Methods and systems for automated identification of protein non-covalent binding

Abstract

Methods and systems for analyzing a sample that includes a protein and a binding ligand, the method including receiving the sample at an ionization device via non-contact sampling, the first sample being in a non-denaturing carrier solvent, ionizing the first sample, generating a deconvoluted mass spectrum for the ionized first sample, and detecting binding between the protein and the binding ligand in the first sample based at least in part on the deconvoluted mass spectrum. The non-contact sampling is performed via a non-contact sample ejector. Methods and systems for analyzing a sample that includes a protein includes receiving the sample via non-contact sampling, the sample being in one of a plurality of non-denaturing carrier solvents, and for non-denaturing carrier solvent, ionizing the sample, generating a deconvoluted mass spectrum for the ionized sample, and detecting protein binding in the first sample based at least in part on the deconvoluted mass spectrum.

Description

[0001] METHODS AND SYSTEMS FOR AUTOMATED IDENTIFICATION OF PROTEIN NON-COVALENT BINDING

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 581,438, filed on September 08, 2023, the content of which is incorporated by reference herein in its entirety.

[0004] BACKGROUND

[0005] Identification of molecules that are non-covalently bound to a protein is a useful tool for use in the development of novel drugs, and mass spectrometry is used to screen such interaction between a target molecule and potential ligands. Mass spectrometry is used to determine whether a ligand is non-covalently bound to a target protein by measuring the mass of a native-like complex and comparing to the unmodified protein.

[0006] SUMMARY

[0007] In one aspect of the present disclosure, a method for analyzing a sample including a protein and a binding ligand includes receiving the first sample at an ionization device via non-contact sampling, the first sample being in a non-denaturing carrier solvent, ionizing the first sample, generating a deconvoluted mass spectrum for the ionized first sample, and detecting an interaction between the protein and the binding ligand in the first sample based at least in part on the deconvoluted mass spectrum.

[0008] In an example of the above aspect, generating the deconvoluted mass spectrum for the ionized first sample includes, capturing a raw mass spectrum of the first sample at a mass analysis device, converting the captured raw mass spectrum to the deconvoluted mass spectrum of the first sample, accessing a reference mass of the protein, and comparing the deconvoluted mass spectrum to the reference mass. Detecting the interaction between the protein and the binding ligand in the first sample includes detecting non-covalent interaction between the protein and the binding ligand based on the comparison. In another example of the above aspect, detecting the non-covalent interaction includes determining a target mass corresponding to a peak of the deconvoluted mass spectrum, calculating a mass difference between the target mass and the reference mass, and detecting the non-covalent interaction between the protein and the binding ligand in the first sample based on the calculated mass difference.

[0009] In other examples of the above aspect, detecting the non-covalent interaction includes identifying the binding ligand non-covalently bound to the protein based on the calculated mass difference. In yet another example, the above method is performed automatically. In a further example, the above method is performed contemporaneously on each of a plurality of samples. In an additional example, the non-denaturing carrier solvent is configured to preserve non-covalent binding between the protein and the binding ligand. In another example, the non-denaturing carrier solvent includes a waterbased solvent. In a further example, the binding ligand includes a drug molecule. In yet a further example, the method further includes receiving a second sample at the ionization device via a denaturing carrier solvent, ionizing the second sample, generating a deconvoluted mass spectrum for the ionized second sample, and determining non-covalent interaction between the protein and the binding ligand in the first sample based at least in part on the deconvoluted mass spectrum for the ionized second sample.

[0010] For example, generating the deconvoluted mass spectrum for the ionized second sample includes capturing a raw mass spectrum of the second sample at the mass analysis device, converting the captured raw mass spectrum to the deconvoluted mass spectrum of the second sample, and comparing the deconvoluted mass spectrum for the ionized second sample to the reference mass of the protein, and determining the non- covalent interaction between the protein and the binding ligand in the first sample includes detecting covalent bonding between the protein and the binding ligand in the second sample based on the comparison, and determining the non-covalent interaction between the protein and the binding ligand in the first sample based on the detected covalent bonding in the second sample. In another example, determining the non- covalent interaction between the protein and the binding ligand in the first sample includes comparing the deconvoluted mass spectrum of the second sample to the deconvoluted mass spectrum of the first sample. For example, the second sample is identical to the first sample. In another example, the denaturing carrier solvent is configured to disrupt the non-covalent interaction between the protein and the binding ligand. For example, the denaturing carrier solvent includes an organic solvent.

[0011] In another aspect of the present disclosure, a sample analyzing system includes a non-contact sample ejector, a sample ionization device coupled to the non-contact sample ejector, a mass analysis device coupled to the sample ionization device, a processor operatively coupled to the sample ionization device and the mass analysis device, and a memory operatively coupled to the processor, the memory storing instructions that, when executed by the processor. In various examples, the memory stores instructions that, when executed by the processor, perform a set of operations. In examples, the set of operations includes receiving, at the ionization device, the first sample via the non-contact sample ejector, the first sample being in a non-denaturing carrier solvent, ionizing, via the sample ionization device, the first sample, generating, via the mass analysis device, a deconvoluted mass spectrum for the ionized first sample, and detecting, via the processor, bonding between the protein and the binding ligand in the first sample based at least in part on the deconvoluted mass spectrum.

[0012] In other examples of the above aspect, the set of instructions includes generating the deconvoluted mass spectrum for the ionized first sample by capturing a raw mass spectrum of the first sample at the mass analysis device, converting the captured raw mass spectrum to the deconvoluted mass spectrum of the first sample, accessing a reference mass of the first sample, and comparing the deconvoluted mass spectrum to the reference mass, and detecting the bonding in the first sample by detecting non-covalent interaction between the protein and the binding ligand based on the comparison.

[0013] In other examples of the above aspect, the set of operations includes detecting the non-covalent interaction by determining a target mass corresponding to a peak of the deconvoluted mass spectrum, calculating a mass difference between the target mass and the reference mass, and detecting the non-covalent interaction between the protein and the binding ligand in the first sample based on the calculated mass difference. In an example, the set of operations includes detecting the non-covalent interaction by identifying the binding ligand non-covalently bound to the protein based on the calculated mass difference. In another example, the non-denaturing carrier solvent is configured to preserve non-covalent interaction between the protein and the binding ligand. For example, the non-denaturing carrier solvent includes a water-based solvent. In other examples of the above aspect, the set of operations further includes contemporaneously detecting non-covalent interaction between the protein and the binding ligand in a plurality of samples. In a further example, the set of operations further includes receiving a second sample at the ionization device via a denaturing carrier solvent, ionizing the second sample, capturing a deconvoluted mass spectrum for the ionized second sample, and determining non-covalent interaction between the protein and the binding ligand in the first sample based at least in part on the deconvoluted mass spectrum for the ionized second sample.

[0014] In other examples, the set of instructions includes generating the deconvoluted mass spectrum for the ionized second sample by capturing a raw mass spectrum of the second sample at the mass analysis device, converting the captured raw mass spectrum to the deconvoluted mass spectrum of the second sample, and comparing the deconvoluted mass spectrum for the ionized second sample to the reference mass of the protein, and determining the non-covalent interaction between the protein and the binding ligand in the first sample by detecting covalent bonding between the protein and the binding ligand in the second sample based on the comparison, and determining the non-covalent interaction between the protein and the binding ligand in the first sample based on the detected covalent bonding in the second sample. In a further example, the set of instructions includes determining the non-covalent interaction between the protein and the binding ligand in the first sample by comparing the deconvoluted mass spectrum of the second sample to the deconvoluted mass spectrum of the first sample. For example, the second sample is identical to the first sample. In yet another example, the denaturing carrier solvent is configured to disrupt the non- covalent interaction between the protein and the binding ligand. For example, the denaturing carrier solvent includes an organic solvent. In other examples of the above aspect, the non-contact sample ejector includes an acoustic droplet ejector.

[0015] Another aspect of the present disclosure includes a method for stress testing a sample, the sample including a protein, the method including receiving the sample at an ionization device via non-contact sampling, the sample being in one of a plurality of carrier solvents, and for each one of the plurality of carrier solvents ionizing the sample, generating a deconvoluted mass spectrum for the ionized sample, and detecting protein configuration in the sample based at least in part on the deconvoluted mass spectrum. In an example, detecting the protein configuration in the sample includes detecting one of a covalent bonding of the protein, a non-covalent interaction of the protein, and a protein unfolding of the protein. In a further example, for each one of the plurality of carrier solvents, generating the deconvoluted mass spectrum for the ionized sample includes capturing a raw mass spectrum of the sample at a mass analysis device, converting the captured raw mass spectrum to the deconvoluted mass spectrum of the sample, accessing a reference mass of the protein, and comparing the deconvoluted mass spectrum to the reference mass, and detecting the protein configuration in the sample includes detecting the protein configuration based on the comparison, and correlating the protein configuration to the carrier solvent.

[0016] In further examples, detecting the protein configuration includes determining a target mass corresponding to a peak of the deconvoluted mass spectrum, calculating a mass difference between the target mass and the reference mass, and detecting the protein configuration in the sample based on the calculated mass difference. In yet another example, a plurality of samples are automatically analyzed according to the above method. For example, a plurality of samples are contemporaneously analyzed according to the above method. In another example, each of the plurality of carrier solvents includes a mixture of a non-denaturing carrier solvent and a denaturing carrier solvent, and any two of the plurality of the carrier solvents have different mixtures of the non-denaturing carrier solvent and the denaturing carrier solvent. In yet another example, each of the plurality of carrier solvents includes a same non-denaturing carrier solvent, the sample in each carrier solvent is maintained at a given temperature, and temperatures of the sample in any two of the plurality of the carrier solvents are different from each other. In a further example, each of the plurality of carrier solvents includes a same denaturing carrier solvent, the sample in each carrier solvent is maintained at a given temperature, and temperatures of the sample in any two of the plurality of the carrier solvents are different from each other.

[0017] In another aspect of the present disclosure, a sample analyzing system includes a non-contact sample ejector, a sample ionization device coupled to the non-contact sample ejector, a mass analysis device coupled to the sample ionization device, a processor operatively coupled to the sample ionization device and the mass analysis device, and a memory operatively coupled to the processor, the memory storing instructions that, when executed by the processor. In various examples, the memory stores instructions that, when executed by the processor, perform a set of operations. In examples, the set of operations includes receiving, at the ionization device, the sample via the non-contact sample ejector, the sample being in one of a plurality of carrier solvents, and for each one of the plurality of carrier solvents, the set of operations include ionizing, via the sample ionization device, the sample, generating, via the mass analysis device, a deconvoluted mass spectrum for the ionized sample, and detecting, via the processor, protein configuration in the sample based at least in part on the deconvoluted mass spectrum.

[0018] In an example, the set of operations includes detecting the protein configuration in the sample by detecting one of a covalent bonding of the protein, a non-covalent interaction of the protein, and a protein unfolding of the protein. In another example, the set of operations further includes, for each one of the plurality of non-denaturing carrier solvents, generating the deconvoluted mass spectrum for the ionized sample by capturing a raw mass spectrum of the sample at the mass analysis device, converting the captured raw mass spectrum to the deconvoluted mass spectrum of the sample, accessing a reference mass of the sample, and comparing the deconvoluted mass spectrum to the reference mass, and detecting the protein configuration in the sample by detecting the protein configuration based on the comparison, and correlating the protein configuration to the non-denaturing carrier solvent.

[0019] In another example, the set of instructions includes detecting the protein bonding by determining a target mass corresponding to a peak of the deconvoluted mass spectrum, calculating a mass difference between the target mass and the reference mass, and detecting the protein bonding in the sample based on the calculated mass difference. In an example, the set of operations further includes contemporaneously detecting the protein bonding in a plurality of samples. In other example, each of the plurality of carrier solvents includes a mixture of a non-denaturing carrier solvent and a denaturing carrier solvent, and any two of the plurality of the carrier solvents have different mixtures of the non-denaturing carrier solvent and the denaturing carrier solvent. In further examples, each of the plurality of carrier solvents includes a same non-denaturing carrier solvent, the set of operations includes maintaining the sample in each carrier solvent at a given temperature, and temperatures of the sample in any two of the plurality of the carrier solvents are different from each other. In further examples, each of the plurality of carrier solvents includes a same denaturing carrier solvent, the set of operations includes maintaining the sample in each carrier solvent at a given temperature, and temperatures of the sample in any two of the plurality of the carrier solvents are different from each other. The details of one or more techniques are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of these techniques is apparent from the description, drawings, and claims.

[0020] BRIEF DESCRITION OF THE DRAWINGS

[0021] FIG. 1 is schematic diagram illustrating another exemplary mass analysis system in accordance with various aspects and examples of the present disclosure.

[0022] FIG. 2 is a schematic diagram illustrating one particular example of the computing device in accordance with various aspects and examples of the present disclosure.

[0023] FIG. 3 depicts a schematic view of an example system combining an acoustic droplet ejection system with a sampling interface and an ion source.

[0024] FIGS. 4A-4C are illustrations of an identification of protein binding, in accordance with various examples of the present disclosure.

[0025] FIG. 5 represents a flowchart illustrating a method for analyzing a sample including a protein and a binding ligand, in accordance with various examples of the disclosure.

[0026] FIG. 6 represents a flowchart illustrating a method for stress testing a sample that includes a protein, in accordance with various examples of the disclosure.

[0027] Before one or more examples of the present teachings are described in detail, one skilled in the art will appreciate that the present teachings are not limited in their application to the details of construction, the arrangements of components, and the arrangement of steps set forth in the following detailed description or illustrated in the drawings. Also, it is to be understood that the terminology used herein is for the purpose of description and should not be regarded as limiting.

[0028] DETAILED DESCRIPTION

[0029] Selected definitions

[0030] For the purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. The definitions set forth below shall supersede any conflicting definitions in any documents incorporated herein by reference.

[0031] As used herein, the singular forms “a,” “an,” and “the,” include both singular and plural referents unless the context clearly dictates otherwise. The terms “comprising,” “comprises,” and “comprised of’ as used herein are synonymous with “including,” “includes,” or “containing,” “contains,” and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. It is appreciated that the terms “comprising,” “comprises,” and “comprised of’ as used herein comprise the terms “consisting of,” “consists,” and “consists of.”

[0032] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0033] Whereas the terms “one or more” or “at least one”, such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6, or >7, etc. of said members, and up to all said members.

[0034] Unless otherwise defined, all terms used in the present disclosure, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present disclosure. In the following passages, different aspects of the present disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary.

[0035] Reference throughout this specification to “one example” or “an example” means that a particular feature, structure or characteristic described in connection with the example is included in at least one example of the present disclosure. Thus, appearances of the phrases “in one example” or “in an example” in various places throughout this specification are not necessarily all referring to the same example, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more examples. Furthermore, while some examples described herein include some, but not other features included in other examples, combinations of features of different examples are meant to be within the scope of the disclosure, and form different examples, as would be understood by those in the art. For example, in the appended claims, any of the claimed examples can be used in any combination. In the present disclosure, reference is made to the accompanying drawings that form a part hereof, and in which are shown by way of illustration only of specific examples in which the present disclosure may be practiced. It is to be understood that other examples may be utilized, and structural or logical changes may be made without departing from the scope of the present disclosure. The following detailed description, therefore, is not to be taken in a limiting sense, and the scope of the present disclosure is defined by the appended claims.

[0036] Ionization devices

[0037] Although the sample ionization process is described above in the context of Acoustic Ejection Mass Spectrometer (AEMS) using ESI, other techniques of generating ionized samples may be used according to various examples of this disclosure. For example, ionized samples may be generated by DESI, which is a combination of ESI and desorption ionization (DI) methods. In DESI, ionization takes place by directing an electrically charged mist to the sample surface that is a few millimeters away. The electrospray mist is pneumatically directed at the sample, thus forming splashed droplets that carry desorbed, ionized analytes. After ionization, the ions travel through air into the atmospheric pressure interface which is connected to the mass spectrometer. Other sample or droplet introduction methods may also be used to introduce a sample or droplet into a continuous carrier liquid flow to the ESI. Such other techniques may include, e.g., introducing nanoliter-sized droplets using I.DOT technology.

[0038] System for mass analysis

[0039] Examples of the present disclosure generally relate to high throughput mass spectrometry (MS) analysis systems and methods for analyzing a collection of substance samples using mass spectrometry. Conventionally, the preparation and introduction of sample into a mass spectrometer is a relatively time-consuming process, particularly where rapid and efficient analysis of a sample pool containing multiple samples, which may or may not be analytically related, is desired. For instance, multiple different systems may have been used that were provided and controlled by separate entities and / or devices. For example, a liquid handling system would be used for preparation of samples, an ejection system would be used for ejecting samples in a non-contact manner into a port or interface, and mass spectrometry system would be used for the actual analysis of the samples. The systems provided in the present disclosure advantageously include a central control system that is able to control the underlying subsystems used in the sample analysis process. For example, a script or set of operations may be generated at the central control system or controller that allows for control of the subsystems such that the subsystems are able to work synchronously across different types of operations performed by each of the subsystems. To accomplish such synchronicity across the subsystems, additional mechanical devices, such as robotics, may be incorporated into the overall system to handle transitions of materials between the systems. Thus, the central controller is able to interface with the various subsystems and transition robotics to more efficiently control each of the operations performed by the subsystems. Furthermore, the present systems advantageously include a computing subsystem and various functional modules thereof configured to efficiently process the data generated from multiple samples, reliably determine the data-sample correlation for a large pool of samples, generate mass spectra for each test sample, analyze the generated mass spectra, and provide real-time feedback to other subsystems.

[0040] Determining non-covalent binding in a target biomolecule or protein

[0041] Targeted inhibitors are currently used in the development of novel drugs, due to the high selectivity and improved potency of these drugs. Mass spectrometry has been used to screen such interaction between the target biomolecule and small molecules, the small molecules being, e.g., drug molecules. For a molecule to have an effect on another molecule, there typically must be an interaction between the two molecules. For many molecules of interest the interaction is non-covalent in nature. As such, evaluating non-covalent interactions generally necessitates the use of a system that preserves the non-covalent interaction. For biomacromolecules such as, e.g., protein / peptide molecules, or DNA / RNA molecules, an aqueous system may be used to preserve the native-like form, e.g., non-denatured form, of the molecules. ESI-MS may be used for the evaluation of non-covalent interactions in an aqueous buffer or carrier solvent. However, most native-like MS methods typically rely on laborious desalting, which may adversely affect the interaction, particularly when small molecules are present that may be removed in the desalting process. For example, a denaturing carrier solvent is configured to disrupt, e.g., a non-covalent interaction between a protein and a binding ligand. A non-denaturing carrier solvent is configured to preserve, e.g., the non-covalent interaction between the protein and the binding ligand. Thus, a technical problem may occur in that the analytical throughput is typically limited to a scale of minute-per-sample on liquid chromatography mass spectrometry (LC-MS), or about 20 seconds per sample with RapidFire technology. Faster throughput, as discussed above, may thus be advantageous. Furthermore, direct infusion, in which proteins are infused directly into an instrument for analysis, typically requires sample cleanup prior to sample analysis, which can negatively affect results. LC and RapidFire technologies integrate parts of the sample cleanup, e.g., by a column in LC or a solid phase extraction (SPE) cartridge in RapidFire, into the experiment. In addition, the removal of ligand from the sample prior to analysis during the sample cleanup may cause drawbacks with the analysis as such cleanup may drive some dissociation of the ligand. AEMS does not require sample cleanup and therefore small ligands are typically not removed prior to analysis.

[0042] In examples, a technical solution to the above technical problem includes using AEMS, which includes the use of an ESI-based high-throughput MS technology. The sample readout speed (1-sec-per-sample or faster) is significantly faster than, e.g., LC- MS and RapidFire technology. The ESI-MS based analysis provides adequate quantitative performance that covers a wide chemical space including for an intact protein. In various examples of this disclosure, AEMS based high-throughput platform and its automated data processing workflow may be used to identify, e.g., the covalent or non-covalent binder of a target molecule, the binder being, e.g. a drug molecule, in faster times. For example, using an open port interface (OPI), ESI may be achieved that may be both fast and minimally disruptive.

[0043] The small molecules such as, e.g., drug molecules, and the target molecule such as, e.g., a protein, may be mixed together and incubated in an assay buffer. During incubation, a protein-compound complex sample may be formed by covalent or non- covalent binding. The sample may be analyzed by, e.g., an AEMS with high throughput readout. The mass scan range may cover, for example, the expected mass-to-charge ratio (m / z) distribution of the target molecule and of the protein-compound complex. In AEMS, each ejection generates about a 1 sec baseline wide signal peak or less. Because the mass spectra information from individual sample ejections of the same plate may be continuously recorded as a single raw data file, examples of the disclosure include isolating the data of a particular well from other ejections and correlating the well position with each MS chronogram. For example, an acoustic ejection log containing the well position and the timing of each ejection event may be used to split the raw data into multiple new data files, each of which contains the information from a single ejection.

[0044] The mass spectra of each sample (e.g., 96, 384 or 1536 samples), which measures the signal intensity with respect to mass-to-charge ratio (m / z), may be deconvoluted to reconstruct the mass by converting the signal from the m / z scale to the mass scale. In various examples, each signal peak may include multiple data points corresponding to multiple mass spectra cycles, and the information to be used for mass deconvolution may be the average or the sum of all the data points, or of a selective single or several data points. In another example, optionally, the mass spectra may also be processed via background subtraction before deconvolution of the extracted mass spectra.

[0045] The binder identification may be based on the mass difference between the target molecule and the covalently or non-covalently modified complex. For example, the intensity of the expected protein mass and protein-compound complex of each sample may be extracted to analyze, e.g., the existence of the target molecule and / or protein-compound complex, and their intensity. In this case, the protein and compound information (e.g., formula, expected mass, etc.) for each well of a well plate may be imported to the data processing module as a sample information table, and it may be possible to automatically validate whether the adducts formed are the expected adducts based on a comparison of the measured mass and a reference mass stored in a sample information table. The protein-compound binding stoichiometry may be analyzed as well to determined, e.g., protein + 1 drug molecule, protein + 2 drug molecules, and the like. The intensity of the expected protein mass and protein-compound complex of each sample may also be extracted to analyze the relative ratio between the unbonded protein and the protein-compound complex which may be calculated automatically. In other examples, a control sample may be included in the analysis, from which the intensity of the total protein may be analyzed, where the ratio of the left-over unbound protein over the total protein may also be calculated for each sample. In addition to the target molecule, in examples, the protein-compound complex, whether a 1:1 complex with one protein covalently or non-covalently bound to one drug molecule, or 1 :N with one protein covalently or non-covalently bonded to N drug molecules, and any additional species with high intensity may be extracted from the reconstructed mass of each sample. Examples of this disclosure include a new application for AEMS that relies on native-like solution conditions, e.g., water or a water-based carrier solvent and an assay buffer, the carrier solvent not including any organic component therein so as to preserve, e.g., non-covalent bonds. In addition, it may be possible to test a molecule, e.g., to test the unfolding of a protein, by submitting the molecule to various stresses such as, e.g., the temperature of the carrier solvent or the denaturing strength of the carrier solvent. Such stresses may affect the viscosity of the protein and buffer solution, which may have an impact on analysis speed. Accordingly, it may be possible to observe heat-induced protein conformational changes with respect to the solution temperature.

[0046] Now referring to FIG. 1, examples of the present disclosure are illustrated and described. The descriptions of FIG. 1 are concurrent unless otherwise noted. In the illustrated examples, the system 10 can each include, in various combinations, pluralities of components, including some or all of: a mass capture and analysis system 100, a sample preparation system 101, an ejector system 102, a computing system 103, a network 104, a database / library 106, and a remote computing device 108.

[0047] In some examples, the mass capture and analysis system 100 may be a mass analysis instrument 100. The mass capture and analysis system 100 may be a mass spectrometer system including a mass analyzer 120 for analyzing ions generated from ionization of a sample. The mass capture and analysis system 100 may also include a capture device or probe 105 that captures the sample and provides the sample to other components of the mass capture and analysis system 100. In other examples, the capture probe 105 may be located externally from the mass analysis instrument 100. For instance, the capture probe 105 may be part of the ejection system 102.

[0048] It will also be appreciated by a person skilled in the art and in light of the teachings herein that the mass analyzer 120 can have a variety of configurations. Generally, the mass analyzer 120 is configured to process (e.g., filter, sort, dissociate, detect, etc.) sample ions generated by the ion source 115. By way of non-limiting example, the mass analyzer 120 can be a triple quadrupole mass spectrometer, or any other mass analyzer known in the art and modified in accordance with the teachings herein. Other non-limiting, exemplary mass spectrometer systems that can be modified in accordance with various aspects of the systems, devices, and methods disclosed herein can be found, for example, in an article entitled “Product ion scanning using a Q-q-Q linear ion trap (Q TRAP) mass spectrometer” (James W. Hager and J. C. Yves Le Blanc; Rapid Communications in Mass Spectrometry; 2003; 17: 1056-1064); and U.S. Pat. No. 7,923,681, the disclosures of which are hereby incorporated by reference herein in their entireties.

[0049] Other configurations, including but not limited to those described herein and others known to those skilled in the art, can also be utilized in conjunction with the systems, devices, and methods disclosed herein. For instance, other suitable mass spectrometers include single quadrupole, triple quadrupole, time-of-flight (ToF), trap, and hybrid analyzers. It will further be appreciated that any number of additional elements can be included in the system 100 including, for example, an ion mobility spectrometer (e.g., a differential mobility spectrometer) that is disposed between the ionization source 115 and the mass analyzer detector 120 and is configured to separate ions based on their mobility difference between in high-field and low-field). Additionally, it is appreciated that the mass analyzer 120 can include a detector 126 that can detect the ions that pass through the analyzer 120 and can, for example, supply a signal indicative of the number of ions per second that are detected.

[0050] The sample preparation system 101 may include a sample source 70 and a sample handler 80. The sample source 70 and a sample handler 80 are operative to retrieve collections of samples from the sample source(s) and to deliver the retrieved collections to capture locations associated with sample capture probes 105. The systems may be operative to independently capture selected ones of the pluralities of samples at the capture locations from the pluralities of samples, to optionally dilute the samples and to transfer the captured samples to mass analysis instruments 100, 120 for mass analysis. In some examples, the sample source 70 may include a set of well plates in a storage housing and / or liquid for adding to well plates. The sample source 70 may include part of a liquid handling system that manipulates and / or injects liquid into the well plates. The sample handler 80 includes one or more electro-mechanical devices (e.g., robotics, conveyor belts, stages, etc.) that are capable of transferring the samples (e.g., well plates) from the sample source to other components of the sample preparation system 101 and / or to other systems, such as the ejection system 102 and / or the capture probe 105. As an example, the sample handler 80 may transfer a well plate from the sample preparation system 101 to the ejection system 102. More specifically, the sample handler 80 may transfer the well plate to a plate handler 95 of the ejection system 102. Accordingly, the sample preparation system 101 may also be referred to as a sample delivery system. In some examples, selected sample information (e.g. sample or compound ID, chemical structure of the target compound, or other sample information) could be obtained during the sample handling steps through the use of sample controller 82 and / or the sample handler 80, and communicated to the computing system 103 or the data processing system 400 thereof.

[0051] In addition to the plate handler 95, the ejection system 102 may include an ejector 90 that ejects droplets from the wells of the well plates. The ejector 90 may be any type of suitable ejector, such as an acoustic ejector, a pneumatic ejector, or other type of contactless ejector. In an example, the plate handler 95 receives a well plate from the sample handler 80. The plate handler 95 transports the plate to a capture location that may be aligned with the capture probe 105. Once in the capture location, the ejector 90 ejects droplets from one or more wells of the well plates. The plate handler 95 may include one or more electro-mechanical devices, such as a translation stage that translates the well plate in an x-y plane to align wells of the well plate with the ejector 90 and / or or the capture probe 105.

[0052] The computing system 103 includes computing resources, components, and modules that are operative to perform various functions including but not limited to: communicating with other subsystems, receiving and transmitting electrical signals with other subsystems or components thereof, receiving, responding to, and executing user instructions, performing calculations, processing raw data received from mass analyzer, performing splitting data, performing sample-dataset correlation, generating and analyzing mass spectrometry data, identifying, annotating, and assigning MS peaks of mass spectra, extracting spectral features from mass spectra, conducting library search, identifying analytes, and outputting analytical report to end users.

[0053] In some examples, the computing system 103 includes a computing device 200, a controller 135, and a data processing system 400. The computing device 200 may be in the form of electronic signal processors and operative to perform various computing functions. The controller 135 may be in the form of electronic signal processors and in electrical communication with other subsystems within the system 10. The controller 135 is further configured to coordinate some or all of the operations of the pluralities of the various components of the system 10. The data processing system 400 may include various components and modules operative to process mass spectrometry data and to provide real-time feedback to end users and other subsystems.

[0054] In some examples, a network 104 may be operably connected to any one or all of the subsystems or components in the system 10. The network 104 is a communication network. In the example, the network 104 is a wireless local area network (WLAN). The network 104 may be any suitable type of network and / or a combination of networks. The network 104 may be wired or wireless and of any communication protocol. The network 104may include, without limitation, the Internet, a local area network (LAN), a wide area network (WAN), a wireless LAN (WLAN), a mesh network, a virtual private network (VPN), a cellular network, and / or any other network that allows system 104 to operate as described herein.

[0055] In some examples, the system 10 may further include one or more library / database 106. The database 106 can be a commercial database, or a private database containing analytical information from previously analyzed samples, or a combination of both. The library / database 106 includes chemical knowledge of standard of known compounds stored therein, including but not limited to chemical formula or elemental composition, neutral mass, monoisotopic mass, or mass of internal fragments thereof. In some examples, the computer system 103 is operative to perform a search using the database 106 and / or to compare data produced by the data processing system 400 to the retrieved data from the database 106 (such as molecular mass information or spectral features) to facilitate mass analysis and / or analyte identification.

[0056] Also illustrated in FIG. 1 are components of a sample delivery system for use in combination with the mass analysis instrument 100. The sample delivery system includes at least a sample source 70 for supplying a plurality of samples, a sample handler 80 for delivering the plurality of samples to a capture location, and a capture probe 105 for independently capturing one or more samples of the plurality of samples. In some aspects, the sample delivery system may further include a stage 95 for locating each sample for the plurality of samples proximate to a capture surface of the capture probe 105 and an ejector 90 for selectively ejecting that located sample into the capture surface of the capture probe.

[0057] In operation, a sample delivery system (including sample source 70 and sample handler 80) can iteratively deliver independent samples from a plurality of samples (e.g., a sample from a well of a well plate 75) to the capture probe 105. The capture probe 105 can dilute and transport each such delivered sample to the ion source 115 disposed downstream of the capture probe 105 for ionizing the diluted sample. A mass analyzer 120 can receive generated ions from the ion source 115 for mass analysis. The mass analyzer 120 is operative to selectively separate ions of interest from generated ions received from the ion source 115 and to deliver the ions of interest to an ion detector 126 that generates a mass spectrometer signal indicative of detected ions to the data processing system 400. In some aspects, the separate ions of interest may be indicated in an analysis instruction associated with that sample. In some aspects, the separate ions of interest may be indicated in an analysis instruction identified by an indicia physically associated with the plurality of samples.

[0058] In some aspects, the system 10 may further include the generation, assignment, and use of identifiers associated with collections of samples and / or individual samples, and incorporation by one or more of components 70, 80, 95, 105, 100, etc. of identifier readers. For instance, an identifier associated with a well plate may be read or scanned by a machine reading device 65 as it leaves the sample source 70 and / or when the well plate is received by the stage 95. In such aspects, the identifiers) may be used by the system to associate a corresponding one or more sets of instructions for use by the mass analysis instrument 100, 120 when analyzing transported sample droplets 125. In some aspects, the identifier may include an indicia physically associated with the plurality of samples. In some aspects, the indicia may be readable by optical, electrical, magnetic or other non-contact reading means. Indicia or identifiers in accordance with such aspects of the disclosure can include any characters, symbols, or other devices suitable for use in adequately identifying samples, sample collections, and / or handling or analysis instructions suitable for use in implementing the various aspects and examples of the present disclosure.

[0059] Additional details regarding implementation and operation of system 10 in accordance with various aspects and examples of the present disclosure can be explained with reference to the Figures. FIGS. 1 and 2 present system diagrams illustrating examples of a system 10, each example including a sample handler 80 and an associated controller 135, which may be, for example, a Biomek computer available from Beckman Coulter Life Sciences, is in operative communication with a mass analysis instrument 100 and a controller for the capture probe 105, which may include, for example, an a SciexOS® computer available from Sciex. The Analyst® or SciexOS® computer includes a control component 107 for the capture probe 105, represented for example by Sciex open port interface (OPI) software, and a control component 127 for the mass analysis instrument 100, which may be the Analyst® computer. The mass analysis instrument 100 and capture probe controller 107 may be further in operative communication with an ejector 90 and an X-Y Well Plate Stage 95 and plate handler controller 96, which may be, for example, a liquid droplet ejector with embedded computer or processor. For the purposes of this application, these distributed controller components may collectively be considered to be a system controller, and depending upon the configuration may be centralized, or distributed as is the case here. For instance, one of the controllers or controller components may send signals to the other controllers to control the respective devices.

[0060] Now referring to FIG. 2, an example of the computing device 200 according to FIG. 1 is illustrated and described. It is noted that the computing system 103 of the system 10 may include a single computing device 200 or may include a plurality of distributed computing devices 200 in operative communication with components of a mass analysis instrument 100. In the illustrated example of FIG. 2, the computing device(s) 200 may include a bus 202 or other communication mechanism of similar function for communicating information, and at least one processing element 204 coupled with bus 202 for processing information. As is appreciated by those skilled in the relevant arts, such at least one processing element 204 may include a plurality of processing elements or cores, which may be packaged as a single processor or in a distributed arrangement. Furthermore, in some examples, a plurality of virtual processing elements 204 may be included in the computing device 200 to provide the control or management operations for the mass analysis instrument 100.

[0061] Computing device 200 may also include one or more volatile memory(ies) 206, which can for example include random access memory(ies) (RAM) or other dynamic memory component(s), coupled to one or more busses 202 for use by the at least one processing element 204. Computing device 200 may further include static, non-volatile memory(ies) 208, such as read only memory (ROM) or other static memory components, coupled to busses 202 for storing information and instructions for use by the at least one processing element 204. A storage component 210, such as a storage disk or storage memory, may be provided for storing information and instructions for use by the at least one processing element 204. As is appreciated, in some examples the computing device 200 may include a distributed storage component 212, such as a networked disk or other storage resource available to the computing device 200.

[0062] Computing device 200 may be coupled to one or more displays 214 for displaying information to a computer user. Optional user input devices 216, such as a keyboard and / or touchscreen, may be coupled to a bus for communicating information and command selections to the at least one processing element 204. An optional graphical input device 218, such as a mouse, a trackball or cursor direction keys for communicating graphical user interface information and command selections to the at least one processing element. The computing device 200 may further include an input / output (I / O) component, such as a serial connection, digital connection, network connection, or other input / output component for allowing intercommunication with other computing components and the various components of the mass analysis instrument 100.

[0063] In various examples, computing device 200 can be connected to one or more other computer systems a network to form a networked system. Such networks can for example include one or more private networks, or public networks such as the Internet. In the networked system, one or more computer systems can store and serve the data to other computer systems. The one or more computer systems that store and serve the data can be referred to as servers or the cloud, in a cloud computing scenario. The one or more computer systems can include one or more web servers, for example. The other computer systems that send and receive data to and from the servers or the cloud can be referred to as client or cloud devices, for example. Various operations of the mass analysis instrument 100 may be supported by operation of the distributed computing systems.

[0064] Computing device 200 may be operative to control operation of the components of the mass analysis instrument 100 and the sample delivery components 70, 80, 95, 105 through controller(s) 135 and to handle data generated by components of the mass analysis instrument 100 through the data processing system 400. In some examples, analysis results are provided by computing device 200 in response to the at least one processing element 204 executing instructions contained in memory 206 or 208 and performing operations on data received from the mass analysis instrument 100. Execution of instructions contained in memory 206 or 208 by the at least one processing element 204 can render the mass analysis instrument 100 and associated sample delivery components operative to perform methods described herein. Alternatively, hard-wired circuitry may be used in place of or in combination with software instructions to implement the present teachings. Thus, implementations of the present teachings are not limited to any specific combination of hardware circuitry and software.

[0065] The term “computer-readable medium” as used herein refers to any media that participates in providing instructions to processor 204 for execution. Such a medium may take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Non-volatile media includes, for example, optical or magnetic disks, such as disk storage 210. Volatile media includes dynamic memory, such as memory 206. Transmission media includes coaxial cables, copper wire, and fiber optics, including the wires that include bus 202.

[0066] Common forms of computer-readable media or computer program products include, for example, a floppy disk, a flexible disk, hard disk, magnetic tape, or any other magnetic medium, a CD-ROM, digital video disc (DVD), a Blu-ray Disc, any other optical medium, a thumb drive, a memory card, a RAM, PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, or any other tangible medium from which a computer can read.

[0067] Various forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to processor 204 for execution. For example, the instructions may initially be carried on the magnetic disk of a remote computer. The remote computer can load the instructions into its dynamic memory and send the instructions over a telephone line using a modem. A modem local to computer system 200 can receive the data on the telephone line and use an infra-red transmitter to convert the data to an infra-red signal. An infra-red detector coupled to bus 202 can receive the data carried in the infra-red signal and place the data on bus 202. Bus 202 carries the data to memory 206, from which processor 204 retrieves and executes the instructions. The instructions received by memory 206 may optionally be stored on storage device 210 either before or after execution by processor 204.

[0068] In accordance with various examples, instructions configured to be executed by a processor to perform a method are stored on a computer-readable medium. The computer-readable medium can be a device that stores digital information. For example, a computer-readable medium includes a compact disc read-only memory (CD-ROM) as is known in the art for storing software. The computer-readable medium is accessed by a processor suitable for executing instructions configured to be executed.

[0069] FIG. 3 depicts a schematic view of an example AEMS system 300 combining an acoustic droplet ejection (ADE) device 302 with an OPI 304 and an ESI device source 314. The system 300 provides an example of an integration and physical connection between the ejection system 102, the capture probe 105, and the mass analysis instrument 100. The ADE 302 includes an acoustic ejector 306 that is configured to eject a droplet 308 from a reservoir 312 into the open end of a sample receiver such as, e.g., sampling OPI 304. The acoustic ejector 306 is one example of the ejector 90, and the sampling OPI 304 is one example of the capture probe 105. As shown in FIG. 3, the example system 300 generally includes the sampling OPI 304 in liquid communication with the ESI source 314 for discharging a liquid containing one or more sample analytes (e.g., via electrospray electrode 316) into an ionization chamber 318, and a mass analyzer detector (depicted generally at 320) in communication with the ionization chamber 318 for downstream processing and / or detection of ions generated by the ESI source 314. The ESI source 314 is an example of the ion source 115, and the mass analyzer detector 320 is an example of the ion detector 126.

[0070] Due to the configuration of the nebulizer probe 338 and electrospray electrode 316 of the ESI source 314, samples ejected therefrom are in the gas phase. A liquid handling system 322 (e.g., including one or more pumps 324 and one or more conduits 325) provides for the flow of a transport liquid from a solvent reservoir 326 to the sampling OPI 304 and from the sampling OPI 304 to the ESI source 314. The solvent reservoir 326 (e.g., containing a liquid, desorption solvent) can be liquidly coupled to the sampling OPI 304 via a supply conduit 327 through which the transport liquid can be delivered at a selected volumetric rate by the pump 324 (e.g., a reciprocating pump, a positive displacement pump such as a rotary, gear, plunger, piston, peristaltic, diaphragm pump, or other pump such as a gravity, impulse, pneumatic, electrokinetic, and centrifugal pump), all by way of non-limiting example. The flow of transport liquid into and out of the sampling OPI 304 occurs within a sample space accessible at the open end such that one or more droplets 308 can be introduced into the liquid boundary 328 at the sample tip and subsequently delivered to the ESI source 314.

[0071] The ADE 302 is configured to generate acoustic energy that is applied to a liquid contained within a well or reservoir 310 of a well plate 312 that causes one or more droplets 308 to be ejected from the reservoir 310 into the open end of the sampling OPI 304. The well plate 312 is an example of the well plates 75 discussed above. The acoustic energy is generated from an acoustic ejector 306, which is an example of the ejector 90 discussed above. The well plate 312 may reside on a movable stage 334, which is an example of the plate stage 95 discussed above.

[0072] A controller 330 can be operatively coupled to the ADE 302 and can be configured to operate any aspect of the ADE 302 (e.g., focusing structures, acoustic ejector 306, automation elements for moving a movable stage 334 so as to position a reservoir 310 into alignment with the acoustic ejector 306 and / or the OPI 304, etc.). This enables the ADE 302 to eject droplets 308 into the sampling OPI 304 as otherwise discussed herein substantially continuously or for selected portions of an experimental protocol by way of non-limiting example. Controller 330 can be, but is not limited to, a microcontroller, a computer, a microprocessor, or any device capable of sending and receiving control signals and data. Wired or wireless connections between the controller 330 and the remaining elements of the system 300 are not depicted but would be apparent to a person of skill in the art. The controller 330 may be any of the controllers discussed above and may be responsible for controlling the mass analysis instrument 100 and / or the sample delivery system 101 as well.

[0073] As shown in FIG. 3, the ESI source 314 can include a source 336 of pressurized gas (e.g., nitrogen, air, or a noble gas) that supplies a high velocity nebulizing gas flow to the nebulizer probe 338 that surrounds the outlet end of the electrospray electrode 316. As depicted, the electrospray electrode 316 protrudes from a distal end of the nebulizer probe 338. The pressured gas interacts with the liquid discharged from the electrospray electrode 316 to enhance the formation of the sample plume and the ion release within the plume for sampling by mass analyzer detector 320, e.g., via the interaction of the high-speed nebulizing flow and jet of liquid sample. The liquid discharged may include discrete volumes of liquid samples LS received from each reservoir 310 of the well plate 312. Each received liquid sample LS is instantly diluted by a solvent after being captured within the OPI, with a dilution factor of about 1000 times (as flow of the solvent moves the liquid samples LS from the OPI 304 to the ESI source 314, the solvent is also referred to herein as the transport liquid). The nebulizer gas can be supplied at a variety of flow rates, for example, in a range from about 0.1 L / min to about 20 L / min, which can also be controlled under the influence of controller 330 (e.g., via opening and / or closing valve 340).

[0074] It is appreciated that the flow rate of the nebulizer gas can be adjusted (e.g., under the influence of controller 330) such that the flow rate of liquid within the sampling OPI 304 can be adjusted based, for example, on suction / aspiration force generated by the interaction of the nebulizer gas and the analyte-solvent dilution as it is being discharged from the electrospray electrode 316 (e.g., due to the Venturi effect). The ionization chamber 318 can be maintained at atmospheric pressure, though in some examples, the ionization chamber 318 can be evacuated to a pressure lower than atmospheric pressure.

[0075] Automatic data processing

[0076] As discussed above, the present systems may be operative to analyze a large collection of substance samples and generate a large quantity of mass spectrometry data in a high throughput fashion. For example, one sample per second, or more than 50,000 samples per day. Systems 10 discussed above according to the present disclosure advantageously provides an auto-triggered data processing function to avoid potential issues arising from the assay throughput bottleneck, to maintain a constant workflow of operations, to perform data processing and acquisition with matched speed as sample analysis, and to improve the overall productivity of the system.

[0077] Identification of Protein Binding

[0078] FIGS. 4A, 4B, and 4C(I-IV) are illustrations of an automated identification of protein binders, in accordance with various examples of the present disclosure. The target molecule, also referred as protein, or unreacted protein, is incubated with specified small molecules for twenty (20) hours (h), and the concentrations of the small molecule and target molecule were 10 micromoles (|1M) and 2 pM, respectively. In examples, FIG. 4A shows a well-defined chromatogram profile with an identifiable peak shape for a given peak 400, and FIG. 4B shows the extracted trace 410 showing the intensity of the measured signal with respect to mass-to-charge ratio (m / z) corresponding to the given peak 400. In examples, the trace 410 corresponds to a 20 second acquisition. From the trace 410, a deconvoluted mass spectrum 405, as illustrated in FIG. 4C, may be derived. The vertical dashed line in the deconvoluted mass spectrum 405 illustrated in FIG. 4C(I) and further discussed below indicates a molecular weight of the unreacted protein of 19.5 kDa as indicated by peak 420.

[0079] FIG. 4C(I) illustrates the deconvoluted zero-charge mass spectrum 405 that is reconstructed based on the trace illustrated in FIG. 4B, and more particularly illustrates the main peak 420 thereof. For example, the deconvoluted mass spectrum 405 corresponds to an unreacted target molecule and shows a relatively high signal-to-noise ratio. In the example illustrated in FIG. 4C(I) with the target mass illustrated by peak 420, the mass measurement accuracy, based on multiple mass measurements of the 19.5 kDa protein, may be measured to within 0.5 Da of the expected molecular weight, and the standard deviation for the measurement illustrated in mass spectrum in FIG. 4B is ±0.02 Da.

[0080] FIG. 4C(II) illustrates the expected mass addition of 125.1 atomic mass units (amu) corresponding to a reactive small molecule. In this example, the reactive small molecule is N-ethylmaleimide, and because this small molecule has a large level of covalent or non-covalent interaction with the target molecule, the small molecule can be easily identified based on the mass shift which corresponds to its molecular weight, the mass shift being observed as an increase in molecular weight of the incubated sample as well. FIG. 4C(II) also shows a good mass accuracy, e.g., a mass accuracy that provides a reliable mass measurement, and high signal-to-noise ratio that are displayed in reconstructed mass spectrum in FIG. 4C(II). In various examples, by measuring the difference between the mass corresponding to the main peak 430 of the reconstructed mass spectrum of the incubated sample and the mass corresponding to the main peak 420 of the unreacted target molecule illustrated in FIG. 4C(I), the mass difference of, e.g., 19.5 kDa, is indicative of the mass or weight of the small molecule or drug that is covalently or non-covalently bound to the target molecule. In this case, the mass of the small molecule that is covalently or non-covalently bound to the target molecule is 125.1 amu (or Da), which corresponds to N-ethylmaleimide. In addition, the fact that FIG. 4C(II) only shows one main peak 430 that corresponds to the covalently or non-covalently bound protein with the N-ethylmaleimide drug molecule, and no peak corresponding to the unreacted target molecule such as peak 420 illustrated in FIG. 4C(I), may be indicative that substantially the entirety of the target molecule is bound to the drug molecule, and substantially no amount of the target molecule is left unreacted or unbonded to N-ethylmaleimide. In another example, the mass shift may be a multiple of the covalently or non-covalently bound drug molecule, where the mass difference may correspond to a multiple of the molecular mass of the drug molecule.

[0081] FIG. 4C(III) illustrates the expected mass addition of 57.1 atomic mass units (amu) for another reactive small molecule. In this example, the other reactive small molecule is lodoacetamide. Because this small molecule has some level of bonding with the target molecule, the small molecule may be identified based on its molecular weight compared to the observed main peak 440 in the illustrated reconstructed mass spectrum. The mass shift may be due to the observed increase in molecular weight of the incubated sample as well as the good mass accuracy and signal-to-noise ratio that are displayed in reconstructed mass spectrum in FIG. 4C(III). FIG. 4C(III) shows that there are two peaks, one peak 440 illustrative of the target molecule, and another peak 445 illustrative of the covalently or non-covalently bound molecule. In various examples, by measuring the difference between the peak 445 of the reconstructed mass spectrum of the incubated sample and the main peak 440 of the unreacted target molecule, it is possible to deduce the mass or weight of the small molecule that is covalently or non-covalently bound to the target molecule. For example, the determined covalent or non-covalent interaction, or determined level of covalent or non-covalent interaction, may be associated with the deconvoluted mass spectrum or the raw mass spectrum and with the sample, e.g., the sample location in the sample source. As such, a record may be created of the covalent or non-covalent interaction, the associated sample location, and the associated deconvoluted mass spectrum or raw mass spectrum. The established record may be rendered accessible and may be used to, e.g., identify or characterize the sample.

[0082] Although the molecular mass of lodoacetamide is 184.96 amu, the measured mass of the small molecule that is covalently or non-covalently bonded to the target molecule is 57.1 amu (or Da). For example, the difference between the molecular mass of lodoacetamide and the measured mass shift may be due to the fact that during the bonding of the target molecule to lodoacetamide, an Iodine atom is removed from the bonded protein, and because the atomic mass of Iodine is 126.90 amu, there is a lower mass shift of 57.1 amu instead of the expected mass shift of 184.96 amu. In yet another example, when, as illustrated in FIG. 4C(III), there are two peaks 440 and 445 corresponding to the unreacted protein molecule and to the covalently or non- covalently bound target molecule, respectively, it can be concluded that not all of the target molecule is bound to the drug molecule. In examples, the peak 440 of the unreacted protein is the peak that corresponds to the molecular mass of the protein, while the peak 445 of the bound protein designate a mass that corresponds to a heavier compound formed by the covalent or non-covalent interaction of the target molecule and the drug molecule, which results in the peak 445 being shifted to the higher masses compared to peak 440. FIG. 4C(III) shows that the intensity of peak 440 illustrating the unreacted target molecule is lower than the intensity of peak 445 representing the target molecule covalently or non-covalently bound to the drug molecule. As such, it can be concluded that more of the target molecule is bound to the drug molecule than is left unreacted. For example, it may be possible to estimate the proportion of the target molecule that is covalently or non-covalently bonded to the drug molecule by comparing the intensity of peaks 440 and 445.

[0083] FIG. 4C(IV) illustrates a deconvoluted mass spectrum for a small molecule- or drug-molecule-modified target molecule. In this case, N-Phenylacrylamide is used as the small molecule, and the deconvoluted mass spectrum on FIG. 4C(IV) shows a main peak 450 that is substantially equal to the main peak 420 corresponding to the target molecule, with no other peak or detectable level of any covalent modification of the target molecule at 19.5 kDa. In examples, the mass of the single peak 450 of the reconstructed mass spectrum of the incubated sample is substantially equal to the mass of the main peak 420 of the unreacted target molecule. In various examples, the result illustrated in FIG. 4C(TV) may indicate that there is no detectable level of covalent or non-covalent interaction between the target molecule and the N-Phenylacrylamide small molecule. Accordingly, based on the result illustrated in FIG. 4C(IV), it may be possible to conclude that the protein did not covalently or non-covalently bond with N- Phenylacrylamide. Accordingly, the deconvoluted mass spectrum of an incubated protein can provide the ability to identify an unknown small molecule such as, e.g., a drug, that is covalently or non-covalently bonded to the protein, as well as, in some cases, the molecular mass of the unknown drug. In other examples, the deconvoluted mass spectrum of an incubated protein can provide the ability to identify the level of bonding of a protein with a small molecule or drug.

[0084] FIG. 5 represents a flowchart illustrating a method for analyzing a sample including a protein and a binding ligand, in accordance with various examples of the disclosure. Operation 510 of the method 500 includes receiving the sample at an ionization device. For example, the sample may be incubated in an assay buffer before being carried via a carrier solvent to the ionization device. The sample may be received via non-contact sampling via a carrier solvent such as, e.g., a non-denaturing carrier solvent. For example, the non-denaturing carrier solvent may be configured to preserve non-covalent interaction between the protein and the binding ligand, which may be or include a drug molecule. In another example, the non-denaturing solvent may include a water-based solvent, and may be configured to preserve non-covalent interaction between the protein and the binding ligand. The non-contact sampling may be achieved via a non-contact sampler such as, e.g., an acoustic droplet ejector.

[0085] Operation 520 includes ionizing the received sample. In an example, ionizing the sample received during operation 510 may include using an ionization device such as, e.g., a DESI device, a matrix-assisted laser desorption ionization (MALDI) device, a liquid-atmospheric pressure (LAP)-MALDI device, a pneumatic ESI device, and an electron ionization (El) device. With respect to FIG. 3, operation 520 includes ionizing the sample LS carried through the conduit 325 to the ionization chamber 318.

[0086] Operation 530 includes generating a deconvoluted mass spectrum for the ionized sample. Generating the deconvoluted mass spectrum may include capturing a raw mass spectrum of the ionized sample at a mass analysis device that is coupled to the ionization device, and converting the captured raw mass spectrum to the deconvoluted mass spectrum of the sample. The mass analysis device may, e.g., an AEMS. The captured raw mass spectrum for the sample may include the measurement of the signal intensity with respect to the mass-to-charge ratio (m / z) of the sample for a series of known charges, as illustrated in FIG. 4(A). With respect to FIGS. 4A-4C, operation 530 includes transitioning from the spectrum illustrated in FIG. 4(A) to the spectrum in FIG. 4(C).

[0087] Operation 540 includes accessing a reference mass of the protein. For example, the reference mass of the protein may be the molecular mass of the protein. Once the reference mass is accessed during operation 540, the method 500 at operation 550 includes comparing the deconvoluted mass spectrum to the reference mass. With reference to FIG. 4C, comparing the generated deconvoluted mass spectrum to the reference mass during operation 550 may be performed by comparing any of the main peaks 430, 440 or 450 to the main peak 420. As illustrated in FIG. 4C(II), comparing the main peak 430 to the main peak 420 shows that there is a mass shift of 125.1 amu. This 125.1 amu mass shift is representative of the increased molecular weight of the covalently or non-covalently bound molecule that accounts for, e.g., a drug that is bound to the protein or target molecule. In this case, the mass of the small molecule that is covalently or non-covalently bound to the protein is 125.1 amu, which corresponds to N-ethylmaleimide. In other examples, other drugs bound to the protein may have different masses and thus different corresponding main peaks. Similarly, as illustrated in FIG. 4C(III), comparing the main peak 440 to the main peak 420 shows that there is a mass shift of 57.1 amu, and the 57.1 amu mass shift is representative of the increased molecular weight of the covalently or non-covalently bonded molecule that accounts of, e.g., a drug that is bonded to the original or target molecule. In this case, the increase in mass of the covalently or non-covalently bonded molecule is 57.1 amu, which corresponds to the protein being bonded to lodoacetamide. In this case, although the molecular mass of lodoacetamide is 184.96 amu, the measured mass shift is 57.1 amu, and the difference between the molecular mass of lodoacetamide and the measured mass shift may be due to the fact that during the bonding of the target molecule to lodoacetamide, an Iodine atom is removed from the bonded protein.

[0088] Operation 560 includes detecting bonding between the protein and the binding ligand in the sample based at least in part on the deconvoluted mass spectrum obtained during operation 530. For example, operation 560 includes detecting non-covalent interaction between the protein and the binding ligand based on the comparison performed during operation 550. Detecting the non-covalent interaction during operation 560 may include determining a target mass corresponding to a peak of the deconvoluted mass spectrum, calculating a mass difference between the target mass and the reference mass, and detecting the non-covalent interaction between the protein and the binding ligand in the sample based on the calculated mass difference. Detecting the non-covalent interaction may include identifying the binding ligand non-covalently bound to the protein based on the calculated mass difference.

[0089] Detecting the protein bonding in the sample during operation 560 may be performed by determining the covalent or non-covalent interaction of a protein present in the sample. In examples, determining the covalent or non-covalent interaction may include determining a target mass corresponding to a peak of the generated deconvoluted mass spectrum, accessing the reference mass that corresponds to the target compound, calculating a mass difference between the target mass and the reference mass, and determining the covalent or non-covalent interaction of the target compound based on the calculated mass difference. Determining the covalent bonding or non-covalent interaction may include identifying a binding ligand such as, e.g., a drug species, that may be bound to the protein. For example, determining the covalent bonding or non-covalent interaction may include determining a number of drugs or drug species that are covalently or non-covalent bound to the protein. Determining the number of binding ligands covalently or non-covalent bound to the protein may be performed by dividing the calculated mass difference by a mass of the binding ligand. In some cases, the calculated difference represents the number of drug molecules that are bound to the protein. In other cases, the calculated mass difference is not equal to a multiple of the molecular mass of the drug that is bound to the protein because, e.g., one or more atoms may be substituted during the bonding of the protein and the drug. Accordingly, the calculated mass difference may not correspond to a multiple of the molecular mass of the drug molecule, but may correspond to a difference between the molecular mass of the protein and the molecular mass of the bonded compound.

[0090] In various examples, as indicated by the dashed arrow connecting operation 560 to operation 510, the above operations 510, 520, 530, 540, 550 and 560 may be performed contemporaneously, simultaneously or automatically for more than one sample of a plurality of samples. For example, the above operations 510, 520, 530, 540, 550 and 560 may be performed contemporaneously, simultaneously or automatically for all of the plurality of samples.

[0091] The method 500 may also include automatically analyzing a plurality of samples by analyzing each sample of the plurality of samples according to operations 510-560 discussed above. The method 500 may also include contemporaneously, or simultaneously, analyzing a plurality of samples by analyzing each sample of the plurality of samples according to operations 510-560 discussed above.

[0092] In various examples, the above operations 510, 520, 530, 540, 550 and 560 may be performed by a sample analyzing system such as the mass analyzing systems described elsewhere herein. In examples, the sample analyzing system may include a non-contact sample ejector, a sample ionization device coupled to the non-contact sample ejector, a mass analysis device coupled to the sample ionization device, a processor operatively coupled to the sample ionization device and the mass analysis device, and a memory operatively coupled to the processor, the memory storing instructions that, when executed by the processor, perform a set of operations. For example, the sample receiver includes an open port interface. The system may further include a well plate having a plurality of wells, each well corresponding to a reservoir of the plurality of reservoirs and including at least a sample. For example, the well plate may include 96 wells, 384 wells, or 1536 wells. The set of operations may also include collecting the mass spectrometry data by receiving an ejected sample from the noncontact sample ejector, and wherein receiving the ejected sample includes introducing, with the non-contact sample ejector, the sample from the well plate into the sample receiver. For example, the non-contact sample ejector includes an acoustic droplet ejector, and a frequency of ejecting the sample is equal to or greater than 1 Hz.

[0093] FIG. 6 represents a flowchart illustrating a method for stress testing a sample that includes a protein by a plurality of carrier solvents, e.g., a plurality of nondenaturing or denaturing carrier solvents, and / or a carrier solvent at a plurality of temperatures, in accordance with various examples of the disclosure. The plurality of carrier solvents may be or include various mixtures of denaturing and non-denaturing carrier solvents and may be or include, e.g., various mixtures of water-based and organic solvents.

[0094] The carrier solvents may have increasing denaturing strengths. For example, a first carrier solvent may have a fully non-denaturing property, a second carrier solvent may have a slightly less non-denaturing property, and subsequent carrier solvents may have successively weaker non-denaturing properties until the last carrier solvent which may be, e.g., a denaturing solvent. Accordingly, the structure of the protein in the sample, e.g., the degree of unfolding of the protein in the sample, may be correlated to the denaturing strength of each carrier solvent.

[0095] In other examples, the plurality of carrier solvents may be a same nondenaturing solvent at varying temperatures. The carrier solvents may thus be waterbased solvents, each water-based solvent being maintained at a given temperature. As such, the protein structure in the sample, e.g., the degree of unfolding of the protein in the sample, may be correlated to the temperature of the carrier solvent.

[0096] In various examples of the disclosure, the method 600 includes operation 610, during which a sample is added to or received in, e.g., an assay buffer. The assay buffer may be or include a non-denaturing assay buffer so as to not to disrupt the configuration, e.g., the unfolding or the bonding, of the protein therein. The method 600 further includes operation 620, which includes receiving the sample from the assay buffer at the ionization device via non-contact sampling, the sample being transported by one of the plurality of different carrier solvents, or being transported in a plurality of the same solvent maintained at one of a plurality of varying temperatures. Each of the plurality of carrier solvents may include a mixture of a non-denaturing carrier solvent and a denaturing carrier solvent. In another example, the plurality of carrier solvents include a same non-denaturing carrier solvent at incrementally increasing temperatures. The plurality of carrier solvents may also include a same denaturing carrier solvent at incrementally increasing temperatures. For each one of the plurality of carrier solvents, e.g., one carrier solvent at a time, operation 630 includes ionizing the sample transported therein. With respect to FIG. 3, operation 630 includes ionizing the sample LS that was carried through the conduit 325 at the ionization chamber 318.

[0097] Operation 640 includes generating a deconvoluted mass spectrum for the ionized sample. For example, the deconvoluted mass spectrum may be generated during operation 640 similarly to operation 530 discussed above. Operation 640 may also include determining a target mass corresponding to a peak of the deconvoluted mass spectrum, calculating a mass difference between the target mass and the reference mass. Operation 650 includes detecting a configuration of the protein structure based on the calculated mass difference between the target mass and the reference mass in the deconvoluted mass spectrum generated during operation 640. The protein configuration may be, e.g., protein covalent bonding or non-covalent interaction with a binding ligand, or may be a degree of protein unfolding when no binding ligand is present. In some examples, operation 650 corresponds to operations 540 to 560 discussed above with respect to FIG. 5. Accordingly, operation 650 includes correlating the thus- detected protein configuration to a given mixture of denaturing and non-denaturing carrier solvents, or to a same carrier solvent at a given temperature. For example, any two of the plurality of the carrier solvents have different mixtures of the non-denaturing carrier solvent and the denaturing carrier solvent. In other examples, temperatures of the sample in any two of the plurality of the carrier solvents are different from each other.

[0098] Once the protein configuration is detected for either or both of a given mixture of denaturing and non-denaturing carrier solvents and the temperature of a same carrier solvent, then during operation 660, a determination is made whether there is another sample to be analyzed. The other sample may also include a mixture of carrier solvents, or another same carrier solvent at another temperature. If that is the case, then the method 600 continues to operations 620-660 for the other sample. The method 600 may also include automatically performing operations 610-660 for a plurality of samples, and for example may include contemporaneously or simultaneously performing operations 610-660 for the plurality of samples.

[0099] Although some aspects have been described in the context of an apparatus, it is clear that these aspects also represent a description of the corresponding method, where a block or device corresponds to a method step or a feature of a method step. Analogously, aspects described in the context of a method step also represent a description of a corresponding block or item or feature of a corresponding apparatus. Some or all of the method steps may be executed by (or using) a hardware apparatus, like for example, a processor, a microprocessor, a programmable computer or an electronic circuit. In some examples, some one or more of the most important method steps may be executed by such an apparatus. Generally, examples of the present disclosure can be implemented through the use of computer program products with program codes, the program codes being operative for performing the operations described herein when the computer program product runs on a computer such as may be used to embody any or all of controllers such as, 135, 82, 96, 107, 127, or 330.

[0100] Although various examples and examples are described herein, those of ordinary skill in the art will understand that many modifications may be made thereto within the scope of the present disclosure. Accordingly, it is not intended that the scope of the disclosure in any way be limited by the examples provided.

Claims

CLAIMSWhat is claimed is:

1. A method for analyzing a first sample comprising a protein and a binding ligand, the method comprising: receiving the first sample at an ionization device via non-contact sampling, the first sample being in a non-denaturing carrier solvent; ionizing the first sample; generating a deconvoluted mass spectrum for the ionized first sample; and detecting an interaction between the protein and the binding ligand in the first sample based at least in part on the deconvoluted mass spectrum.

2. The method of claim 1, wherein: generating the deconvoluted mass spectrum for the ionized first sample comprises: capturing a raw mass spectrum of the first sample at a mass analysis device; converting the captured raw mass spectrum to the deconvoluted mass spectrum of the first sample; accessing a reference mass of the protein; and comparing the deconvoluted mass spectrum to the reference mass; and detecting the interaction between the protein and the binding ligand in the first sample comprises: detecting non-covalent interaction between the protein and the binding ligand based on the comparison.

3. The method of claim 2, wherein detecting the non-covalent interaction comprises: determining a target mass corresponding to a peak of the deconvoluted mass spectrum; calculating a mass difference between the target mass and the reference mass; and detecting the non-covalent interaction between the protein and the binding ligand in the first sample based on the calculated mass difference.

4. The method of claim 3, wherein detecting the non-covalent interaction comprises identifying the binding ligand non-covalently bound to the protein based on the calculated mass difference.

5. A method for automatically analyzing a plurality of samples, the method comprising analyzing each of the plurality of samples according to the method of any one of claims 1-4.

6. A method for contemporaneously analyzing a plurality of samples, the method comprising contemporaneously analyzing each of the plurality of samples according to the method of any one of claims 1-5.

7. The method of any one of claims 1-6, wherein the non-denaturing carrier solvent is configured to preserve non-covalent binding between the protein and the binding ligand.

8. The method of any one of claims 1-7, wherein the non-denaturing carrier solvent comprises a water-based solvent.

9. The method of any one of claims 1-8, wherein the binding ligand comprises a drug molecule.

10. The method of any one of claims 2-9, further comprising: receiving a second sample at the ionization device via a denaturing carrier solvent; ionizing the second sample; generating a deconvoluted mass spectrum for the ionized second sample; and determining non-covalent interaction between the protein and the binding ligand in the first sample based at least in part on the deconvoluted mass spectrum for the ionized second sample.

11. The method of any one of claims 1-10, wherein: generating the deconvoluted mass spectrum for the ionized second sample comprises:capturing a raw mass spectrum of the second sample at the mass analysis device; converting the captured raw mass spectrum to the deconvoluted mass spectrum of the second sample; and comparing the deconvoluted mass spectrum for the ionized second sample to the reference mass of the protein; and determining the non-covalent interaction between the protein and the binding ligand in the first sample comprises: detecting covalent bonding between the protein and the binding ligand in the second sample based on the comparison; and determining the non-covalent interaction between the protein and the binding ligand in the first sample based on the detected covalent bonding in the second sample.

12. The method of claim 11, wherein determining the non-covalent interaction between the protein and the binding ligand in the first sample comprises comparing the deconvoluted mass spectrum of the second sample to the deconvoluted mass spectrum of the first sample.

13. The method of claim 11 or claim 12, wherein the second sample is identical to the first sample.

14. The method of any one of claims 11-13, wherein the denaturing carrier solvent is configured to disrupt the non-covalent interaction between the protein and the binding ligand.

15. The method of any one of claims 11-14, wherein the denaturing carrier solvent comprises an organic solvent.

16. A sample analyzing system comprising: a non-contact sample ejector; a sample ionization device coupled to the non-contact sample ejector; a mass analysis device coupled to the sample ionization device;a processor operatively coupled to the sample ionization device and the mass analysis device; and a memory operatively coupled to the processor, the memory storing instructions that, when executed by the processor, perform a set of operations comprising: receiving, at the ionization device, the first sample via the non-contact sample ejector, the first sample being in a non-denaturing carrier solvent; ionizing, via the sample ionization device, the first sample; generating, via the mass analysis device, a deconvoluted mass spectrum for the ionized first sample; and detecting, via the processor, bonding between the protein and the binding ligand in the first sample based at least in part on the deconvoluted mass spectrum.

17. The system of claim 16, wherein the set of operations further comprises: generating the deconvoluted mass spectrum for the ionized first sample by: capturing a raw mass spectrum of the first sample at the mass analysis device; converting the captured raw mass spectrum to the deconvoluted mass spectrum of the first sample; accessing a reference mass of the first sample; and comparing the deconvoluted mass spectrum to the reference mass; and detecting the bonding in the first sample by detecting non-covalent interaction between the protein and the binding ligand based on the comparison.

18. The system of claim 17, wherein the set of instructions comprises detecting the non- covalent interaction by: determining a target mass corresponding to a peak of the deconvoluted mass spectrum; calculating a mass difference between the target mass and the reference mass; and detecting the non-covalent interaction between the protein and the binding ligand in the first sample based on the calculated mass difference.

19. The system of claim 18, wherein the set of operations comprises detecting the non- covalent interaction by identifying the binding ligand non-covalently bound to the protein based on the calculated mass difference.

20. The system of any one of claims 16-19, wherein the non-denaturing carrier solvent is configured to preserve non-covalent interaction between the protein and the binding ligand.

21. The system of any one of claims 16-20, wherein the non-denaturing carrier solvent comprises a water-based solvent.

22. The system of any one of claims 16-21, wherein the set of operations further comprises contemporaneously detecting non-covalent interaction between the protein and the binding ligand in a plurality of samples.

23. The system of any one of claims 17-22, wherein the set of operations further comprises: receiving a second sample at the ionization device via a denaturing carrier solvent; ionizing the second sample; capturing a deconvoluted mass spectrum for the ionized second sample; and determining non-covalent interaction between the protein and the binding ligand in the first sample based at least in part on the deconvoluted mass spectrum for the ionized second sample.

24. The system of claim 23, wherein the set of instructions comprises: generating the deconvoluted mass spectrum for the ionized second sample by: capturing a raw mass spectrum of the second sample at the mass analysis device; converting the captured raw mass spectrum to the deconvoluted mass spectrum of the second sample; and comparing the deconvoluted mass spectrum for the ionized second sample to the reference mass of the protein; anddetermining the non-covalent interaction between the protein and the binding ligand in the first sample by: detecting covalent bonding between the protein and the binding ligand in the second sample based on the comparison; and determining the non-covalent interaction between the protein and the binding ligand in the first sample based on the detected covalent bonding in the second sample.

25. The system of claim 24, wherein the set of instructions comprises determining the non-covalent interaction between the protein and the binding ligand in the first sample by comparing the deconvoluted mass spectrum of the second sample to the deconvoluted mass spectrum of the first sample.

26. The system of claim 25, wherein the second sample is identical to the first sample.

27. The system of any one of claims 23-26, wherein the denaturing carrier solvent is configured to disrupt the non-covalent interaction between the protein and the binding ligand.

28. The system of any one of claims 23-27, wherein the denaturing carrier solvent comprises an organic solvent.

29. The system of any one of claims 16-28, wherein the non-contact sample ejector comprises an acoustic droplet ejector.

30. A method for stress testing a sample, the sample comprising a protein, the method comprising: receiving the sample at an ionization device via non-contact sampling, the sample being in one of a plurality of carrier solvents; and for each one of the plurality of carrier solvents: ionizing the sample; generating a deconvoluted mass spectrum for the ionized sample; and detecting protein configuration in the sample based at least in part on the deconvoluted mass spectrum.

31. The method of claim 30, wherein detecting the protein configuration in the sample comprises detecting one of a covalent bonding of the protein, a non-covalent interaction of the protein, and a protein unfolding of the protein.

32. The method of claim 30 or claim 31, wherein, for each one of the plurality of carrier solvents: generating the deconvoluted mass spectrum for the ionized sample comprises: capturing a raw mass spectrum of the sample at a mass analysis device; converting the captured raw mass spectrum to the deconvoluted mass spectrum of the sample; accessing a reference mass of the protein; and comparing the deconvoluted mass spectrum to the reference mass; and detecting the protein configuration in the sample comprises: detecting the protein configuration based on the comparison; and correlating the protein configuration to the carrier solvent.

33. The method of claim 32, wherein detecting the protein configuration comprises: determining a target mass corresponding to a peak of the deconvoluted mass spectrum; calculating a mass difference between the target mass and the reference mass; and detecting the protein configuration in the sample based on the calculated mass difference.

34. A method for automatically analyzing a plurality of samples, the method comprising analyzing each of the plurality of samples according to the method of any one of claims 30-33.

35. A method for contemporaneously analyzing a plurality of samples, the method comprising contemporaneously analyzing each of the plurality of samples according to the method of any one of claims 30-34.

36. The method of any one of claims 30-35, wherein:each of the plurality of carrier solvents includes a mixture of a non-denaturing carrier solvent and a denaturing carrier solvent; and any two of the plurality of the carrier solvents have different mixtures of the non-denaturing carrier solvent and the denaturing carrier solvent.

37. The method of any one of claims 30-36, wherein: each of the plurality of carrier solvents includes a same non-denaturing carrier solvent; the sample in each carrier solvent is maintained at a given temperature; and temperatures of the sample in any two of the plurality of the carrier solvents are different from each other.

38. The method of any one of claims 30-37, wherein: each of the plurality of carrier solvents includes a same denaturing carrier solvent; the sample in each carrier solvent is maintained at a given temperature; and temperatures of the sample in any two of the plurality of the carrier solvents are different from each other.

39. A sample analyzing system comprising: a non-contact sample ejector; a sample ionization device coupled to the non-contact sample ejector; a mass analysis device coupled to the sample ionization device; a processor operatively coupled to the sample ionization device and the mass analysis device; and a memory operatively coupled to the processor, the memory storing instructions that, when executed by the processor, perform a set of operations comprising: receiving, at the ionization device, the sample via the non-contact sample ejector, the sample being in one of a plurality of carrier solvents; and for each one of the plurality of carrier solvents, the set of operations comprise: ionizing, via the sample ionization device, the sample; generating, via the mass analysis device, a deconvoluted mass spectrum for the ionized sample; anddetecting, via the processor, protein configuration in the sample based at least in part on the deconvoluted mass spectrum.

40. The method of claim 39, wherein the set of operations comprises detecting the protein configuration in the sample by detecting one of a covalent bonding of the protein, a non-covalent interaction of the protein, and a protein unfolding of the protein.

41. The system of claim 39 or claim 40, wherein the set of operations further comprises, for each one of the plurality of non-denaturing carrier solvents: generating the deconvoluted mass spectrum for the ionized sample by: capturing a raw mass spectrum of the sample at the mass analysis device; converting the captured raw mass spectrum to the deconvoluted mass spectrum of the sample; accessing a reference mass of the sample; and comparing the deconvoluted mass spectrum to the reference mass; and detecting the protein configuration in the sample by: detecting the protein configuration based on the comparison; and correlating the protein configuration to the non-denaturing carrier solvent.

42. The system of claim 41, wherein the set of instructions comprises detecting the protein bonding by: determining a target mass corresponding to a peak of the deconvoluted mass spectrum; calculating a mass difference between the target mass and the reference mass; and detecting the protein bonding in the sample based on the calculated mass difference.

43. The system of any one of claims 39-42, wherein the set of operations further comprises contemporaneously detecting the protein bonding in a plurality of samples.

44. The system of any one of claims 39-43, wherein:each of the plurality of carrier solvents includes a mixture of a non-denaturing carrier solvent and a denaturing carrier solvent; and any two of the plurality of the carrier solvents have different mixtures of the non-denaturing carrier solvent and the denaturing carrier solvent.

45. The system of any one of claims 39-44, wherein: each of the plurality of carrier solvents includes a same non-denaturing carrier solvent; the set of operations comprises maintaining the sample in each carrier solvent at a given temperature; and temperatures of the sample in any two of the plurality of the carrier solvents are different from each other.

46. The system of any one of claims 39-45, wherein: each of the plurality of carrier solvents includes a same denaturing carrier solvent; the set of operations comprises maintaining the sample in each carrier solvent at a given temperature; and temperatures of the sample in any two of the plurality of the carrier solvents are different from each other.

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