Systems and computer-implemented methods for generating a deconvoluted mass spectrum

Abstract

Disclosed herein are systems and computer-implemented methods for generating a deconvoluted mass spectrum including receiving, via one or more computing devices, a data set comprising at least one mass spectrum, where the data set was generated via analysis of a sample by a mass spectrometer; selecting a target ion peak and one or more target ion adduct peaks from the at least one mass spectrum; and generating at least one deconvoluted spectrum by associating the target ion peak and the one or more target ion adduct peaks.

Description

Sciex Ref. No. 2025-23492-P-WO Lathrop Ref. No. 776086: DASC-003PCSYSTEMS AND COMPUTER-IMPLEMENTED METHODS FOR GENERATING A DECONVOLUTED MASS SPECTRUMRELATED APPLICATIONS

[0001] The present patent application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63 / 788,273, filed April 14, 2025, the content of which is hereby incorporated by reference in its entirety into this disclosure.BACKGROUND

[0002] The instant disclosure relates generally to mass spectrometry systems and methods for deconvoluting mass spectra. More specifically, the disclosure relates to using computer-implemented methods on a mass spectrometer system or stand-alone processing system to generate deconvoluted mass spectra.

[0003] Mass spectrometry (MS) is an analytical technique for measuring mass-to-charge ratios of molecules, with both qualitative and quantitative applications. MS can be useful for identifying unknown compounds, determining the structure of a particular compound by observing its fragmentation patterns and quantifying the amount of a particular compound in a sample. Mass spectrometers separate and detect chemical entities as ions, such that a conversion of the analytes to charged ions must occur prior to entering the mass spectrometer.

[0004] High-throughput experimentation (HTE) has emerged as an important tool in drug discovery, providing a platform for preparing large compound libraries and enabling swift reaction screening over wide-ranging conditions. Recent advances in automated high-density, material- sparing HTE have necessitated the development of rapid analytics with sufficient sensitivity and resolution to identify products and / or assess reaction performance in a timely and data-rich manner. The combination of an ultra-throughput (UT) reader platform with Acoustic Droplet Ejection-Open Port Interface-Mass Spectrometry (ADE-OPI-MS) or direct liquid transfer into the OPI-MS provides the requisite speed and sensitivity.BRIEF SUMMARY

[0005] Further and alternative aspects and features of the disclosed principles will be appreciated from the following detailed description. As will be appreciated, the compositions and methods disclosed herein are capable of being carried out and used in other and different embodiments and capable of being modified in various respects. Accordingly, it is to beSciex Ref. No. 2025-23492-P-WO Lathrop Ref. No. 776086: DASC-003PCunderstood that both the foregoing general description and the following detailed description are exemplary and explanatory only and do not restrict the scope of the appended claims.

[0006] In one aspect, the disclosure features a computer-implemented method for generating a deconvoluted mass spectrum including receiving, via one or more computing devices, a data set comprising at least one mass spectrum, where the data set was generated via analysis of a sample by a mass spectrometer; selecting a target ion peak and one or more target ion adduct peaks from the at least one mass spectrum; and generating at least one deconvoluted spectrum by associating the target ion peak and the one or more target ion adduct peaks. In some embodiments, correlating the target ion peak and the one or more target ion adduct peaks includes adding, via the one or more computing devices, a signal from the target ion peak to signals from the one or more target ion adduct peaks.

[0007] In some embodiments, the sample is substantially free of salt or buffer reduction step prior to introducing the sample. In some embodiments, the salt or buffer reduction (and / or removal) step that is eliminated from the methods disclosed herein comprises liquid chromatography, a spin column, or dialysis for the case where it is desired to use such reduction and / or removal of salts and / or buffers from the sample, in which the reduction steps are known and not further described in detail, prior to introducing the sample into the mass spectrometer. In some embodiments, the sample comprises from about 10 nM to about 100 mM salt or buffer. In some embodiments, the salt or buffer comprises Na+, K+, NH4+, or combinations thereof. In some embodiments, the computer- implemented method further comprises obtaining the at least one mass spectrum from the mass spectrometer run in negative mode. In some embodiments, for the case where at least one mass spectrum is obtained from the mass spectrometer run in negative mode, the one or more target ion adducts comprises Na+, K+, NH4+, or combinations thereof. In some embodiments, the target ion peak is a polynucleotide. In some embodiments, the polynucleotide comprises from about 10 to about 120 nucleobases. In some embodiments, the computer-implemented method further comprises obtaining the at least one mass spectrum from the mass spectrometer run in positive mode. In some embodiments, for the case where the mass spectrometer is run in positive mode, the one or more target ion adducts comprises Na+, K+, NH4+, or combinations thereof.

[0008] In some embodiments, the selecting further comprises selecting one or more target ion isotopic peaks and wherein adding further comprises adding together signals from the one or more target ion isotopic peaks to the signals from the target ion peak and the one or more target ion adduct peaks. In some embodiments, the at least one deconvoluted spectrumSciex Ref. No. 2025-23492-P-WO Lathrop Ref. No. 776086: DASC-003PCcomprises at least one deconvoluted target ion peak and wherein a signal-to-noise (S / N) ratio, an area under a curve, a peak height, or a combination thereof, of the deconvoluted target ion peak are increased by a factor of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 100, 250, 1000, or more compared to the target ion peak in the at least one mass spectrum.

[0009] In some embodiments, the signals comprise only signals above a predetermined threshold. In some embodiments, the predetermined threshold is a signal-to-noise (S / N) ratio, an area under the curve, the peak height, or a combination thereof. In some embodiments, the predetermined threshold comprises a baseline correction or baseline interpolation. In some embodiments, the method is initiated by a user via a graphical user interface. In some embodiments, one or more adduct ions are selected for associating via the graphical user interface. In some embodiments, the selecting the target ion peak and one or more target ion adduct peaks are selected from the at least one mass spectrum comprising peaks with different charge states. In some embodiments, the computer- implemented method further comprises deconvoluting the at least one mass spectrum, where the at least one mass spectrum comprises the target ion peak and one or more target ion adduct peaks having a neutral charge state. In some embodiments, the computer-implemented method further comprises repeating the steps of receiving, selecting, and generating, wherein repeating is 1, 2, 5, 10, 16, 32, 64, 96, 128, 256, 384, 1,536, 5,000, 25,000, 100,000 times, or more.

[0010] In one aspect, the disclosure provides a mass spectrometry system for analyzing a sample using adduct peak deconvolution including a sample introduction device, a mass spectrometer comprising a mass analyzer, and a computer system, wherein the computer system comprises machine-executable or machine-readable code that generates a deconvoluted mass spectrum by: instructing the sample introduction device to deliver a sample to the mass spectrometer; instructing the mass analyzer to detect a signal for each ion in the sample; receiving a data set comprising the signal for the each ion, thereby forming a mass spectrum; selecting a target ion peak and one or more target ion adduct peaks from the mass spectrum; and generating at least one deconvoluted spectrum by associating the target ion peak and the one or more target ion adduct peaks. In some embodiments, the mass analyzer comprises any one of a sector mass analyzer, a single quadrupole mass analyzer, a triple quadrupole mass analyzer, a QToF mass analyzer, a ToF mass analyzer, a mass analyzer having an ion trap, and a Fourier-transform ion cyclotron resonance-based mass analyzer. In some embodiments, the sample introduction device comprises an autosampler, an open port interface, or an acoustic droplet ejection open port interface.Sciex Ref. No. 2025-23492-P-WO Lathrop Ref. No. 776086: DASC-003PC

[0011] In one aspect, the disclose provides a memory unit comprising computerexecutable code configured to execute the computer-implemented method of any preceding embodiment.

[0012] In one aspect, the disclose provides a mass spectrum formed by the mass spectrometry system of any preceding embodiment.

[0013] In one aspect, the disclosure provides a method for confirming the manufacture of a unit of chemical material using information obtained from a mass spectrum formed by the mass spectrometry system of any preceding embodiment.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings:

[0015] FIG. 1A depicts a total ion chromatogram showing 36 peaks correlating with the introduction of 36 samples into the mass spectrometer in less than 1.3 minutes.

[0016] FIG. IB depicts a deconvoluted mass spectrum showing the isotopic and adduct mass peaks of a single sample with a 30-mer polynucleotide from FIG. 1A.

[0017] FIG. 2 depicts an enlarged, annotated, and deconvoluted mass spectrum showing the isotopic and adduct mass peaks of the same spectrum (30-mer polynucleotide) from FIG. IB.

[0018] FIG. 3 depicts a deconvoluted mass spectrum showing the monoisotopic peak distribution of the spectrum from FIG.2.

[0019] FIG. 4 depicts a further deconvoluted mass spectrum showing the resulting monoisotopic peak after the adduct peaks are combined from FIG.3.

[0020] FIG. 5A depicts a mass spectrum showing the peak distribution of an about 120-mer polynucleotide.

[0021] FIG. 5B depicts a deconvoluted mass spectrum showing the average mass peak distribution and adduct peaks of the polynucleotide from FIG.5A.

[0022] FIG. 5C depicts a further deconvoluted mass spectrum showing the average monoisotopic peak after the adduct peaks are combined from FIB.5B.

[0023] FIG. 6A depicts an enlarged total ion chromatogram showing a single peak correlating to the introduction of a sample containing a peptide.Sciex Ref. No. 2025-23492-P-WO Lathrop Ref. No. 776086: DASC-003PC

[0024] FIG. 6B depicts a mass spectrum showing the peak distribution of the peptide depicted in FIG. 6A. The higher mass peak cluster, centered about 1255 m / z, represents the doubly charged peptide. The lower mass peak cluster, centered about 880 m / z, represents the triply charged peptide.

[0025] FIG. 6C depicts a deconvoluted mass spectrum showing the monoisotopic peaks depicted in FIG. 6B.

[0026] FIG. 6D depicts a further deconvoluted mass spectrum showing the monoisotopic peak after the adduct peaks are combined depicted in FIG. 6C.

[0027] FIG. 7A depicts a mass spectrum showing the peak distribution of a sample from a combinatorial labeling experiment. The sample includes a combinatorial small molecule moiety and a DNA-encoded tag.

[0028] FIG. 7B depicts a mass spectrum showing the monoisotopic peaks (isotopic and adduct peaks added together) of the expected reaction product (combinatorial small molecule moiety) and DNA encoded tag at the higher mass (5096 Da); and the unreacted DNA encoded tag at the smaller mass (4934.9 Da).

[0029] FIGS. 8A-8C depicts mass spectra showing the monoisotopic peaks (isotopic and adduct peaks added together) of three additional samples from the combinatorial labeling experiment. Each spectrum shows the expected reaction product (combinatorial small molecule moiety) and DNA-encoded tag at a higher (greater) mass, as well as the unreacted DNA-encoded tag at a lower (smaller) mass. The relative ratios of the peaks indicate the reaction efficiency. FIG. 8A shows that the reaction efficiency was low, resulting in about only 10% conversion to the desired product. FIG. 8B shows a moderate reaction efficiency, yielding about 40% conversion. FIG. 8C shows a high reaction efficiency, yielding about 90% conversion.DETAILED DESCRIPTIONI. Introduction

[0030] The demand for high-throughput quality assurance and quality control (QA / QC) on proteins, small molecules, and nucleic acids (e.g., synthetic DNA) has surged due to the expanding synthetic biology and biocatalysis markets. Currently, the method employed for analyzing synthetic DNA is liquid chromatography-mass spectrometry (LC-MS), which accurately determines the molecular mass of DNA molecules for comparison against theoretical values. However, the analysis speed of LC-MS systems currently remains constrained to a few minutes per sample, resulting, unfortunately, in a throughput of fewerSciex Ref. No. 2025-23492-P-WO Lathrop Ref. No. 776086: DASC-003PCthan approximately 500 samples per day. To address this undesirable limitation and the need for higher throughput, a new method and platform described herein have been developed, which preferably includes a relatively high-throughput Mass Spectrometer (MS) platform, which preferably does not rely on liquid chromatography (LC). Advantageously, this alternative method and platform significantly enhances analysis speed, thereby providing a more efficient workflow for the quality assurance and quality control (QA / QC) of samples (e.g., nucleic acid standards).

[0031] Due to the high sample volume (amount) and the high concentration of the target analyte, a high-throughput MS platform (e.g., Echo® MS platform manufactured by AB SCIEX LLC based in the U.S.A.) emerged as a promising candidate for conducting this assay. However, a notable challenge arose from the absence of a chromatographic step for eliminating salts. This absence led to intricate and convoluted spectra displaying multiple adducts of DNA with salts, as demonstrated in FIG. IB. This particular challenge introduced technical difficulty in distinguishing the signals originating from the target DNA versus those stemming from DNA salt adducts. Furthermore, when considering the sodium-free form (the first group of peaks in the figure), the signal intensity is notably reduced, which may lead to difficulties in accurately determining mass.

[0032] It will be appreciated that the phrase “substantially free of salt or buffer reduction step” means that the sample preparation process, in accordance with some preferred embodiments, does not include removal steps to remove salts or buffers before introducing the sample into the mass spectrometer for generation of a mass spectrum to be analyzed. In other words, the sample is used in its near-original state without undergoing extensive purification processes for the elimination of salts or buffers from the sample. This phrase “substantially free of salt or buffer reduction step” describes a step where the sample is analyzed by the mass spectrometer without a prior step to reduce or remove salts or buffers from the sample. By avoiding the purification step prior to generating the mass spectrum, this arrangement can be a significant improvement because traditional or known methods often involve purification steps such as liquid chromatography, spin columns, or dialysis to purify the sample prior to introducing the sample into a mass spectrum for the generation of a mass spectrum. By avoiding the purification step(s), the “substantially free of salt or buffer reduction step” aims to streamline the process and reduce preparation time, which can be particularly beneficial for high-throughput applications. By avoiding these purification steps prior to introduction to the mass spectrometer, at least one of the following advantages may be achieved: (A) improved efficiency by eliminating the need for salt or buffer reductionSciex Ref. No. 2025-23492-P-WO Lathrop Ref. No. 776086: DASC-003PCsteps can significantly speed up the sample preparation process, (B) improved high-throughput mass spectrometry, where large numbers of samples can be processed more quickly, and / or (C) while this approach can lead to more complex spectra due to the presence of salts in the sample, this step may further include steps or techniques to deconvolute these more complex spectra to more accurately identify the target ions in the sample.

[0033] Conventionally, spectra have merged signals originating from diverse charge states of a single signal, requiring a consolidated representation through mass deconvolution. In addition, the consolidation of signals from distinct isotopes into a singular peak, referred to as deisotoping or isotope clustering, has also been established. Disclosed herein are methods and platforms that combine this concept with the signals emerging from different adducts of the target molecules.

[0034] While complex spectra are commonly encountered from the deconvoluted mass, ions associated with the target analyte can still be identified utilizing known information, including isotopic and adduct masses (e.g., sodium, potassium, ammonia). Therefore, the signals originating from these target analytes are deconvoluted into a single peak. This consolidation enables the determination of the mass devoid of adducts, resulting in enhanced accuracy and sensitivity for mass determination. Additionally, the consolidated spectrum is much simpler, allowing actual impurity peaks to be more easily spotted by visual examination.

[0035] In some cases, the salt- free form and the adducts may share a common elevated baseline. In this case, it is not desirable to merge the raw total intensity of the adduct peak into the salt- free form but just the portion above the baseline. The baseline can be estimated in various ways, for example, by using the minimum intensity at either the start or end of the detected peak, a linear interpolation between these points, or other more advanced baseline detection algorithms (e.g., filter-based and penalized least squares-based algorithms).

[0036] In general, the mass spectrum, obtained through mass spectrometry, is configured to provide information used for identifying and designing a chemical material or substance. While it doesn’t directly make a chemical, it provides vital information that helps chemists create or analyze compounds. Here are some, but not limited to, examples of how the information contained in the mass spectrum may be used: (A) Molecular Composition: chemists can use a mass spectrum to detect the molecular weight of components of a test sample and confirm composition before manufacturing or identifying unknown substances; (B) Structural Information: chemists can analyze fragmentation patterns displayed in a mass spectrum that they can use to infer atomic arrangements within a molecule, which is essentialSciex Ref. No. 2025-23492-P-WO Lathrop Ref. No. 776086: DASC-003PCfor designing and manufacturing a chemical structure; (C) Purity Assessment: chemical impurities in a test sample can be detected in a mass spectrum, confirming purity before manufacturing; (D) Reaction Monitoring: during synthesis, the mass spectrum can track intermediates and confirm reactions have proceeded as planned; (E) Quantification: by comparing the intensity of signals in a mass spectrum, chemists can quantify the amount of a compound in a test sample, ensuring that correct proportions have been used during chemical synthesis.II. Definitions

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein belong. Any reference to standard methods refers to the most recent available version of the method at the time of filing of this disclosure unless otherwise indicated.

[0038] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0039] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading unless so specified.

[0040] The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0041] The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0042] The singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. These articles refer to “one” or to “more than one” (i.e., to at least one). The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed itemsSciex Ref. No. 2025-23492-P-WO Lathrop Ref. No. 776086: DASC-003PC(A or B) or any combination of two or more of the listed items (A and B) unless otherwise indicated herein or clearly contradicted by context. As used herein, the term "or" is generally employed in its usual sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means any one or more of the items in the list joined by "and / or". As an example, "x and / or y" means any element of the three-element set {(x), (y), (x, y) } . In other words, "x and / or y" means "one or both of x and y". As another example, "x, y, and / or z" means any element of the seven-element set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z) } . In other words, "x, y and / or z" means "one or more of x, y and z".

[0043] Where ranges are given, endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Herein, "up to" a number (for example, up to 50) includes the number (for example, 50). The term "in the range" or "within a range" (and similar statements) includes the endpoints of the stated range.

[0044] Reference throughout this specification to “one aspect (or embodiment),” “an aspect (or embodiment),” “certain aspects (or embodiments),” or “some aspects (or embodiments),” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the aspect is included in at least one aspect of the disclosure. Thus, the appearance of such phrases in various places throughout this specification does not necessarily refer to the same embodiment of the disclosure.Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects.

[0045] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." As used herein in connection with a measured quantity, the term "about" refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term "about" is used in connection with a numerical value throughout the specification and the claims denote an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such an interval of accuracy is + / -10%. Thus, "about" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, 0.1 %,Sciex Ref. No. 2025-23492-P-WO Lathrop Ref. No. 776086: DASC-003PC0.05%, 0.01 %, or 0.001 % greater or less than the stated value. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0046] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0047] The term "exemplary" means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms "e.g.," and "for example" set off lists of one or more non-limiting aspects, examples, instances, or illustrations.

[0048] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.Biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, "substantially" may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest.

[0049] As used herein, the term “fixedly positioned” as referring to an element indicates that the position of that element is not adjustable by a user.

[0050] As used herein, the term “monoisotopic mass” refers to the calculated mass considering the exact mass of the more abundant isotopes, e.g.,JH = 1.007825,12C = 12.000000,14N = 14.003074,16O = 15.994915,31P = 30.973762,32S = 31.972070).III. Methods for Generating Deconvoluted Mass Spectra

[0051] In general, the methods disclosed herein can be used as part of an integrated system (e.g., an integrated mass spectrometry system), that is able to introduce samples into the mass spectrometer, analyze the samples, record the data, and perform post-acquisitionSciex Ref. No. 2025-23492-P-WO Lathrop Ref. No. 776086: DASC-003PCdata processing methods. Alternatively, the methods disclosed herein are used as part of a stand-alone computer system that performs post-acquisition data processing methods. In one aspect, the method is a computer-implemented method that generates a deconvoluted mass spectrum from a dataset produced by a mass spectrometer from the analysis of a sample. The method includes and is not limited to choosing a target molecule and associating (e.g., adding together) the target ion peaks and the target- adduct peaks.

[0052] For many applications, the number of samples generated is large, and one bottleneck in advancing the experiment is the preparation time of each sample for analysis. The presence of salts and other constituents in a sample, typically present after manufacturing a chemical or a biochemical, can result in the presence of additional peaks in a mass spectrum, leading to convoluted mass spectra. The convoluted mass spectra require additional processing time to analyze and may result in several unidentified peaks. For example, dialysis, column separation, and spin filtration are common sample preparation techniques used to remove salts or other constituents that can significantly prolong a test or an experiment. Disclosed herein are methods that can reduce, or preferably eliminate, sample preparation steps and may take a sample directly from a sample generation step to introduction into a mass spectrometer. In some embodiments, the sample is free of any sample preparation steps (e.g., column chromatography, dialysis, spin column filtration). In some embodiments, the sample includes a salt or buffer at a concentration ranging from about 10 nM (nanomolar) to about 100 mM (millimolar). In principle, any salt that forms an adduct with a target ion may be present. In some embodiments, Na+, K+, NH4+, or combinations thereof are present in the sample.

[0053] In some embodiments, the target ion peaks may include single, doubly, triply, or larger-charged state ions. In some embodiments, the target ion peaks are associated with isotopic target ion peaks. In some embodiments, the peak signals are added together. In some embodiments, the peak signals are weighted (e.g., multiplied by a coefficient), and then added together. The weighting coefficient may be based on one or more of the differences between the expected mass and the observed mass, the signal-to-noise ratio, the charge state, the isotopic abundance, and the type and / or number of adducts. In some embodiments, the mass spectrometer is run in negative mode. In mass spectrometry, “negative mode” refers to the ionization process where negatively charged ions are generated and analyzed. This mode is used to detect and measure the mass-to-charge ratio of anions (negatively charged ions) in a sample. In some embodiments, the mass spectrometer is run in positive mode. In mass spectrometry, “positive mode” refers to the ionization process where positively charged ionsSciex Ref. No. 2025-23492-P-WO Lathrop Ref. No. 776086: DASC-003PC(cations) are generated and analyzed, and this mode is used to detect and measure the mass-to-charge ratio of cations in a sample.

[0054] In some embodiments, the sample includes a polynucleotide. In some embodiments, the polynucleotide ranges in length from about 10 to about 150 nucleobases. In some embodiments, the polynucleotide ranges in length from about 20 to about 120 nucleobases. In some embodiments, the sample includes a polypeptide. In some embodiments, the sample includes a small molecule.

[0055] In some embodiments, the spectrum is further deconvoluted by associating (e.g., adding together) the signals from the target ion peak and the isotopic target ion peak, resulting in a monoisotopic peak. In some embodiments, associating the target ion peaks with the adduct peaks increases the signal-to-noise (S / N) ratio by a factor of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 100, 250, 1000, or more compared to the target ion peak in the mass spectrum.

[0056] In some embodiments, a peak-picking or baseline correction algorithm is used. In some embodiments, the signals of the peaks used for associating are above a predetermined threshold value, which can be a value above a S / N ratio, an area under the curve, a peak height (absolute or relative), or a combination thereof. In some embodiments, the threshold value determination includes a baseline correction or baseline interpolation.

[0057] In some embodiments, the target ion peak and one or more target ion adduct peaks are selected for associating via a graphical user interface. In some embodiments, the associating also includes peaks with different charge states. In some embodiments, the associating deconvolutes the spectrum such that the combined target ion peak and adduct peaks have a neutral charge state. A mass spectrum with a neutral charge state refers to a mass spectrum where the ions being analyzed have no net electrical charge. This means that the ions are neither positively charged (cations) nor negatively charged (anions). It will be appreciated that the mass spectrometer separates and analyzes positively or negatively charged ions; moreover, the term “ion” implies a non-neutral charge. The hardware of the mass spectrometer acquires a positive spectrum or a negative spectrum in the usual way, and then a software ‘deconvolution’ step or ‘reconstruction’ step analyzes the mass spectrum and converts the mass / charge data points to mass. The deconvoluted spectrum is then used as input to the embodiments.

[0058] The methods disclosed herein are compatible with high-throughput methods. For example, a sample can be introduced into a mass spectrometer, the data collected, and the spectrum deconvoluted within a time range from about 1 to about 10 seconds, from about 1 to about 5 seconds, or from about 1.5 to about 3 seconds per sample. In some embodiments, theSciex Ref. No. 2025-23492-P-WO Lathrop Ref. No. 776086: DASC-003PCsteps of introducing a sample into a mass spectrometer, collecting the data, and deconvoluting the spectrum are completed in about 1 second, about 1.5 seconds, about 2 seconds, about 2.5 seconds, or about 3 seconds per sample. In some embodiments, the steps of introducing a sample into a mass spectrometer, collecting the data, and deconvoluting the spectrum are repeated 1, 2, 5, 10, 16, 32, 64, 96, 128, 256, 384, 1,536, 5,000, 25,000, 100,000 or more times.

[0059] In one example, the methods disclosed herein can be applied to screening DNA-encoded chemical libraries (DECL), which is a technology that enablesthe synthesis and screening on an unprecedented scale of collections of smallmolecule compounds. DECL technology involves the conjugation of chemical compounds or building blocks to short DNA fragments that serve as identification barcodes and, in some cases, also direct and control chemical synthesis. The technique enables the mass creation and interrogation of libraries via affinity selection, typically on an immobilized protein target. These libraries may include more than a million different chemical entities.

[0060] Medicinal and process research chemists have long experimented with automated methods for rapidly screening reaction conditions. Many of these methods can be adapted to producing libraries of compounds, thereby serving a dual purpose in the synthesis laboratory. Recently, impressive advances in reaction screening technology have been made by the advancement of high-throughput experimentation (HTE) technologies. Importantly, these methods can employ microgram or even nanogram amounts per reaction, thereby sparing precious templates and reducing the cost of the overall reaction optimization process. Many of these methods utilize plate-based or segmented flow-based reactors, offering, for the first time, the ability to survey hundreds of reaction variables in a single experiment. The output of these experiments is a plot of reaction yields across a landscape of reaction conditions. Collecting the analytical data, however, is often a significant bottleneck.

[0061] Coupling a mass spectrometry (MS) system with an HPLC (High-Performance Liquid Chromatography) system or a Liquid Chromatography-Mass Spectrometry (LC-MS) system is a common method for analyzing such reactions. This approach requires a relatively small amount of material (typically about 2 to 15 pL (microliter)), allows simultaneous detection of starting material, product, and side products, and enables data to be collected in a matter of minutes for each reaction under testing and / or study. Approaches to analyzing reactions in situ are being pursued; however, the material requirements are typically much higher than what is used in microscale reactions for parallel medicinal chemistry (PMC). Although analysis times are relatively short for a single well, when aggregated over hundredsSciex Ref. No. 2025-23492-P-WO Lathrop Ref. No. 776086: DASC-003PCof compounds, several hours or even days can be required to analyze a high-density experiment. Therefore, techniques that accelerate analysis significantly increase the feasibility of HTE.

[0062] In one embodiment, the ADE-OPI-MS method also offers significant speed advantages: with an average analysis time of 1-2 s per sample, such that a typical 384-well plate can be analyzed in under 15 min. Finally, the ADE-OPI is compatible with both nominal and high-resolution mass spectrometers, allowing rapid quantification with the former, and extensive analyte identification with the latter. However, data analysis and interpretation remain a bottleneck.

[0063] The methods disclosed herein provide a high-throughput screening method that reduces data analysis and interpretation time enabling more rapid determination of whether the synthesis of each chemical entity was successful. For example, FIGS. 7A - 7B show the deconvolution of the mass spectrum depicted in FIG. 7A by associating the isotopic target peaks and adduct target peaks with the target peaks. FIG. 7B shows the expected monoisotopic peak at about 5096 Da (Daltons) and the unreacted DNA at about 4935 Da. FIGS. 8A to 8C depict the deconvolution results of additional samples. FIG. 8A shows that only about 10% of the expected product was manufactured in that sample. FIG. 8B shows that about 40% of the expected product was manufactured in that sample. FIG. 8C shows that about 90% of the expected product was manufactured in that sample.IV. Systems for Generating Deconvoluted Mass Spectra

[0064] In one aspect, the present disclosure provides a mass spectrometry system for analyzing a sample using adduct peak deconvolution. In some embodiments, the system comprises a sample introduction device, a mass spectrometer with a mass analyzer, and a computer system, where the computer system includes machine-executable (computerexecutable) code and / or machine-readable (computer-executable) code that directs or urges a programmable computer (machine) to generate a deconvoluted mass spectrum. The machineexecutable code (instructions) and / or machine-readable code (instructions) are configured to: instruct, for instance, the sample introduction device to deliver a sample to the mass spectrometer, instruct the mass analyzer to detect a signal for each ion in the sample, receive a data set comprising the signal for each ion, thereby forming a mass spectrum, select a target ion peak and one or more target ion adduct peaks from the mass spectrum, and generate atSciex Ref. No. 2025-23492-P-WO Lathrop Ref. No. 776086: DASC-003PCleast one deconvoluted spectrum by associating the target ion peak and the one or more target ion adduct peaks.

[0065] In some embodiments, the mass analyzer can include a sector mass analyzer, a single quadrupole mass analyzer, a triple quadrupole mass analyzer, a Quadrupole Time-of-Flight (QToF) mass analyzer, a Time-of-Flight (ToF) mass analyzer, a mass analyzer having an ion trap, and / or a Fourier-transform ion cyclotron resonance-based mass analyzer, and any equivalent thereof. In some embodiments, a test-sample introduction device can include an autosampler, an open port interface, and / or an acoustic droplet ejection open port interface, and any equivalent thereof.

[0066] The following examples further illustrate the invention but should not be construed as limiting its scope in any way.EXAMPLESExample 1

[0067] A series of samples with a 30-mer polynucleotide, shown in FIG. 1A, was introduced into an Acoustic Ejection Mass Spectrometry (AEMS) system using an open port interface. The samples were introduced at approximately two (2) second intervals.

[0068] FIG. IB shows the deconvoluted mass spectrum of one of the sample peaks. FIG.2 shows an annotated reproduction of FIG. IB, which identifies the target peak (M), the associated isotopic target peaks (Ml - M10), and the associated target adduct (M+Na, M+2Na, etc...) / isotopic target adduct peaks (Ml+Na to MIO+Na, Ml+2Na to M10+2Na, etc...).

[0069] The target peak and the isotopic target peaks were associated (deconvoluted) from FIG. 2 and FIG. 3, which shows the target peak along with several target adduct (Na) peaks. The target peak and the target adduct peaks that were associated (deconvoluted) are shown in FIG. 4, which shows the deconvoluted monoisotopic peak.Example 2

[0070] A sample with an about 120-mer polynucleotide was introduced into an Acoustic Ejection Mass Spectrometry (AEMS) system using an open port interface. FIG 5A shows the convoluted mass spectrum of the sample. The target peak and the isotopic target peaks were associated (deconvoluted) from FIG. 5A and shown in FIG. 5B, which shows the target peak along with several target adduct (Na) peaks. The target peak and the target adduct peaks wereSciex Ref. No. 2025-23492-P-WO Lathrop Ref. No. 776086: DASC-003PCassociated (deconvoluted) from FIG. 5B and shown in FIG. 5C, which shows the deconvoluted monoisotopic peak.Example 3

[0071] A sample containing a peptide was introduced into an Acoustic Ejection Mass Spectrometry (AEMS) system using an open-port interface. FIG. 6A shows a total ion chromatogram of the sample after introduction into the mass spectrometer. FIG. 6B shows the mass spectrum at a single time point from FIG. 6A. The peaks in FIG. 6B centered about 1255 Da represent the doubly charged peak envelope and the peaks centered about 890 Da represent the triply charged peak envelope. The target peak and the isotopic target peaks were associated (deconvoluted) from FIG. 6B and shown in FIG. 6C, which shows the target peak along with several target adduct (Na) peaks. The target peak and the target adduct peaks were associated (deconvoluted) from FIG. 6C and shown in FIG. 6D, which shows the deconvoluted monoisotopic peak.Example 4

[0072] A sample with a polynucleotide and a linked chemical moiety was introduced into an Acoustic Ejection Mass Spectrometry (AEMS) system using an open port interface. FIGS.7A and 7B show the deconvolution of the mass spectrum in FIG. 7A by associating the isotopic target peaks and adduct target peaks with the target peaks. FIG. 7B shows the expected monoisotopic peak at about 5096 Da and the impurity (e.g., unsuccessful reaction product) at about 4935 Da. FIGS. 8A to 8C show the deconvolution results of additional samples. FIG. 8A shows that only about 10% of the expected product was manufactured in that sample. FIG. 8B shows that about 40% of the expected product was manufactured in that sample. FIG. 8C shows that about 90% of the expected product was manufactured in that sample.

[0073] All features disclosed in the specification, including the claims, abstracts, and drawings, and all the steps in any method or process disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. Each feature disclosed in the specification, including the claims, abstract, and drawings, can be replaced by alternative features serving the same, equivalent or similar purpose unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.Sciex Ref. No. 2025-23492-P-WO Lathrop Ref. No. 776086: DASC-003PC

[0074] It will be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Sciex Ref. No. 2025-23492-P-WO Lathrop Ref. No. 776086: DASC-003PCClaims:

1. A computer- implemented method for generating a deconvoluted mass spectrum, the method comprising:receiving, via one or more computing devices, a data set comprising at least one mass spectrum, wherein the data set was generated via analysis of a sample by a mass spectrometer;selecting a target ion peak and one or more target ion adduct peaks from the at least one mass spectrum;correlating the target ion peak and the one or more target ion adduct peaks, which comprises combining, via the one or more computing devices, a signal from the target ion peak to signals from the one or more target ion adduct peaks; and generating at least one deconvoluted spectrum by associating the target ion peak and the one or more target ion adduct peaks.

2. The computer- implemented method of claim 1, wherein the sample is substantially free of salt or buffer reduction step prior to introducing the sample.

3. The computer- implemented method of claim 2, wherein the salt or buffer reduction step comprises liquid chromatography, a spin column, or dialysis.

4. The computer- implemented method of any one of claims 1 to 3, wherein the sample comprises from about 10 nM to about 100 mM salt or buffer.

5. The computer-implemented method of any one of claims 1 to 4, wherein the salt or buffer comprises Na+, K+, NH4+, or combinations thereof.

6. The computer-implemented method of any one of claims 1 to 5 further comprising obtaining at least one mass spectrum from the mass spectrometer run in negative mode.

7. The computer-implemented method of claim 6, wherein the one or more target ion adducts comprises Na+, K+, NH4+, or combinations thereof.Sciex Ref. No. 2025-23492-P-WO Lathrop Ref. No. 776086: DASC-003PC8. The computer- implemented method of any one of claims 6 to 7, wherein the target ion peak is a polynucleotide.

9. The computer-implemented method of claim 8, wherein the polynucleotide comprises from about 10 to about 120 nucleobases.

10. The computer- implemented method of any one of claims 1 to 5 further comprising obtaining at least one mass spectrum from the mass spectrometer run in positive mode.

11. The computer- implemented method of claim 10, wherein the one or more target ion adducts comprises Na+, K+, NH4+, or combinations thereof.

12. The computer- implemented method of any one of claims 1 to 11, wherein the selecting further comprises selecting one or more target ion isotopic peaks and wherein combining further comprises adding together signals from the one or more target ion isotopic peaks to the signals from the target ion peak and the one or more target ion adduct peaks.

13. The computer-implemented method of any one of claims 1 to 12, wherein the at least one deconvoluted spectrum comprises at least one deconvoluted target ion peak and wherein a signal-to-noise (S / N) ratio, an area under a curve, a peak height, or a combination thereof, of the deconvoluted target ion peak are increased by a factor of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 100, 250, 1000, or more compared to the target ion peak in the at least one mass spectrum.

14. The computer-implemented method of any one of claims 1 to 12, wherein the signals comprise only signals above a predetermined threshold.

15. The computer-implemented method of claim 14, wherein the predetermined threshold is a signal-to-noise (S / N) ratio, an area under the curve, the peak height, or a combination thereof.Sciex Ref. No. 2025-23492-P-WO Lathrop Ref. No. 776086: DASC-003PC16. The computer-implemented method of claim 15, wherein the predetermined threshold comprises a baseline correction or baseline interpolation.

17. The computer-implemented method of any one of claims 1 to 16, wherein the computer-implemented method is initiated by a user via a graphical user interface.

18. The computer- implemented method of claim 17, wherein one or more adduct ions are selected for associating via the graphical user interface.

19. The computer- implemented method of any one of claims 1 to 18, wherein the selecting the target ion peak and one or more target ion adduct peaks are selected from the at least one mass spectrum comprising peaks with different charge states.

20. The computer- implemented method of any one of claims 1 to 18, further comprising deconvoluting the at least one mass spectrum, wherein the at least one mass spectrum comprises the target ion peak and one or more target ion adduct peaks having a neutral charge state.

21. The computer- implemented method of any one of claims 1 to 20, further comprising repeating the steps of receiving, selecting, and generating, wherein the repeating is 1, 2, 5, 10, 16, 32, 64, 96, 128, 256, 384, 1,536, 5,000, 25,000, 100,000 times, or more.

22. A mass spectrometry system for analyzing a sample using adduct peak deconvolution, the system comprising:a sample introduction device,a mass spectrometer comprising a mass analyzer, anda computer system, wherein the computer system comprises machine-executable or machine-readable code that generates a deconvoluted mass spectrum by:instructing the sample introduction device to deliver a sample to the mass spectrometer;instructing the mass analyzer to detect a signal for each ion in the sample; receiving a data set comprising the signal for each ion, thereby forming a mass spectrum;Sciex Ref. No. 2025-23492-P-WO Lathrop Ref. No. 776086: DASC-003PCselecting a target ion peak and one or more target ion adduct peaks from the mass spectrum; andgenerating at least one deconvoluted spectrum by associating the target ion peak and the one or more target ion adduct peaks.

23. The mass spectrometry system of claim 22, wherein the mass analyzer comprises any one of a sector mass analyzer, a single quadrupole mass analyzer, a triple quadrupole mass analyzer, a QToF mass analyzer, a ToF mass analyzer, a mass analyzer having an ion trap, and a Fourier-transform ion cyclotron resonance-based mass analyzer.

24. The mass spectrometry system of claim 22 or claim 23, wherein the sample introduction device comprises an autosampler, an open port interface, or an acoustic droplet ejection open port interface.

25. The mass spectrometry system of any one of claims 22 to 24, wherein the sample is substantially free of salt or buffer reduction step prior to introducing the sample.

26. The mass spectrometry system of claim 25, wherein the salt or buffer reduction step comprises liquid chromatography, a spin column, or dialysis.

27. The mass spectrometry system of any one of claims 22 to 26, wherein the sample comprises from about 10 nM to about 100 mM salt or buffer.

28. The mass spectrometry system of any one of claims 22 to 27, wherein the salt or buffer comprises Na+, K+, NH4+, or combinations thereof.

29. The mass spectrometry system of any one of claims 22 to 28 further comprising obtaining the at least one mass spectrum from the mass spectrometer run in negative mode.

30. The mass spectrometry system of any one of claims 22 to 28 further comprising obtaining the at least one mass spectrum from the mass spectrometer run in positive mode.Sciex Ref. No. 2025-23492-P-WO Lathrop Ref. No. 776086: DASC-003PC31. The mass spectrometry system of claim 29 or claim 30, wherein the one or more target ion adducts comprises Na+, K+, NELd, or combinations thereof.

32. A memory unit comprising computer-executable code configured to execute the computer- implemented method of claim 1.

33. A mass spectrum formed by the mass spectrometry system of claim 32.

34. A method for confirming the manufacture of a unit of chemical material using information obtained from a mass spectrum formed by the mass spectrometry system of any one of claims 22 to 31.

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