WO2025250525A1

WO2025250525A1 - Systems and methods for performing multiplexed mass spectrometry for quantitation

Info

Publication number: WO2025250525A1
Application number: PCT/US2025/031011
Authority: WO
Inventors: Graeme C. Mcalister; Lilian HEIL
Original assignee: Thermo Finnigan LLC
Current assignee: Thermo Finnigan LLC
Priority date: 2024-05-31
Filing date: 2025-05-27
Publication date: 2025-12-04
Anticipated expiration: 2026-11-30

Abstract

A system for mass spectrometry generates, based on an observed elution peak for a precursor m/z: a first simulated elution peak and a second simulated elution peak that contribute to the observed elution peak and that represent elution of analytes labeled with a deuterated isobaric mass tag and elution of analytes labeled with a non-deuterated isobaric mass tag, respectively. The system determines, based on the first simulated elution peak, the second simulated elution peak, and a timing of acquiring an MSn spectrum for the precursor m/z, a first correction factor and a second correction factor. The system adjusts, based on the first correction factor, a first observed signal, within the MSn spectrum, for a first reporter ion from the deuterated isobaric mass tag and adjusts, based on the second correction factor, a second observed signal, within the MSn spectrum, for a second reporter ion from the non-deuterated isobaric mass tag.

Description

SYSTEMS AND METHODS FOR PERFORMING MULTIPLEXED MASS SPECTROMETRY FOR QUANTITATION

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/654,818, filed May 31, 2024, the contents of which is hereby incorporated by reference in its entirety.

BACKGROUND INFORMATION

[0002] A mass spectrometer is a sensitive instrument that may be used to detect, identify, and/or quantify molecules based on the mass-to-charge ratio (m/z) of ions produced from the molecules. A mass spectrometer generally includes an ion source for producing ions from molecules included in a sample, a mass analyzer for separating the ions based on their m/z, and an ion detector for detecting the separated ions. The mass spectrometer may include or be connected to a computer-based software platform that uses data from the ion detector to construct a mass spectrum that shows a relative abundance of each of the detected ions as a function of m/z. The mass spectrum may be used to detect and quantify molecules in simple and complex mixtures. A separation system, such as a liquid chromatograph (LC), gas chromatograph (GO), or capillary electrophoresis (CE) system, may be coupled to the mass spectrometer in a combined system (e.g., LC-MS, GC-MS, or CE-MS system) to separate components (e.g., analytes) in the sample before the components are introduced to the mass spectrometer.

[0003] One application of mass spectrometry is the identification, quantification, and structural elucidation of peptides, proteins, and related molecules in complex biological samples. In some such experiments, often referred to as tandem mass spectrometry (MS/MS or MS2) or multi-stage mass spectrometry (MSn where n is 2 or more), certain ions are fragmented in a controlled manner to yield product ions. A mass analysis (referred to as an MS/MS, MS2, or MSn analysis) is then performed on the product ions to generate mass spectra of the product ions. The mass spectra of the product ions provide information that may be used to identify, determine quantity, and/or derive structural details regarding analytes of interest.

[0004] Various techniques may be used to acquire mass spectra using tandem mass spectrometry and MSn mass spectrometry. One commonly used technique is data-dependent acquisition (DDA), which uses data acquired in one mass analysis to select, based on predetermined criteria, one or more ion species or a narrow m/z range for mass isolation and fragmentation. For example, the mass spectrometer may perform a full MS survey scan of precursor ions over a wide precursor m/z range and then select one or more precursor ion species from the resulting spectra for subsequent MS/MS or MSn analysis. The criteria for selection of precursor ion species may include intensity, charge state, m/z, inclusion/exclusion lists, or isotopic patterns. The main disadvantage of the DDA technique is the inherently random nature of the results. When technical replicates of the same sample or comparative analysis on other samples is performed, some analytes may be measured in one experiment but not in others. This frustrates attempts to perform reproducible analyses and is known as the “missing value problem”.

[0005] In contrast to DDA, data-independent acquisition (DIA) is a technique in which all precursor ion species within a wide precursor m/z range (e.g., 500 m/z - 900 m/z) are isolated and fragmented via a sequentially advancing isolation window of a fixed m/z width (e.g., 10 m/z, 4 m/z, or 2 m/z) to generate product ions. An MS2 or MSn analysis is then performed on the product ions in a methodical and unbiased manner. The acquisition of the set of spectra spanning the full precursor m/z range constitutes one cycle, which is repeated to generate MS2 or MSn mass spectra of the product ions. The cycle time, or time required to acquire the spectra in a cycle, is typically set such that at least a certain number of cycles will be executed per chromatography peak width, such that area of the peaks may be properly integrated. In the DIA technique, isolation and fragmentation of one or more precursor ion species is not dependent on data acquired in a survey mass analysis, as in DDA, and is much more suitable for comparing results across different samples than DDA.

[0006] In contrast to DDA and DIA, targeted mass spectrometry performs analysis of a fixed list of analytes. Targeted mass spectrometry comes in many forms, such as selected reaction monitoring (SRM), multiple reaction monitoring (MRM), and parallel reaction monitoring (PRM). Generally, the introduction of the sample to the mass spectrometer is performed using a separation system, and to increase experiment capacity the operator schedules analysis of each compound only during a narrow period of time around the expected elution times of each analyte of interest. Targeted mass spectrometry is advantageous because of the high data quality (quantitative precision and sensitivity) that can be produced when the instrument is dedicated to the analysis of a smaller group of analytes of interest, each with a narrow or even customized precursor isolation window. Narrow scheduled retention time windows may produce results with low limits of detection and high dynamic range, as well as allow for increasing a number of targets in an assay.

[0007] The selective detection and quantification of a specific analyte of interest in a complex mixture is often very difficult, even with targeted acquisition. For example, in proteomics research, a peptide of interest may be included in a complex biological matrix composed of a mixture of tens of thousands of peptides with abundances spanning many orders of magnitude. Multiplexing with the use of isobaric mass tags, such as Tandem Mass Tag® (TMT®) reagents (produced by Electrophoretics Limited and available from Thermo Fisher Scientific, Waltham, MA) and/or isobaric tags for relative and absolute quantitation (iTRAQ®) (AB Sciex Pte. Ltd.), may increase sample throughput. Isobaric mass tagging reagents are compounds that react with and attach to analytes, such as peptides, and have a structure comprised of a reporter region and a balance region, each containing heavy stable isotopes, and a reactive group (e.g., an amine-reactive group, a cysteine-reactive group, or a carbonylreactive group) for derivatization of functional groups. Versions of isobaric mass tags have been created that all have substantially the same total mass of reporter region plus balance region, but the reporter region mass and the balance region mass for each version is different using various different combinations and positions of stable isotopes (e.g., ¹³C and ¹⁵N isotopes). Multiple individual samples may be multiplexed by labeling analytes (e.g., peptides) in each sample with a different version of the isobaric mass tag, combining all the samples together, and analyzing the combined samples via LC-MS or GC-MS in one experiment. The same isobaric mass tag-labeled peptides across the various individual samples all have substantially the same m/z, but when they are fragmented during acquisition of an MSn spectrum, the reporter region of the isobaric mass tag is cleaved off and the reporter ions, which may have different m/z values, are generated and may be measured. The relative intensity of the reporter ions at their various m/z are indicative of the relative concentrations of the analytes in each individual sample.

SUMMARY

[0008] The following description presents a simplified summary of one or more aspects of the methods and systems described herein to provide a basic understanding of such aspects. This summary is not an extensive overview of all contemplated aspects and is intended to neither identify key or critical elements of all aspects nor delineate the scope of any or all aspects. Its sole purpose is to present some concepts of one or more aspects of the methods and systems described herein in a simplified form as a prelude to the more detailed description that is presented below.

[0009] In some illustrative examples, a system for multiplexed mass spectrometry comprises: one or more processors; and memory storing executable instructions that, when executed by the one or more processors, cause a computing device to perform a process including: generating, based on an observed elution peak for a precursor m/z and an elution peak simulation model: a first simulated elution peak that contributes to the observed elution peak and that represents elution of analytes labeled with a deuterated isobaric mass tag; and a second simulated elution peak that contributes to the observed elution peak and that represents elution of analytes labeled with a non-deuterated isobaric mass tag; determining, based on the first simulated elution peak and a timing of acquiring an MSn spectrum for the precursor m/z relative to the observed elution peak, a first correction factor; determining, based on the second simulated elution peak and the timing of acquiring the MSn spectrum for the precursor m/z relative to the observed elution peak, a second correction factor; adjusting, based on the first correction factor, a first observed signal, within the MSn spectrum, for a first reporter ion derived from the deuterated isobaric mass tag; and adjusting, based on the second correction factor, a second observed signal, within the MSn spectrum, for a second reporter ion derived from the non-deuterated isobaric mass tag.

[0010] In some illustrative examples, a non-transitory computer-readable medium stores instructions that, when executed, direct at least one processor of a computing device for mass spectrometry to perform a method comprising: generating, based on an observed elution peak for a precursor m/z and an elution peak simulation model: a first simulated elution peak that contributes to the observed elution peak and that represents elution of analytes labeled with a deuterated isobaric mass tag; and a second simulated elution peak that contributes to the observed elution peak and that represents elution of analytes labeled with a non-deuterated isobaric mass tag; determining, based on the first simulated elution peak and a timing of acquiring an MSn spectrum for the precursor m/z relative to the observed elution peak, a first correction factor; determining, based on the second simulated elution peak and the timing of acquiring the MSn spectrum for the precursor m/z relative to the observed elution peak, a second correction factor; adjusting, based on the first correction factor, a first observed signal, within the MSn spectrum, for a first reporter ion derived from the deuterated isobaric mass tag; and adjusting, based on the second correction factor, a second observed signal, within the MSn spectrum, for a second reporter ion derived from the non-deuterated isobaric mass tag.

[0011] In some illustrative examples, a system for multiplexed mass spectrometry, comprises: one or more processors; and memory storing executable instructions that, when executed by the one or more processors, cause a computing device to perform a process comprising: obtaining mass spectra data representative of: an observed elution profile for a population of ions having a precursor m/z, wherein the population of ions includes a first population of ions labeled with a deuterated isobaric mass tag and a second population of ions labeled with a non-deuterated isobaric mass tag; and an MSn spectrum for the precursor m/z, wherein the MSn spectrum includes a first observed signal for reporter ions derived from ions included in the first population of ions and a second observed signal for reporter ions derived from ions included in the second population of ions; determining an elution time separation between an elution time of the first population of ions and an elution time of the second population of ions; determining a first correction factor and a second correction factor based on the observed elution profile for the population of ions, a timing of acquiring the MSn spectrum relative to an observed elution time of the population of ions, and the elution time separation; adjusting the first observed signal based on the first correction factor; and adjusting the second observed signal based on the second correction factor.

[0012] In some illustrative examples, a system for multiplexed mass spectrometry comprising: one or more processors; and memory storing executable instructions that, when executed by the one or more processors, cause a computing device to perform a process including: obtaining a mass spectrum including, for each channel of a plurality of isobaric mass tag channels, an observed reporter ion signal for analytes labeled with an isobaric mass tag, wherein the plurality of isobaric mass tag channels includes a first set of channels for deuterated isobaric mass tags and a second set of channels for non-deuterated isobaric mass tags; determining a correction factor based on a ratio of the observed reporter ion signal for a first bridge channel included in the first set of channels to the observed reporter ion signal for a second bridge channel included in the second set of channels, wherein analytes labeled with the deuterated isobaric mass tag of the first bridge channel and analytes labeled with the nondeuterated isobaric mass tag of the second bridge channel are derived from a same sample; and adjusting, based on the correction factor, the observed reporter ion signal for each channel included in the second set of channels or for each channel included in the first set of channels.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings illustrate various embodiments and are a part of the specification. The illustrated embodiments are merely examples and do not limit the scope of the disclosure. Throughout the drawings, identical or similar reference numbers designate identical or similar elements.

[0014] FIG. 1 shows a functional diagram of an illustrative LC-MS system.

[0015] FIG. 2 shows a functional diagram of an illustrative implementation of a mass spectrometer that may be included in the LC-MS system of FIG. 1.

[0016] FIG. 3 shows a diagram that illustrates the retention time shift of deuterated isobaric mass tag-labeled analytes relative to non-deuterated isobaric mass tag-labeled analytes.

[0017] FIG. 4 shows a functional diagram of an illustrative MS control system.

[0018] FIG. 5 shows a flowchart of an illustrative data analysis method for multiplexed mass spectrometry using isobaric mass tags.

[0019] FIG. 6 illustrates the principles of the method of FIG. 5 and includes an observed elution peak for a precursor m/z, a first simulated elution peak, and a second simulated elution peak.

[0020] FIG. 7 shows a flowchart of another illustrative data analysis method for multiplexed mass spectrometry using isobaric mass tags.

[0021] FIG. 8 shows a flowchart of an illustrative data analysis method according to the “bridge” technique for multiplexed mass spectrometry using isobaric mass tags.

[0022] FIG. 9 shows an illustrative computing device that may be specifically configured to perform one or more of the processes described herein.

DETAILED DESCRIPTION

[0023] The reporter ion intensities produced when analyzing a mixture of deuterated and non-deuterated isobaric mass tags may be distorted depending upon the timing of the MSn acquisition relative to the differing elution profiles for the deuterated and non-deuterated compounds. Described herein are methods of performing multiplexed mass spectrometry for relative quantitation by adjusting reporter ion intensities to account for retention time shifts between deuterated and non-deuterated isobaric mass tags. The novel methods described herein allow more accurate quantitation using a set of deuterated and non-deuterated isobaric mass tags.

[0024] Various embodiments will now be described in more detail with reference to the figures. The systems and methods described herein may provide one or more of the benefits mentioned above and/or various additional and/or alternative benefits that will be made apparent herein.

[0025] Multiplexed mass spectrometry for quantitation is performed with a combined separation-mass spectrometry system, such as a liquid chromatography-mass spectrometry (LC-MS) system. Accordingly, an LC-MS system will now be described. The described LC-MS system is illustrative and not limiting. The methods and systems described herein may operate as part of or in conjunction with the LC-MS system described herein and/or with any other suitable separation-mass spectrometry system, including a high-performance liquid chromatography-mass spectrometry (HPLC-MS) system, a gas chromatography-mass spectrometry (GC-MS) system, a capillary electrophoresis-mass spectrometry (CE-MS) system, or an ion mobility-mass spectrometry system (e.g., a source-based field asymmetric ion mobility spectrometry (FAIMS) system). For example, an ion mobility cell may induce gas-phase separation of deuterated and non-deuterated isobaric mass tag-labeled analytes in the time domain.

[0026] FIG. 1 shows a functional diagram of an illustrative LC-MS system 100. LC-MS system 100 includes a liquid chromatograph 102, a mass spectrometer 104, and a controller 106. Liquid chromatograph 102 is configured to separate, over time, components (e.g., analytes) within a sample 108 that is injected into liquid chromatograph 102. Sample 108 may include, for example, chemical components (e.g., molecules, ions, etc.) and/or biological components (e.g., metabolites, proteins, peptides, lipids, etc.) for detection and analysis by LC-MS system 100. Liquid chromatograph 102 may be implemented by any liquid chromatograph as may suit a particular implementation. In liquid chromatograph 102, sample 108 is injected into a mobile phase (e.g., a solvent), which carries sample 108 through a column 110 containing a stationary phase (e.g., an adsorbent packing material). As the mobile phase passes through column 110, components within sample 108 elute from column 110 at different times based on, for example, size, affinity to the stationary phase, polarity, and/or hydrophobicity of the components.

[0027] A detector (e.g., an ion detector component of mass spectrometer 104, an ionelectron converter and electron multiplier, etc.) may measure the relative intensity of a signal modulated by each separated component in eluate 112 from column 110. Data generated by the detector may be represented as a chromatogram, which plots retention time on the X-axis and a signal representative of the relative intensity on the Y-axis. The retention time of an analyte is generally measured as the period of time between injection of sample 108 into the mobile phase and the relative intensity peak maximum after chromatographic separation. In some examples, the relative intensity may be correlated to or representative of relative abundance of the separated components. Data generated by liquid chromatograph 102 is output to controller 106. [0028] In some cases, particularly in analyses of complex mixtures, multiple different components in sample 108 co-elute from column 110 at approximately the same time, and thus may have the same or similar retention times. As a result, determination of the relative intensity of the individual components within sample 108 requires further separation of signals attributable to the individual components. To this end, liquid chromatograph 102 directs components included in eluate 112 to mass spectrometer 104 for further separation, identification, and/or quantification of one or more of the components.

[0029] Mass spectrometer 104 is configured to produce ions from the components received from liquid chromatograph 102 and sort or separate the produced ions based on m/z of the ions. Mass spectrometer 104 may be implemented by a multi-stage mass spectrometer configured to perform multi-stage mass spectrometry (denoted MSn where n is 2 or more) or a tandem mass spectrometer configured to perform tandem mass spectrometry (a form of multi-stage mass spectrometry denoted MS/MS or MS2 (where n is 2)). A detector in mass spectrometer 104 measures the intensity of the signal produced by the ions. As used herein, “intensity” or “signal intensity” refers to the response of the detector and may represent absolute abundance, relative abundance, ion count, intensity, relative intensity, ion current, or any other suitable measure of ion detection. Data acquired by mass spectrometer 104 is output to controller 106. Data generated by the detector may be represented by mass spectra, which plot the intensity of the observed signal as a function of m/z of the detected ions.

[0030] FIG. 2 shows a functional diagram of an illustrative implementation of mass spectrometer 104. As shown, mass spectrometer 104 includes an ion source 202, a first mass analyzer 204-1, a collision cell 204-2, a second mass analyzer 204-3, and a controller 206. Mass spectrometer 104 may further include any additional or alternative components not shown as may suit a particular implementation (e.g., ion optics, filters, ion stores, an autosampler, a detector, etc.).

[0031] Ion source 202 is configured to produce a stream 208 of ions from the separated components in sample 108 and deliver the ions to first mass analyzer 204-1. Ion source 202 may use any suitable ionization technique, including without limitation electron ionization, chemical ionization, matrix assisted laser desorption/ionization, electrospray ionization, atmospheric pressure chemical ionization, atmospheric pressure photoionization, inductively coupled plasma, and the like. Ion source 202 may include various components for producing ions from components included in sample 108 and delivering the ions to first mass analyzer 204- 1. [0032] First mass analyzer 204-1 is configured to receive ion stream 208, isolate precursor ions of a precursor m/z, and deliver a beam 210 of the precursor ions to collision cell 204-2. Collision cell 204-2 is configured to receive beam 210 of precursor ions and produce product ions (e.g., fragment ions) via controlled dissociation processes. Collision cell 204-2 is further configured to direct a beam 212 of product ions to second mass analyzer 204-3. Second mass analyzer 204-3 is configured to filter and/or perform a mass analysis of the product ions.

[0033] Mass analyzers 204-1 and 204-3 are configured to isolate or separate ions according to m/z of each of the ions. Mass analyzers 204-1 and 204-3 may be implemented by any suitable mass analyzer, such as a quadrupole mass filter, an ion trap (e.g., a three-dimensional quadrupole ion trap, a cylindrical ion trap, a linear quadrupole ion trap, a toroidal ion trap, etc.), a time-of-flight (TOF) mass analyzer, an electrostatic trap mass analyzer (e.g. an orbital electrostatic trap such as an Orbitrap mass analyzer, a Kingdon trap, etc.), a Fourier transform ion cyclotron resonance (FT-ICR) mass analyzer, and the like. Mass analyzers 204-1 and 204-3 need not be implemented by the same type of mass analyzer. In some examples, mass analyzer 204-3 is an asymmetric track lossless (Astral™) mass analyzer, from Thermo Fisher Scientific™.

[0034] Collision cell 204-2 may be implemented by any suitable collision cell. As used herein, “collision cell” may encompass any structure or device configured to produce product ions via controlled dissociation processes and is not limited to devices employed for collisionally- activated dissociation. For example, collision cell 204-2 may be configured to fragment precursor ions using collision induced dissociation, electron transfer dissociation, electron capture dissociation, photo induced dissociation, surface induced dissociation, ion/molecule reactions, and the like.

[0035] An ion detector (not shown) is configured to detect ions at each of a variety of different m/z and responsively generate an electrical signal representative of ion intensity. The electrical signal is transmitted to controller 206 for processing, such as to construct a mass spectrum of the analyzed ions. For example, mass analyzer 204-3 may emit an emission beam of separated ions to the ion detector, which is configured to detect the ions in the emission beam and generate or provide data that can be used by controller 206 to construct a mass spectrum of the analyzed ions. The ion detector may be implemented by any suitable detection device, including without limitation an electron multiplier, a Faraday cup, and the like. In other examples, such as where second mass analyzer 204-3 is implemented by an orbital electrostatic trap mass analyzer, second mass analyzer 204-3 functions as both a mass analyzer and a detector.

[0036] Controller 206 may be communicatively coupled with, and configured to control operations of, mass spectrometer 104. For example, controller 206 may be configured to control operation of various hardware components included in ion source 202 and/or mass analyzers 204-1 and 204-3. To illustrate, controller 206 may be configured to control an accumulation time of ion source 202 and/or mass analyzers 204, control an oscillatory voltage power supply and/or a DC power supply to supply an RF voltage and/or a DC voltage to mass analyzers 204, adjust values of the RF voltage and DC voltage to select an effective m/z (including a mass tolerance window) for analysis, and adjust the sensitivity of the ion detector (e.g., by adjusting the detector gain).

[0037] Controller 206 may include any suitable hardware (e.g., a processor, circuitry, etc.) and/or software as may serve a particular implementation. While FIG. 2 shows that controller 206 is included in mass spectrometer 104, controller 206 may alternatively be implemented in whole or in part separately from mass spectrometer 104, such as by a computing device communicatively coupled to mass spectrometer 104 by way of a wired connection (e.g., a cable) and/or a network (e.g., a local area network, a wireless network (e.g., Wi-Fi), a wide area network, the Internet, a cellular data network, etc.). In some examples, controller 206 is implemented in whole or in part by controller 106.

[0038] In the example of FIG. 2, mass spectrometer 104 is tandem-in-space (e.g., has multiple mass analyzers) and has two stages for performing tandem mass spectrometry. However, mass spectrometer 104 is not limited to this configuration but may have any other suitable configuration. For example, mass spectrometer 104 may be tandem-in-time. Additionally or alternatively, mass spectrometer 104 may be a multi-stage mass spectrometer and may have any suitable number of mass analyzers and stages (e.g., three or more) for performing multi-stage mass spectrometry (e.g., MS/MS/MS).

[0039] Referring again to FIG. 1, controller 106 is communicatively coupled with, and configured to control operations of, LC-MS system 100 (e.g., liquid chromatograph 102 and mass spectrometer 104). Controller 106 may include any suitable hardware (e.g., a processor, circuitry, etc.) and/or software configured to control operations of and/or interface with the various components of LC-MS system 100 (e.g., liquid chromatograph 102 or mass spectrometer 104).

[0040] Controller 106 and/or controller 206 may also include and/or provide a user interface configured to enable user interaction with LC-MS system 100 or mass spectrometer 104. The user may interact with controller 106 and/or controller 206 via the user interface by tactile, visual, auditory, and/or other sensory type communication. For example, the user interface may include a display device (e.g., liquid crystal display (LCD) display screen, a touch screen, etc.) for displaying information (e.g., mass spectra, notifications, etc.) to the user. The user interface may also include an input device (e.g., a keyboard, a mouse, a touchscreen device, etc.) that allows the user to provide input to controller 106 and/or controller 206. In other examples, the display device and/or input device may be separate from, but communicatively coupled to, controller 106 and/or controller 206. For instance, the display device and the input device may be included in a computer (e.g., a desktop computer, a laptop computer, etc.) communicatively connected to controller 106 and/or controller 206 by way of a wired connection (e.g., by one or more cables) and/or a wireless connection. [0041] Controller 106 acquires data acquired over time by LC-MS system 100. The data may include a series of mass spectra (e.g., MS1 spectra or MS2 spectra) including intensity values of ions produced from the analytes present in sample 108 (e.g., precursor ions or product ions) as a function of m/z of the ions. The series of mass spectra may be represented in a three- dimensional map in which elution time (e.g., retention time) is plotted along an X-axis of the map, m/z is plotted along a Y-axis of the map, and intensity is plotted along a Z-axis of the map. Spectral features on the map (e.g., Z-axis peaks of intensity) represent detection by LC-MS system 100 of ions produced from various analytes included in sample 108. The X-axis and Z- axis of the map may be used to generate an elution profile (e.g., a mass chromatogram) that plots detected intensity as a function of time for the precursor m/z.

[0042] As used herein, “precursor m/z” refers to a specific m/z, with or without a mass tolerance window (e.g., +/- 0.5 m/z), or a narrow range of m/z (e.g., an isolation window with a width or range such as 10 m/z, 4 m/z, 3 m/z, 2 m/z, etc.), which is isolated for MS2 analysis. In an MS2 or MSn analysis, such as a data dependent MS2, selected reaction monitoring (SRM) analysis, a multiple reaction monitoring (MRM) analysis, or a parallel reaction monitoring (PRM) analysis, the precursor m/z corresponds to m/z of the precursor ion(s) that is/are isolated and subsequently fragmented to generate reporter ions for relative quantitation.

[0043] As used herein, an “acquisition” refers to a mass analysis performed at a discrete point in time to acquire a single mass spectrum, wherein precursor ions of a precursor m/z are isolated, fragmented, and mass analyzed to generate an MS2 or MSn spectrum. In some embodiments, the mass spectrum is acquired based on a DDA analysis. In other examples, the mass spectrum is acquired based on an MS2 analysis or DIA analysis. In other embodiments, there may be multiple rounds of fragmentation and m/z selection, sometimes involving the simultaneous selection of multiple m/z values, all for the sake of performing a higher order MSn acquisition (e.g., an MS3 acquisition).

[0044] As mentioned, multiplexed mass spectrometry experiments may be designed to identify and/or gather quantitative information about a set of analytes. An isobaric mass tag- based mass spectrometry experiment may be used to identify and/or determine a relative quantity of a particular analyte (e.g., peptide) across multiple different samples. For example, an isobaric mass tag-based mass spectrometry experiment may determine a quantity of an analyte in each of multiple different samples relative to the quantity of the analyte in a reference sample. To this end, analytes in each sample, including the reference sample, are labeled with an isobaric mass tag. An isobaric mass tag-labeled analyte (precursor ion) will have substantially the same m/z across all samples (varying only slightly, e.g., on the order of a few mDa, due to the difference, for example, between heavy nitrogen and carbon for TMT® reagents or heavy oxygen for iTRAQ® reagents) so that it is hard to distinguish all versions of the isobaric mass tag-labeled analyte that co-elute from column 110 at the same time by MS1 alone. However, when the isobaric mass tag-labeled analytes are fragmented by MSn (e.g., by MS2 or MS3), the reporter regions are cleaved off and reporter ions are generated and can be measured by an MSn (e.g., MS2 or MS3) analysis. The relative intensity of the reporter ions at their respective m/z are indicative of the relative quantities of the analyte in each sample.

[0045] Conventional sets of isobaric mass tags, such as TMT® reagents, provide multiple different channels for multiplexing for relative quantitation using various different combinations and positions of stable isotopes (e.g., ¹³C, ¹⁵N, and/or ¹⁸O isotopes) to produce, when fragmented, reporter ions of varying mass. For example, TMT® reagents produce reporter ions having a mass between about 126 Da and about 135 Da. Isobaric mass tags are now provided that increase the number of channels for multiplexing by also using deuterated forms of the isobaric mass tags (incorporating ²H isotopes) and non-deuterated forms of the isobaric mass tags (incorporating only ¹H isotopes). For example, a deuterated isobaric mass tag and a nondeuterated isobaric mass tag may have a same composition of isotopes (e.g., ¹³C and ¹⁵N isotopes in the same combination and positions). Using these deuterated and non-deuterated isobaric mass tags, many more samples can be multiplexed for relative quantitation than is possible with conventional isobaric mass tags.

[0046] However, deuterated isobaric mass tag-labeled analytes elute at slightly different times during pre-MS separations (e.g., reversed-phase LC (RPLC) separations) than non- deuterated isobaric mass tag-labeled analytes due to the ²H isotope. FIG. 3 shows a diagram 300 that illustrates the retention time shift of deuterated isobaric mass tag-labeled analytes relative to non-deuterated isobaric mass tag-labeled analytes. Chart 300 includes an illustrative elution profile 302 of reporter ions derived from MS2 acquisitions of a peptide (LKPDPNTLCDEFK (+3), 830.47 m/z) labeled with a deuterated TMT® 127d reagent and an illustrative elution profile 304 of reporter ions derived from MS2 acquisitions of the peptide labeled with a non-deuterated TMT® 127c reagent. The peptide labeled with the deuterated reagent elutes at approximately 52 minutes 26 seconds, as indicated by dashed line 306, and the same peptide labeled with the non-deuterated reagent elutes at approximately 52 minutes 28 seconds, as indicated by dashed line 308. Thus, the peptide labeled with the deuterated reagent elutes approximately 2 seconds before the peptide labeled with the non-deuterated reagent. This phenomenon occurs in both online RPLC-MS analysis and offline RPLC fractionation.

[0047] The reporter ion intensities produced when analyzing a mixture of deuterated and non-deuterated isobaric mass tag-labeled analytes may distort quantitative interpretation depending upon the timing of the MSn acquisition relative to the differing elution profiles for the deuterated and non-deuterated compounds. Typical isobaric mass tag-based MS methods rely on data-dependent acquisition (DDA) techniques to trigger an MSn analysis of the labeled analytes. These data-dependent MS methods only provide a single MSn “snapshot” of the reporter ion intensities at a given LC retention time. Given the shifts in the retention times between the deuterated and non-deuterated isobaric mass tag-labeled analytes, measured reporter ion intensities in this MSn “snapshot” can skew relative to when the MSn analysis is performed during the LC elution profile (or between offline LC fractions). Results can vary according to whether the observed intensities in the MSn spectra are acquired closer to the “leading” edge of the elution peak, where deuterated reagents dominate, or closer to the “trailing” edge of the elution peak, where non-deuterated reagents dominate.

[0048] Consider the example of FIG. 3. If an MSn analysis is performed at 52 minutes 20 seconds, as indicated by dashed line 310, the observed intensity of the peptide labeled with the deuterated reagent (indicated by dashed line 312) is significantly higher than the intensity of the peptide labeled with the non-deuterated reagent (indicated by dashed line 314), even though the apexes of elution profiles 302 and 304 are substantially the same. This problem is referred to herein as “reporter ion ratio distortion.”

[0049] Improved methods of multiplexed mass spectrometry for relative quantitation using deuterated and non-deuterated isobaric mass tags compensate for reporter ion ratio distortion by adjusting reporter ion intensities to account for retention time shifts between deuterated and non-deuterated isobaric mass tag-labeled analytes. In some examples, a first correction factor is determined for the deuterated reporter ions and the observed intensity of the deuterated reporter ions is adjusted based on the first correction factor and/or a second correction factor is determined for the non-deuterated reporter ions and the observed intensity of the non- deuterated reporter ions is adjusted based on the second correction factor. Illustrative examples of determining the first correction factor and the second correction factor will be described below in more detail. By correcting the intensity of the observed signal of one or both of the deuterated and non-deuterated reporter ions, reporter ion intensities can be normalized and/or estimated to reflect what would have been observed if the MSn spectra had been acquired at the elution peak apex for the deuterated isobaric mass tag-labeled analytes and non-deuterated isobaric mass tag-labeled analytes. The examples described herein use a simple Gaussian peak shape. However, any other non-Gaussian peak shape could be used, including peak shapes based on the observed LC peaks. These and other illustrative methods and systems for multiplexed mass spectrometry for quantitation will be described in more detail below.

[0050] One or more operations associated with improved methods of multiplexed mass spectrometry for relative quantitation using deuterated and non-deuterated isobaric mass tags may be performed by a mass spectrometry control system in conjunction with a mass spectrometry system (e.g., an LC-MS system 100, a GC-MS system, or a CE-MS system). The mass spectrometry control system may control and/or perform one or more operations described herein. FIG. 4 shows a functional diagram of an illustrative MS control system 400 (“system 400”). System 400 may be implemented entirely or in part by a mass spectrometry system, such as LC-MS system 100 (e.g., by controller 106 and/or controller 206). Alternatively, system 400 may be implemented separately from the mass spectrometry system (e.g., a remote computing system or server separate from but communicatively coupled to controller 106 and/or controller 206 of LC-MS system 100).

[0051] System 400 may include, without limitation, a memory 402 and a processor 404 selectively and communicatively coupled to one another. Memory 402 and processor 404 may each include or be implemented by hardware and/or software components (e.g., processors, memories, communication interfaces, instructions stored in memory for execution by the processors, etc.). In some examples, memory 402 and processor 404 are distributed between multiple devices and/or multiple locations as may serve a particular implementation.

[0052] Memory 402 may maintain (e.g., store) executable data used by processor 404 to perform any of the operations described herein. For example, memory 402 may store instructions 406 that may be executed by processor 404 to perform any of the operations described herein. Instructions 406 may be implemented by any suitable application, software, code, and/or other executable data instance. Memory 402 may also maintain any data acquired, received, generated, managed, used, and/or transmitted by processor 404. For example, memory 402 may maintain LC-MS data.

[0053] Processor 404 is configured to perform (e.g., execute instructions 406 stored in memory 402 to perform) various processing operations described herein. It will be recognized that the operations and examples described herein are merely illustrative of the many different types of operations that may be performed by processor 404. In the description herein, any references to operations performed by system 400 may be understood to be performed by processor 404 of system 400. Furthermore, in the description herein, any operations performed by system 400 may be understood to include system 400 directing, commanding, or instructing another system or device to perform the operations.

[0054] FIG. 5 shows a flowchart of an illustrative data analysis method 500 for multiplexed mass spectrometry using isobaric mass tags. Method 500 may be performed post-acquisition, e.g., after performing a multiplexed mass spectrometry experimental run using isobaric mass tags, to process and analyze data generated by the multiplexed mass spectrometry experiment. While FIG. 5 shows illustrative operations according to one embodiment, other embodiments may omit, add to, reorder, and/or modify any of the operations shown in FIG. 5. One or more of the operations shown in FIG. 5 may be performed by LC-MS system 100 and/or system 400, any components included therein, and/or any implementations thereof.

[0055] At operation 502, system 400 generates, based on an observed elution peak for a precursor m/z and an elution peak simulation model, a first simulated elution peak and a second simulated elution peak. The observed elution peak represents the actual, observed elution of isobaric mass tag-labeled analytes all having substantially the same total mass. However, due to retention time differences between deuterated and non-deuterated isobaric mass tag-labeled analytes, the observed elution peak is the result of the contributions of two components: an elution peak for deuterated isobaric mass tag-labeled analytes and an elution peak for non- deuterated isobaric mass tag-labeled analytes. The first simulated elution peak contributes to the observed elution peak and represents elution of analytes labeled with a deuterated isobaric mass tag. The second simulated elution peak contributes to the observed elution peak and represents elution of analytes labeled with a non-deuterated isobaric mass tag.

[0056] In some examples, the observed elution peak for the precursor m/z is based on a series of MS1 spectra acquired over time for a precursor m/z (e.g., a trace or extracted ion chromatogram (XIC) for the precursor m/z). For example, in a DDA analysis a set of MS1 spectra are acquired over time and an acquisition of an MSn spectrum (e.g., an MS2 or MS3 spectrum) is triggered when the observed MS1 signal satisfies certain criteria, such as a minimum intensity threshold. The observed elution peak in the DDA analysis is the extracted ion chromatogram for the precursor m/z. Thus, in the MS1 analysis the elution peak is based on direct MS1 measurements of the precursor ion over time.

[0057] In other examples, the observed elution peak for the precursor m/z is based on a series of MSn spectra (e.g., MS2 or MS3 spectra) acquired over time by an MSn analysis (e.g., an MS2 analysis or MS3 analysis), such as a DIA or PRM analysis. In MSn analyses, the observed elution peak for the precursor m/z is based on indirect measurement of the precursor m/z by MS2 (or MS3) measurements of product ions derived from and attributable to precursor ions having the precursor m/z. In DIA analyses, chromatographic data typically represents a total ion current based on the combination of all co-isolated precursor ions. PRM analyses often use narrower isolation windows than DIA analyses but may still result in co-isolation of precursor ions. To remove the influence of co-isolated precursor ions in both DIA and PRM analyses, in operation 502 the observed elution peak for the precursor m/z is based on sequence-specific product ions (e.g., b- and y-type product ions that are sequence-specific) that are directly attributable to the precursor ion having the precursor m/z.

[0058] The elution peak simulation model is configured to take varying items of input data and output the first simulated elution peak and the second simulated elution peak. For a workflow for multiplexed mass spectrometry for quantitation, a peptide sequence is known or can be determined post-acquisition, such as by spectral matching or database or library searching. Accordingly, the first simulated elution peak and the second simulated elution peak can be generated by providing, as inputs to the elution peak simulation model, data such as, but not limited to, the peptide sequence, the observed elution peak data, previous modeling, historical data for the deuterated and non-deuterated isobaric mass tags, and/or historical data for the same peptides and isobaric mass tags. The first simulated elution peak and the second simulated elution peak can be generated by assuming a certain peak shape, such as a Gaussian peak shape. Elution peaks having a simple Gaussian profile are represented by the following formula: where I is the estimated intensity at time t, t_max is the time at the peak apex, l_max is the estimated maximum intensity at time t_max, and o is the standard deviation of the elution peak. Method 500 is not limited to Gaussian peak shapes, as other non-Gaussian peak shapes may be used. [0059] In some examples, generating the first simulated elution peak and the second simulated elution peak includes determining a position (in time) of the simulated elution peaks relative to the observed elution peak. For example, system 400 may estimate the retention times of the first simulated elution peak and the second simulated elution peak and/or determine an estimated retention time difference between the first simulated elution peak and the second simulated elution peak. Retention time estimation is an extensive field, with many approaches known to those of skill in the art. In some examples, the elution peak simulation model includes or is implemented by any suitable technique, algorithm, or model, including those based on or implementing machine learning or artificial intelligence, to estimate or predict the retention times of the first simulated elution peak and second elution peak and/or to determine the estimated retention time difference between the first simulated elution peak and the second simulated elution peak. Based on the estimated retention times and/or the estimated retention time difference between the first simulated elution peak and the second simulated elution peak, system 400 may determine positions of the first simulated elution peak and the second simulated elution peak relative to the observed elution peak (e.g., relative to an apex of the observed elution peak). A variety of techniques known to those of skill in the art, such as nonnegative least squares, may be used for determining positions of the first simulated elution peak and the second simulated elution peak relative to the observed elution peak. In some examples, system 400 determines the positions of the first simulated elution peak and the second simulated elution peak so that the apexes of the first simulated elution peak and the second simulated elution peak are equidistant from the apex of the observed elution peak (e.g., one-half the estimated retention time difference is positioned on either side of the apex of the observed elution peak).

[0060] At operation 504, system 400 determines a first correction factor based on the first simulated elution peak and a timing of an MSn acquisition for the precursor m/z relative to the observed elution peak. Similarly, at operation 506, system 400 determines a second correction factor based on the second simulated elution peak and the timing of the MSn acquisition for the precursor m/z relative to the observed elution peak. As will be explained below, the first correction factor may be used to adjust observed reporter ion signals for deuterated analytes and the second correction factor may be used to adjust observed reporter ion signals for nondeuterated analytes. The first correction factor and the second correction factor are based on one or more of the observed elution peak, the first simulated elution peak, the second simulated elution peak, and the timing of the MSn acquisition.

[0061] In some examples, the first correction factor is the ratio of the maximum intensity of the first simulated elution peak (e.g., the intensity at the apex of the first simulated elution peak) to the intensity of the first simulated elution peak at a time of the MSn acquisition for the precursor m/z. The first correction factor may be determined by formula (2): where li(timax) is the estimated intensity of the first simulated elution peak at time ti_max(e.g., the maximum intensity of the first simulated elution peak), ti_max is the estimated time of the apex of the first simulated elution peak, (tiMSn) is the intensity at time t sn, and time t sn is the time at which the MSn spectrum is acquired. Similarly, the second correction factor is the ratio of the maximum intensity of the second simulated elution peak (e.g., the intensity at the apex of the second simulated elution peak) to the intensity of the second simulated elution peak at the time of the MSn acquisition for the precursor m/z. The second correction factor may be determined by formula (3): where h(t2m_ax) is the estimated intensity of the second simulated elution peak at time t2m_ax(e.g., the maximum intensity of the second simulated elution peak), t2m_ax is the estimated time of the apex of the second simulated elution peak, l₂(t2MSn) is the intensity at time t2MSn, and time t2MSn is the time at which the MSn spectrum is acquired. As used herein, “maximum” does not necessarily mean the absolute highest value but may include a value within a relatively small tolerance, e.g., 1%, 3%, 5%, or 10%, of the absolute highest value.

[0062] When the first simulated elution peak is generated using a Gaussian profile according to formula (1), the first correction factor Ci becomes: where tMSn is the time of the MSn acquisition, ti_max is the estimated time of the apex of the first simulated elution peak, and 01 is the standard deviation of the first simulated elution peak. Similarly, when the second simulated elution peak is generated using a Gaussian profile according to formula (1), the second correction factor C2 becomes: where tMSn is the time of the MSn acquisition, t2m_ax is the estimated time of the apex of the second simulated elution peak, and 02 is the standard deviation of the second simulated elution peak. As can be seen from formulas (4) and (5), the first correction factor and the second correction factor may be obtained by determining, based on the timing of acquiring the MSn spectrum relative to the observed elution peak, a timing of acquiring the MSn spectrum relative to the first simulated elution peak and a timing of acquiring the MSn spectrum relative to the second simulated elution peak.

[0063] It will be recognized that these methods for determining the first correction factor and the second correction factor are not limiting, as the first correction factor and the second correction factor may be determined in various other ways based on one or more of the observed elution peak, the first simulated elution peak, the second simulated elution peak, and the timing of the MSn acquisition.

[0064] At operation 508, system 400 adjusts, based on the first correction factor, a first observed signal, within an MSn spectrum acquired by the MSn acquisition, for a first reporter ion derived from the deuterated isobaric mass tag. For example, system 400 multiplies the first observed signal by the first correction factor Ci as follows: f deuterated. ⁼ ^deuterated * ^C1 (6) where Ideuterated is the observed reporter ion signal for deuterated reporter ions, Ci is the first correction factor, and I deuterated is the adjusted reporter ion signal for the deuterated reporter ions. Similarly, at operation 510, system 400 adjusts, based on the second correction factor, a second observed signal, within the MSn spectrum acquired by the MSn acquisition, for a second reporter ion derived from the non-deuterated isobaric mass tag. For example, system 400 multiplies the second observed signal by the second correction factor C2 as follows: f non-deuterated ⁼ non- deuterated * ^c2 (7) where Ideuterated is the observed reporter ion signal for non-deuterated reporter ions, C2 is the second correction factor, and I'non-deuterated is the adjusted reporter ion signal for the non- deuterated reporter ions. In some examples, all observed reporter ion signals, within the MSn spectrum, for the deuterated analytes are adjusted by the first correction factor and all observed reporter ion signals, within the MSn spectrum, for the non-deuterated analytes are adjusted by the second correction factor. In this way, the observed reporter ion signals for the deuterated analytes are scaled up to what would have been observed if the MSn spectrum had been acquired at the apex of the first simulated elution peak, and the observed reporter ion signals for the non-deuterated analytes are scaled up to what would have been observed if the MSn spectrum had been acquired at the apex of the second simulated elution peak.

[0065] FIG. 6 illustrates the principles of method 500. FIG. 6 shows a diagram 600 that includes an observed elution peak 602 for a precursor m/z, a first simulated elution peak 604-1, and a second simulated elution peak 604-2. Observed elution peak 602 is based on actual MS1 measurements (a series of MS1 acquisitions represented by markers 606) of deuterated and non-deuterated precursor ions having the precursor m/z. For example, observed elution peak 602 represents observed elution of analytes labeled with a deuterated isobaric mass tag and analytes labeled with a non-deuterated isobaric mass tag. First simulated elution peak 604-1 and second simulated elution peak 604-2 are generated based on observed elution peak 602 and an elution peak simulation model, as described above, and have a simple Gaussian profile. First simulated elution peak 604-1 contributes to observed elution peak 602 and represents estimated or theoretical MS1 measurements of deuterated precursor ions (e.g., analytes labeled with a deuterated isobaric mass tag). Second simulated elution peak 604-2 contributes to observed elution peak 602 and represents estimated or theoretical MS1 measurements of non- deuterated precursor ions (e.g., analytes labeled with a non-deuterated isobaric mass tag). [0066] The estimated retention time difference between first simulated elution peak 604-1 and second simulated elution peak 604-2 is labeled ART. First simulated elution peak 604-1 and second simulated elution peak 604-2 are aligned with observed elution peak 602 such that one- half the estimated retention time difference ART is positioned on either side of an apex of observed elution peak 602, which occurs at time tobs- Thus, an apex of first simulated elution peak 604-1 and an apex of second simulated elution peak 604-2 are equidistant from the apex of observed elution peak 602. The apex of the first simulated elution peak 604-1 (e.g., the estimated retention time of the deuterated analytes) occurs at time timax and the apex of the second simulated elution peak 604-2 (e.g., the estimated retention time of the non-deuterated analytes) occurs at time bmax. In this example, the MSn acquisition for the precursor m/z is triggered by MS1 acquisition 606a and occurs at time tMSn.

[0067] A first correction factor Ci is determined by the following formula (8): where hmax is the maximum intensity of first simulated elution peak 604-1 at time timax and I sn is the intensity of first simulated elution peak 604-1 at the time of the MSn acquisition. A second correction factor C2 is determined by the following formula (9): where hmax is the maximum intensity of second simulated elution peak 604-2 at time t2max and hMSn is the intensity of second simulated elution peak 604-2 at the time of the MSn acquisition. [0068] FIG. 7 shows a flowchart of another illustrative data analysis method 700 for multiplexed mass spectrometry using isobaric mass tags. Method 700 may be performed postacquisition, e.g., after performing a multiplexed mass spectrometry experimental run using isobaric mass tags, to process and analyze data generated by the multiplexed mass spectrometry experiment. While FIG. 7 shows illustrative operations according to one embodiment, other embodiments may omit, add to, reorder, and/or modify any of the operations shown in FIG. 7. One or more of the operations shown in FIG. 7 may be performed by LC-MS system 100 and/or system 400, any components included therein, and/or any implementations thereof.

[0069] At operation 702, system 400 obtains mass spectra data representative of an observed elution profile for a population of ions having a precursor m/z and an MSn spectrum for the precursor m/z. The population of ions includes a first population of ions labeled with a deuterated isobaric mass tag and a second population of ions labeled with a non-deuterated isobaric mass tag. The MSn spectrum includes a first observed signal for reporter ions derived from ions included in the first population of ions and a second observed signal for reporter ions derived from ions included in the second population of ions. [0070] At operation 704, system 400 determines an elution time separation between an elution time of the first population of ions and an elution time of the second population of ions. Operation 704 may be performed in any way described herein, such as by using an elution peak simulation model.

[0071] At operation 706, system 400 determines a first correction factor and a second correction factor based on the observed elution profile for the population of ions, a timing of acquiring the MSn spectrum relative to an observed elution time of the population of ions, and the elution time separation. Operation 706 may be performed in any described herein.

[0072] In some examples, determining the first correction factor includes determining, based on the elution time separation and the timing of acquiring the MSn spectrum relative to the observed elution time of the population of ions, a timing of acquiring the MSn spectrum relative to the elution time of the first population of ions. With the timing of acquiring the MSn spectrum relative to the observed elution time of the population of ions and the timing of acquiring the MSn spectrum relative to the elution time of the first population of ions, the first correction factor may be determined, such as by using formula (4).

[0073] In some examples, determining the second correction factor includes determining, based on the elution time separation and the timing of acquiring the MSn spectrum relative to the observed elution time of the population of ions, a timing of acquiring the MSn spectrum relative to the elution time of the second population of ions. With the timing of acquiring the MSn spectrum relative to the observed elution time of the population of ions and the timing of acquiring the MSn spectrum relative to the elution time of the second population of ions, the second correction factor may be determined, such as by using formula (5).

[0074] At operation 708, system 400 adjusts the first observed signal based on the first correction factor. Similarly, at operation 710, system 400 adjusts the second observed signal based on the second correction factor.

[0075] Various modifications can be made to the examples described above. In the examples described above, the observed reporter ion signals are adjusted to normalize the reporter ion signal relative to an estimated or predicted elution peak apex for each of the deuterated and non-deuterated isobaric mass tag-labeled analytes. In other examples, the observed reporter ion signals may be normalized to any other estimated or predicted point in the elution profiles. In some examples, the observed elution peak is based on any observed signal that is not within the isobaric mass tag envelope (e.g., any signal that is not representative of reporter ion intensities).

[0076] The examples described above are described for on-line LC-MS analysis. However, the method of correcting the reporter ion intensities described above can also be applied to offline LC fractionation with various adjustments for offline fractionation. For example, in the case of offline fractionation, the modeling of the retention time shift of the deuterated isobaric mass tag-labeled analytes and non-deuterated isobaric mass tag-labeled analytes is updated to reflect the method of offline fractionation. Similarly, in some examples the observed LC fractionation “peak shape” is utilized when calculating the first correction factor and the second correction factor. Furthermore, instead of positioning in time the first simulated elution peak and second simulated elution peak relative to the observed LC-MS signal, the first simulated elution peak and second simulated elution peak are correlated to the integrated precursor extracted ion chromatogram (XIC) between fractions.

[0077] There are alternative ways to address the problem of reporter ion ratio distortion besides the approach described with reference to method 500. In a first approach, a set of two or more isobaric mass tag channels is used as a “bridge” between deuterated and nondeuterated isobaric mass tag-labeled analytes. Each channel corresponds to a unique isobaric mass tag. In the bridge technique, analytes from the same sample are labeled using a deuterated version and a non-deuterated version of an isobaric mass tag. Given that analytes from the same sample are labeled with the deuterated and non-deuterated versions of the isobaric mass tags, the ratio of the reporter ion signal for the deuterated analytes to the reporter ion signal for the non-deuterated analytes should be one-to-one. However, as explained above, the observed ratio is not one-to-one due to the retention time difference between the deuterated analytes and non-deuterated analytes. Accordingly, a correction factor is determined and used to adjust the reporter ion signal for the non-deuterated isobaric mass tag-labeled analytes or the reporter ion signal for the deuterated isobaric mass tag-labeled analytes.

[0078] In some examples, the correction factor is based on the ratio of the observed reporter ion signal for the deuterated isobaric mass tag-labeled “bridge” to the observed reporter ion signal for the non-deuterated isobaric mass tag-labeled ’’bridge”. The observed reporter ion signals for the non-deuterated isobaric mass tag-labeled analytes may then be adjusted in one or more other channels in the reporter ion spectrum, such as by multiplying the observed reporter ion signals by the correction factor derived from the ratio of the “bridge” channels. This bridge technique normalizes the observed reporter ion signals with each other as opposed to normalizing the observed reporter ion signals with respect to an estimated or predicted elution peak maximum for each deuterated and non-deuterated species, as in method 500. Method 500 has the advantage that no channels are lost or used for signal correction, as in the bridge technique.

[0079] FIG. 8 shows a flowchart of an illustrative data analysis method 800 according to the “bridge” technique for multiplexed mass spectrometry using isobaric mass tags. Method 800 may be performed post-acquisition, e.g., after performing a multiplexed mass spectrometry experimental run using isobaric mass tags, to process and analyze data generated by the multiplexed mass spectrometry experiment. While FIG. 8 shows illustrative operations according to one embodiment, other embodiments may omit, add to, reorder, and/or modify any of the operations shown in FIG. 8. [0080] At operation 802, system 400 obtains a mass spectrum comprising, for each channel of a plurality of isobaric mass tag channels, an observed reporter ion signal for analytes labeled with an isobaric mass tag. The plurality of isobaric mass tag channels includes a first set of channels for deuterated isobaric mass tags and a second set of channels for non-deuterated isobaric mass tags.

[0081] At operation 804, system 400 determines a correction factor based on a ratio of the observed reporter ion signal for a first bridge channel included in the first set of channels to the observed reporter ion signal for a second bridge channel included in the second set of channels. In some examples, the correction factor is the ratio of the observed reporter ion signal for the first bridge channel included in the first set of channels to the observed reporter ion signal for the second bridge channel included in the second set of channels. Analytes labeled with the deuterated isobaric mass tag of the first bridge channel and analytes labeled with the nondeuterated isobaric mass tag of the second bridge channel are derived from a same sample.

[0082] At operation 806, system 400 adjusts, based on the correction factor, the observed reporter ion signal for each channel included in the second set of channels or for each channel included in the first set of channels.

[0083] Another approach for isobaric mass tag-based mass spectrometry for quantitation utilizes a DIA-type method, wherein multiple reporter ion spectra are acquired across the entire LC-MS elution profile. The observed reporter ion signals across the entire profile are then integrated for each channel to determine the relative abundance of each reporter ion. This technique removes any influence caused by retention time difference.

[0084] In certain embodiments, one or more of the systems, components, and/or processes described herein may be implemented and/or performed by one or more appropriately configured computing devices. To this end, one or more of the systems and/or components described above may include or be implemented by any computer hardware and/or computer- implemented instructions (e.g., software) embodied on at least one non-transitory computer- readable medium configured to perform one or more of the processes described herein. In particular, system components may be implemented on one physical computing device or may be implemented on more than one physical computing device. Accordingly, system components may include any number of computing devices, and may employ any of a number of computer operating systems.

[0085] In certain embodiments, one or more of the processes described herein may be implemented at least in part as instructions embodied in a non-transitory computer-readable medium and executable by one or more computing devices. In general, a processor (e.g., a microprocessor) receives instructions, from a non-transitory computer-readable medium, (e.g., a memory, etc.), and executes those instructions, thereby performing one or more processes, including one or more of the processes described herein. Such instructions may be stored and/or transmitted using any of a variety of known computer-readable media. [0086] A computer-readable medium (also referred to as a processor-readable medium) includes any non-transitory medium that participates in providing data (e.g., instructions) that may be read by a computer (e.g., by a processor of a computer). Such a medium may take many forms, including, but not limited to, non-volatile media, and/or volatile media. Non-volatile media may include, for example, optical or magnetic disks and other persistent memory. Volatile media may include, for example, dynamic random access memory (“DRAM”), which typically constitutes a main memory. Common forms of computer-readable media include, for example, a disk, hard disk, magnetic tape, any other magnetic medium, a compact disc read-only memory (“CD-ROM”), a digital video disc (“DVD”), any other optical medium, random access memory (“RAM”), programmable read-only memory (“PROM”), electrically erasable programmable readonly memory (“EPROM”), FLASH-EEPROM, any other memory chip or cartridge, or any other tangible medium from which a computer can read.

[0087] FIG. 9 shows an illustrative computing device 900 that may be specifically configured to perform one or more of the processes described herein. As shown in FIG. 9, computing device 900 may include a communication interface 902, a processor 904, a storage device 906, and an input/output (“I/O”) module 908 communicatively connected one to another via a communication infrastructure 910. While an illustrative computing device 900 is shown in FIG. 9, the components illustrated in FIG. 9 are not intended to be limiting. Additional or alternative components may be used in other embodiments. Components of computing device 900 shown in FIG. 9 will now be described in additional detail.

[0088] Communication interface 902 may be configured to communicate with one or more computing devices. Examples of communication interface 902 include, without limitation, a wired network interface (such as a network interface card), a wireless network interface (such as a wireless network interface card), a modem, an audio/video connection, and any other suitable interface.

[0089] Processor 904 generally represents any type or form of processing unit capable of processing data and/or interpreting, executing, and/or directing execution of one or more of the instructions, processes, and/or operations described herein. Processor 904 may perform operations by executing computer-executable instructions 912 (e.g., an application, software, code, and/or other executable data instance) stored in storage device 906.

[0090] Storage device 906 may include one or more data storage media, devices, or configurations and may employ any type, form, and combination of data storage media and/or device. For example, storage device 906 may include, but is not limited to, any combination of the non-volatile media and/or volatile media described herein. Electronic data, including data described herein, may be temporarily and/or permanently stored in storage device 906. For example, data representative of computer-executable instructions 912 configured to direct processor 904 to perform any of the operations described herein may be stored within storage device 906. In some examples, data may be arranged in one or more databases residing within storage device 906.

[0091] I/O module 908 may include one or more I/O modules configured to receive user input and provide user output. One or more I/O modules may be used to receive input for a single virtual experience. I/O module 908 may include any hardware, firmware, software, or combination thereof supportive of input and output capabilities. For example, I/O module 908 may include hardware and/or software for capturing user input, including, but not limited to, a keyboard or keypad, a touchscreen component (e.g., touchscreen display), a receiver (e.g., an RF or infrared receiver), motion sensors, and/or one or more input buttons.

[0092] I/O module 908 may include one or more devices for presenting output to a user, including, but not limited to, a graphics engine, a display (e.g., a display screen), one or more output drivers (e.g., display drivers), one or more audio speakers, and one or more audio drivers. In certain embodiments, I/O module 908 is configured to provide graphical data to a display for presentation to a user. The graphical data may be representative of one or more graphical user interfaces and/or any other graphical content as may serve a particular implementation.

[0093] In some examples, any of the systems, computing devices, and/or other components described herein may be implemented by computing device 900. For example, memory 402 may be implemented by storage device 906, and processor 404 may be implemented by processor 904.

[0094] It will be recognized by those of ordinary skill in the art that while, in the preceding description, various illustrative embodiments have been described with reference to the accompanying drawings. It will, however, be evident that various modifications and changes may be made thereto, and additional embodiments may be implemented, without departing from the scope of the invention as set forth in the claims that follow. For example, certain features of one embodiment described herein may be combined with or substituted for features of another embodiment described herein. The description and drawings are accordingly to be regarded in an illustrative rather than a restrictive sense.

[0095] Advantages and features of the present disclosure can be further described by the following examples:

[0096] Example 1. A system for multiplexed mass spectrometry, comprising: one or more processors; and memory storing executable instructions that, when executed by the one or more processors, cause a computing device to perform a process comprising: generating, based on an observed elution peak for a precursor m/z and an elution peak simulation model: a first simulated elution peak that contributes to the observed elution peak and that represents elution of analytes labeled with a deuterated isobaric mass tag; and a second simulated elution peak that contributes to the observed elution peak and that represents elution of analytes labeled with a non-deuterated isobaric mass tag; determining, based on the first simulated elution peak and a timing of acquiring an MSn spectrum for the precursor m/z relative to the observed elution peak, a first correction factor; determining, based on the second simulated elution peak and the timing of acquiring the MSn spectrum for the precursor m/z relative to the observed elution peak, a second correction factor; adjusting, based on the first correction factor, a first observed signal, within the MSn spectrum, for a first reporter ion derived from the deuterated isobaric mass tag; and adjusting, based on the second correction factor, a second observed signal, within the MSn spectrum, for a second reporter ion derived from the non-deuterated isobaric mass tag.

[0097] Example 2. The system of any of the preceding examples, wherein generating the first simulated elution peak and the second simulated elution peak comprises: determining a retention time difference between the first simulated elution peak and the second simulated elution peak; and determining, based on the retention time difference between the first simulated elution peak and the second simulated elution peak, a position of the first simulated elution peak and a position of the second simulated elution peak relative to the observed elution peak.

[0098] Example 3. The system of any of the preceding examples, wherein determining the first correction factor and determining the second correction factor comprises: determining, based on the timing of acquiring the MSn spectrum relative to the observed elution peak, a timing of acquiring the MSn spectrum relative to the first simulated elution peak and a timing of acquiring the MSn spectrum relative to the second simulated elution peak.

[0099] Example 4. The system of any of the preceding examples, wherein: the first correction factor is a ratio of a maximum intensity of the first simulated elution peak to an intensity of the first simulated elution peak at the timing of acquiring the MSn spectrum; and the second correction factor is a ratio of a maximum intensity of the second simulated elution peak to an intensity of the second simulated elution peak at the timing of acquiring the MSn spectrum. [0100] Example 5. The system of any of the preceding examples, wherein one or both of the first simulated elution peak and the second simulated elution peak has a Gaussian profile.

[0101] Example 6. The system of any of the preceding examples, wherein one or both of the first simulated elution peak and the second simulated elution peak has a non-Gaussian profile. [0102] Example 7. The system of any of the preceding examples, wherein the observed elution peak is based on an MS1 mass analysis of the precursor m/z.

[0103] Example 8. The system of any of examples 1-6, wherein the observed elution peak is based on a DIA analysis.

[0104] Example 9. The system of any of examples 1-6, wherein the observed elution peak comprises a parallel reaction monitoring (PRM) trace.

[0105] Example 10. The system of any of the preceding examples, wherein the deuterated isobaric mass tag and the non-deuterated isobaric mass tag comprise at least one of ¹³C, ¹⁵N, or ¹⁸O isotopes. [0106] Example 11. The system of any of the preceding examples, wherein a reporter region of each of the deuterated isobaric mass tag and the non-deuterated isobaric mass tag has a mass between about 126 Da and about 135 Da.

[0107] Example 12. A non-transitory computer-readable medium storing instructions that, when executed, direct at least one processor of a computing device for mass spectrometry to perform a method comprising: generating, based on an observed elution peak for a precursor m/z and an elution peak simulation model: a first simulated elution peak that contributes to the observed elution peak and that represents elution of analytes labeled with a deuterated isobaric mass tag; and a second simulated elution peak that contributes to the observed elution peak and that represents elution of analytes labeled with a non-deuterated isobaric mass tag; determining, based on the first simulated elution peak and a timing of acquiring an MSn spectrum for the precursor m/z relative to the observed elution peak, a first correction factor; determining, based on the second simulated elution peak and the timing of acquiring the MSn spectrum for the precursor m/z relative to the observed elution peak, a second correction factor; adjusting, based on the first correction factor, a first observed signal, within the MSn spectrum, for a first reporter ion derived from the deuterated isobaric mass tag; and adjusting, based on the second correction factor, a second observed signal, within the MSn spectrum, for a second reporter ion derived from the non-deuterated isobaric mass tag.

[0108] Example 13. The non-transitory computer-readable medium of example 12, wherein generating the first simulated elution peak and the second simulated elution peak comprises: determining a retention time difference between the first simulated elution peak and the second simulated elution peak; and determining, based on the retention time difference between the first simulated elution peak and the second simulated elution peak, a position of the first simulated elution peak and a position of the second simulated elution peak relative to the observed elution peak.

[0109] Example 14. The non-transitory computer-readable medium of any of examples 12 and 13, wherein determining the first correction factor and determining the second correction factor comprises: determining, based on the timing of acquiring the MSn spectrum relative to the observed elution peak, a timing of acquiring the MSn spectrum relative to the first simulated elution peak and a timing of acquiring the MSn spectrum relative to the second simulated elution peak.

[0110] Example 15. The non-transitory computer-readable medium of example 14, wherein: the first correction factor is a ratio of a maximum intensity of the first simulated elution peak to an intensity of the first simulated elution peak at the timing of acquiring the MSn spectrum; and the second correction factor is a ratio of a maximum intensity of the second simulated elution peak to an intensity of the second simulated elution peak at the timing of acquiring the MSn spectrum. [0111] Example 16. The non-transitory computer-readable medium of any of examples 12-

15, wherein one or both of the first simulated elution peak and the second simulated elution peak has a Gaussian profile.

[0112] Example 17. The non-transitory computer-readable medium of any of examples 12-

16, wherein one or both of the first simulated elution peak and the second simulated elution peak has a non-Gaussian profile.

[0113] Example 18. The non-transitory computer-readable medium of any of examples 12-

17, wherein the observed elution peak is based on an MS1 mass analysis of the precursor m/z. [0114] Example 19. The non-transitory computer-readable medium of any of examples 12- 17, wherein the observed elution peak is based on a DIA analysis.

[0115] Example 20. The non-transitory computer-readable medium of any of examples 12- 17, wherein the observed elution peak comprises a parallel reaction monitoring (PRM) trace.

[0116] Example 21. The non-transitory computer-readable medium of any of examples 12-

20, wherein the deuterated isobaric mass tag and the non-deuterated isobaric mass tag comprise at least one of ¹³C, ¹⁵N, or ¹⁸O isotopes.

[0117] Example 22. The non-transitory computer-readable medium of any of examples 12-

21, wherein a reporter region of each of the deuterated isobaric mass tag and the nondeuterated isobaric mass tag has a mass between about 126 Da and about 135 Da.

[0118] Example 23. A method comprising: generating, based on an observed elution peak for a precursor m/z and an elution peak simulation model: a first simulated elution peak that contributes to the observed elution peak and that represents elution of analytes labeled with a deuterated isobaric mass tag; and a second simulated elution peak that contributes to the observed elution peak and that represents elution of analytes labeled with a non-deuterated isobaric mass tag; determining, based on the first simulated elution peak and a timing of acquiring an MSn spectrum for the precursor m/z relative to the observed elution peak, a first correction factor; determining, based on the second simulated elution peak and the timing of acquiring the MSn spectrum for the precursor m/z relative to the observed elution peak, a second correction factor; adjusting, based on the first correction factor, a first observed signal, within the MSn spectrum, for a first reporter ion derived from the deuterated isobaric mass tag; and adjusting, based on the second correction factor, a second observed signal, within the MSn spectrum, for a second reporter ion derived from the non-deuterated isobaric mass tag.

[0119] Example 24. A system for multiplexed mass spectrometry, comprising: one or more processors; and memory storing executable instructions that, when executed by the one or more processors, cause a computing device to perform a process comprising: obtaining mass spectra data representative of: an observed elution profile for a population of ions having a precursor m/z, wherein the population of ions includes a first population of ions labeled with a deuterated isobaric mass tag and a second population of ions labeled with a non-deuterated isobaric mass tag; and an MSn spectrum for the precursor m/z, wherein the MSn spectrum includes a first observed signal for reporter ions derived from ions included in the first population of ions and a second observed signal for reporter ions derived from ions included in the second population of ions; determining an elution time separation between an elution time of the first population of ions and an elution time of the second population of ions; determining a first correction factor and a second correction factor based on the observed elution profile for the population of ions, a timing of acquiring the MSn spectrum relative to an observed elution time of the population of ions, and the elution time separation; adjusting the first observed signal based on the first correction factor; and adjusting the second observed signal based on the second correction factor.

[0120] Example 25. The system of example 24, wherein: the determining the first correction factor comprises determining, based on the elution time separation and the timing of acquiring the MSn spectrum relative to the observed elution time of the population of ions, a timing of acquiring the MSn spectrum relative to the elution time of the first population of ions; and the determining the second correction factor comprises determining, based on the elution time separation and the timing of acquiring the MSn spectrum relative to the observed elution time of the population of ions, a timing of acquiring the MSn spectrum relative to the elution time of the second population of ions.

[0121] Example 26. A system for multiplexed mass spectrometry, comprising: one or more processors; and memory storing executable instructions that, when executed by the one or more processors, cause a computing device to perform a process comprising: obtaining a mass spectrum comprising, for each channel of a plurality of isobaric mass tag channels, an observed reporter ion signal for analytes labeled with an isobaric mass tag, wherein the plurality of isobaric mass tag channels includes a first set of channels for deuterated isobaric mass tags and a second set of channels for non-deuterated isobaric mass tags; determining a correction factor based on a ratio of the observed reporter ion signal for a first bridge channel included in the first set of channels to the observed reporter ion signal for a second bridge channel included in the second set of channels, wherein analytes labeled with the deuterated isobaric mass tag of the first bridge channel and analytes labeled with the non-deuterated isobaric mass tag of the second bridge channel are derived from a same sample; and adjusting, based on the correction factor, the observed reporter ion signal for each channel included in the second set of channels or for each channel included in the first set of channels.

Claims

CLAIMS What is claimed is:

1. A system for multiplexed mass spectrometry, comprising: one or more processors; and memory storing executable instructions that, when executed by the one or more processors, cause a computing device to perform a process comprising: generating, based on an observed elution peak for a precursor m/z and an elution peak simulation model: a first simulated elution peak that contributes to the observed elution peak and that represents elution of analytes labeled with a deuterated isobaric mass tag; and a second simulated elution peak that contributes to the observed elution peak and that represents elution of analytes labeled with a non-deuterated isobaric mass tag; determining, based on the first simulated elution peak and a timing of acquiring an MSn spectrum for the precursor m/z relative to the simulated elution peak, a first correction factor; determining, based on the second simulated elution peak and the timing of acquiring the MSn spectrum for the precursor m/z relative to the simulated elution peak, a second correction factor; adjusting, based on the first correction factor, a first observed signal, within the MSn spectrum, for a first reporter ion derived from the deuterated isobaric mass tag; and adjusting, based on the second correction factor, a second observed signal, within the MSn spectrum, for a second reporter ion derived from the non-deuterated isobaric mass tag.

2. The system of claim 1 , wherein generating the first simulated elution peak and the second simulated elution peak comprises: determining a retention time difference between the first simulated elution peak and the second simulated elution peak; and determining, based on the retention time difference between the first simulated elution peak and the second simulated elution peak, a position of the first simulated elution peak and a position of the second simulated elution peak relative to the observed elution peak.

3. The system of claim 1 , wherein determining the first correction factor and determining the second correction factor comprises: determining, based on the timing of acquiring the MSn spectrum relative to the observed elution peak, a timing of acquiring the MSn spectrum relative to the first simulated elution peak and a timing of acquiring the MSn spectrum relative to the second simulated elution peak.

4. The system of claim 3, wherein: the first correction factor is a ratio of a maximum intensity of the first simulated elution peak to an intensity of the first simulated elution peak at the timing of acquiring the MSn spectrum; and the second correction factor is a ratio of a maximum intensity of the second simulated elution peak to an intensity of the second simulated elution peak at the timing of acquiring the MSn spectrum.

5. The system of claim 1 , wherein one or both of the first simulated elution peak and the second simulated elution peak has a Gaussian profile.

6. The system of claim 1 , wherein one or both of the first simulated elution peak and the second simulated elution peak has a non-Gaussian profile.

7. The system of claim 1, wherein the observed elution peak is based on an MS1 mass analysis of the precursor m/z.

8. The system of claim 1 , wherein the deuterated isobaric mass tag and the nondeuterated isobaric mass tag comprise at least one of ¹³C, ¹⁵N, or ¹⁸O isotopes.

9. The system of claim 1 , wherein a reporter region of each of the deuterated isobaric mass tag and the non-deuterated isobaric mass tag has a mass between about 126 Da and about 135 Da.

10. A non-transitory computer-readable medium storing instructions that, when executed, direct at least one processor of a computing device for mass spectrometry to perform a method comprising: generating, based on an observed elution peak for a precursor m/z and an elution peak simulation model: a first simulated elution peak that contributes to the observed elution peak and that represents elution of analytes labeled with a deuterated isobaric mass tag; and a second simulated elution peak that contributes to the observed elution peak and that represents elution of analytes labeled with a non-deuterated isobaric mass tag; determining, based on the first simulated elution peak and a timing of acquiring an MSn spectrum for the precursor m/z relative to the first simulated elution peak, a first correction factor; determining, based on the second simulated elution peak and the timing of acquiring the MSn spectrum for the precursor m/z relative to the second simulated elution peak, a second correction factor; adjusting, based on the first correction factor, a first observed signal, within the MSn spectrum, for a first reporter ion derived from the deuterated isobaric mass tag; and adjusting, based on the second correction factor, a second observed signal, within the MSn spectrum, for a second reporter ion derived from the non-deuterated isobaric mass tag.

11. The non-transitory computer-readable medium of claim 10, wherein generating the first simulated elution peak and the second simulated elution peak comprises: determining a retention time difference between the first simulated elution peak and the second simulated elution peak; and determining, based on the retention time difference between the first simulated elution peak and the second simulated elution peak, a position of the first simulated elution peak and a position of the second simulated elution peak relative to the observed elution peak.

12. The non-transitory computer-readable medium of claim 10, wherein determining the first correction factor and determining the second correction factor comprises: determining, based on the timing of acquiring the MSn spectrum relative to the observed elution peak, a timing of acquiring the MSn spectrum relative to the first simulated elution peak and a timing of acquiring the MSn spectrum relative to the second simulated elution peak.

13. The non-transitory computer-readable medium of claim 12, wherein: the first correction factor is a ratio of a maximum intensity of the first simulated elution peak to an intensity of the first simulated elution peak at the timing of acquiring the MSn spectrum; and the second correction factor is a ratio of a maximum intensity of the second simulated elution peak to an intensity of the second simulated elution peak at the timing of acquiring the MSn spectrum.

14. The non-transitory computer-readable medium of claim 10, wherein one or both of the first simulated elution peak and the second simulated elution peak has a Gaussian profile.

15. The non-transitory computer-readable medium of claim 10, wherein one or both of the first simulated elution peak and the second simulated elution peak has a non-Gaussian profile.

16. The non-transitory computer-readable medium of claim 10, wherein the observed elution peak is based on an MS1 mass analysis of the precursor m/z.

17. The non-transitory computer-readable medium of claim 10, wherein the deuterated isobaric mass tag and the non-deuterated isobaric mass tag comprise at least one of ¹³C, ¹⁵N, or ¹⁸O isotopes.

18. The non-transitory computer-readable medium of claim 10, wherein a reporter region of each of the deuterated isobaric mass tag and the non-deuterated isobaric mass tag has a mass between about 126 Da and about 135 Da.

19. A system for multiplexed mass spectrometry, comprising: one or more processors; and memory storing executable instructions that, when executed by the one or more processors, cause a computing device to perform a process comprising: obtaining mass spectra data representative of: an observed elution profile for a population of ions having a precursor m/z, wherein the population of ions includes a first population of ions labeled with a deuterated isobaric mass tag and a second population of ions labeled with a non-deuterated isobaric mass tag; and an MSn spectrum for the precursor m/z, wherein the MSn spectrum includes a first observed signal for reporter ions derived from ions included in the first population of ions and a second observed signal for reporter ions derived from ions included in the second population of ions; determining an elution time separation between an elution time of the first population of ions and an elution time of the second population of ions; determining a first correction factor and a second correction factor based on the observed elution profile for the population of ions, a timing of acquiring the MSn spectrum relative to an observed elution time of the population of ions, and the elution time separation; adjusting the first observed signal based on the first correction factor; and adjusting the second observed signal based on the second correction factor.

20. The system of claim 19, wherein: the determining the first correction factor comprises determining, based on the elution time separation and the timing of acquiring the MSn spectrum relative to the observed elution time of the population of ions, a timing of acquiring the MSn spectrum relative to the elution time of the first population of ions; and the determining the second correction factor comprises determining, based on the elution time separation and the timing of acquiring the MSn spectrum relative to the observed elution time of the population of ions, a timing of acquiring the MSn spectrum relative to the elution time of the second population of ions.