a molecule, an antibody, or other biologies that may have a therapeutic effect.

[0100] In some examples, the molecule or compound may comprise a molecular mass of 1500 Daltons or less. For example, the molecule or compound may comprise a molecularmass of about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, or 1500 Daltons or less, and useful ranges may be selected between any of these values (for example, about 50 to about 1500, about 100 to about 1400, about 200 to about 1300, about 300 to about 1200, about 400 to about 1000, about 500 to about 800 Daltons).

[0101] The molecule or compound may comprise 5 hydrogen bond donors or less. For example, the molecule or compound may comprise 5, 4, 3, 2, 1, or 0 hydrogen bond donors, and useful ranges may be selected between any of these values.

[0102] The molecule or compound may comprise 10 hydrogen bond acceptors or less. For example, the molecule or compound may comprise 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 hydrogen bond acceptors, and useful ranges may be selected between any of these values.

[0103] The candidate compounds are not limited to any particular type, source or class of compounds.

[0104] The molecule or compound may be a synthetic product.

[0105] The molecule or compound may be a natural product or derived therefrom, including but not limited to, macrolides and peptides.

[0106] Molecules or compounds derived from natural sources may be introduced directly as complex compound mixtures dissolved in solvents, in the form of natural product extracts.

[0107] The molecule or compound may be soluble in aqueous solutions for compatibility with assay buffers. In some examples, the candidate compound may be introduced into the sample using a small volume of an organic solvent (such as methanol).

[0108] The candidate compound may comprise a compound library. The compound library may comprise about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more candidate compounds. The candidate compounds in the compound library may be known to bind biomaterials.

[0109] The biomaterial may compromise any biological material or substance containing a drug target or potential drug target (such as a protein target). For example, the biomaterial may comprise a nucleic acid, a genetic material, or a gene product.

[0110] The protein target can be any class of proteins that are soluble in an assay solution when identifying binding compounds with a selected affinity to the protein target is desirable.

[0111] The biomaterial may be a mixture of proteins, a complex mixture, or natural product extract.

[0112] The gene product may comprise any product resulting from gene expression. For example, the gene product may be a ribonucleic acid (RNA), a peptide, a polypeptide, or a protein.

[0113] The protein may be a purified protein or a recombinant protein. The protein may be a complex of one more proteins.

[0114] The biomaterial may comprise a cell lysate containing a drug target or potential drug target. The cell lysate may comprise a transcriptome and / or proteome.

[0115] The cell lysate may be prokaryotic or eukaryotic. For example, the cell lysate may comprise a bacterial cell lysate or a mammalian cell lysate.

[0116] The proteome may comprise soluble proteome and / or insoluble membrane proteomes. Insoluble membrane proteomes may be solubilised. The proteomes may comprise other sub proteomes such as nuclear, mitochondrial, and cytoskeletal proteomes.

[0117] The biomaterial may comprise a panel of cell lysates. The panel of cell lysates may be derived from one or more cell lines.

[0118] Each cell line may have a known transcriptome and / or proteome.

[0119] The panel of cell lysates may comprise soluble proteome, and / or solubilised membrane proteome from each cell line. For example, each cell line in the panel may comprise a soluble proteome cell lysate and a solubilised membrane proteome cell lysate.

[0120] The cells for cell lysates may be obtained by suitable cell culture methods. Nucleic acid for a target may be transfected into cells to obtain cell lines over-expressing.

[0121] In other examples, the cell lysate may be derived from normal or mutant or diseased cells.

[0122] The cells may be lysed using any suitable techniques. In some examples, the cells are lysed by mechanical disruption including but not limited to, liquid homogenisation,high frequency sound waves, freeze-thaw cycles, and manual grinding. The cell lysis may be performed using any suitable equipment including but not limited to sonicators or pressuredropping devices such as a French press.

[0123] Lysis preparation of the cell pellets may include resuspending the washed cell pellets in a lysis buffer containing ammonium acetate and protease inhibitors under appropriate lysis buffer condition. It will be appreciated by the person skilled that the lysis buffer conditions including type of protease inhibitors used, appropriate pH, temperature, salt identities and concentrations, and buffering agents identities and concentrations may vary depending on the protein targets.

[0124] The separation of lysed from unlysed cells and cellular debris may be achieved by centrifugation. It will be appreciated by the person skilled that this may involve subjecting the cell lysate to centrifugation under specific centrifugation speed, time and temperature depending on the sample and assay requirements.

[0125] In other examples, the separation of lysed from unlysed cells and cellular debris may involve suitable filtration methods, and / or collecting the supernatant from the centrifugation step and discarding the precipitated materials.

[0126] The method according to the present disclosure may be used to screen known compounds against a target by analysing a mixture of proteins and ligands. The method may also be used to screen natural products against a target.

[0127] The sample (or assay mixture) comprising the biomaterial-candidate complexes, including unbound biomaterial and candidate compounds, may be transferred into a mass spectrometer through an ionisation source.(b) Sample introduction step - ionisation

[0128] The ionisation source ionises the biomaterial-candidate complexes. This may cause them to break up into positively or negatively charged fragments or becoming positively or negatively charged without fragmenting. The charged particles are separated by their mass-to-charge ratio, and are then detected by an ion detection system. The results are displayed as a mass spectra which depicts the signal intensity of the detected ions as a function of the m / z of the ions.

[0129] Ionisation can be achieved through a wide variety of techniques which will be appreciated by the skilled person. Different techniques are suitable for different types of samples. Electron or chemical ionisation may be particularly suitable for gas samples.Electrospray ionisation or matrix-assisted laser desorption / ionisation may be particularly suitable for liquid and solid biological samples.

[0130] The sample may be introduced or injected into the mass spectrometer while maintaining the native state of the biomaterial-candidate complex. Preserving the native state of the ligand-protein complex enables the protein-ligand complex to be directly observed using a mass spectrometer while better reflecting the structure, and functionalities of the biomolecule prior to or during ionisation.

[0131] In some examples, electrospray ionisation (ESI) to introduce the sample into a mass spectrometer. ESI is a soft ionisation technique that preserves protein-ligand complexes in their native state. ESI produces ions using an electrospray where a high voltage is applied to liquid to produce an aerosol.

[0132] The sample may be introduced into the ESI source by direct injection, or chromatography-based techniques.

[0133] In some examples, the capillary voltage may be set to about 1.0, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 2.0, 3.1, 3.2, 3.3, 3.4 or 3.5 kV in negative mode or positive mode, between any of these values.

[0134] In some examples, the capillary temperature may be set to about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, or 280 °C, while the desolvation temperature may be set to about 250, 260, 270, 280, 290, 300, 310, 320, 330, 340 or 350 °C, and suitable ranges may be selected between any of these values.

[0135] In certain high throughput screening applications, the sample may be transferred from vials or well plates into an ionisation source via a high-performance liquid chromatograph autosampler operating with or without a chromatography column.

[0136] In some examples, the autosampler may operate at a flow rate of 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 pL / min, and suitable ranges may be selected between any of these values.

[0137] In other examples, the autosampler may operate at a flow rate of 0.1, 0.5, 1.0,1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0,11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, or 15.0 pL / min, and suitable ranges may be selected between any of these values.

[0138] In some examples, the method may only require about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 pL of sample per injection via an automated LC-MS autosampler, and useful ranges may be selected between any of these values (for example, about 1 to about 15, about 2 to about 11, about 3 to about 14, about 4 about 13, about 5 to about 12, about 6 to about 11, about 7 to about 10, about 8 to about 9 pL of sample per injection.

[0139] Following sample injection / introduction, the biomaterial-candidate complexes and unbound candidate compounds may be separated to select the biomaterial-candidate complex for analysis. The separation of the ions can be achieved through a number of different types of separating apparatuses which will be appreciated by the skilled person.

[0140] A mass analyser maybe used to separate the biomaterial-candidate complex from the unbound candidate compounds by configuring the mass analyser to capture ions above certain mass-to-charge ratio (m / z) and discarding the unbound candidate compound by exhausting the ions below certain m / z into vacuum.

[0141] For example, when screening a plurality of candidate compounds with a molecular weight of less than 1000 Dalton, the mass analyser may be configured to isolate ions above m / z 1000 such that all unbound molecules may be filtered out.

[0142] The biomaterial-candidate complex may be separated from unbound candidate compounds by selecting for ions having a high mass range.

[0143] The high mass range may be a mass range of above 1 ,000, 1,100, 1,200,I,300, 1 ,400, 1,500, 1,600, 1,700, 1 ,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500,2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,600, 3,700, 3,800,3,900, 4,000, 4,100, 4,200, 4,300, 4,400, 4,500, 4,600, 4,700, 4,800, 4,900, 5,000, 5,100,5,200, 5,300, 5,400, 5,500, 5,600, 5,700, 5,800, 5,900, 6,000, 7,000, 8,000, 9,000, 10,000,I I ,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21 ,000, 22,000, 23,000, 24,000 or 25,000 m / z, and useful ranges may be selected between any of these values (for example, about 1 , 100 to about 25,000, about 1 ,500 to about 5,500, about 2,000 to about 4,500, about 2,500 to about 5,000, about 3,000 to about 3,500 m / z).

[0144] In some embodiments, the high mass range corresponds to the mass of the target protein in the biomaterial or the mass of the protein-ligand complex.(c) Mass analyser

[0145] Broadly, a mass analyser is a component of a mass spectrometer that separates ionised molecules based on their mass-to-charge ratios and directs the selected molecules to a detector to be detected and converted into a signal (e.g. a mass spectrum).

[0146] The mass analyser may be selected from an Orbitrap, a quadrupole, linear ion trap, and time of flight.

[0147] An Orbitrap mass analyser generally comprises a central electrode and two outer electrodes. The central electrode is a coaxial spindle-like structure while the outer electrode forms a barrel like structure around the central electrode. The Orbitrap traps ions in an orbital motion around the central electrode in between the two outer electrodes. The ions oscillate at different frequencies enabling different ions to be separated based on the different oscillation frequencies.

[0148] A quadrupole generally comprises a four parallel cylindrical metallic rods which carry electrical current in a vacuum. Quadrupole analysers use oscillating electrical fields to selectively stabilize or destabilize the paths of ions passing through a radio frequency (RF) quadrupole field created between the four parallel rods.

[0149] A linear ion trap (LIT) generally comprises four hyperbolic rods arranged around a central axis. The rods may be segmented and include openings for ion injection. LIT confines ions radially using two-dimensional radio frequency field and axially by stopping potentials to end electrodes.

[0150] A sector field mass analyser uses static electric and / or magnetic fields to affect the path and / or velocity of the charged particles.

[0151] Time-of-flight (TOF) analysers use an electric field to accelerate ions through the same potential and then measures the time it takes the particles to reach the detector. The velocity of the ions correlates to the mass of the ions.

[0152] In some examples, the method may employ two or more mass analysers. For example, the mass spectrometer maybe configured in a tribid configuration in which Orbitrap is used in combination with LIT and quadrupole mass analysers. Other examples combinations of mass analysers include but are not limited to Q-TOF comprising quadrupole and time of flight, Q-Trap comprising quadrupole and ion trap, and Q Exactive comprising quadrupole and Orbitrap.

[0153] The combination of mass analysers may be selected based on the requirements of the assay. For example, Orbitrap or ion trap may be selected as the detectorwhere high sensitivity, resolution, and mass accuracy is required. Similarly, the Orbitrap and ion trap may be selected when detecting ions of higher mass range of up to m / z 6000 and m / z 4000, respectively.

[0154] When an Orbitrap is used, the mass analyser may comprise automatic gain control (AGO) target for mass analysis. In such examples, the AGO target value may be increased if higher sensitivity is required and decreased to speed up fill times.

[0155] In some examples, the Orbitrap mass analyser may be configured to have an automatic gain control (AGO) target of about 50, 100, 150, 200, 250, 300, 350, 400, 450 500, or 550%, and useful ranges may be selected between any of these values (for example about 50 to about 550, about 100 to about 500, or about 200 to about 400%).

[0156] In some examples, the Orbitrap mass analyser may be configured to specific a maximum injection time for AGO. In some examples, the maximum injection time may be about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 millisecond (ms), and useful ranges may be selected between any of these values (for example, about 10 to about 250, about 50 to about 200, about 100 to about 150 ms). In such examples, the ion routing multipole fills with ions until it reaches the AGO target and then the mass spectrometer transfers the ions to the ion trap or Orbitrap.(d) Dissociation step - CID / HCD

[0157] A next step may be to dissociate the biomaterial-candidate complex. Dissociating the candidate compound from the biomaterial-candidate complex may be achieved by colliding ions transmitted and accelerated from the mass analyser with neutral gas molecules in a collision cell of a mass spectrometer such that the ions into dissociate into smaller charged product ions.

[0158] The method according to the present disclosure uses collision-induced dissociation (CID) or higher energy collisional dissociation (HCD) to dissociate candidates from the biomaterial-candidate complex.

[0159] CID is a mass spectrometry technique for fragmenting molecular ions in the gas phase. The molecular ions may be accelerated by electrical potential to generate high kinetic energy in the vacuum chamber of the mass spectrometer and allowed to collide with neutral gas molecules. The collision may result in the conversion of kinetic energy into an internal energy that may result in bond breakage and fragmentation.

[0160] For CID to achieve collision energy on an ion trap instrument, ions must be accelerated relatively slowly from their starting energy to the final energy. The accelerated ions collide with the inert gas during the excitation process. CID may approximate an equilibrium process in which each collision may heat or cool the stored precursor ion. The evolution of this equilibrium may be vary based on the ion kinetic energy. For example, in an ion trap, resonant excitation will accelerate the precursor ions such that after dissociation has occurred, fragment collisions with the gas molecules will cool the ions, reducing or eliminating further fragmentation into smaller pieces.

[0161] Without wishing to be bound to theory, HCD is a non-resonant activation technique in Orbitrap instruments in which ions do not experience a long series of “equilibrating” collisions with gas molecules. In HCD, a single bolus of energy may be imparted on the ions in a short time such that both weak bonds and stronger bonds may be broken, depending on which ones are excited by the smaller number of collisions with gas molecules. Product ions remain excited, but do not experience enough collisions to cool such that if the ions further collide with gas molecules, the ions may be further fragmented.

[0162] In HCD, the collision energy applied to achieve fragmentation is higher, and the activation time may be less than resonance-CID. Additionally, HCD also does not have the limitation of a low mass cut-off like ion trap-based CID.

[0163] The method provides enhanced control of isolation window. In mass spectrometry, an isolation window defines the range of mass-to-charge ratios (m / z) that are selected for fragmentation. This enhancement may be advantageous for ligand screening in cell lysates, where sample complexity is high. The ability to isolate a narrower m / z range of the method of the present disclosure may not only exclude unbound ligands but may also minimise interference from non-specific protein-ligand clusters and background noise, thereby improving signal specificity and detection sensitivity.

[0164] In some examples, the method may comprise applying a narrow mass filter isolation range based on the expected location of a biomaterial-candidate complex. The narrow mass filter isolation range may be within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% of the expected location of a biomaterialcandidate complex.

[0165] The expected location of a biomaterial-candidate complex may be determined by scanning adjacent or overlapping m / z windows. For example, by scanning adjacent m / z windows of 1000-2000 m / z, 2000-3000 m / z, and 3000-4000 m / z or scanning overlapping windows of 1000-2000 m / z, 1500-2500 m / z, and 2000-3000 m / z. In other examples, theexpected location of a biomaterial-candidate complex may be determined by continuous scanning of defined window from low to high m / z.

[0166] The HCD may be configured to fragment the one or more dissociated candidate compounds into smaller fragments during dissociation from the biomaterial-candidate complex.

[0167] The smaller fragments may be 1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, 300 or less, 200 or less, or 100 or less m / z and useful ranges may be selected between any of these values (for example, about 100 to about 1000, about 200 to about 800, about 400 to about 600, about 450 to about 550 m / z).

[0168] HCD is able fragment dissociated compounds into smaller fragments to achieve better ion distribution than conventional resonance-based CID. This enables better structural identification of small molecules. In some examples, HCD may be performed with a plurality of collision energies to obtain a plurality of fragmentation patterns of the dissociated biomaterial and / or dissociated candidate compound. For example, the HCD may be performed using 2, 3, 4, 5, 6, 7, 8, 9, or 10 collision energies.

[0169] Different collision energies may be used to generate different fragmentation patterns. A plurality of fragmentation patterns may be used to determine other characteristics or features of a candidate compound that may be useful in drug development. Greater fragmentation achieved using different HCD collision energies may improve elucidation of characteristics of the candidate compound, for example the structure of the candidate compound, and site of attachment of the compound to a target. This may help in annotating complex samples such certain types of post translationally modified peptides such as glycopeptides, particularly where a candidate compound is dissociated from the biomaterial and fragmented as a result of sufficiently high collision energy.

[0170] The plurality of collision energies may comprise at least one low collision energy, and at least one high collision energy.

[0171] In some examples, the low collision energy may be about 1 , 3, 6, 9, 12, 15, 18, 21 , 24, 27, 30, 33, 36, 39, 42, 45, 49% of a total available voltage of the CID or HCD, and useful ranges may be selected between any of these values (for example, about 1% to about 49%, about 5% to about 40%, about 10% to about 30%, about 15% to about 25% of a total available voltage of the CID or HCD).

[0172] In some examples, the high collision energy may be about 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 99, or 100 of the total available voltage of theCID or HCD, and useful ranges may be selected between any of these values (for example, about 50% to 100%, about 60% to about 95%, about 70% to about 90%, about 80% to about 85% of the total available voltage of the CID or HCD).

[0173] In some examples, the collision energy may be increased at a predetermined rate to determine dissociation to obtain a plurality of fragmentation patterns of the dissociated candidate compound. For example, the predetermined rate may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 % of the collision energy.

[0174] In some examples, a voltage of the CID or HCD may be increased incrementally (ramped) to determine dissociation behaviour between the biomaterial and candidate compound.

[0175] Dissociation behaviour may be broadly described as the condition in which a candidate compound bound to a target may dissociate. For example, the level of collision energy or HCD voltage that may be required dissociate different candidate compounds with different binding affinities to the targets from the biomaterial-candidate compound complex.

[0176] The more strongly a ligand or compound binds to a target, the higher the energy required to dissociate the ligand from the complex. As a result, ligand with strong binding affinity may come off relatively slowly as the collision energy or HCD voltage increases and the dissociation should persist at higher voltages when compared to a ligand with a weaker binding affinity to the same target.

[0177] The voltage of HCD may be incrementally increased at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 volt increments, and useful ranges may be selected between any of these values (for example, about 1 to about 20, about 2 to about 18, about 4 to about 16, about 6 to about 14, about 8 to about 12 volts).

[0178] The voltage of HCD may be incremented about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times, and useful ranges may be selected between any of these values (for example, about 5 to about 50, about 10 to about 40, about 20 to about 30 times).

[0179] For example, biomaterial-candidate compound complex may be subjected to voltage stepping starting at 10 volts and increased at 10 volt increments over 10 increments.

[0180] The voltage or collision energy stepping may be performed within a single analytical run.(e) Separation step

[0181] Following dissociation of biomaterial and candidate, an optional separation step may be performed to capture low mass range ions. This may involve detecting a low m / z range through selecting a specific mass range in the mass detector such as an ion trap or Orbitrap detector. The low mass range ions may correspond to the mass range of the candidate compounds.

[0182] The low mass range may be about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1050, 1100, 1200, 1300, 1400, 1500 m / z, and useful ranges may be selected between any of these values (for example, about 50 to about 1500, about 100 to about 1400, about 200 to about 1300, about 300 to about 1200, about 400 to about 1100, about 500 to about 1000, about 600 to about 900, about 700 to about 800 m / z).

[0183] In some examples, TOF analysers may be used to capture low mass range ions by adjusting voltages and other parameters of the pusher or electrode system directly upstream of the TOF tubing such that only low m / z ions are pushed into the TOF analyser.

[0184] Following dissociation, the captured dissociated compound may be detected by an ion detector. The signals measured by the ion detector may be processed to obtain a mass spectrum. The mass spectrum plots the ions’ mass-to-charge ratios on the x-axis and the relative abundance of the ions on the y-axis.

[0185] Generally, the mass-to-charge ratio and / or isotopic distribution of ions for a compound or molecule provides a unique identifier that may be used to identify and quantify the compound or molecule in a sample. This enables the captured dissociated compound to be determined based on their mass-to-charge ratio (m / z). The dissociated compound may be detected in protonated or deprotonated forms, and / or as an adduct to a large ion such as but not limited to Na+ or K+. Depending on the detected form, dissociated compound may have different m / z values and isotopic distributions. The compound may be detected in any such forms and the detected m / z and / or isotopic distribution is processed to determine the original mass of the compound.

[0186] Determination of the dissociated compound may be achieved by correlating the mass-to-charge ratio and / or isotopic distribution of ions of the dissociated compound with a reference or database of compounds. The m / z and / or isotopic distribution of ions of the dissociated candidate compound maybe correlated with the mass-to-charge ratio and / or isotopic distribution of ions of a compound in a reference or established database to identify the identity of compound that bound to and formed a complex with a target in the biomaterial.

[0187] The method may further comprise generating a reference for the candidate compounds. The reference may be generated by measuring the mass-to-charge ratios of the candidate compounds and generating a list of mass-to-charge ratios that may be correlated with the mass-to-charge of the dissociated compound to determine binding to a target.

[0188] In some examples, reference mass-to-charge ratios and / or isotopic distribution may be calculated where the when the atomic composition of the compound is known.

[0189] Individual molecules have relatively unique fragmentation patterns. The greater the degree of fragmentation patterns, the greater the scope of information derivable when determining a compound. HCD is able to produce greater number of smaller fragments during dissociation using HCD such that there more spectral peaks in the mass spectrum. The greater number of spectral peaks enables greater amount of information to be derived from by comparing the differences in m / z between the base or tallest peaks (corresponding to the unfragmented compound) and smaller or fragment peaks (corresponding to fragments of the unfragmented compound).

[0190] Generating a plurality of smaller fragments allows for a greater degree of fragmentation patterns to be produced. The greater degree of fragmentation patterns may allow additional information to be derived such that the structure and / or function of a candidate compound may be better characterised for drug development applications.(f) Phenotype screening

[0191] Also described is a method for screening a phenotypic panel. A first sample is prepared by incubating a first cell lysate derived from cells overexpressing a protein target with a candidate compound such that the protein target within the first cell lysate forms a complex with the candidate compound. A second sample is prepared by incubating a second cell lysate derived from cells not overexpressing the target protein with the candidate compound. Each sample is introduced to a mass spectrometer under conditions that retain the protein-candidate complex, when present, in their native state. The protein-candidate complex, when present, is separated from unbound candidate compounds by selecting for ions having a high mass range corresponding to the protein target using a mass analyser configured to capture the one or more biomaterial-candidate complexes and excluding unbound candidate compounds based on a difference in oscillation frequencies of their respective ions. The protein-candidate complex is dissociated using higher energy collisional dissociation (HCD) to obtain one or more dissociated candidate compounds. The dissociated candidate compound is captured by selecting for ions having a low mass range. The mass spectrum of the first sample is compared to the mass spectrum of the secondsample to determine the difference in binding of the candidate compound to the first and second cell lysates.

[0192] The phenotypic panel screening according to the present disclosure may assist in identifying the mechanism of action in the diseased model by comparing the binding of a candidate to compound to a diseased cell line with target protein overexpression and a nondiseased cell line (same type of cells, same type of endogenous proteins in the background) with no target protein expression.

[0193] Figure 2 illustrates a method for screening a phenotypic panel according to the present disclosure. The method may broadly comprise preparing two samples of cell lysates incubated with a candidate compound. One of the cell lysates is derived from cells overexpressing a target, while the other cell lysate is derived from cells. The samples are ionised and individually transferred into a mass analyser for mass selection. The high mass range ions corresponding to protein-candidate compound complex are trapped while the low mass range ions are excluded.

[0194] The steps of sample introduction, separation the protein-candidate complex, dissociation of the protein-candidate complex, and capturing the dissociated candidate compound is described in more detail elsewhere in the specification.

[0195] The captured protein-candidate compound complex may be dissociated using HCD and the low mass range ions are captured for detection. The mass spectra from the respective samples are then compared to determine the difference in binding between the first and second samples.

[0196] Where cells overexpressing a protein target is derived from a diseased cell, the candidate compound may be known to bind a target in the diseased cell lysate. In such examples, a result may show that the diseased model has spectral peaks corresponding to dissociated bound drug candidate, and the non-diseased model will have negligible or no peaks corresponding to the drug candidate, which indicates that the target protein in the diseased model is responsible for binding to the drug candidate. Generally, the signal may be higher in the diseased model but a signal may still be present in the non-diseased model due to non-specific binding between the compounds or between the compound and other proteins)

[0197] In other examples, the diseased cells overexpressing a target may be screened with a plurality of candidate compounds to identify a compound that binds to the overexpressed target in the diseased cell. The candidate compound is then screenedagainst the normal or non-diseased cells. Comparing the binding of the compound in the diseased and non-diseased cells may indicate whether the binding between the overexpressed protein in the diseased cell is target specific.(g) Voltage stepping

[0198] Precise evaluation of protein-ligand interactions is an important consideration for elucidating biochemical pathways and optimizing the activity of bioactive molecules. The method of the present disclosure enables such analyses by modulating the energy applied during collisional activation, thereby controlling the extent of ligand dissociation from protein complexes.

[0199] By modulating the collision energy, the method enables differentiation between weak and strong interactions, as lower-affinity complexes dissociate at lower collision energies, whereas high-affinity complexes resist dissociation until subjected to higher activation voltages. Further, modulating the collision energy during dissociation of a sample containing a biomaterial (e.g. a cell lysate containing a target protein) and a pool of candidate compounds enables analysis of dissociation behaviour within a single experimental run compared to existing methods.

[0200] In some examples, the method may further comprise:• dissociating biomaterial-candidate compound complex by applying a plurality of collision energies,• capturing the dissociated candidate compounds at each of the plurality of collision energies, and• determining dissociation constant of the dissociated candidate compounds.

[0201] In such examples, the collision energy may be configured to increase at a predetermined rate. For example, the collision energy or voltage may increase at a rate of 5 V per increment over 5 increments.

[0202] The dissociation constant may be determined by comparing the relative amounts of a dissociated candidate at a plurality of dilution factors of the biomaterial and plurality of collision energies. For example, during sample preparation a cell lysate may be serially diluted and incubated a predetermined amount of candidate compound, and each serial dilution containing biomaterial-candidate compound complexes may be dissociated by applying a plurality of collision energies at various increments.

[0203] Comparing the relative amounts of a candidate compound at each dilution and collision energy provides an indication of the dissociation constant of the candidate compound and biomaterial. Dilution that does not result in a reduction of detected candidate compounds may be indicative of a low dissociation constant. Conversely, dilution that results in a decrease in detected candidate compounds may be indicative of a high dissociation constant.(h) Ranking dissociation constants

[0204] In primary screening during the drug discovery process, the data relating to the dissociation maybe analysed in all voltage channels to identify the dissociated candidate compounds where no prior knowledge of the optimal voltage to for dissociation the candidate compounds in a pool of compounds. By applying a plurality of collision energies or voltages the increases the probability of identifying hits.

[0205] For the identified hits, a dissociation profile as a function of applied voltage may be generated to rank the strength and / or stability of the protein-ligand interactions.

[0206] The method may further comprise ranking at least two dissociated candidate compounds based on their respective dissociation constants. For example, where detection of a first candidate compound shows a linear increase across the decreasing dilution factors for a cell lysate overexpressing a protein, and a second candidate shows a non-linear increase (e.g. a plateau at higher lysate concentrations), this indicates that the second candidate compound has a lower dissociation constant to the overexpressed / upregulated target protein.

[0207] In some examples, a biomaterial such as a cell lysate overexpressing a target protein may be diluted in a control cell lysate with no overexpression. Such a step ensures that only those proteins that were overexpressed in the cell lysate relative to the control lysate were serially diluted. In such examples, dilution of the overexpressed target protein may be performed without purification from the cell lysate.

[0208] The method according to the present disclosure may be applied directly in a cell lysate without the need for protein purification.

[0209] The method according to the present disclosure consists of a sequence of steps which can be individually optimised or synergistically optimised to achieve greater sensitivity or specificity of the detection of analytes of interest.

[0210] The method according to the present disclosure comprises the following steps, which can be individually or synergistically optimised:• the preparation of a cell lysate that contains a proteome;• the fractionation of the of the lysate into sub-proteomes such as the soluble and / or membrane-bound proteome(s);• the adjustment of the lysis buffer composition to stabilise proteins, protein complexes, and protein-compound complexes in their native states, whereby such adjustment can be performed before or after cell lysis, before or after the fractionation of the lysate into sub-proteomes, and before or after addition of compounds to the lysate;• the dilution of the proteome or the mixing of proteomes at pre-determined ratios;• the mixing of the proteome with one or more compounds of interest;• the incubation conditions of such mixture to promote protein-ligand interaction;• the controlled injection of the sample into a mass spectrometer, whereby a predetermined sample injection volume and flow rate are achieved, and which can be achieved with the use of a liquid chromatography system combined with an autosampler;• the choice of chromatography column, including the option of bypassing a chromatography column;• the choice of material, length, and inner diameter of the tubing used in the liquid chromatography system;• the choice of temperatures, voltages, radio frequencies, pressures, gas flow rates, and other such parameters across the mass spectrometer to achieve desolvation, ionisation, guiding, trapping, accumulation, dissociation, fragmentation, m / z measurement, and any other biophysical analysis available to the mass spectrometer; the choice and parameters of ion filtering to remove unbound small molecules, including the option of screening adjacent m / z isolation windows, screeningoverlapping m / z windows, or applying a single m / z cut-off that retains large m / z ions.• the choice and parameters of dissociation and fragmentation method, such as CID or HCD, when the choice is available, including the option of performing CID, HCD, or other analyte dissociation method, at different energy values;• the choice of mass analyser, such as an Orbitrap, time-of-flight analyser, linear ion trap analyser, or quadrupole, when the choice is available;• the choice of mass spectrometry data time and m / z range acquisition;• the preparation of reference mass spectra, which may be empirically obtained or theoretically calculated, for inspection of the mass spectra recorded using the method as described in the present disclosure.

[0211] In some examples, one or more steps can be omitted, such as the preparation of a dilution series of the cell lysate(s), the mixing of different cell lysates, the inclusion of m / z isolation windows, or the inclusion of energy steps during ligand dissociation by CID, HCD (or any method relying on the collision of biomolecular ions with inert gases), or the use of CID or HCD (or any method relying on the collision of biomolecular ions with inert gases).

[0212] The proceeding examples demonstrate the following aspects of methods of the present disclosure:• Example 1 demonstrates identification of interactions between individual compounds or compounds within a pool with a purified target protein• Example 2 demonstrates identification of interactions between individual compounds or compounds within a pool with a target within a bacterial cell lysate• Example 3 demonstrates phenotypic screening panel in mammalian cell lysate overexpressing a target protein• Example 4 demonstrates identification of protein-ligand interactions in various buffer systemsExample 5 compares sensitivity achieved by control of isolation in the present method with CIAS-MS• Example 6 demonstrates identification of membrane protein-ligand interactions within a complex sample mixture using HCD• Example 7 demonstrates collision energy modulation in CID or HCD for analysing ligand dissociation from protein complexes.• Example 8 demonstrates ranking of ligand dissociation constants directly in cell lysate.EXAMPLE 1 - IDENTIFICATION OF HUMAN CARBONIC ANHYDRASE I (HCAI) PROTEIN INTERACTION WITH KNOWN LIGANDS

[0213] The present example describes the application of the method for screening analytes of interest against soluble protein targets in a drug discovery context. The workflow includes several optional optimization steps designed to enhance analytical performance.These include modifications aimed at increasing sensitivity and specificity, reducing required sample volume, minimizing sample preparation time, and shortening mass spectrometry acquisition time. The optimized protocol enables efficient detection of protein-ligand interactions, facilitating high-throughput screening with minimal material consumption and rapid turnaround.

[0214] This example demonstrates identification of hCAI interactions with• furosemide• diclofemanide• furosemide within pooled compounds• diclofemanide within pooled compounds.(a) Materials and Methods

[0215] Protein and ligands. hCAI protein, furosemide and diclofenamide were sourced from Merck. hCAI was resuspended in 100 mM ammonium acetate to a stock concentration of 100 pM prior to MS sample preparation. Ligands were dissolved in methanol to prepare 1 mM stock solutions, which were subsequently diluted in 100 mM ammonium acetate for use in experiments.

[0216] Pooled compounds. 100 compounds sourced from MedChem Express were dissolved in DMSO to a stock concentration of 10 mM. The compounds were pooled, vacuum-dried, and resuspended in methanol to a final stock concentration of 100 pM (each compound).

[0217] MS sample preparation. Each 5 pL sample consisted of hCAI (5 pM) incubated with either furosemide or diclofenamide (5 pM). Where applicable, pooled compounds (5 pM each) were included. All samples were prepared in 100 mM ammonium acetate (pH 8.0, adjusted with ammonia). The final methanol content ranged from 0% to 5% (v / v).

[0218] Experimental conditions: MS experiment was performed with a Waters Vion mass spectrometer equipped with an autosampler operating at a flow rate of 100 pL / min. Samples were introduced via an automated LC autosampler, followed by electrospray ionization and transfer into the mass spectrometer. The capillary voltage was set at 1.5 kV in negative mode, the capillary temperature was set to 120 °C, and the desolvation temperature was set to 300 °C. The quadrupole was configured to pass ions with a mass-to- charge ratio between m / z 2,400 to 15,000. CID was performed using voltages up to 15 V per scan. The mass spectrometer was operating under negative ESI mode.

[0219] Native MS Conditions: Native MS analysis was conducted by using the same conditions described, except the quadrupole was configured to pass all ions and CID was turned off by setting the voltage to 0 V.(b) Results - identification of hCAI interactions with furosemide

[0220] Ions corresponding to the molecular weight of furosemide were detected with high intensity at m / z 328 - 334 and corresponding to the major isotopes of deprotonated furosemide under negative ESI mode (Figure 3A, inset) indicating the detection of dissociated bound furosemide from the hCAI-furosemide complex. No ligand could be detected when CID was turned off (Figure 3D).

[0221] Control experiments were performed using furosemide (5 pM) or hCAI (5 pM) alone, both with CID turned on (Figures 3B and 3C) and turned off (Figures 3E and 3F). In all control spectra, ion signals corresponding to furosemide could not be detected, or were only detected with negligible intensities. These control experiments confirmed that the quadrupole settings effectively excluded unbound ligand ions.

[0222] The native MS spectra of hCAI, the hCAI-furosemide complex, and furosemide I shown in Figure SI 1A-C.(c) Results - detection of diclofemanide interaction with hCAI

[0223] Ions corresponding to the molecular weight of diclofenamide were detected with high intensity within the 302 to 309 m / z range, corresponding to the major isotopes of deprotonated diclofenamide (Figure 5A, inset). Without CID, the ligand could not be detected (Figure 5D).

[0224] Control experiments were performed using diclofenamide (5 pM) or hCAI (5 pM) alone, both with CID turned on (Figures 5B and 5C) and turned off (Figures 5E and 5F). In all control spectra, ion signals corresponding to diclofenamide could not be detected, or were only detected with negligible intensities.

[0225] The native MS spectra of hCAI, the hCAI-diclofenamide complex, and diclofenamide alone are shown Figure S1A, F, and G.(d) Results - identification of hCAI interactions with furosemide within pooled compounds

[0226] Furosemide was detected with high intensity among the dissociated analytes (Figure 4A, inset) confirming the identification of hCAI - furosemide interaction in a complex sample.

[0227] Control experiments were conducted by omitting hCAI and / or disabling CID (Figures 4B-D). In the absence of hCAI, furosemide was effectively excluded by quadrupole filtering, and CID did not yield detectable furosemide (Figure 4B). When CID was disabled, no ligand dissociation was observed under any condition (Figures 4C and 4D), confirming that both protein binding and collisional activation are required for ligand release and detection.(e) Results - identification of hCAI interactions with diclofemanide within pooled compounds

[0228] Diclofenamide could be detected with high intensity among the dissociated analytes (Figure 6A, inset). When analysing the 101 -compound mixture sample without hCAI (Figure 6B), ions corresponding to deprotonated diclofenamide could be detected at low intensities, possibly due to clustering between compounds enabling their passing through the quadrupole. This control experiment establishes the baseline signal for diclofenamide in the absence of protein targets. In the presence of hCAI, the signal for diclofenamide is several-fold higher due to the hCAI-diclofenamide interaction (Figure 6A).

[0229] When analysed without CID, no ion signals corresponding to diclofenamide were observed from either sample (Figure 6C and 6D).EXAMPLE 2 - IDENTIFICATION OF LIGAND INTERACTION IN BACTERIAL CELL LYSATE

[0230] The purpose of this study is to demonstrate the identification of ligand binding to a target protein within a sample mixture containing one protein target in bacterial cell lysateand one compound and a complex sample mixture containing one protein target in bacterial cell lysate and a mixture of compounds.

[0231] The method incorporates several optional optimization steps to improve analytical performance for drug discovery applications conducted directly in crude bacterial lysates. These optimizations include: the adjustments to improve sensitivity and specificity of detection; a tailored liquid chromatograph autosampler protocol that reduces the required sample volume; a single mass spectrometer acquisition run to enable higher-throughput analysis with simultaneous acquisition of MS spectra with both collision energy turned on and off; which is particularly beneficial for complex lysate samples. Together, these modifications enable robust and efficient detection of protein-ligand interactions under physiologically relevant conditions, supporting high-throughput screening with minimal material input and rapid data acquisition.

[0232] The example demonstrates identification of hCAI within a bacterial cell lysate interacting with:• furosemide• diclofemanide• furosemide within pooled compounds• diclofemanide within pooled compounds(a) Materials and Methods

[0233] Cell Lysate Preparation: E. coli cells were harvested following cultivation by resuspending the cell pellet in ice-cold phosphate-buffered saline (PBS), followed by centrifugation to remove the supernatant. The resulting pellet was resuspended in ice-cold 100 mM ammonium acetate (pH 8.0). Cell lysis was performed via sonication using short pulses to minimize heat generation. Lysates were clarified by centrifugation and filtered through 0.2 pm syringe filters. Final lysates were stored at -80 °C until use.

[0234] Protein and Ligands: Human carbonic anhydrase I (hCAI), furosemide, and diclofenamide were obtained from Merck. hCAI was resuspended in 100 mM ammonium acetate to a stock concentration of 100 pM. Ligands were dissolved in methanol at 1 mM and further diluted in 100 mM ammonium acetate as needed.

[0235] Buffer Exchange: Prior to MS analysis, lysates (lacking hCAI) were thawed on ice and buffer-exchanged into 100 mM ammonium acetate (pH 8.0) using NAP-5 columns (Cytiva).

[0236] Pooled Compound Library: A library of 100 small molecules was sourced from MedChem Express. Each compound was initially dissolved in DMSO at 10 mM. The compounds were pooled, vacuum-dried, and resuspended in methanol to maintain the same final concentration. Lysate containing hCAI was incubated with a pooled compound library consisting of 101 different molecules (each at 5 pM), spiked with furosemide or diclofenamide.

[0237] MS Sample Preparation: For each 5 pL sample injection, lysate (final protein concentration 1 mg / mL) was combined with or without hCAI (5 pM), furosemide or diclofenamide (5 pM), and, where applicable, pooled compounds (5 pM each). Control samples were prepared identically but without added hCAI. All samples were prepared in 100 mM ammonium acetate (pH 8.0, adjusted with ammonia), with final methanol content ranging from 0-5% (v / v).

[0238] Experimental Conditions: Samples were subject to electrospray ionisation of the complex, followed by transmission into the mass spectrometer. Samples were introduced via autosampler at a flow rate of 100 pL / min. The waters VION mass spectrometer was operated in negative ion mode with a capillary voltage of 1.5 kV, a capillary temperature of 120 °C, and a desolvation temperature of 300 °C. The quadrupole was configured to transmit ions in the m / z 2,400-15,000 range to filter out small unbound molecules. The selected complexes were transferred to the collision cell to trigger ligand dissociation. CID was performed with voltages ramped up to 15 V per scan.

[0239] Native MS Conditions: Native mass spectrometry was conducted under identical source conditions. However, the quadrupole was set to transmit all ions, and CID was disabled by setting the collision voltage to 0 V.(b) Results - identification of hCAI interactions with furosemide within a bacterial lysate

[0240] Ions corresponding to the molecular weight of furosemide were detected with high intensity at m / z 328 - 334 and corresponding to the major isotopes of deprotonated furosemide (Figure 7A, inset) indicating dissociation of furosemide from the cell lysate containing hCAI was detected. Without CID, the ligand could not be detected (Figure 7D).

[0241] Control experiments included control lysate (lacking hCAI) mixed with furosemide, or lysate containing hCAI but lacking furosemide, both with (Figures 7B and 7C) and without (Figures 7E and 7F) CID. In all control spectra, no ions were observed within the 327-335 m / z range (corresponding to the major isotopes of deprotonated furosemide, orwere observed with negligible intensities, confirming that unbound ligand ions were effectively excluded by the quadrupole settings.(c) Results - identification of hCAI interactions with diclofenamide within a bacterial lysate

[0242] Ions corresponding to the molecular weight of diclofenamide were detected with high intensity within the m / z 302-309 range, corresponding to the major isotopes of deprotonated diclofenamide (Figure 9A, inset). Without CID, the ligand could not be detected (Figure 9D).

[0243] Control experiments included lysate (lacking hCAI) mixed with diclofenamide, or lysate containing hCAI but lacking diclofenamide, both with (Figures 9B and 9C) and without (Figures 9E and 9F) CID. In all control spectra, no ions were observed within the 327- 335 m / z range (corresponding to the major isotopes of deprotonated furosemide, or were observed with negligible intensities, confirming that unbound ligand ions were effectively excluded by the quadrupole settings.

[0244] Native MS spectra of the hCAI-containing lysate, hCAI-containing lysate mixed with diclofenamide, and the control cell lysate lacking hCAI incubated with diclofenamide are shown in Figure S2 2A, 2F-G.(d) Results - identification of hCAI interactions with furosemide in a pooled compound within a bacterial lysate

[0245] A high-intensity dissociated ligand signal (Figure 8A, inset) corresponding to furosemide was detected. In contrast, no dissociated ions were observed when CID was disabled (Figure 8C).

[0246] In the controls containing the same 101-compound mixture (including furosemide) incubated with cell lysate lacking hCAI, no furosemide ions were detected in the target m / z range, regardless of whether CID was enabled or disabled (Figures 8B and 8D).

[0247] These results demonstrate that the method selectively identified furosemide as a ligand binding to hCAI directly within a complex sample containing pooled small molecules and unpurified lysate-derived protein.(e) Results - identification of hCAI interactions with diclofenamide in a pooled compound within a bacterial lysate

[0248] Diclofenamide could be detected as a ligand with high intensity (Figure 10A, inset). When analysed without CID, no ion signals were observed (Figure 10C).

[0249] As a control, the pooled mixture containing diclofenamide in the cell lysate (but lacking hCAI) was analysed with CID either enabled or disabled. In the absence of hCAI, ions corresponding to deprotonated diclofenamide could be detected at low intensities (Figure 8B), possibly due to clustering between compounds or low affinity interactions with other proteins enabling their passing through the quadrupole. This control experiment establishes the baseline signal for diclofenamide in the absence of protein targets. In the presence of hCAI, the signal for diclofenamide is several-fold higher due to the hCAI- diclofenamide interaction (Figure 10A).

[0250] When analysed without CID, no ion signals corresponding to diclofenamide were observed from either sample (Figure 10C and 10D).EXAMPLE 3 - PHENOTYPIC SCREENING PANEL IN MAMMALIAN CELL LYSATE OVEREXPRESSING HCAI OR BCAII

[0251] The purpose of the study is to demonstrate a phenotypic screening panel in mammalian cell lysate overexpressing hCAI or bCAII.

[0252] The method includes a range of enhancements designed to improve analytical sensitivity, specificity, and overall efficiency when applied directly to crude mammalian lysates with target protein overexpression. These enhancements may involve adjusting experimental parameters to strengthen detection of protein-ligand interactions and implementing a refined LC autosampler method to minimize sample volume requirements. In addition, a single MS acquisition run is used to capture both activated collision energy data and collision energy off control data, supporting streamlined and higher-throughput analysis. Together, these optimizations enable reliable and rapid detection of target engagement in physiologically relevant conditions, making the workflow highly suitable for phenotypic drug screening with minimal material input.

[0253] This example demonstrates identification of• hCAI-furosemide interactions in mammalian cell lysate overexpressing hCAI• hCAI -diclofemanide interactions in mammalian cell lysate overexpressing hCAI• hCAI-furosemide interactions in a complex mixture of overexpression cell lysate and pooled small molecules• hCAI-diclofemanide interactions in a complex mixture of overexpression cell lysate and pooled small molecules bCAII-furosemide interactions in mammalian cell lysate overexpressing bCAII• bCAII-furosemide interactions in a complex mixture of overexpression cell lysate and pooled small molecules• bCAII-diclofemanide interactions in mammalian cell lysate overexpressing bCAII• bCAII-diclofemanide interactions in a complex mixture of overexpression cell lysate and pooled small molecules(a) Materials and Methods

[0254] Cell lysate preparation: CHO cells were transfected with plasmids overexpressing the hCAI or bCAII protein. 100 mL cell cultures of each cell line were prepared alongside a control non-transfected cell line. Lysates were prepared by harvesting the cells by centrifugation, resuspending the cells in 10 mL of ice cold 100 mM ammonium acetate, followed by sonication using short pulses to avoid solvent heating. Lysates were clarified by centrifugation and filtered through 0.2 pm syringe filters. The final lysates were aliquoted and stored at -80 °C prior to sample preparation for mass spectrometry (MS) analyses. Aliquots were either thawed on ice prior to sample preparation or further desalted after thawing into into 100 mM ammonium acetate (pH 8.0, adjusted with ammonia) using NAP-5 columns (Cytiva) before MS analysis. Where relevant, saline buffers were added to the lysates. The identity and final concentrations of salts and buffer agents in lysates were NaCI (50 mM) and Tris-HCI (20 mM) or Na2SO4 (10 mM) and HEPES (10 mM). The saline buffer stocks (10X concentrations) were adjusted to pH 7.4 with sodium hydroxide prior to mixing with lysates.

[0255] Pooled compounds: A total of 100 compounds (MedChem Express) were dissolved and mixed in DMSO, vacuum-dried, and redissolved in methanol to prepare 100 pM (each compound) stock solutions. In hCAI experiments, both the hCAI-overexpressing lysate and the control lysate (no overexpression) were incubated with a pooled compound library consisting of 101 different molecules (each at 5 pM), including furosemide or diclofemanide. In bCAII experiments, both the bCAII-overexpressing lysate and the control lysate (no overexpression) were incubated with a pooled compound library consisting of 101 different molecules (each at 5 pM), including furosemide or diclofemanide.

[0256] MS sample preparation: Each 5 pL injection contained cell lysate (2 mg / mL final), furosemide or diclofenamide (5 pM final), and, where relevant, the pooled compound mixture (5 pM each). All samples were prepared in 100 mM ammonium acetate (pH 8.0, adjusted with ammonia), with final methanol concentrations ranging from 0-5% (v / v).

[0257] Experimental conditions: The autosampler operated at a flow rate of100 pL / min. The sample containing a mixture of furosemide or diclofenamide and cell lysates were subject to electrospray ionisation followed by transmission of ions into through the mass spectrometer. Capillary voltage was set to 1.5 kV in negative mode. Capillary and desolvation temperatures were 120 °C and 300 °C, respectively. The quadrupole was set to isolate ions in the 2,400-15,000 m / z range to isolate large molecular entities and filter out small unbound molecules. CID was performed with voltages up to 15 V per scan.

[0258] Native MS: Native MS was performed under the same conditions as described in the experimental conditions above, except the quadrupole passed all ions and CID was disabled by setting the voltage to 0 V.(b) Results - Identification of hCAI-furosemide interactions in mammalian cell lysate overexpressing hCAI

[0259] Ions within the 327-335 m / z range and corresponding to the major isotopes of deprotonated furosemide were detected with high intensity (Figure 11A).

[0260] A control experiment performed with CID turned off (Figure 11 D). Control samples were a control lysate (no overexpression) mixed with furosemide and the hCAI- overexpressing lysate (without ligand), both analysed with (Figures 11 B and 11C) and without (Figures 11 E and 11 F) CID. In all control spectra, ions within the 327-335 m / z range and corresponding to the major isotopes of deprotonated furosemide were either not detected or detected with negligible intensity.(c) Results - Identification of hCAI-diclofemanide interactions in mammalian cell lysate overexpressing hCAI

[0261] Ions within the 301-309 m / z range and corresponding to the major isotopes of deprotonated diclofenamide were detected with high intensity (Figure 13A).

[0262] A control experiment performed using the control lysate (no overexpression) and ions corresponding to deprotonated diclofenamide could be detected with substantially lower intensity (Figure 13B), indicating that some diclofenamide interacted with one or more proteins in the lysate, but that overexpression of hCAI resulted in the affinity selection of diclofenamide during the quadrupole isolation stage of the method, effectively detecting diclofenamide as an hCAI binder in the hCAI-overexpression lysate relative to the control lysate.

[0263] Other control experiments included the method analysis of the hCAI- overexpressing lysate (without ligand) (Figure 13C), and the analysis of all samples with CIDturned off (Figures 13D-F). In these control experiments, ions corresponding to deprotonated diclofenamide were either not detected or detected with negligible intensity.(d) Results - Identification of hCAI-furosemide interactions in a complex mixture of overexpression cell lysate and pooled small molecules

[0264] Dissociated furosemide ions were detected with high intensity in the hCAI- overexpressing lysate (Figure 12A) and with low intensity in the control lysate (Figure 12B), indicating that some furosemide interacted with one or more proteins in the lysate (Figure 10B), but that overexpression of hCAI resulted in the affinity selection of furosemide during the quadrupole isolation stage of the method, effectively detecting furosemide as an hCAI binder in the hCAI-overexpression lysate relative to the control lysate.

[0265] Control experiments with CID turned off yielded negligible signal of deprotonated furosemide ions (Figure 12C-D).(e) Results - Identification of hCAI-diclofemanide interactions in a complex mixture of overexpression cell lysate and pooled small molecules

[0266] Dissociated diclofenamide ions were detected with high intensity in the hCAI- overexpressing lysate (Figure 14A) and with low intensity in the control lysate (Figure 14B), indicating that some diclofenamide interacted with one or more proteins in the lysate (Figure 12B), but that overexpression of hCAI resulted in the affinity selection of diclofenamide during the quadrupole isolation stage of the method, effectively detecting diclofenamide as an hCAI binder in the hCAI-overexpression lysate relative to the control lysate.

[0267] Control experiments with CID turned off yielded negligible signal of deprotonated diclofenamide ions (Figure 14C-D).(f) Results - Identification of bCAII-furosemide interactions in mammalian cell lysate overexpressing bCAII

[0268] The dissociation of furosemide from the bCAII-overexpressing cell lysate was also detected using mass spectrometry (Figure 15A). Ions within the 327-335 m / z range and corresponding to the major isotopes of deprotonated furosemide were detected with high intensity.

[0269] A control experiment was performed using the control lysate (no overexpression) and ions corresponding to deprotonated furosemide could be detected with substantially lower intensity (Figure 15B), indicating that some furosemide interacted with one or more proteins in the lysate, but that overexpression of bCAII resulted in the affinity selection of furosemide during the quadrupole isolation stage of the method, effectivelydetecting furosemide as a bCAII binder in the bCAII-overexpression lysate relative to the control lysate.

[0270] Other control experiments included analysis of the bCAII-overexpressing lysate (without ligand) (Figure 15C), and the analysis of all samples with CID turned off (Figures 15D-F). In these control experiments, ions corresponding to deprotonated furosemide were either not detected or detected with negligible intensity.(g) Results - identification of bCAII-furosemide interactions in a complex mixture of overexpression cell lysate and pooled small molecules

[0271] Dissociated furosemide ions were detected with high intensity in the bCAII- overexpressing lysate (Figure 16A) and with low intensity in the control lysate (Figure 16B), indicating that some furosemide interacted with one or more proteins in the lysate (Figure 16B), but that overexpression of bCAII resulted in the affinity selection of furosemide during the quadrupole isolation stage of the method, effectively detecting furosemide as a bCAII binder in the bCAII-overexpression lysate relative to the control lysate.

[0272] Control experiments with CID turned off yielded negligible signal of deprotonated furosemide ions (Figure 16C-D).(h) Results - Identification of bCAII-diclofemanide interactions in mammalian cell lysate overexpressing bCAII

[0273] The dissociation of diclofenamide from the bCAII-overexpressing cell lysate was also detected using mass spectrometry (Figure 17A). Ions within the 302-309 m / z range and corresponding to the major isotopes of deprotonated diclofenamide were detected with high intensity.

[0274] A control experiment was performed using the control lysate (no overexpression) and ions corresponding to deprotonated diclofenamide could be detected with substantially lower intensity (Figure 17B), indicating that some diclofenamide interacted with one or more proteins in the lysate, but that overexpression of bCAII resulted in the affinity selection of diclofenamide during the quadrupole isolation stage of the method, effectively detecting diclofenamide as a bCAII binder in the bCAII-overexpression lysate relative to the control lysate.

[0275] Other control experiments included the analysis of the bCAII-overexpressing lysate (without ligand) (Figure 17C), and the analysis of all samples with CID turned off (Figures 17D-F). In these control experiments, ions corresponding to deprotonated diclofenamide were either not detected or detected with negligible intensity.(i) Results - identification of bCAII-diclofemanide interactions in a complex mixture of overexpression cell lysate and pooled small molecules

[0276] Dissociated diclofenamide ions were detected with high intensity in the bCAII- overexpressing lysate (Figure 18A) and with very low intensity in the control lysate (Figure 18B), indicating that a small portion of diclofenamide interacted with one or more proteins in the lysate (Figure 18B), but that overexpression of bCAII resulted in the affinity selection of diclofenamide during the quadrupole isolation stage of the method, effectively detecting diclofenamide as a bCAII binder in the bCAII-overexpression lysate relative to the control lysate.

[0277] Control experiments with CID turned off yielded negligible signal of deprotonated diclofenamide ions (Figure 18C-D).EXAMPLE 4 - BUFFER COMPATIBILITY MAMMALIAN LYSATE OVEREXPRESSING HCAI PROTEIN IN VARIOUS BUFFER SOLUTION

[0278] The purpose of this study is to demonstrate that protein-ligand interactions can be reliably detected by mass spectrometry in the presence of various common salts, thereby addressing the conventional limitation of using ammonium acetate as the sole buffer system for native condition MS screening. This approach expands the method’s applicability to a broader range of protein targets, including those that do not perform optimally in ammonium acetate. By accommodating alternative buffer systems, the method enables screening under conditions that more closely reflect native biological environments, without compromising detection sensitivity or data quality.

[0279] The hCAI-overexpressing lysate was complemented with either 50 mM NaCI, 20 mM Tris (pH 7.4) or with 10 mM Na2SC>4, 10 mM HEPES (pH 7.4), incubated with furosemide, and analysed by the method (Figures 19A-C), or incubated with a 101- compound mixture (including furosemide), and analysed by the method (Figures 19D-F).

[0280] In all scenarios, the method could detect furosemide as an hCAI binder, in spite of some ion suppression caused by the presence of salts and buffering agents.EXAMPLE 5 - CONTROL OF ISOLATION WINDOW

[0281] The purpose of this study is to demonstrate the improved sensitivity achieved by precise control of the isolation window in the present method compared to CIAS-MS method. Unlike a simple cutoff for mass filtering in CIAS-MS, this method employs a narrowly defined isolation range with cutoffs on both sides of the target m / z. This rangebased filtering more effectively excludes ions outside the desired window, thereby increasingdetection sensitivity. Such precise isolation is particularly valuable when analyzing complex samples, such as cell lysates, where filtering out irrelevant ion clusters enhances signal clarity and overall analytical performance.We show that control over the isolation range (the ability to isolate in narrower, predetermined m / z ranges) is advantageous compared to a broad isolation that only filters out small molecules ahead of CID / HCD.(a) Materials and Methods

[0282] Cell lysate preparation: Cell lysate was prepared as previously described.

[0283] MS sample preparation: Each injection contained cell lysate (32 pg / mL), incubated with PFOSA (5 pM). All samples were prepared in 100 mM ammonium acetate (pH 8.0, adjusted with ammonia), with final methanol concentrations ranging from 0-5% (v / v).

[0284] Experimental conditions: Nano-electrospray ionisation (nESI) emitters were fabricated from borosilicate glass capillaries (Harvard Apparatus, 1.2 mm o.d., 0.68 mm i.d. , 500 nm bore). Mass spectrometry was performed using a Thermo Fisher Scientific LIHMR mass spectrometer. Capillary voltage was set between 1.4 kV and 1.6 kV relative to the heated capillary entrance (280 °C). A maximum ion injection time of 100 ms was used. The quadrupole mass filter was configured to transmit ions within the following m / z ranges of 2,500 - 4,000, 2,500 - 25,000, 3,000 - 4,000, 2,000 - 3,000, and 4,000 - 5,000. Higher- energy collisional dissociation (HCD) was applied with a normalised collision energy (NCE) 35 V. Spectra were acquired by averaging 100 scans with five microscans each. MS was conducted under negative mode.

[0285] The sensitivity of the method was benchmarked against CIAS-MS in regard to the control of isolation window, and otherwise using identical lysate and ligand concentrations under the same buffer conditions and same amount of collision energy, with HCD used for dissociation of the ligand (Figure 20).

[0286] Using a controlled quadrupole isolation window, the amount of dissociated ligand detected by the present method was approximately eightfold higher than that observed using a CIAS-MS relying on cut-off filtering.

[0287] To illustrate the importance of applying a narrow mass filter isolation range, three isolation ranges were evaluated. The m / z range of 3,000-4,000 — corresponding to the expected location of the protein-ligand complex — yielded optimal detection of thedissociated ligand (Figure 21A). In contrast, no ligand signal was observed in the preceding range of 2,000-3,000 (Figure 21 B), and only minimal signal was detected in the subsequent range of 4,000-5,000 (Figure 21C).

[0288] These results highlight the critical role of precise mass filtering in enhancing detection sensitivity and specificity in complex lysate samples.

[0289] This enhancement is particularly critical for ligand screening in cell lysates, where sample complexity is high. The ability to isolate a narrower m / z range not only excludes unbound ligands but also minimizes interference from non-specific protein-ligand clusters and background noise, thereby improving signal specificity and detection sensitivity.EXAMPLE 6 - IDENTIFICATION OF LIGAND INTERACTION WITH MEMBRANE PROTEIN

[0290] The purpose of the study is to demonstrate detection of membrane proteinligand interactions within a complex sample mixture containing one membrane protein target and one compound and a complex sample mixture containing one membrane protein target and a mixture of compounds.

[0291] The study described herein incorporates the use of HCD with voltage stepping as part of the mass spectrometry workflow — a combination that has not been previously reported to the best of our knowledge — enabling characterization of membrane protein binding events in complex matrices.

[0292] The example demonstrates:• detection of dissociation of ertapenem from the OmpF-detergent-ertapenem complex• detection of membrane protein-ligand interactions in complex mixtures of pooled compounds(a) Materials and methods

[0293] Protein preparation: Recombinant Pseudomonas aeruginosa OmpF protein was purchased from Abeam. The protein was detergent-exchanged into 200 mM ammonium acetate buffer (pH 8) containing 2* the critical micelle concentration (CMC) of C8E4 and allowed to solubilise overnight at 4 °C. The solubilised membrane protein was then concentrated using a 50 kDa MWCO concentrator (Millipore) to a working concentration of 5 pM prior to mass spectrometry (MS) analysis.

[0294] Ligand and pooled compounds: Ertapenem was sourced from Merck and used at a screening concentration of 5 pM. A library of 100 small molecules(MedChem Express) was dissolved and mixed in DMSO, vacuum-dried, and reconstituted in methanol to form 100 pM stock solutions. To assess the applicability of the method for detecting membrane protein-ligand interactions in complex mixtures, OmpF (5 pM) was incubated with a pooled compound library comprising 100 small molecules (each at 5 pM), with ertapenem spiked into the mixture at 5 pM.

[0295] MS sample preparation: Each sample consisted of ~5 pM OmpF protein mixed with 5 pM sodium ertapenem (MW 497.5 g / mol), and where applicable, the pooled compound mixture (5 pM each). All samples were prepared in 200 mM ammonium acetate, pH 8.0 (adjusted with ammonia).

[0296] Experiment conditions: Electrospray ionization (ESI) was used to introduce the complex into the gas phase, followed by in-source dissociation to liberate the membrane protein complex from detergent micelles. Nano-electrospray ionisation (nESI) emitters were fabricated from borosilicate glass capillaries (Harvard Apparatus, 1.2 mm o.d., 0.68 mm i.d. , 500 nm bore). Mass spectrometry was performed using a Thermo Fisher Scientific UHMR mass spectrometer. Capillary voltage was set between 1.4 kV and 1.6 kV relative to the heated capillary entrance (280 °C). A maximum ion injection time of 100 ms was used. Insource dissociation was set to 125 V. The quadrupole mass filter was configured to transmit ions within a m / z range of 3,000-7,000. Higher-energy collisional dissociation (HCD) was applied with a normalised collision energy (NCE) above 15 V. Spectra were acquired by averaging 100 scans with one microscan each. MS was conducted under positive mode unless stated otherwise.

[0297] Native MS: Native MS analysis was conducted under the same conditions, with the quadrupole configured to transmit all ions and HCD set below 15 NCE to enable gentle collisional activation and liberation of membrane protein complexes from detergent micelles.(b) Results - detection of dissociation of ertapenem from the OmpF- detergent-ertapenem complex

[0298] The dissociation of ertapenem from the OmpF-detergent-ertapenem complex was detected under experimental conditions of the method (Figure 22A). A prominent ion at m / z 498 was observed (Figure 22A, inset), consistent with the protonated molecular ion of ertapenem in positive ion mode.

[0299] Control experiments were performed using samples containing either ertapenem (5 pM) or OmpF (5 pM) alone, analyzed with and without HCD (Figures 22B,22C, 22E, and 22F). In all cases, no signal corresponding to ertapenem was detected, confirming effective exclusion of free ligand by the quadrupole and supporting the specificity of ligand release from the intact complex.(c) Results - detection of membrane protein-ligand interactions in complex mixtures of pooled compounds

[0300] Ertapenem could be detected with high intensity among the dissociated analytes (Figure 23A, inset).

[0301] Control experiments were performed using the 101-compound mixture (including ertapenem) in the absence of OmpF, with and without higher-energy collisional dissociation (HCD) (Figures 23B and 23D). Without the protein present, ertapenem was effectively excluded by quadrupole filtering, and application of HCD did not result in detectable release of the ligand (Figure 23D). Furthermore, when HCD was disabled, no ligands were released or detected, even in the presence of OmpF (Figure 23C), confirming that activation energy is required for ligand dissociation and detection in the method.EXAMPLE 7 - VOLTAGE STEPPING

[0302] The purpose of this study is to demonstrate evaluation of protein-ligand interactions by modulating the energy applied during collisional activation, thereby controlling the extent of ligand dissociation from protein complexes.

[0303] The modulation of energy during collisional activation is achieved using either CID or HCD at different energy values within those available in the mass spectrometer.

[0304] The different energy values can be applied in successive analysis of a sample sample, each analysis being performed at a discrete voltage, or each analysis being performed at a different voltage ramp, when voltage ramps are available, during the collisional activation, such as CID or HCD, step.

[0305] The different energy values can be applied in a single analysis by extending the mass spectrometer duty cycle to incorporate multiple scans at different voltages or voltage ramps during the collisional activation, such as CID or HCD, step.(a) Materials and Methods

[0306] Protein preparation: Recombinant Pseudomonas aeruginosa OmpF protein, mammalian lysate overexpressing hCAI are prepared as described previously.

[0307] Ligand and pooled compounds: Ertapenem, PFOSA, diclofenamide and furosemide were sourced from Merck and prepared as described previously.

[0308] MS conditions: The MS parameters for voltage stepping were identical to those described in the previous example. In the case of OmpF and ertapenem, a varying HCD NCE voltage were applied from 15 V to 70 V with a NCE 5 increment. For hCAI lysate against PFOSA, diclofenamide or furosemide, a varying CID voltage from 0 V to 25 V were applied with increment of 5V.(b) Results

[0309] In samples containing OmpF-ertapenem complexes, dissociation of bound ertapenem began at normalized collision energy (NCE) >15 (Figure 24A). Under the current experimental conditions, NCE 15 represented the threshold for liberating the complex from the detergent micelle and initiating ligand dissociation. Signal intensity increased with higher NCE, reaching a plateau at NCE 35.

[0310] The voltage stepping experiment was also performed using hCAI- overexpressing lysates incubated with PFOSA, diclofenamide, or furosemide (Figures 24C- D). Collision energy stepping was performed within a single analytical run.

[0311] Dissociation of all three ligands increased with rising collision energy. Among the binders, PFOSA and diclofenamide reached dissociation plateaus at lower voltages compared to furosemide. The hCAI-furosemide complex continued to release ligand at voltages up to 25 V, suggesting greater complex stability in the context of a heterogeneous lysate, which is consistent with known dissociation constant values for the three ligands against hCAI.EXAMPLE 8 - RANKING OF LIGAND DISSOCIATION CONSTANTS DIRECTLY IN LYSATE

[0312] The purpose of this study is to demonstrate ranking of dissociation constants of protein ligands directly in cell lysate, without protein purification, using the method described in the present disclosure.

[0313] The ranking of dissociation constants is achieved with simple adjustment of sample preparation, whereby a cell lysate containing a proteome is either: serially diluted in an appropriate buffer or solvent, or;• mixed with another cell lysate containing a proteome at pre-determined ratios while keeping total protein concentration constant.(a) Materials and Methods

[0314] Cell lysate preparation: All CHO cell lysates (hCAI over expression, bCAII overexpression, no overexpression) were adjusted to 2 mg / mL concentration in 100 mM ammonium acetate. 1 -in-2 serial dilutions of hCAI- or bCAII-overexpression cell lysates were prepared using either 100 mM ammonium acetate as diluent (resulting in total protein dilution) or using the control lysate (no overexpression) as diluent (resulting in the specific dilution of overexpressed proteins relative to the control lysate). Each dilution was incubated with furosemide, diclofenamide, or perfluorooctanesulfonamide (PFOSA) (5 pM final) and analysed by the method.

[0315] In a first experiment, serial dilutions in 100 mM ammonium acetate of hCAI- overexpressing lysate were prepared and separately incubated with 5 pM furosemide or 5 pM PFOSA. Analysis was then performed on each sample with voltage stepping.

[0316] In a second experiment, the serial dilutions in 100 mM ammonium acetate of hCAI-overexpressing lysate (no overexpression) to ensure only those proteins that were overexpressed in the bCAII-overexpression lysate relative to the control lysate were serially diluted. In essence, this resulted in the dilution of bCAII without requiring its purification from the lysate. The samples were incubated with 5 pM furosemide or 5 pM diclofenamide and analysed by the method.(b) Results - serial dilutions in 100 mM ammonium acetate of hCAI- overexpressing lysate

[0317] Dilution of the lysate (and therefore of any ligand protein target in the lysate) did not incur in a decrease of detected furosemide during performance of the method (Figure 25A), indicating that furosemide exhibits a very low dissociation constant to the hCAI- overexpression lysate.

[0318] Conversely, dilution of the lysate incurred in a proportional decrease of in detected PFOSA, indicating that the concentration of the hCAI-PFOSA complex decreased with dilution of hCAI-overexpressing lysate. Therefore, the method can rank the apparent dissociation constants of ligands towards complex lysates that contain at least one protein target for the ligand.(c) Results - serial dilutions in 100 mM ammonium acetate of hCAI- overexpressing lysate

[0319] Detection of furosemide showed a linear increase with the fraction of bCAII- overexpressing lysate, whereas detection of diclofenamide showed the start of the plateau. These results indicated that diclofenamide exhibited a lower dissociation constant to the upregulated fraction of the bCAII-overexpressing lysate proteome relative to the control proteome and demonstrates ranking the apparent dissociation constants of ligands towards specific proteome fractions according to a method of the present disclosure.

[0320] Although embodiments have been described with reference to a number of illustrative embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

[0321] Many modifications will be apparent to those skilled in the art without departing from the scope of the present invention as herein described with reference to the accompanying drawings.

Claims

WE CLAIM1. A method for analysing analytes of interest comprising: a) mixing a biomaterial containing a protein target, with one or more candidate compounds for binding to the biomaterials to obtain a sample comprising one or more biomaterial-candidate complexes, b) introducing the sample to a mass spectrometer by ionising the sample under conditions that retain the one or more biomaterial-candidate complex in their native state, c) separating the one or more biomaterial-candidate complex from unbound candidate compounds by selecting for ions having a high mass range using a mass analyser configured to capture the one or more biomaterial-candidate complexes and excluding unbound candidate compounds based on a difference in oscillation frequencies of their respective ions, d) dissociating the biomaterial-candidate complex using collisional dissociation to obtain one or more dissociated candidate compounds, and e) determining the dissociated candidate compound based on mass-to-charge ratio (m / z).

2. The method of claim 1, further comprising a sample preparation step comprising one or more of: a. buffering the sample with a buffer system comprising on or more of ammonium acetate, NaCI, Tris, Na2SO4, and HEPES, b. preparing a plurality of dilutions of the biomaterial, and c. transferring the sample into an ionisation source using a high-performance liquid chromatograph autosampler operating with or without a chromatography column.

3. The method of claim 1 or 2 wherein the collision dissociation is CID or HCD.

4. The method of any one of claims 1 to 3, further comprising capturing the dissociated candidate compound by selecting for ions having a low mass range.

5. The method of claim 3, wherein the HCD is configured to fragment the one or more dissociated candidate compounds into smaller fragments during dissociation from the biomaterial-candidate complex.

6. The method of claim 5, wherein the smaller fragments are less than 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 m / z.

7. The method of any one of claims 3 to 6, wherein the CID or HCD is performed with a plurality of collision energies to obtain a plurality of fragmentation patterns of the one or more dissociated candidate compounds.

8. The method of claim 7, wherein the plurality of collision energies comprise at least one low collision energy, and at least one high collision energy.

9. The method of claim 8, wherein the low collision energy corresponds to about 1 to about 49% of a total available voltage of the HCD, and the high collision energy corresponds to about 50 to 100% of the total available voltage of the HCD.

10. The method of any one of claims 1 to 9, wherein the HCD collision energy is increased at a predetermined rate to determine dissociation to obtain a plurality of fragmentation patterns of the dissociated biomaterial and / or dissociated candidate compound.

11. The method of claim 10, wherein the predetermined rate is about 5% to about 10% collision energy.

12. The method of any one of claims 1 to 11 , wherein a voltage of the HCD is increased incrementally to determine dissociation behaviour of the biomaterial and candidate compound.

13. The method of claim 12, wherein the voltage of HCD is increased incrementally from about 10, 20, 30, 40, 50, 60, 70, 80, 90 to 100 volts.

14. The method of claim 12 or 13, further comprising comparing the binding strength of two or more candidate compounds to the biomaterial containing the target proteins.

15. The method of any one of claims 1 to 14, wherein the high mass range is about 2000 to about 6000 m / z.

16. The method of any one of claims 1 to 15, wherein the low mass range is about 100 to about 1000 m / z.

17. The method of any one of claims 1 to 16, wherein the mass analyser comprises a central electrode and two outer electrodes, wherein ions of the biomaterial-candidate complex and unbound candidate compounds oscillate around the central electrode betweenthe outer electrodes such that different ions oscillate at different frequencies that used to separate the ions.

18. The method of any one of claims 1 to 17, wherein the mass analyser is selected from the group comprising an Orbitrap, a quadrupole, linear ion trap, and time of flight.

19. The method of claim 18, wherein when the mass analyser is an Orbitrap, the mass analyiser is configured to have an automatic gain control (AGO) target of about 100 to about 500%.

20. The method of claim 19, wherein the AGO is configured to have a maximum injection time of about 50 to about 200ms.

21. The method of any one of claims 1 to 20, wherein the biomaterial is derived from a cell lysate.

22. The method of claim 21, wherein the cell lysate contains an overexpressed protein target.

23. The method of claim 21 or 22, wherein the cell lysate is prepared by a. lysing cells by mechanical disruption, b. separating lysed cells from unlysed cells and cellular debris by centrifugation at predetermined speed and time, and c. retaining the supernatant containing the cell lysate.

24. The method of claim 23, wherein the mechanical disruption comprises sonication.

25. The method of any one of claims 1 to 24, wherein the candidate compounds may comprise a library of compounds.

26. The method of any one of claims 1 to 25, wherein the candidate compounds are 1000 Daltons or less.

27. The method of any one of claims 1 to 26, wherein the one or more candidate compounds are soluble in an aqueous solution.

28. The method of any one of claims 1 to 27, wherein the sample is introduced into the mass spectrometer via electrospray ionisation (ESI).

29. The method of any one of claims 1 to 28, wherein the sample is introduced into an electrospray ionisation source by direction injection.

30. The method of any one of claims 1 to 29, wherein the sample is introduced into an electrospray ionisation source by a high-performance liquid chromatograph autosampler.

31. The method of any one of claims 1 to 30, wherein determination of the dissociated compound is based on comparing the mass-to-charge ratio of the dissociated compound with a reference or database of compounds.

32. The method of any one of claims 7 to 31 , further comprising determining one or more characteristics of the dissociated compound based on the plurality of fragmentation patterns.

33. A method for screening a phenotypic panel comprising: a) preparing a first sample by incubating a first cell lysate derived from diseased cells overexpressing a protein target with a candidate compound such that the protein target within the first cell lysate forms a complex with the candidate compound, b) preparing a second sample by incubating a second cell lysate derived from nondiseased cells with the candidate compound, wherein for each sample, c) introducing the sample to a mass spectrometer by ionising the sample under conditions that retain the protein-candidate complex, when present, in their native state, d) separating the protein-candidate complex, when present, from unbound candidate compounds by selecting for ions having a high mass range corresponding to the protein target using a mass analyser configured to capture the one or more biomaterial-candidate complexes and excluding unbound candidate compounds based on a difference in oscillation frequencies of their respective ions, e) dissociating the protein-candidate complex using higher energy collisional dissociation (HCD) to obtain one or more dissociated candidate compounds, f) capturing the dissociated candidate compound by selecting for ions having a low mass range, and g) comparing a mass spectrum of the first sample, and a mass spectrum of the second sample to determine the difference in binding of the candidate compound to the first and second cell lysates.

34. The method of any one of claim 33, wherein the candidate compound is known to bind the overexpressed protein in the diseased cell.

35. The method of any one of claim 33, comprising incubating the cell lysate derived from diseased cells overexpressing the protein target with a plurality of candidate compounds, and incubating the cell lysate derived from non-diseased cells with a candidate compound from the plurality of candidate compound which binds to a target in cell lysate of the diseased cell.

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