Mass spectrometry-based method and system for enzyme engineering

Abstract

Examples of this disclosure include methods and systems of enzyme engineering, including preparing a plurality of DNA samples, combining at least one of the plurality of DNA samples with a host cell, incubating the combined at least one of the plurality of DNA samples and the host cell at one of a desired first temperature and for a desired first period of time to generate a plurality of first enzymes, adding a first cell lysis reagent to the combined at least one of the plurality of DNA samples and the host cell to release the plurality of first enzymes, adding a first catalyzing substrate to the plurality of first enzymes to generate a first product and a first by-product for each first enzyme via catalysis, evaluating a first reaction yield for each first enzyme, and selecting a first enzyme from the plurality of first enzymes based on the evaluation.

Description

[0001] MASS SPECTROMETRY-BASED METHOD AND SYSTEM FOR ENZYME ENGINEERING

[0002] Cross-Reference To Related Application

[0003] This application is being filed as a PCT International application and claims the benefit of and priority to U.S. Provisional Application No. 63 / 594,701, filed October 31, 2023, the disclosure of which is hereby incorporated by reference in its entirety.

[0004] Background

[0005] Enzyme engineering, also referred to herein as protein engineering or biocatalysis, is the process of designing enzymes for a specific or desired catalysis activity. Enzyme engineering typically includes the modification or mutation of a DNA sequence, and the DNA sequence is then transcribed and translated to an enzyme or protein. Enzyme engineering is widely used in various industries such as, e.g., the pharmaceutical, food, detergent, and biotransformation industries. The process of enzyme engineering and the analysis thereof is a process that includes recombinant DNA mutation, incubation with a medium or cell, enzyme extraction, and an assessment of enzyme activity.

[0006] Summary

[0007] In one aspect, the technology relates to a method of enzyme engineering, the method including generating a plurality of first enzymes from at least one of a plurality of first DNA samples, performing a catalysis reaction of at least one of the plurality of first enzymes to generate a first product for each of the at least one of the plurality of first enzymes, performing high-throughput mass spectrometry on at least one of the first products to evaluate a first reaction yield thereof, and selecting a first enzyme from the at least one of the plurality of first enzymes based on the performed high- throughput mass spectrometry.

[0008] In an example, evaluating the first reaction yield includes evaluating a yield of the first product. In another example, selecting the first enzyme includes generating a first high-throughput mass spectrum for the at least one of the first products, determining a peak height and a peak area from the first high-throughput mass spectrum for the at least one of the first products, and selecting the first enzyme that corresponds to one of a first desired peak height and a first desired peak area. In yet another example, the method further includes performing a random mutation on the selected first enzyme to generate a plurality of second DNA samples, generating a plurality of second enzymes from at least one of the plurality of second DNA samples, performing a catalysis reaction of at least one of the plurality of second enzymes to generate a second product for the at least one of the plurality of second enzymes, performing high-throughput mass spectrometry on at least one of the second products to evaluate a second reaction yield thereof, and selecting a second enzyme from the at least one of the plurality of second enzymes based on the performed high-throughput mass spectrometry.

[0009] In a further example, evaluating the second reaction yield includes evaluating a yield of the second product. In yet another example, selecting the second enzyme includes generating a second high-throughput mass spectrum for the at least one of the second products, determining a peak height and a peak area from the second high- throughput mass spectrum for the at least one of the second products, and selecting the second enzyme that corresponds to one of a second desired peak height and a second desired peak area. In another example, at least one of the first product and the second product include a protein. In a further example, a yield of the second product is greater than a yield of the first product. In another example, generating the plurality of first enzymes from at least one of a plurality of first DNA samples includes preparing a plurality of first DNA samples, separately combining at least one of the plurality of first DNA samples with a first host cell, incubating the combined at least one of the plurality of first DNA samples and the first host cell to generate the plurality of first enzymes in the host cell, and adding a first cell lysis reagent to the first host cell to release the plurality of first enzymes from the host cell. For example, preparing the plurality of first DNA samples includes synthesizing the plurality of first DNA samples via random mutation of an original DNA sample, purifying the plurality of first DNA samples, and assigning a unique ID to each of the plurality of first DNA samples.

[0010] In another aspect, an integrated system for enzyme selection includes a DNA synthesis apparatus, a catalysis apparatus, a high-throughput mass analysis device coupled to a sample ionization device, a processor operatively coupled to the high- throughput mass analysis device, and a memory coupled to the processor, the memory storing instructions. The instructions, when executed by the processor, perform a set of operations including generating, via the DNA synthesis apparatus, a plurality of first enzymes from at least one of a plurality of first DNA samples, performing, via the catalysis apparatus, a catalysis reaction of at least one of the plurality of first enzymes to generate a first product for each of the at least one of the plurality of first enzymes, performing, via the high-throughput mass analysis device, high-throughput mass spectrometry on at least one of the first products to evaluate a first reaction yield thereof, and selecting, via the processor, a first enzyme from the at least one of the plurality of first enzymes based on the performed high-throughput mass spectrometry.

[0011] In another example, the system further includes a plurality of sample containers, wherein the set of operations includes at least one of generating the plurality of first enzymes in at least one of the plurality of sample containers, and performing the catalysis reaction in the at least one of the plurality of sample containers. In a further example, the set of operations includes evaluating the first reaction yield by evaluating a yield of the first product. In yet another example, the set of operations includes selecting the first enzyme by generating a first high-throughput mass spectrum for the at least one of the first products, determining a peak height and a peak area from the first high-throughput mass spectrum for the at least one of the first products, and selecting the first enzyme that corresponds to one of a first desired peak height and a first desired peak area. In a further example, the set of operations further includes performing a random mutation on the selected first enzyme to generate a plurality of second DNA samples, generating a plurality of second enzymes from at least one of the plurality of second DNA samples, performing a catalysis reaction of at least one of the plurality of second enzymes to generate a second product for the at least one of the plurality of second enzymes, performing high-throughput mass spectrometry on at least one of the second products to evaluate a second reaction yield thereof, and selecting a second enzyme from the at least one of the plurality of second enzymes based on the performed high-throughput mass spectrometry.

[0012] In yet another example, the set of operations includes evaluating the second reaction yield by evaluating a yield of the second product. In a further example, the set of operations includes selecting the second enzyme by generating a second high- throughput mass spectrum for the at least one of the second products, determining a peak height and a peak area from the second high-throughput mass spectrum for the at least one of the second products, and selecting the second enzyme that corresponds to one of a second desired peak height and a second desired peak area. In yet a further example, a yield of the second product is greater than a yield of the first product. In an additional example, the plurality of sample containers are included in one of a well plate and a microarray. In another example, at least one of the first product and the second product include a protein.

[0013] Brief Description of the Drawings

[0014] FIG. 1 is schematic diagram illustrating another exemplary mass analysis system in accordance with various aspects and examples of the present disclosure.

[0015] 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.

[0016] FIG. 3 is an illustration of an integrated system for enzyme selection, in accordance with various examples of the present disclosure.

[0017] FIG. 4 represents a flowchart illustrating a method for enzyme engineering, in accordance with various examples of the disclosure.

[0018] FIG. 5 represents a flowchart illustrating a method of enzyme engineering, in accordance with various examples of the disclosure.

[0019] 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.

[0020] Detailed Description

[0021] The technologies described herein may be utilized in high-throughput enzyme engineering workflows. One way to assess enzyme activity is to utilize mass spectrometry (MS) detection. This MS detection monitors the catalysis efficiency that may be, e.g., the conversion from substrate to product, in a label free manner. When there is a large amount of sample, where a large amount of DNA modification or mutations that lead to the various enzymes may take place, a high-throughput mass spectrometry (HT-MS) technology is advantageous, and is even more advantageous when utilized with automatic data processing. In addition, one way to increase throughput is to utilize HT-MS. As a result of an automated integrated HT-MS evaluation of enzymes, it may be possible to select one or more enzymes with a desired activity. For example, HT-MS evaluation may uncover one or more enzymes that generate a highest product yield. Enzymes that generate high product yields are useful in any of pharmaceutical, food, detergent, and biotransformation industries, or in industries otherwise known to persons of skill in the art.

[0022] In various examples, an integrated system and method of a high-throughput enzyme engineering workflow may be designed, and may include a plurality of operations or stages. A first operation or stage may include generating a plurality of enzymes from DNA samples. The enzymes may be generated from the DNA samples via a plurality of operations such as combining the DNA samples with a host cell and an incubating medium, incubating the combination to generate enzymes, and adding a cell lysis reagent to release the enzymes.

[0023] A second operation may include performing a catalysis reaction by adding a catalyzing substrate the released enzymes to generate a product and / or a by-product for released enzyme via catalysis. When the released enzyme has sufficient catalyzing activity, the catalysis reaction of the substrate generates a product and / or by-product. In some cases, the product and by-product may be small molecules. In other cases, the product may be, e.g., a protein, and the by-product may be, e.g., an isomer of the protein. For example, a catalyzing material, also referred to herein as a catalyzing substrate, is added to each sample so that a catalysis reaction may occur in the isolated compartment that holds the sample. The catalysis reaction may convert the substrate to a product and / or a by-product. Each substrate may be converted to a given product via the catalysis, so that different catalyzing substrates added to the same sample may generate different products.

[0024] A third operation or stage may include the analysis of the product to evaluate the reaction yield of the product. During this third operation or stage, a product generated via the first and second operations discussed above is analyzed by, e.g., HT- MS. In examples, the HT-MS result may be used to analyze the catalysis efficiency of a given sample. Evaluating the reaction yield during this third operation may include evaluating a yield of the product and / or a yield of the by-product for each enzyme. In another example, evaluating the yield of the product and / or the yield of the by-product for each enzyme may be achieved by generating a high-throughput mass spectrum for each enzyme via, e.g., a HT-MS device, and analyzing the resulting signal. Analyzing the resulting signal may include determining any one or more of a peak height, an area under the peak, a signal-to-noise ratio, and the like, from the resulting signal, for the product and / or for the by-product resulting from the enzyme.

[0025] A fourth operation or stage includes selecting an enzyme having a most desired yield. This operation includes correlating the MS measurement results, which are representative of catalysis efficiency of a given enzyme, with the ID of the same DNA sample that produced the enzyme. Given the large number of DNA samples, e.g., hundreds or even thousands of DNA samples, that are prepared as the result of the random mutation of the original DNA sample, using HT-MS may be substantially advantageous in obtaining automated and rapid analysis of the catalysis efficiency of each enzyme. In various examples, during this fourth operation or stage, a yield of the product may be determined for each DNA sample. The DNA sample that generates the highest product yield may be selected, and a new workflow cycle, including the first stage through the third stage, may be performed a new on the selected DNA sample. For example, the selected DNA sample may be subjected to a random mutation to generate a plurality of DNA samples, and the stages discussed above may be performed on each resulting randomly mutated DNA sample. As noted above, continued utilization of HT-MS to analyze the product yield of each enzyme via catalysis of the substrate is advantageous.

[0026] Examples of the above workflow, as well as other workflows described herein, may be performed with HT-MS analysis systems. 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 is desired. For instance, a number of different systems may have been used that were provided and controlled by separate entities and / or devices. A liquid handling system may be used for preparation of samples. An acoustic ejection system may be used for combining DNA samples with host cells, or transferring these combined DNA / host cells into an incubation medium. A mass spectrometry system may be used for the actual analysis of the samples.

[0027] 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. Specific implementations of HT-MS system are further described in the context of the following FIGS. 1 and 2.

[0028] Now referring to FIG. 1, an example of an HT-MS system that may be utilized in the performance of the workflows described herein are illustrated and described.

[0029] 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. In one example, an open port interface (OPI) may be utilized as the capture probe 105.

[0030] 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. 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.

[0031] The sample preparation system 101 may include a sample source 70 and a sample handler 80. For example, the sample source 70 may be or include a well plate, the well plate having a plurality of wells, at least one of the wells including a source of DNA samples. The sample source 70 may include DNA samples that are combined with a host cell, the combined DNA samples and host cell may be incubated in the sample source 70 together with an incubation medium, a cell lysis reagent may be added to the incubated DNA samples in the sample source 70 to release enzymes, and / or the sample source 70 may receive a catalyzing substrate configured to catalyze the substrate in reaction with the released enzymes into generating a product, the product and any by-product being held therein. As such, the sample source 70 and the 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. In other examples, the well plates of the sample source 70 may be pre-filled with the combination of the DNA samples, the host cell, the incubating medium, the cell lysis reagent, and the catalyzing substrate, and then may be transferred to the ion source 115 or the mass analyzer 120.

[0032] The sample handler 80 may be or include a transporter configured to transport the DNA samples from the source 70 to, e.g., the ion source 115 and / or the mass analyzer 120. The transporter may be robot or other system to move a prepare well plate 75 from the sample source 70 to the well plate stage 95. Any combination of the DNA samples, the host cell, the incubating medium, the cell lysis reagent and the catalyzing substrate may be transferred from the well plate 75 to the mass analyzer 120 or the ion source 115 via the acoustic ejector 90.

[0033] 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) may 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 203 thereof.

[0034] 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.

[0035] The computing system 103 includes computing resources, components, and modules that are operative to perform various functions including controlling the process of DNA synthesis, providing instructions to combine DNA samples with a host cell, to add the incubating medium, to add a cell lysis reagent, and to add a catalyzing substrate, providing a timing of incubation of the combined DNA samples and host cell, providing an ID for each DNA sample or each resulting product, and the like. Other functions may include but are 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.

[0036] In some examples, the computing system 103 includes a computing device 200, a controller 135, and a data processing system 203. The computing device 200 may be in the form of electronic signal processors and operative to perform various computing functions, and is described in further detail in FIG. 2. 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 203 may include various components and modules operative to process mass spectrometry data and to provide real-time feedback to end users and other subsystems.

[0037] 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.

[0038] 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 may include, e.g., the IDs of each DNA sample, or of each product, present in the sample source 70, or present in each well of the well plate 75. 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 203 to the retrieved data from the database 106 (such as molecular mass information or spectral features) to facilitate mass analysis and / or analyte identification.

[0039] 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.

[0040] 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 ion source 115 may be or include, e.g., desorption electrospray ionization (DESI), which is a combination of electrospray ionization (ESI) and desorption ionization (DI), matrix assisted desorption ionization (MALDI), rapid-fire mass spectrometry, liquid atmospheric pressure (LAP) MALDI, pneumatic ESI, and the like. 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 203. 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.

[0041] 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 identifier(s) 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.

[0042] 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. The controller includes a control component 107 for the capture probe 105, represented for example by an open port interface (OPI) software, and a control component 127 for the mass analysis instrument 100, which may be the MS 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.

[0043] Now referring to FIG. 2, an example of the computing device 200 depicted in 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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 203. 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. The methods described herein, as further discussed below, may include any combination of generating a plurality of enzymes from at least one of a plurality of DNA samples, performing a catalysis reaction of at least one of the enzymes to generate a product for enzyme, performing high-throughput mass spectrometry on the product to evaluate a reaction yield thereof, and selecting a preferred one of the plurality of enzymes based on the performed high-throughput mass spectrometry. Operations related to the described methods or required or desired to, e.g., prepare the DNA samples, may also be controlled by the computing device. 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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, 10,000 to 25,000 samples per day. The system 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. FIG. 3 is an illustration of an integrated system for enzyme selection, in accordance with various examples of the present disclosure. In FIG. 3, the system 300 includes a DNA synthesis apparatus 310, a mass analysis device coupled to a sample ionization device 320, and a processor 340. The DNA synthesis apparatus 310 may be, e.g., a machine used to custom-build DNA molecules to contain a particular sequence of nucleotides by, e.g., undergoing random mutations of an original DNA sample. In another example, a DNA synthesizer can create specific DNA molecules for use in the treatment of a variety of diseases by replacing a faulty or damaged section of DNA with a repaired section. When DNA samples are prepared at the DNA synthesis apparatus 310, the DNA samples, or any combination of the DNA samples, the host cell, the incubating medium, the cell lysis reagent and the catalyzing substrate that results in a product, may be transported to, e.g., a sample analysis device 320. The sample analysis device 320 may be or include, e.g., a mass analysis device such as an HT-MS coupled to a sample ionization device. In an integrated system, the DNA samples or resulting product may be automatically transported to the sample analysis device 320 without human intervention. Alternatively, the DNA samples or resulting product may be manually transported to the sample analysis device 320. For example, the mass analysis device or HT-MS device may be similar to the mass analysis instrument 100 illustrated in FIG. 1, and the sample ionization device may be similar to the ion source 115 described therein. The network 330 may be a direct hardware communication between the sample ionization device 320 and the processor 340 or between the DNA synthesis apparatus 310 and the processor 340, or may be or include the cloud, internet, or other wireless communication technique or system. Accordingly, the processor 340 may be used to control operation of the DNA synthesis apparatus 310 and of the sample analysis device 320. The processor 340 may be coupled to a memory 350 and may be, e.g., similar to the computing system 103 discussed above with respect to FIG. 1, or similar to the processor 204 discussed above with respect to FIG. 2. The memory 350 may be similar to the RAM 206 or the ROM 208 also discussed above with respect to FIG. 2.

[0053] In operation, the memory 350 stores instructions that, when executed by the processor 340, perform a set of operations including generating, via the DNA synthesis apparatus 310, a plurality of enzymes from at least one of a plurality of DNA samples via, e.g., random mutation of an original DNA sample. The instructions may also include controlling the DNA synthesis apparatus 310 to perform a catalysis reaction of at least one of the plurality of first enzymes. The stored instructions may also control the sample analysis device 320 so as to perform high-throughput mass spectrometry on at least one of the first products to evaluate a first reaction yield thereof. Data obtained from that analysis may be used by the processor to select a first enzyme from the at least one of the plurality of first enzymes.

[0054] While described above as an integrated system 300, the system 300 may consist of several discrete components (e.g., the DNA synthesis apparatus 310 and of the sample analysis device 320 may be discrete system controlled in whole or in part by a processor 340 of a linked computer) operating to obtain the required or desired results. In another example, the integrated system 300 for enzyme selection may be a single, integrated system where all the operations discussed above are performed within a same system, and the operations may be performed in an automated process. Automation of the above operations provide substantial advantages in terms of the large amount of DNA samples and enzymes that can be analyzed as well as in the accuracy of the analysis provided by the use of a same HT-MS device, sample transfer systems and transfer storage systems for all DNA samples.

[0055] FIG. 4 represents a flowchart illustrating a method for enzyme engineering, in accordance with various examples of the disclosure. In various examples, the method 400 includes operation 410, which includes generating a plurality of first enzymes from at least one of a plurality of first DNA samples such as, e.g., recombinant DNA samples. For example, generating the plurality of first enzymes from at least one of a plurality of first DNA samples may include preparing a plurality of first DNA samples, separately combining at least one of the plurality of first DNA samples with a first host cell, incubating the combined at least one of the plurality of first DNA samples and the first host cell to generate the plurality of first enzymes in the host cell, and adding a first cell lysis reagent to the first host cell to release the plurality of first enzymes from the host cell. Preparing the plurality of first DNA samples may include synthesizing the plurality of DNA samples via a random mutation of an original DNA sample, purifying the plurality of randomly mutated DNA samples, and assigning a unique ID to each of the plurality of randomly mutated DNA samples. Once the plurality of first enzymes are generated during operation 410, operation 420 includes performing a catalysis reaction of at least one of the plurality of first enzymes to generate a first product for each of the at least one of the plurality of first enzymes. The first product may be, e.g., a protein, and a first by-product generated with the first product may be, e.g., an isomer of the protein.

[0056] Operation 430 includes performing HT-MS on at least one of the first products to evaluate a first reaction yield thereof. In examples, the HT-MS result may be used to analyze the catalysis efficiency of a given sample. Evaluating the reaction yield during operation 430 may include evaluating a yield of the first product and / or a yield of the first by-product for each enzyme. In another example, evaluating the yield of the first product and / or the yield of the first by-product for each enzyme may be achieved by generating a high-throughput mass spectrum for each first enzyme via, e.g., a HT-MS device, and analyzing the resulting signal. Analyzing the resulting signal may include determining any one or more of a peak height, an area under the peak, a signal-to-noise ratio, and the like, from the resulting signal, for the first product and / or for the first byproduct resulting from the first enzyme.

[0057] Operation 440 includes selecting a first enzyme from the at least one of the plurality of first enzymes based on the performed high-throughput mass spectrometry. Evaluating the first reaction yield may include evaluating a yield of the first product. In an example, selecting the first enzyme may include generating a first high-throughput mass spectrum for the at least one of the first products, determining a peak height and a peak area from the first high-throughput mass spectrum for the at least one of the first products, and selecting the first enzyme that corresponds to one of a first desired peak height and a first desired peak area.

[0058] In other examples of the disclosure, the method 400 may further include operation 450, which includes performing a random mutation on the selected first enzyme to generate a plurality of second DNA samples and to generate a plurality of second enzymes. Operation 450 may also include performing a catalysis reaction of at least one of the plurality of second enzymes to generate a second product for the at least one of the plurality of second enzymes.

[0059] Operation 460 includes performing high-throughput mass spectrometry on at least one of the second products to evaluate a second reaction yield thereof. Evaluating the reaction yield during operation 460 may include evaluating a yield of the second product and / or a yield of the second by-product for each second enzyme. In another example, evaluating the yield of the second product and / or the yield of the second byproduct for each second enzyme may be achieved by generating a high-throughput mass spectrum for each second enzyme via, e.g., a HT-MS device, and analyzing the resulting signal. Analyzing the resulting signal may include determining any one or more of a peak height, an area under the peak, a signal-to-noise ratio, and the like, from the resulting signal, for the second product and / or for the second by-product resulting from the second enzyme.

[0060] Operation 470 includes selecting a second enzyme from the at least one of the plurality of second enzymes based on the performed high-throughput mass spectrometry. Selecting the second enzyme may include generating a second high- throughput mass spectrum for the at least one of the second products, determining a peak height and a peak area from the second high-throughput mass spectrum for the at least one of the second products, and selecting the second enzyme that corresponds to one of a second desired peak height and a second desired peak area. The first product and / or the second product may include a protein. For example, due to the fact that the second product is selected based on random mutations of the first product, the yield of the second product may be greater than the yield of the first product.

[0061] As a result of operations 410-470, the selected second enzyme has an even higher yield than the first enzyme selected during operation 440. In various examples, the method 400 may also include repeating operations 410-440 or 450-470 by starting from the second enzyme selected during operation 470 in order to obtain a third enzyme that may have an even higher yield than the selected second enzyme. This example is illustrated in FIG. 4 as the dashed line linking operation 470, during which a second enzyme is selected, back to operation 450, during which random permutations may be performed on the selected second enzyme.

[0062] FIG. 5 represents a flowchart illustrating a method for enzyme engineering, in accordance with various examples of the disclosure. The method illustrated in FIG. 5 may be applied to the selection of the first enzymes discussed in operations 410-440, or to the selection of second enzymes discussed in operations 450-460. The method 500 describes in greater detail the manner in which an enzyme is selected using HT-MS. In FIG. 5, operation 510 includes preparing a plurality of DNA samples such as, e.g., recombinant DNA samples. Recombinant DNA samples are synthesized, purified and each assigned with a specific identification marker, type, or code (ID). The recombinant DNA samples may be prepared via random mutations of an initial DNA sample, resulting in a randomly mutated plurality of DNA samples. Each randomly mutated DNA sample may be stored or contained in, e.g., a multi-well plate, a microarray, or other sample container. When the DNA samples are prepared as the result of a random mutation of the original DNA sample, hundreds or even thousands of DNA samples may be generated.

[0063] Operation 520 includes separately combining the plurality of DNA samples, or separately combining each of the plurality of DNA samples, with a host or a host cell. The host cell may be or include, e.g., a bacteria, combining the plurality of DNA samples during operation 520 may include combining the DNA samples with the bacteria. Operation 520 may also include adding an incubation medium to the combined DNA sample and host cell. In some instances, the host cell includes the incubation medium or is configured to generate the enzyme from the DNA sample. These combinations may be performed in individual containers, in order to isolate particular samples for further processing. The individual containers may be isolated compartments such as, e.g., a sample well of a multi-well plate or a discrete liquid droplet. During operation 520, combining the DNA with the host cell and the incubation medium may be achieved automatically via acoustic droplet ejection, pipetting, microfluidic droplet merging, and the like.

[0064] Operation 530 includes incubating each of the DNA samples combined with the host cell and the incubation medium in a separate compartment for a desired period of time to generate a plurality of enzymes in the host cell. The incubation periods of time may vary for various DNA samples. During incubation, the DNA sample combined with the host cell in the incubation medium produces proteins, e.g., enzymes, within the host cell.

[0065] Operation 540 includes adding a cell lysis reagent to the plurality of enzymes to release the enzymes generated during the incubation period from the host cell, also referred to herein as protein extraction. Releasing the protein or enzyme may be achieved via automatic liquid handling techniques such as acoustic droplet dispensing, pipetting, microfluidic droplet merging, and the like.

[0066] Operation 550 includes adding a catalyzing substrate to the released enzymes to generate a product and / or a by-product for released enzyme via catalysis. The catalysis reaction may convert the enzyme to a product and / or a by-product. Each substrate may generate a given product, so that different catalyzing substrates added to the same sample may generate different products. The product may be, e.g., a protein, and the by-product may be, e.g., an isomer of the protein. Operation 560 includes performing HT-MS to evaluate a reaction yield for each enzyme. Performing HT-MS during operation 560 may be similar to performing HT- MS during operation 460 discussed above.

[0067] Operation 570 includes selecting an enzyme from the plurality of enzymes based on the evaluation. For example, operation 570 includes selecting the enzyme by comparing a peak height and / or a peak area from the mass spectrum area for the plurality of enzymes to one another, and selecting the enzyme that has a desired peak height and / or a desired peak area. The desired peak height and / or desired peak area may be a largest peak height, a largest peak area, a largest ratio between peak height of the product and peak height of the by-product, a largest ratio between peak area of the product and peak area of the by-product, and the like.

[0068] This disclosure described some examples of the present technology with reference to the accompanying drawings, in which only some of the possible examples were shown. Other aspects can, however, be embodied in many different forms and should not be construed as limited to the examples set forth herein. Rather, these examples were provided so that this disclosure was thorough and complete and fully conveyed the scope of the possible examples to those skilled in the art.

[0069] Although specific examples were described herein, the scope of the technology is not limited to those specific examples. One skilled in the art will recognize other examples or improvements that are within the scope of the present technology. Therefore, the specific structure, acts, or media are disclosed only as illustrative examples. Examples according to the technology may also combine elements or components of those that are disclosed in general but not expressly exemplified in combination, unless otherwise stated herein. The scope of the technology is defined by the following claims and any equivalents therein.

Claims

CLAIMS1. A method of enzyme engineering, the method comprising: generating a plurality of first enzymes from at least one of a plurality of first DNA samples; performing a catalysis reaction of at least one of the plurality of first enzymes to generate a first product for each of the at least one of the plurality of first enzymes; performing high-throughput mass spectrometry on at least one of the first products to evaluate a first reaction yield thereof; and selecting a first enzyme from the at least one of the plurality of first enzymes based on the performed high-throughput mass spectrometry.

2. The method of claim 1, wherein evaluating the first reaction yield comprises evaluating a yield of the first product.

3. The method of claim 1 or claim 2, wherein selecting the first enzyme comprises: generating a first high-throughput mass spectrum for the at least one of the first products; determining a peak height and a peak area from the first high-throughput mass spectrum for the at least one of the first products; and selecting the first enzyme that corresponds to one of a first desired peak height and a first desired peak area.

4. The method of any one of claims 1-3, further comprising: performing a random mutation on the selected first enzyme to generate a plurality of second DNA samples; generating a plurality of second enzymes from at least one of the plurality of second DNA samples; performing a catalysis reaction of at least one of the plurality of second enzymes to generate a second product for the at least one of the plurality of second enzymes; performing high-throughput mass spectrometry on at least one of the second products to evaluate a second reaction yield thereof; and selecting a second enzyme from the at least one of the plurality of second enzymes based on the performed high-throughput mass spectrometry.

5. The method of claim 4, wherein evaluating the second reaction yield comprises evaluating a yield of the second product.

6. The method of claim 4 or claim 5, wherein selecting the second enzyme comprises: generating a second high-throughput mass spectrum for the at least one of the second products; determining a peak height and a peak area from the second high-throughput mass spectrum for the at least one of the second products; and selecting the second enzyme that corresponds to one of a second desired peak height and a second desired peak area.

7. The method of any one of claims 4-6, wherein at least one of the first product and the second product comprise a protein.

8. The method of any one of claims 4-7, wherein a yield of the second product is greater than a yield of the first product.

9. The method of any one of claims 1-8, wherein generating the plurality of first enzymes from at least one of a plurality of first DNA samples comprises: preparing a plurality of first DNA samples; separately combining at least one of the plurality of first DNA samples with a first host cell; incubating the combined at least one of the plurality of first DNA samples and the first host cell to generate the plurality of first enzymes in the host cell; and adding a first cell lysis reagent to the first host cell to release the plurality of first enzymes from the host cell.

10. The method of claim 9, wherein preparing the plurality of first DNA samples comprises: synthesizing the plurality of first DNA samples via random mutation of an original DNA sample; purifying the plurality of first DNA samples; and assigning a unique ID to each of the plurality of first DNA samples.

11. An integrated system for enzyme selection, the system comprising: a DNA synthesis apparatus; a catalysis apparatus; a high-throughput mass analysis device coupled to a sample ionization device; a processor operatively coupled to the high-throughput mass analysis device; and a memory coupled to the processor, the memory storing instructions that, when executed by the processor, perform a set of operations comprising: generating, via the DNA synthesis apparatus, a plurality of first enzymes from at least one of a plurality of first DNA samples; performing, via the catalysis apparatus, a catalysis reaction of at least one of the plurality of first enzymes to generate a first product for each of the at least one of the plurality of first enzymes; performing, via the high-throughput mass analysis device, high- throughput mass spectrometry on at least one of the first products to evaluate a first reaction yield thereof; and selecting, via the processor, a first enzyme from the at least one of the plurality of first enzymes based on the performed high-throughput mass spectrometry.

12. The system of claim 11, further comprising a plurality of sample containers, wherein the set of operations comprises at least one of: generating the plurality of first enzymes in at least one of the plurality of sample containers; and performing the catalysis reaction in the at least one of the plurality of sample containers.

13. The system of claim 11 or claim 12, wherein the set of operations comprises evaluating the first reaction yield by evaluating a yield of the first product.

14. The system of any one of claims 11-13, wherein the set of operations comprises selecting the first enzyme by:generating a first high-throughput mass spectrum for the at least one of the first products; determining a peak height and a peak area from the first high-throughput mass spectrum for the at least one of the first products; and selecting the first enzyme that corresponds to one of a first desired peak height and a first desired peak area.

15. The system of any one of claims 11-14, wherein the set of operations further comprises: performing a random mutation on the selected first enzyme to generate a plurality of second DNA samples; generating a plurality of second enzymes from at least one of the plurality of second DNA samples; performing a catalysis reaction of at least one of the plurality of second enzymes to generate a second product for the at least one of the plurality of second enzymes; performing high-throughput mass spectrometry on at least one of the second products to evaluate a second reaction yield thereof; and selecting a second enzyme from the at least one of the plurality of second enzymes based on the performed high-throughput mass spectrometry.

16. The system of claim 15, wherein the set of operations comprises evaluating the second reaction yield by evaluating a yield of the second product.

17. The system of claim 16, wherein the set of operations comprises selecting the second enzyme by: generating a second high-throughput mass spectrum for the at least one of the second products; determining a peak height and a peak area from the second high-throughput mass spectrum for the at least one of the second products; and selecting the second enzyme that corresponds to one of a second desired peak height and a second desired peak area.

18. The system of any one of claims 15-17, wherein a yield of the second product is greater than a yield of the first product.

19. The system of any one of claims 11-18, wherein the plurality of sample containers are included in one of a well plate and a microarray.

20. The system of any one of claims 15-19, wherein at least one of the first product and the second product comprise a protein.

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