Optimization of acquisition window width for targeted mass spectrometry

Abstract

To enable generation of an acquisition schedule for a targeted assay of a sample.SOLUTION: The acquisition schedule schedules acquisition, by a mass spectrometer during an acquisition window having a dynamic acquisition window width, of a set of mass spectra for each target analyte included in a set of target analytes included in the sample as the target analytes elute from a separation system. Generating the acquisition schedule includes determining the dynamic acquisition window width based on an acquisition cycle period for the targeted assay. The mass spectrometer is directed to acquire each set of mass spectra in accordance with the acquisition schedule.SELECTED DRAWING: Figure 3A

Description

[Technical Field]

[0001] The present invention relates to optimizing acquisition window width for targeted mass analysis. [Background technology]

[0002] Mass spectrometers can be used to detect, identify, and / or quantify molecules based on the mass-to-charge ratio (m / z) of ions generated from the molecules. Mass spectrometers generally include an ion source for generating ions from molecules contained in a sample, a mass analyzer for separating the ions based on their m / z, and an ion detector for detecting the separated ions. Mass spectrometers can include or be connected to a computer-based software platform that uses data from the ion detector to construct a mass spectrum showing the relative abundance of each of the detected ions as a function of m / z. Mass spectra can be used to detect and quantify molecules in simple and complex mixtures. In some configurations, mass spectrometers are used in combination with liquid chromatographs (LCs), gas chromatographs (GMSs), and other similar systems. A separation system, such as a chromatograph (GC) or capillary electrophoresis (CE) system, is coupled to a mass spectrometer in a hybrid system (e.g., an LC-MS, GC-MS, or CE-MS system) to separate the analytes in a sample before the analytes are introduced into 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 multistage mass spectrometry (MSn, where n is 2 or greater) or tandem mass spectrometry (MS / MS or MS2 (n=2)), specific ions (called precursor ions) are isolated and fragmented in a controlled manner to yield product ions. Mass spectrometry is then performed on the product ions to generate a mass spectrum of the product ions. The mass spectrum of the product ions provides information that can be used to confirm the identity, determine the quantity, and / or derive structural details about the analyte of interest.

[0004] Various techniques can be used to acquire mass spectra using multistage or tandem mass spectrometry. One commonly used technique is data-dependent acquisition (DDA), which uses data acquired in a single mass analysis to select one or more ion species, or narrow m / z ranges, for isolation and fragmentation based on predetermined criteria. For example, a mass spectrometer may perform a full MS survey scan of precursor ions over a wide precursor m / z range and select one or more precursor ion species from the resulting mass spectrum for isolation, fragmentation, and mass analysis based on the MS survey scan. Precursor ion species selection criteria may include, for example, intensity, charge state, m / z, an inclusion / exclusion list, or isotopic pattern. A major drawback of DDA techniques is the inherently random nature of their results. When technical repeats of the same sample or comparative analyses of other samples are performed, some analytes may be measured in one experiment but not in another. This frustrates attempts to perform reproducible analyses and is known as the "missing value problem."

[0005] In contrast to DDA, data independent acquisition DIA (Distributed Infrared Acquisition) is a technique in which all precursor ion species within a wide precursor m / z range (e.g., 600-900 m / z) are isolated and fragmented via sequentially advancing an isolation window of fixed m / z width (e.g., 20 m / z) to generate product ions. Mass analysis is then performed on the product ions in a systematic and unbiased manner. The isolation of precursor ions across the entire precursor m / z range, fragmentation of the isolated precursor ions, and mass analysis of the product ions constitute one acquisition cycle, which is repeated to generate a mass spectrum of product ions. In DIA techniques, the isolation and fragmentation of one or more precursor ion species does not rely on data acquired in survey mass analysis as in DDA, and DIA techniques do not suffer from missing data problems, making them much more suitable for comparing results across different samples than DDA.

[0006] In contrast to DDA and DIA, targeted mass spectrometry is a technique in which mass spectrometry is performed on a fixed, known list of analytes contained in a sample. Targeted mass spectrometry experiments are designed to gather quantitative information about a set of analytes, the identities of which are known before the experiment begins. Typically, a sample is introduced into a separation system (e.g., an LC system), which separates the analytes and introduces them into a mass spectrometer as they elute from the separation system. Given some knowledge of the expected elution times of the analytes from the separation system, an acquisition schedule can be created that specifies which analytes the mass spectrometer will target for mass analysis at which times during the experiment. Analytes that are part of such a targeted assay are often referred to as "targets" or "target analytes." During an experiment, a target is said to be "active" if the elapsed time is between the target's elution start and stop times. This helps utilize instrument resources more efficiently than if mass spectra were acquired sequentially for all targets in the assay.

[0007] However, due to time-dependent variations in the separation system, such as the solvent composition and stationary phase conditions in an LC system, the analyte elution time can change, a phenomenon known as retention time shift or elution time drift. To accommodate elution time drift, various techniques have been developed for adjusting scheduled acquisition windows in real time. A conventional technique for correcting elution time drift involves adjusting the acquisition schedule in real time using data acquired during an experiment. However, such techniques can be adversely affected during periods of low analyte concentration, which can result in poor and / or insufficient data being generated for elution time drift correction. Summary of the Invention

[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 not intended to identify key or critical elements of all aspects or to 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 non-transitory computer-readable medium stores instructions that, when executed, instruct at least one processor of a computing device for mass spectrometry to: generate an acquisition schedule for a target assay of the sample, the acquisition schedule scheduling acquisition by a mass spectrometer of a set of mass spectra of each target analyte included in a set of target analytes included in the sample as the target analytes elute from a separation system, within an acquisition window having a dynamic acquisition window width, wherein generating the acquisition schedule includes determining the dynamic acquisition window width based on an acquisition cycle period of the target assay; and instructing the mass spectrometer to acquire each set of mass spectra in accordance with the acquisition schedule.

[0010] In some illustrative examples, a method for performing targeted mass analysis includes generating an acquisition schedule for a target assay of a sample, the acquisition schedule scheduling acquisition by a mass spectrometer of a set of mass spectra of each target analyte included in a set of target analytes included in the sample as the target analytes elute from a separation system within an acquisition window having a dynamic acquisition window width, wherein generating the acquisition schedule includes determining the dynamic acquisition window width based on an acquisition cycle period of the target assay, and instructing the mass spectrometer to acquire each set of mass spectra in accordance with the acquisition schedule.

[0011] In some illustrative examples, a system for mass spectrometry comprises one or more processors and a memory storing executable instructions that, when executed by the one or more processors, cause a computing device to: generate an acquisition schedule for a target assay of a sample, the acquisition schedule scheduling acquisition by a mass spectrometer of a set of mass spectra of each target analyte included in a set of target analytes contained in a sample as the target analytes elute from a separation system within an acquisition window having a dynamic acquisition window width, wherein generating the acquisition schedule includes determining the dynamic acquisition window width based on an acquisition cycle period of the target assay; and instructing the mass spectrometer to acquire each set of mass spectra in accordance with the acquisition schedule. [Brief explanation of the drawings]

[0012] The accompanying drawings illustrate various embodiments and are a part of this specification. The illustrated embodiments are merely examples and are not intended to limit the scope of the present disclosure. Throughout the drawings, the same or similar reference numbers refer to the same or similar elements. [Figure 1] FIG. 1 shows a functional diagram of an exemplary LC-MS system. [Figure 2] 2 shows a functional diagram of an exemplary implementation of a mass spectrometer included in the LC-MS system of FIG. 1. [Figure 3A] A portion of an exemplary elution profile for selected m / z is shown. [Figure 3B] 3B shows a frequency domain representation of the elution profile of FIG. 3A. [Figure 4] Three exemplary heat maps are shown depicting the cross-correlation of real-time mass spectral data with reference mass spectral data for three different experiments performed in close succession on a set of 2,972 target peptides. [Figure 5]Graphs are shown plotting the number of simultaneous precursor ions as a function of time and the instrument time required to mass analyze all simultaneous precursor ions during an acquisition cycle for the target assay of FIG. [Figure 6] FIG. 1 shows a functional diagram of an exemplary target MS control system. [Figure 7] 1 illustrates an exemplary method for performing targeted mass analysis using an optimized acquisition window width. [Figure 8] 1 illustrates an exemplary method for determining a dynamic acquisition window width. [Figure 9] 10 illustrates another exemplary method for determining a dynamic acquisition window width. [Figure 10] 10 illustrates another exemplary method for determining a dynamic acquisition window width. [Figure 11] Graphs plotting instrument time as a function of elapsed time and acquisition window width as a function of elapsed time are shown for both constant and dynamic acquisition window widths. [Figure 12] 1 illustrates an exemplary computing device. DETAILED DESCRIPTION OF THE INVENTION

[0013] Described herein are systems and methods for performing targeted mass analysis with improved acquisition scheduling and optimized acquisition window widths. The acquisition window width of a target assay is optimized by using a dynamic acquisition window width (e.g., a time-dependent or time-varying acquisition window width) determined based on the assay's acquisition cycle duration. The dynamic acquisition window width based on the acquisition cycle duration compensates for variations in elution time drift during periods of low analyte concentration while ensuring that the instrument time required to mass analyze all active targets at any given time during the assay does not exceed the assay's acquisition cycle duration. Compared to a constant acquisition window width, the dynamic acquisition window width improves correction of elution time drift without sacrificing data quality or target analyte throughput.

[0014] Various examples will now be described in more detail with reference to the drawings. The systems and methods described herein may provide one or more of the advantages set forth above and / or various additional and / or alternative advantages as set forth herein.

[0015] Targeted mass analysis with improved acquisition scheduling and optimized acquisition window widths is performed using a combined separation-mass spectrometry system, such as an LC-MS system. Accordingly, an LC-MS system will now be described. The described LC-MS system is exemplary and non-limiting. The methods and systems described herein may operate as part of or in conjunction with the LC-MS systems described herein and / or any other suitable separation-mass spectrometry system, such as a high-performance liquid chromatography-mass spectrometry (HPLC-MS) system, a gas chromatography-mass spectrometry (GC-MS) system, or a capillary electrophoresis-mass spectrometry (CE-MS) system. The methods and systems described herein can also work in conjunction with any other continuous flow sample source, such as a flow-injection mass spectrometry system (FI-MS), in which analytes are injected into a mobile phase (without separation in a column) and enter the mass spectrometer with time-dependent intensity fluctuations (e.g., Gaussian-like peaks).

[0016] FIG. 1 shows a functional diagram of an exemplary LC-MS system 100. The LC-MS system 100 includes a liquid chromatograph 102, a mass spectrometer 104, and a controller 106. The liquid chromatograph 102 is configured to separate analytes over time in a sample 108 injected into the liquid chromatograph 102. The sample 108 may include, for example, chemical analytes (e.g., molecules, ions, etc.) and / or biological analytes (e.g., metabolites, proteins, peptides, lipids, etc.) for detection and analysis by the LC-MS system 100. The liquid chromatograph 102 may be implemented by any liquid chromatograph compatible with a particular implementation. In the liquid chromatograph 102, the sample 108 is injected into a mobile phase (e.g., a solvent), which carries the sample 108 through a column 110 containing a stationary phase (e.g., an adsorbent packing material). As the mobile phase passes through the column 110, the analytes in the sample 108 elute from the column 110 at different times based on, for example, their size, affinity for the stationary phase, polarity, and / or hydrophobicity.

[0017] A detector (e.g., an ion detector component of the mass spectrometer 104, an ion-electron converter, and an electron multiplier) can measure the relative intensities of signals modulated by the separated analytes in the eluate 112 from the column 110. The data generated by the detector can be represented as a chromatogram plotting retention time on the x-axis and a signal representing relative intensity on the y-axis. The retention time of an analyte is generally measured as the period between injection of the sample 108 into the mobile phase and the relative intensity peak maximum after chromatographic separation. In some examples, the relative intensity can correlate to or represent the relative abundance of the separated analytes. The data generated by the liquid chromatograph 102 is output to the controller 106.

[0018] In some cases, particularly in the analysis of complex mixtures, multiple different analytes in sample 108 may co-elute from column 110 at approximately the same time and therefore have the same or similar retention times. As a result, determining the relative intensities of individual analytes in sample 108 requires further separation of signals attributable to individual analytes. To this end, liquid chromatograph 102 directs the analytes contained in eluate 112 to mass spectrometer 104 for further separation, identification, and / or quantification of the analytes.

[0019] The mass spectrometer 104 generates ions from the analytes received from the liquid chromatograph 102 and classifies or separates the generated ions based on their m / z. The mass spectrometer 104 may be implemented by a multi-stage mass spectrometer configured to perform multi-stage mass spectrometry (also denoted MSn, where n is 2 or greater) 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 within the mass spectrometer 104 measures the intensity of signals generated by the ions. As used herein, "intensity" or "signal strength" refers to the detector response and may represent absolute abundance, relative abundance, ion number, intensity, relative intensity, ion current, or any other suitable measure of ion detection. Data acquired by the mass spectrometer 104 is output to the controller 106. The data generated by the detector may be represented by a mass spectrum, which plots the intensity of the observed signal as a function of the m / z of the detected ions.

[0020] 2 shows a functional diagram of an exemplary implementation of mass spectrometer 104. 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 that may be compatible with a particular embodiment (e.g., ion optics, filters, ion storage, autosamplers, detectors, etc.).

[0021] The ion source 202 generates a stream of ions 208 from the analytes received from the column 110 and delivers the ions to the first mass analyzer 204-1. The ion source 202 may use any suitable ionization technique, including, but not limited to, electron ionization, chemical ionization, matrix-assisted laser desorption / ionization, electrospray ionization, atmospheric pressure chemical ionization, atmospheric pressure photoionization, inductively coupled plasma, etc. The ion source 202 may include various components for generating ions from the analytes contained in the sample 108 and delivering the ions to the first mass analyzer 204-1.

[0022] The first mass analyzer 204-1 receives the ion stream 208, isolates precursor ions in a selected m / z range, and delivers a beam 210 of precursor ions to the collision cell 204-2. The collision cell 204-2 receives the beam 210 of precursor ions and generates product ions (e.g., fragment ions) through a controlled dissociation process. The collision cell 204-2 directs a beam 212 of product ions to the second mass analyzer 204-3. The second mass analyzer 204-3 filters and / or performs mass analysis on the product ions.

[0023] Mass analyzers 204-1 and 204-3 isolate or separate ions according to the m / z of each ion. Mass analyzers 204-1 and 204-3 can 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 or a Kingdon trap), a Fourier transform ion cyclotron resonance (FT-ICR) mass analyzer, etc. Mass analyzers 204-1 and 204-3 do not have to be implemented by the same type of mass analyzer.

[0024] 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 generate product ions via a controlled dissociation process, but is not limited to devices used for collision-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, etc.

[0025] An ion detector (not shown) detects ions at each of a variety of different m / z and accordingly generates an electrical signal representing the ion intensity. This electrical signal is sent to the controller 206 for processing, such as constructing a mass spectrum of the analyzed ions. For example, the mass analyzer 204-3 can emit an ejected beam of separated ions to an ion detector, which is configured to detect ions in the ejected beam and generate or provide data that the controller 206 can use to construct a mass spectrum. The ion detector can be implemented by any suitable detection device, including, but not limited to, an electron multiplier, a Faraday cup, or the like. In other examples, such as when the second mass analyzer 204-3 is implemented by an orbital electrostatic trap mass analyzer, the second mass analyzer 204-3 functions as both a mass analyzer and a detector.

[0026] The controller 206 is communicatively coupled to the mass spectrometer 104 and configured to control the operation of the mass spectrometer. For example, the controller 206 may be configured to control the operation of various hardware components included in the ion source 202 and / or the mass analyzers 204-1 and 204-3. By way of example, the controller 206 may be configured to control the accumulation time of the ion source 202 and / or the mass analyzer 204, control the oscillating voltage power supply and / or the DC power supply to supply RF and / or DC voltage to the mass analyzer 204, adjust the values ​​of the RF and DC voltages to select a valid m / z (including a mass acceptance window) for analysis, and adjust the sensitivity of the ion detector (e.g., by adjusting the detector gain).

[0027] The controller 206 may include any suitable hardware (e.g., processor, circuitry, etc.) and / or software as may be useful for a particular implementation. While Figure 2 illustrates the controller 206 being included within the mass spectrometer 104, the controller 206 may alternatively be implemented completely or partially separate from the mass spectrometer 104, such as by a computing device communicatively coupled to the mass spectrometer 104 via a wired connection (e.g., 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, the controller 206 is implemented completely or partially by the controller 106.

[0028] 2, the mass spectrometer 104 is tandem in space (e.g., has multiple mass analyzers) and has two stages for performing tandem mass analysis. However, the mass spectrometer 104 is not limited to this configuration and may have any other suitable configuration. For example, the mass spectrometer 104 may be tandem in time. Additionally or alternatively, the 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 tandem mass analysis (e.g., MS / MS / MS).

[0029] 1 , controller 106 is communicatively coupled to and configured to control the operation of LC-MS system 100 (e.g., liquid chromatograph 102 and mass spectrometer 104). Controller 106 may include any suitable hardware (e.g., processor, circuitry, etc.) and / or software configured to control the operation of and / or interface with the various components of LC-MS system 100 (e.g., liquid chromatograph 102 or mass spectrometer 104).

[0030] 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. A user may interact with controller 106 and / or 206 via a user interface that utilizes tactile, visual, auditory, and / or other sensory communication. For example, the user interface may include a display device (e.g., a liquid crystal display (LCD) screen, a touch screen, etc.) for displaying information (e.g., mass spectra, notifications, etc.) to the user. The user interface may also include input devices (e.g., a keyboard, a mouse, a touch screen device, etc.) that enable a 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 controller 106 and / or controller 206 but may be communicatively coupled to the controller. For example, the display device and input device may be included in a computer (e.g., a desktop computer, a laptop computer, etc.) that is communicatively connected to the controllers 106 and / or 206 via a wired connection (e.g., by one or more cables) and / or a wireless connection.

[0031] The controller 106 acquires data acquired over time by the LC-MS system 100. The data may include a series of mass spectra including intensity values ​​of ions generated from analytes in the sample 108 as a function of the ions' m / z. The series of mass spectra may be represented in a three-dimensional map in which elution time (e.g., retention time) is plotted along the map's X-axis, m / z is plotted along the map's Y-axis, and intensity is plotted along the map's Z-axis. Spectral features on the map (e.g., Z-axis peaks in intensity) represent the detection by the LC-MS system 100 of ions generated from various analytes contained in the sample 108. The map's X- and Z-axes may be used to generate an elution profile (e.g., a mass chromatogram) that plots detected intensity as a function of time for a selected m / z.

[0032] As used herein, "selected 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 isolated window with a width or range of 20 m / z, 10 m / z, 4 m / z, 3 m / z, etc.). In targeted MS2 or MSn analyses, such as selected reaction monitoring (SRM) analyses, multiple reaction monitoring (MRM) analyses, or parallel reaction monitoring (PRM) analyses, the selected m / z corresponds to the m / z of the product ions of distinct transitions (precursor ion / product ion pairs), and the recorded intensity as a function of time vector (e.g., trace) represents the elution profile of the distinct transitions. The Y and Z axes of the map may be used to generate mass spectra, and each mass spectrum may plot intensity as a function of m / z for a particular acquisition.

[0033] As described above, targeted mass spectrometry experiments are designed to gather quantitative information about a set of analytes, the identities of which are known before the experiment begins. The amount of an analyte can be determined by integrating the area under its elution peak. In some examples, quantifying an analyte involves summing the detected signals for multiple different selected m / z values ​​for product ions characteristic of the analyte of interest and integrating the area under the summed signals. For example, an analyte of interest may have multiple characteristic transitions, each of which can be summed to form an elution profile with an increased signal-to-noise ratio. Thus, as used herein, a "selected m / z" can also be a combination of multiple distinct m / z values ​​or m / z ranges. For example, the selected m / z value for the analyte of interest can be a combination of multiple different m / z values ​​or m / z ranges for each ion characteristic of the analyte of interest, and the elution profile for the selected m / z value can be the sum signal of each different m / z value or m / z range. In a further example, the multiple distinct m / z values ​​or m / z ranges span the entire m / z spectrum, and the elution profile is a total ion current (TIC).

[0034] As used herein, "acquisition" refers to mass analysis performed at discrete time points to acquire a single mass spectrum across an m / z range of interest (e.g., a selected m / z). It is recognized that in some targeted MS2 analyses, a true "spectrum" is not acquired, in that the detected intensity as a function of time is acquired or recorded only for a selected m / z, rather than for a broad m / z spectrum. Nevertheless, for ease of discussion herein, the recorded intensity versus time vector for such targeted MS2 analyses will be referred to herein as a mass spectrum.

[0035] Multiple acquisitions for a selected m / z may be acquired at a sampling rate. The sampling rate of an elution profile for a selected m / z will now be described with reference to FIGS. 3A and 3B. FIG. 3A shows a portion of an exemplary elution profile 300 (indicated by the solid curve) for a selected m / z. The elution profile 300 is generated from data acquired by a mass spectrometer, such as multiple MS2 acquisitions. The elution profile 300 plots intensity amplitude (arbitrary units) as a function of time. Time is generally measured starting from an initialization event, e.g., injection of a sample into a separation system. As shown in FIG. 3A, the elution profile 300 includes multiple acquisition points 302, each obtained by a separate acquisition. As an analyte elutes from the separation system, the detected intensity of ions generated from the analyte forms an elution peak 304 having an approximately Gaussian profile. However, the elution peak 304 and / or other elution peaks (not shown) in the elution profile 300 may have other, non-Gaussian profiles.

[0036] As used herein, "sampling rate" refers to the number of acquisitions per unit time for a selected m / z. In the example of FIG. 3A, the sampling rate is approximately 0.2 Hz (4 acquisitions every 20 seconds). The sampling rate may also be expressed as the number of acquisitions per elution peak. An elution peak may be defined as any detected signal above a threshold (e.g., 6% or 10% above the baseline signal). In FIG. 3A, the elution peak 304 has a peak width W of approximately 30 seconds (e.g., 35 seconds to 65 seconds). peak The sampling rate in FIG. 3A can be expressed as 6 acquisitions per peak.

[0037] As used herein, an "acquisition cycle period" is the duration between successive acquisitions (e.g., between successive acquisition points 302) in the elution profile of a selected m / z. In the example of FIG. 3A, the acquisition cycle period is approximately 6 seconds. During an acquisition cycle period, mass analysis (e.g., acquisition) is performed for each of a plurality of different selected m / z (e.g., for each "active" target analyte), generally each at the same sampling rate. Thus, multiple acquisitions are performed for each distinct selected m / z (e.g., distinct target analyte) during an acquisition cycle period, referred to as an acquisition cycle. The acquisition cycle is repeated multiple times to generate an elution profile for each distinct selected m / z.

[0038] As used herein, "instrument speed" refers to the amount of time required for a mass spectrometer to perform one acquisition for a selected m / z. Instrument speed is generally based on mass spectrometer characteristics and parameters, such as mass analysis time and ion injection time and / or dwell time. The instrument speed and acquisition cycle duration determine the number of target analytes or distinct selected m / z that can be analyzed during an acquisition cycle and, therefore, over the course of an experiment.

[0039] Many experiments have sampling rate requirements. As used herein, "sampling rate requirement" refers to the minimum number of acquisitions per unit time for each selected m / z or the minimum number of acquisitions across each elution peak for each selected m / z, as required by the method parameters for the particular experiment being performed. The sampling rate requirement may be informed by, but is not necessarily the same as, the Nyquist limit. In some examples, the Nyquist limit is determined based on a frequency domain representation of the elution profile of a selected m / z. FIG. 3B shows a frequency domain representation 306 of elution profile 300. FIG. 3B may be generated from elution profile 300 in any suitable manner, such as by performing a Fourier transform on elution profile 300. Frequency domain representation 306 includes peak 308 corresponding to elution peak 304. Based on peak 308, the highest frequency to digitally recover may be determined to be 0.1 Hz, as indicated by dashed line 310. Thus, the Nyquist limit is 0.2 Hz (e.g., twice the highest frequency), giving a 6-second acquisition cycle period and a sampling rate of 6 acquisitions across the eluting peak 304. A sampling rate that meets or exceeds the Nyquist limit is estimated to produce data with sufficient precision to accurately determine peak intensities and peak areas, and thus accurately quantify target analytes. It will be recognized that the Nyquist limit may be determined in other ways, and that the sampling rate requirement may differ from the Nyquist limit (e.g., be greater or less than the Nyquist limit). For example, the sampling rate requirement for a particular method may be determined experimentally.

[0040] For most experiments, the sampling rate requirement is at least six acquisitions per elution peak, with many experiments ranging from six to fifteen acquisitions per elution peak. In some instances, such as in the case of a Gaussian-shaped elution profile, the sampling rate requirement is five acquisitions per peak. In other instances, the sampling rate requirement is six acquisitions per peak. In a further instance, the sampling rate requirement is eight acquisitions per peak. In yet a further instance, the sampling rate requirement is ten acquisitions per peak. As discussed above, the sampling rate can also be expressed as the number of acquisitions per unit time. Thus, in some instances, the sampling rate requirement is 0.25 Hz or greater. In a further instance, the sampling rate requirement is 0.30 Hz or greater. In yet a further instance, the sampling rate requirement is 0.40 Hz or greater. In yet a further instance, the sampling rate requirement is 0.50 Hz or greater.

[0041] As used herein, "acquisition window" refers to a segment of time during which mass analysis is performed for a selected m / z (one or more active target analytes) to acquire a set of mass spectra for the selected m / z. The acquisition window is positioned across the elution peak for the selected m / z such that multiple acquisitions are performed for the selected m / z. Typically, the sampling rate for the multiple acquisitions is set to meet the sampling rate requirements for the selected m / z. The width of the acquisition window ("acquisition window width") is typically greater than the elution peak width for the selected m / z, e.g., 2, 3, or 4 times, to capture data both before and after the elution peak. For example, as shown in FIG. 3A, the selected m / z is acquired within an acquisition window width w of approximately 95 seconds (from 0 seconds to 95 seconds). window The acquisition window width w in Figure 3A window It will be appreciated that is merely an example and may take any other suitable value.

[0042] As used herein, "instrument time" refers to the amount of time that a mass spectrometer would need to perform an acquisition cycle for all target analytes scheduled during that acquisition cycle. Instrument time depends on the number of simultaneous targets in an acquisition cycle (e.g., targets scheduled for acquisition during that acquisition cycle) and the instrument speed. Generally, the number of simultaneous targets scheduled for acquisition during an acquisition cycle increases as the acquisition window width increases, because a wider acquisition window can encompass additional eluting peaks.

[0043] In targeted experiments, mass analysis is performed on a fixed, known list of analytes contained in a sample. Given some knowledge of the expected elution times of the analytes from the separation system, an acquisition schedule is created that specifies which analytes the mass spectrometer will target for mass analysis at which times during the experiment (such analytes are referred to as "target analytes"). In chromatographic separation applications, the elution time of an analyte refers to its retention time, which is typically measured as the period between the injection of the sample into the mobile phase and the relative intensity peak maximum after chromatographic separation. In capillary electrophoresis applications, where the analytes are not retained but instead move continuously, the elution time of an analyte refers to its migration time. Migration time is typically measured as the period it takes for the analyte to travel from the beginning of the capillary to the detection location. In ion mobility separations, the elution time of an analyte refers to its drift time through the buffer gas, which can occur either in space (e.g., a drift tube) or in time (e.g., a trapping ion mobility cell).

[0044] Due to time-dependent variations in separation system characteristics, such as solvent composition and stationary phase conditions in an LC separation system, elution times can fluctuate and shift from the expected elution time during a run. This phenomenon of elution time change is called elution time drift. Various techniques have been developed to address elution time drift in real time during an experiment and adjust the acquisition schedule; these techniques are called elution time drift correction. Some techniques involve measuring reference compounds and updating target scheduling windows in real time. For example, a set of standard compounds with reference elution times can be spiked into a sample. Observation of the standard at a new elution time allows the scheduled acquisition window to be updated to account for any elution time drift. Another technique, called elution time ordering, associates target analytes with specific high-intensity analytes or standards. Observation of the standard activates the acquisition window for the associated target. Another technique, called elution time ordering, determines a ranked elution order for all target analytes. Observation of a specific target analyte activates the acquisition windows for nearby target analytes in the ranking. Another technique, called SureQuant (developed by Thermo Fisher Scientific, Waltham, MA), spikes a set of heavy-labeled standards into the sample. Once a specific heavy standard is detected in MS query mode, the mass spectrometer acquires data on the heavy endogenous target analytes in quantitative mode.

[0045] Another technique, developed by Thermo Fisher Scientific, estimates elution time drift and updates the target acquisition window based on cross-correlation of real-time mass spectral data with reference mass spectral data, regardless of the identity of the analyte. Figure 4 shows three exemplary heat maps 400A, 400B, and 400C, depicting the cross-correlation of real-time mass spectral data with reference mass spectral data for three different experiments performed in close succession on a set of 2,972 target peptides. Bright areas in heat map 400 represent high correlations. The deviation of maximum correlation from zero on the y-axis indicates elution time drift relative to the reference mass spectral data.

[0046] As shown in Figure 4, the largest differences between the data sets represented by heat maps 400A, 400B, and 400C are at the beginning of the experiment (e.g., 0 to approximately 7 minutes), when very little target analyte enters the mass spectrometer. These early elution time differences can be problematic for any type of scheduled targeted mass spectrometry experiment, even one that involves real-time elution time drift correction, because there may be insufficient data to use for elution time drift correction. These regions of low analyte concentration and insufficient data for elution time drift correction may also occur at other times during the experiment.

[0047] A simple solution to the problem of insufficient data for correction of elution time drift involves increasing the scheduled acquisition window width for all target analytes in the target assay. However, this approach is unnecessarily overly wide and requires a significant sacrifice of assay capacity because, as explained with reference to Figure 5, a wider acquisition window reduces the total number of target analytes that can be measured in the assay.

[0048] Figure 5 shows graph 500A plotting the number of simultaneous precursor ions as a function of time, and graph 500B plotting the instrument time required to mass analyze all simultaneous precursor ions during an acquisition cycle for the target assay of Figure 4. Assuming an instrument speed of approximately 16.7 milliseconds (ms) and a fixed acquisition window width of 0.4 minutes, the maximum number of simultaneous precursor ions is approximately 100, as shown by curve 502 in graph 500A, and the instrument time required to mass analyze a maximum of 100 precursor ions is 1,670 ms, as shown by curve 504 in graph 500B. Increasing the acquisition window width from 0.4 minutes to 1.4 minutes increases the maximum number of simultaneous precursors to approximately 350, as shown by curve 506 in graph 500A, and correspondingly increases the instrument time required to analyze all precursor ions to approximately 5,000 ms, as shown by curve 508 in graph 500B. Considering that assay capacity is limited by the acquisition cycle duration (based on elution peak width and sampling rate requirements) and instrument speed, data quality must be sacrificed by acquiring 3.3-fold fewer points per elution peak, or assay capacity must be sacrificed by reducing the number of target peptides in the assay 3.3-fold from 2,972 to approximately 900. And even a 1.4-minute acquisition window width may not be wide enough for some of the highly variable early-eluting peptides. For these reasons, adjusting the global acquisition window width is not preferred.

[0049] To address these issues, the acquisition schedule for a target assay specifies a dynamic acquisition window width. The dynamic acquisition window width optimizes the acquisition window width based on the acquisition cycle duration of the target assay. For example, the acquisition window width for one or more target analytes is maximized while keeping the instrument time for mass analysis of all active target analytes at any time during the assay equal to or less than the acquisition cycle duration of the assay. The improved method reduces problems during periods of low analyte concentration, such as at the beginning and end of a target assay. Optimizing the acquisition window width for targeted mass analysis and methods for performing targeted mass analysis using optimized acquisition window widths are described in more detail below.

[0050] As used herein, "optimize" and variations thereof mean seeking an improved or optimal solution from among a set of possible solutions, although the best solution may not necessarily be obtained when the optimization process terminates before finding the best solution, when multiple solutions exist that meet predefined criteria, when a solution meets a minimum criterion, or when the selected optimization technique is unable to converge to the best solution, etc. Similarly, as used herein, an "optimum" parameter (e.g., the "maximum" or "minimum" value of a parameter) refers to a solution obtained as a result of performing an optimization process, and thus may not necessarily be the absolute extreme value of the parameter (e.g., the absolute maximum or minimum value), but may still result in adjusting the parameter to provide an improvement.

[0051] One or more operations associated with optimizing the acquisition window width for a targeted mass analysis may be performed by a target MS control system in conjunction with an MS system (e.g., LC-MS system 100, a GC-MS system, or a CE-MS system). The target MS control system may control and / or perform one or more operations described herein. FIG. 6 shows a functional diagram of an exemplary target MS control system 600 (“system 600”). System 600 may be implemented in whole or in part by an MS system such as LC-MS system 100 (e.g., by controller 106 and / or controller 206). Alternatively, system 600 may be implemented separately from the MS 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).

[0052] System 600 may include, but is not limited to, a memory 602 and a processor 604 selectively and communicatively coupled to each other. Memory 602 and processor 604 may each include or be implemented by hardware and / or software components (e.g., a processor, memory, a communication interface, instructions stored in memory for execution by the processor, etc.). Memory 602 and processor 604 may be distributed among multiple devices and / or multiple locations as may be useful for a particular implementation.

[0053] Memory 602 may maintain (e.g., store) executable data used by processor 604 to perform any of the operations described herein. For example, memory 602 may store instructions 606 that may be executed by processor 604 to perform any of the operations described herein. Instructions 606 may be implemented by any suitable application, software, code, and / or other executable data instance. Memory 602 may also maintain any data acquired, received, generated, managed, used, and / or transmitted by processor 604. For example, memory 602 may maintain LC-MS data, such as an acquisition schedule, as described in more detail below.

[0054] The processor 604 is configured to perform (e.g., execute instructions 606 stored in memory 602 to perform) various processing operations described herein. It will be recognized that the operations and examples described herein are merely illustrative of many different types of operations that may be performed by the processor 604. In the description herein, any reference to an operation performed by the system 600 may also be understood to be performed by the processor 604 of the system 600. Furthermore, in the description herein, any operation performed by the system 600 may be understood to include the system 600 instructing or commanding another system or device to perform the operation.

[0055]

[0033] Figure 7 illustrates an exemplary method 700 for performing targeted mass analysis using an optimized acquisition window width. While Figure 7 illustrates exemplary operations according to one embodiment, other embodiments may omit, add, reorder, and / or modify any of the operations illustrated in Figure 7. One or more of the operations illustrated in Figure 7 may be performed by LC-MS system 100 and / or system 600, any components included therein, and / or any implementation thereof (e.g., mass spectrometer 104, one or more components of mass spectrometer 104, and / or a remote computing system separate from but communicatively coupled to mass spectrometer 104).

[0056] In operation 702, an acquisition schedule is generated for a target assay. The acquisition schedule schedules the acquisition, by a mass spectrometer, of a set of mass spectra for each target analyte in a set of target analytes contained in a sample as the target analytes elute from the separation system in an acquisition window having a dynamic acquisition window width. Generating the acquisition schedule includes determining the dynamic acquisition window width based on an acquisition cycle period for the target assay. Operation 702 is described in more detail below.

[0057] In operation 704, the mass spectrometer is instructed to acquire a set of mass spectra for a set of target analytes according to an acquisition schedule.

[0058] Various illustrative examples of performing operation 702 are now described. A sample typically contains more potential target analytes than can be mass analyzed at any point during a target assay based on sampling rate requirements and instrument speed for the assay. Thus, the set of target analytes is the set of analytes selected as targets for mass analysis from a list of potential target analytes contained in the sample. The set of target analytes may be selected in any suitable manner. In some examples, the set of target analytes is selected based on the load balancing techniques described in U.S. Patent Application No. 18 / 668,638, filed May 20, 2024, which is incorporated herein by reference in its entirety.

[0059] The dynamic acquisition window width for a target assay is determined based on the acquisition cycle duration of the assay, which is based on the sampling rate requirements for the target assay and the fixed or time-varying elution peak width for the assay.

[0060] The sampling rate requirement may be specified by the method parameters for a particular experiment. For example, the method parameters may specify a sampling rate requirement of six acquisitions across each peak. The method parameters may be accessed from local storage (e.g., memory 602), from a remote computing system, or from user input provided by a user.

[0061] The elution peak width for the target assay is determined based on elution peak width data representing the expected elution times of analytes included in a list of known analytes contained in the sample. In some examples, the elution peak width data is accessed from public data, which may be accessed, for example, from local storage (e.g., memory 602) or from a remote computing system. In other examples, the elution peak width data is generated by performing a characterization analysis of the sample prior to operation 702. In some examples, the characterization analysis is a data-dependent acquisition (DDA) analysis of the sample. In an alternative example, the characterization analysis includes a set of multiple narrow, isolated-window data-independent acquisition (DIA) experiments that together span the m / z range of interest. An illustrative example of performing multiple narrow, isolated-window DIA experiments to characterize a sample is described in U.S. Patent Application Serial No. 18 / 668,638.

[0062] Based on the sampling rate requirement and elution peak width data, the acquisition cycle duration can be calculated. For example, given a sampling rate requirement of 8 acquisitions across each elution peak and an elution peak width of 10 seconds, the acquisition cycle duration is 1.25 seconds.

[0063] In some instances, the acquisition cycle duration is fixed (e.g., always constant) throughout the entire experiment. For example, the elution peak width of an assay may be determined as the mean, median, maximum, minimum, weighted average, etc., of the elution peak widths of the analytes in a sample or set of target analytes.

[0064] In other examples, the acquisition cycle duration is dynamic (e.g., varies over time), such as when elution peak widths change over time. The time-varying acquisition cycle duration can be determined in any suitable manner, including any of the methods described in U.S. Patent Application No. 18 / 668,638, filed May 20, 2024.

[0065] The dynamic acquisition window width, based on the acquisition cycle duration of the target assay, varies over time during the assay to satisfy the constraint on the acquisition cycle duration. The maximum width of the acquisition window that still allows acquisition of a set of mass spectra for all scheduled target analytes at the required sampling rate is constrained by the acquisition cycle duration for the assay. In some examples, the acquisition cycle duration constraint requires that the maximum instrument time ratio for the assay be less than or equal to a threshold ratio value. The threshold ratio value is less than or equal to 1. For example, the threshold ratio value can be 1.0, 0.99, 0.97, 0.95, or any other suitable value.

[0066] The instrument time ratio is the ratio of the instrument time required to perform an acquisition cycle at a particular time point during the assay (e.g., during a particular acquisition window) to the acquisition cycle duration at that particular time point. Because a target assay includes multiple separate acquisition windows, the maximum instrument time ratio will be the maximum instrument time ratio for that assay.

[0067] In some instances, such as when the acquisition cycle duration is fixed throughout the target assay, the maximum instrument time ratio can be determined as the ratio of the maximum instrument time required to perform an acquisition cycle during a scheduled assay to the acquisition cycle duration of the assay. For example, the acquisition cycle duration constraint is satisfied (e.g., the maximum instrument time ratio is less than or equal to a threshold ratio value) when the acquisition window width is set for a subset of one or more target analytes such that the maximum instrument time required to perform an acquisition cycle during a scheduled assay does not exceed the acquisition cycle duration.

[0068] In other examples, such as when the acquisition cycle duration is dynamic (e.g., varies over time), an instrument time ratio is determined for each acquisition window so that a maximum instrument time ratio can be identified. The acquisition cycle duration constraint is satisfied when the acquisition window width is set for a subset of one or more target analytes such that, at all times during the scheduled assay, the instrument time required to perform an acquisition cycle does not exceed the acquisition cycle duration (e.g., the maximum instrument time ratio is less than or equal to a threshold ratio value).

[0069] In some examples, the dynamic acquisition window width is determined on a per-target basis, where the acquisition window width is determined individually for each target analyte based on the acquisition cycle duration for the target assay. In other examples, the dynamic acquisition window width is determined on a group basis, where the set of target analytes is divided into multiple groups of multiple target analytes, and a single acquisition window width is determined for each respective group of multiple target analytes. The set of target analytes may be divided into groups of target analytes in any suitable manner. In some examples, the set of target analytes is divided into groups of target analytes based on the estimated elution time of each target analyte. For example, target analyte groups may be defined based on overlapping elution times or based on elution volume during predefined or user-defined time segments (e.g., 1-minute segments, 2-minute segments, etc.).

[0070] The methods and concepts described herein apply equally to individual target analytes and groups of multiple target analytes, and thus the term "subset of one or more target analytes" refers to both individual target analytes and groups of multiple target analytes.

[0071] The dynamic acquisition window width based on the acquisition cycle duration may be determined in any suitable manner. In some examples, the acquisition window widths for all scheduled target analytes are initially set to an acquisition window width default value such that the constraints on the acquisition cycle duration are met (e.g., the maximum instrument time ratio is less than or equal to a threshold ratio value). The default acquisition window width may be determined in any suitable manner, such as based on a predetermined value (e.g., 0.1 minutes, 0.2 minutes, 0.3 minutes, etc.), based on method parameters of the target assay, based on or during a load balancing step to determine the set of target analytes, and / or based on user input. Then, after increasing the acquisition window width for a subset of one or more target analytes, the acquisition window widths for the subset of one or more target analytes are increased such that the constraints on the acquisition cycle duration are met (e.g., the maximum instrument time ratio is less than or equal to a threshold ratio value).

[0072] In some examples, the acquisition window width for a subset of one or more target analytes is iteratively increased in order of increasing estimated elution time, as described herein with reference to FIG. 8 . FIG. 8 illustrates an exemplary method 800 for determining a dynamic acquisition window width. While FIG. 8 illustrates exemplary operations according to one embodiment, other embodiments may omit, add, reorder, and / or modify any of the operations illustrated in FIG. 8 . One or more of the operations illustrated in FIG. 8 may be performed by LC-MS system 100 and / or system 600, any components included therein, and / or any implementation thereof (e.g., mass spectrometer 104, one or more components of mass spectrometer 104, and / or a remote computing system separate from but communicatively coupled to mass spectrometer 104).

[0073] In operation 802, a set A of n target analytes is sorted based on their estimated elution times (e.g., from lowest to highest elution time). The sorted set A is a set of n target analytes a for scheduled assays with acquisition cycle duration c. iwhere i ranges from 1 to n (A={a1, a2,..., a n The acquisition cycle period c can be determined as described herein based on the sampling rate requirements for the assay and the elution peak width for the assay. i is the estimated elution time r, which may be determined as described herein. i It has.

[0074] In operation 804, the acquisition window width w for all target analytes in set A is calculated. i is set to a default value w0 so that the acquisition cycle duration constraint is satisfied (e.g., the maximum ratio of instrument time T(A) to acquisition cycle duration c at all times during the assay (max(T(A) / c)) is less than or equal to 1 (or some other threshold ratio value)). i is the time at which the mass spectrometer detects the target analyte a i is a time segment in which mass spectral data is acquired for a target analyte a i Acquisition window period for p i teeth,

number

[0075] In operation 806, an acquisition window width w for the target analyte is determined. i The process for determining i is initialized by setting i to 1 so that the first target analyte in sorted set A is considered first. If set A is sorted by decreasing elution time, target analyte a1 has the shortest expected elution time.

[0076] In operation 808, the target analyte a i is selected for processing.

[0077] In operation 810, the selected target analyte a i Trial acquisition window width w' for iis increased by an acquisition window width increment δw. The increment δw may be any suitable value. In some examples, the increment δw is predefined, set by a user, or set as a fraction of a default value w (e.g., 6%, 10%, 15%, 20%, 25%, etc.).

[0078] In operation 812, a trial maximum instrument time ratio max(T(A) / c)' is determined. The trial maximum instrument time ratio max(T(A) / c)' is the time required for the selected target analyte a i Trial acquisition window width w' for i , the current acquisition window width w for all other target analytes i , and the instrument speed.

[0079] In operation 814, it is determined whether the trial maximum instrument time ratio max(T(A) / c)' is less than or equal to 1. If the trial maximum instrument time ratio max(T(A) / c)' is less than or equal to 1, the selected target analyte a i Get window width for w i The gradual increase of x ensures that the acquisition window width is not too large, and that the acquisition cycle period c is sufficient to mass analyze all target analytes in the assay. Processing of method 800 then continues to operation 816.

[0080] In operation 816, the selected target analyte a i Get window width for w i is the selected target analyte a i Trial acquisition window width w' for i Processing then continues to operation 818.

[0081] In operation 814, if the trial maximum instrument time ratio max(T(A) / c)' is greater than 1, the selected target analyte a i Get window width for w i makes the acquisition window width too large, resulting in too many target analytes being mass analyzed within the acquisition cycle period c. iTrial acquisition window width w' for i and discard the selected target analyte a i Get window width for w i remains unchanged. Processing of method 800 then continues to operation 818.

[0082] In operation 818, the selected target analyte a i It is determined whether i is the last target analyte in set A (e.g., whether i=n). i If i is not the last target analyte (i≠n), processing continues to operation 820 .

[0083] At operation 820, i is incremented by +1 and processing returns to operation 808 to find the next target analyte a in set A. i Select .

[0084] In operation 818, the selected target analyte a i If i is the last target analyte (i=n), then processing returns to operation 806 to initialize i and perform another iteration including operations 808-820. This iterative process iterates through the target analyte a i Additionally or alternatively, the iterative process may be capped at a maximum number of iterations (e.g., 3, 6, 10, 20, etc.).

[0085] Various modifications can be made to method 800. In some examples, instead of incrementally increasing the acquisition window width for each target (e.g., for each individual target analyte), the acquisition window width is incrementally increased for a group of multiple target analytes. For example, in operation 808, a group of target analytes may be selected, and in operation 810, the acquisition window width for each target analyte in the group may be increased by an increment δw. Thus, method 800 can be performed for a subset of one or more target analytes (e.g., for each target analyte on a target-by-target basis, or for groups of target analytes on a group-by-group basis).

[0086] In a further example, the trial acquisition window width w' i may be capped at a maximum value. Figure 9 shows an example method 900 for performing operation 702 for determining a dynamic acquisition cycle width. Method 900 is similar to method 800, except that method 900 includes a maximum width check procedure 902 after operation 810. In operation 902, a trial acquisition window width w' i w is the maximum acquisition window width value w max In some examples, the maximum acquisition window width value w max is a multiple (e.g., 2x, 3x, 4x, 10x, 15x, etc.) of the default value w0. max is a predetermined width value such as 2 minutes, 3 minutes, 4 minutes, 5 minutes, etc. The maximum acquisition window width value w max is the acquisition window width w i is chosen so that it is not too large, which may limit the maximum injection time and therefore the lower limit of quantitation of the assay.

[0087] In some examples, the maximum acquisition window width w max changes over time. For example, the maximum acquisition window width w max may be larger during periods of low target analyte concentration, such as at the beginning and / or end of an elution run, and smaller during periods of high target analyte concentration, such as the middle of an elution run. Since there is often sufficient data for correction of elution time drift, a relatively large acquisition window width w during periods of high analyte concentration is often not required. Furthermore, a smaller maximum acquisition window width w during periods of high analyte concentration may be used. max prevents the injection or dwell time for each acquisition from becoming too small, which would result in a loss of sensitivity, while periods of low analyte concentration allow for a larger maximum acquisition window width w without a significant reduction in injection or dwell time and a corresponding loss of sensitivity. max In some examples, the time-varying maximum acquisition window width w maxis determined based on the estimated elution time of each target analyte or group of target analytes and / or based on the estimated number of coincident precursors at various times or time segments during the elution run. In a further example, the time-varying maximum acquisition window width w max is a function of time (eg, a linear function, a piecewise function, or a polynomial function of second or higher order).

[0088] Trial acquisition window width w' i w is the maximum acquisition window width value w max If not, processing continues at operation 812. i W is the maximum acquisition window width value w max If the selected target analyte a i Trial acquisition window width w' for i and discard the selected target analyte a i Acquisition window width w i remains unchanged. Processing of method 900 then continues to operation 818.

[0089] In other variations of method 800, the increment δw may be decreased for successive iterations. Figure 10 shows an example method 1000 that performs operation 702 for determining the dynamic acquisition cycle width. Method 1000 is similar to method 800, except that method 1000 includes operation 1002 after operation 818. In operation 818, the selected target analyte a i If i is the last target analyte (i=n), processing continues to operation 1002. In operation 1002, the increment δw is decreased, such as by a step decrease (e.g., 1 ms, 3 ms, 6 ms, 20 ms, 60 ms, 100 ms) or by a fraction of the current increment δw or the initial increment δw0, e.g., ½, ⅓, ¼, etc.). Processing then returns to operation 806 to reinitialize i and perform another iteration, including operations 808-820 and 1002.

[0090] 10 shows the increment δw decreasing with each successive iteration, the increment δw may decrease at any other frequency, such as every other iteration, every third iteration, every fourth iteration, etc. Furthermore, it will be appreciated that method 900 and method 1000 may be combined (e.g., by including both operations 902 and 1002 in the same method).

[0091] To illustrate the acquisition window optimization technique, consider an illustrative example using the same set of 2,972 target peptides shown in FIGS. 4 and 5. FIG. 11 shows graph 1100A, which plots instrument time as a function of elapsed time, and graph 1100B, which plots acquisition window width as a function of elapsed time. Data acquired using a fixed acquisition window width of 0.4 minutes is represented by curve 1102 in graph 1100A and curve 1104 in graph 1100B. Data obtained using a variable acquisition window width is represented by curve 1106 in graph 1100A and curve 1108 in graph 1100B. The variable acquisition window width is determined by setting the maximum width factor to 10, the increment δw to 0.2, and performing five iterations. Having determined the acquisition window width in this manner, the acquisition window width is no longer constant but rather has the value represented by curve 1108 in graph 1100B. Because less analyte elutes in the early and late stages of the assay, the acquisition window width can be increased from 0.4 minutes up to 4 minutes (based on a maximum width factor of 10). As shown by curve 1106 of graph 1100A, the instrument time for the dynamic acquisition window width is always less than the acquisition cycle period of 1,670 ms (the period represented by dashed line 1110).

[0092] The maximum acquisition window width of 4.0 minutes in this example compensates for much of the variability in elution times observed in Figures 4 and 5, where the initial elution time drift is on the order of about 2 minutes. This technique neatly solves the problem of how to increase the acquisition window width in regions where it is possible, which occurs early and late in the assay, where elution time drift correction algorithms may struggle to perform. The middle portion of the assay, where more target analytes are eluting and there is typically more information to perform elution time drift correction, does not allow for as large an increase in their acquisition window width.

[0093] In certain embodiments, one or more of the systems, components, and / or processes described herein may be implemented and / or executed 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 on two or more physical computing devices. Thus, system components may include any number of computing devices but may employ any of several computer operating systems.

[0094] In certain embodiments, one or more of the processes described herein are executable, at least in part, as instructions embodied in a non-transitory computer-readable medium and executable by one or more computing devices. Generally, a processor (e.g., a microprocessor) receives instructions from a non-transitory computer-readable medium (e.g., a memory, etc.) and executes those instructions to thereby perform 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.

[0095] Computer-readable media (also referred to as processor-readable media) include any non-transitory media that participate in providing data (e.g., instructions) that may be read by a computer (e.g., by a processor of a computer). Such media 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 the main memory. Common forms of computer-readable media include, for example, disks, hard disks, magnetic tape, any other magnetic media, compact disc read-only memory ("CD-ROM"), digital video disc ("DVD"), any other optical media, random access memory ("RAM"), programmable read-only memory ("PROM"), erasable programmable read-only memory ("EPROM"), FLASH-EEPROM, any other memory chip or cartridge, or any other tangible medium from which a computer can read.

[0096] FIG. 12 illustrates an exemplary computing device 1200 that may be specifically configured to perform one or more of the processes described herein. As shown in FIG. 12, computing device 1200 may include a communication interface 1202, a processor 1204, a storage device 1206, and an input / output ("I / O") module 1208 that are communicatively coupled to each other via a communication infrastructure 1210. While FIG. 12 illustrates an exemplary computing device 1200, the components illustrated in FIG. 12 are not intended to be limiting. In other embodiments, additional or alternative components may be used. The components of computing device 1200 illustrated in FIG. 12 will now be described in further detail.

[0097] Communications interface 1202 may be configured to communicate with one or more computing devices. Examples of communications interface 1202 include, but are not limited to, 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.

[0098] Processor 1204 generally represents any type or form of processing unit capable of processing data and / or interpreting, executing, and / or directing the execution of one or more of the instructions, processes, and / or operations described herein. Processor 1204 may perform operations by executing computer-executable instructions 1212 (e.g., applications, software, code, and / or other executable data instances) stored on storage device 1206.

[0099] Storage device(s) 1206 may include one or more data storage media, devices, or configurations, but may employ any type, form, and combination of data storage media and / or devices. For example, storage device(s) 1206 may include, but are not limited to, any combination of non-volatile and / or volatile media described herein. Electronic data, including data described herein, may be temporarily and / or permanently stored in storage device(s) 1206. For example, data representing computer-executable instructions 1212 configured to direct processor 1204 to perform any of the operations described herein may be stored in storage device(s) 1206. In some examples, data may be located in one or more databases residing in storage device(s) 1206.

[0100] I / O module(s) 1208 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(s) 1208 may include any hardware, firmware, software, or combination thereof that supports input and output capabilities. For example, I / O module 1208 may include hardware and / or software for capturing user input, including, but not limited to, a keyboard or keypad, a touchscreen component (e.g., a touchscreen display), a receiver (e.g., an RF or infrared receiver), a motion sensor, and / or one or more input buttons.

[0101] I / O module 1208 may include one or more devices for presenting output to a user, examples of which include, but are not limited to, a graphics engine, a display (e.g., a display screen), one or more output drivers (e.g., a display driver), one or more audio speakers, and one or more audio drivers. In a particular embodiment, I / O module 1208 is configured to provide graphical data to a display for presentation to a user. The graphical data may represent one or more graphical user interfaces and / or any other graphical content as may be useful in a particular implementation.

[0102] In some examples, any of the systems, computing devices, and / or other components described herein may be implemented by computing device 1200. For example, memory 602 may be implemented by storage device 1206, and processor 604 may be implemented by processor 1204.

[0103] Meanwhile, those skilled in the art will recognize that in the foregoing description, various exemplary embodiments have been described with reference to the accompanying drawings. However, it will be apparent 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 following claims. For example, certain features of one embodiment described herein may be combined with, or substituted for, features of another embodiment described herein. Accordingly, the specification and drawings should be considered in an illustrative, and not a restrictive, sense.

[0104] The advantages and features of the present disclosure can be further illustrated by the following examples.

[0105] Example 1. A non-transitory computer-readable medium storing instructions that, when executed, direct at least one processor of a computing device for mass spectrometry to: generate, for a target assay of a sample, an acquisition schedule that schedules the acquisition, by a mass spectrometer, of a set of mass spectra of each target analyte in a set of target analytes contained in the sample as the target analytes elute from a separation system, in an acquisition window having a dynamic acquisition window width, wherein the acquisition schedule includes determining the dynamic acquisition window width based on an acquisition cycle period of the target assay; and instructing the mass spectrometer to acquire each set of mass spectra in accordance with the acquisition schedule.

[0106] Example 2. The non-transitory computer-readable medium of Example 1, wherein determining a dynamic acquisition window width based on an acquisition cycle duration for the target assay comprises individually determining an acquisition window width for each target analyte included in the set of target analytes based on the acquisition cycle duration for the target assay.

[0107] Example 3. The non-transitory computer-readable medium of Example 1, wherein determining the dynamic acquisition window width comprises dividing the set of target analytes into a plurality of groups of target analytes, and determining, for each group of target analytes included in the plurality of groups of target analytes, an acquisition window width based on an acquisition cycle duration for each group of target analytes.

[0108] Example 4. The non-transitory computer-readable medium of Example 3, wherein the set of target analytes is divided into a plurality of groups of target analytes based on an estimated elution time of each target analyte included in the set of target analytes.

[0109] Example 5. The non-transitory computer-readable medium of Example 1, wherein the acquisition cycle period for the target assay is fixed.

[0110] Example 6. The non-transitory computer-readable medium of Example 1, wherein the acquisition cycle period for the target assay is dynamic.

[0111] Example 7. The non-transitory computer-readable medium of Example 1, wherein determining a dynamic acquisition window width further comprises determining an acquisition cycle duration for the target assay based on a sampling rate requirement for the target assay and a fixed or time-varying elution peak width for the target assay.

[0112] Example 8. The non-transitory computer-readable medium of Example 1, wherein determining a dynamic acquisition window width based on an acquisition cycle duration for the target assay comprises: determining, for each subset of one or more target analytes included in the set of target analytes, an acquisition window width default value such that a maximum instrument time ratio for the target assay is less than or equal to a threshold ratio value, wherein the maximum instrument time ratio for the target assay is a maximum ratio of instrument time performing an acquisition cycle to an acquisition cycle duration for the acquisition cycle, and wherein the threshold ratio value is less than or equal to 1; and increasing the acquisition window width for the subset of one or more target analytes such that after increasing the acquisition window width for the subset of one or more target analytes, the maximum instrument time ratio for the target assay is less than or equal to the threshold ratio.

[0113] Example 9. The non-transitory computer-readable medium of Example 8, wherein determining a dynamic acquisition window width based on an acquisition cycle duration for the target assays further comprises sorting the set of target analytes based on an estimated elution time of each target analyte included in the set of target analytes, and wherein increasing the acquisition window width for the subset of one or more target analytes comprises increasing the acquisition window width for each subset of one or more target analytes included in the plurality of subsets of the one or more target analytes in order of increasing estimated elution time based on the acquisition window width increment.

[0114] Example 10. The non-transitory computer-readable medium of Example 9, wherein the acquisition window width increment decreases with successive iterations.

[0115] Example 11. The non-transitory computer-readable medium of Example 1, wherein determining the dynamic acquisition window width is further based on a maximum acquisition window width value.

[0116] Example 12. A method for performing targeted mass analysis, comprising: generating an acquisition schedule for a target assay of a sample that schedules the acquisition by a mass spectrometer of a set of mass spectra of each target analyte in a set of target analytes contained in the sample as the target analytes elute from a separation system in an acquisition window having a dynamic acquisition window width, wherein generating the acquisition schedule comprises determining the dynamic acquisition window width based on an acquisition cycle period for the target assay; and instructing the mass spectrometer to acquire each set of mass spectra in accordance with the acquisition schedule.

[0117] Example 13. The method of Example 12, wherein determining a dynamic acquisition window width further comprises determining an acquisition cycle duration for the target assay based on a sampling rate requirement for the target assay and a fixed or time-varying elution peak width for the target assay.

[0118] Example 14. The method of Example 12, wherein determining a dynamic acquisition window width based on an acquisition cycle duration for a target assay comprises: determining, for each subset of one or more target analytes included in the set of target analytes, an acquisition window width default value such that a maximum instrument time ratio for the target assay is less than or equal to a threshold ratio value, wherein the maximum instrument time ratio for the target assay is a maximum ratio of instrument time performing an acquisition cycle to an acquisition cycle duration for the acquisition cycle, and wherein the threshold ratio value is less than or equal to 1; and increasing the acquisition window width for the subset of one or more target analytes such that after increasing the acquisition window width for the subset of one or more target analytes, the maximum instrument time ratio for the target assay is less than or equal to the threshold ratio.

[0119] Example 15. The method of Example 14, wherein determining a dynamic acquisition window width based on an acquisition cycle duration for the target assay further comprises sorting the set of target analytes based on an estimated elution time of each target analyte included in the set of target analytes, and wherein increasing the acquisition window width for the subset of one or more target analytes comprises increasing the acquisition window width for each subset of one or more target analytes included in the plurality of subsets of the one or more target analytes in order of increasing estimated elution time based on the acquisition window width increment.

[0120] Example 16 The method of example 15, wherein the acquisition window width increment decreases with successive iterations.

[0121] Example 17. The method of example 12, wherein determining the dynamic acquisition window width is further based on a maximum acquisition window width value.

[0122] Example 18. A system for mass spectrometry, comprising one or more processors and a memory storing executable instructions, which, when executed by the one or more processors, cause a computing device to: generate an acquisition schedule for a target assay of a sample, the acquisition schedule scheduling acquisition by a mass spectrometer of a set of mass spectra for each target analyte in a set of target analytes contained in the sample as the target analytes elute from a separation system, within an acquisition window having a dynamic acquisition window width, wherein generating the acquisition schedule includes determining the dynamic acquisition window width based on an acquisition cycle period of the target assay; and instructing the mass spectrometer to acquire each set of mass spectra in accordance with the acquisition schedule.

[0123] Example 19. The system of Example 18, wherein determining a dynamic acquisition window width based on an acquisition cycle duration for a target assay comprises: determining, for each subset of one or more target analytes included in the set of target analytes, a default acquisition window width value such that a maximum instrument time ratio for the target assay is less than or equal to a threshold ratio value, wherein the maximum instrument time ratio for the target assay is a maximum ratio of instrument time performing an acquisition cycle to an acquisition cycle duration for the acquisition cycle, and wherein the threshold ratio value is less than or equal to 1; and increasing the acquisition window width for the subset of one or more target analytes such that after increasing the acquisition window width for the subset of one or more target analytes, the maximum instrument time ratio for the target assay is less than or equal to the threshold ratio.

[0124] Example 20. The system of example 18, further comprising a mass spectrometer.

Claims

1. A non-transitory computer-readable medium storing instructions that, when executed, cause at least one processor of a computing device for mass spectrometry to: generating an acquisition schedule for a target assay of a sample, the acquisition schedule scheduling acquisition by a mass spectrometer of a set of mass spectra for each target analyte in the set of target analytes contained in the sample as the target analytes elute from a separation system within an acquisition window having a dynamic acquisition window width, wherein generating the acquisition schedule includes determining the dynamic acquisition window width based on an acquisition cycle duration for the target assay; instructing the mass spectrometer to acquire each set of mass spectra according to the acquisition schedule.

2. determining the dynamic acquisition window width based on the acquisition cycle duration of the target assay, 2. The non-transitory computer-readable medium of claim 1, further comprising individually determining an acquisition window width for each target analyte included in the set of target analytes based on the acquisition cycle duration of the target assay.

3. determining the dynamic acquisition window width Dividing the set of target analytes into a plurality of groups of target analytes; and determining, for each target analyte group included in the plurality of target analyte groups, an acquisition window width based on an acquisition cycle duration for each of the target analyte groups.

4. 4. The non-transitory computer-readable medium of claim 3, wherein the set of target analytes is divided into a plurality of groups of the target analytes based on an estimated elution time of each target analyte included in the set of target analytes.

5. 10. The non-transitory computer-readable medium of claim 1, wherein the acquisition cycle period for the target assay is fixed.

6. 10. The non-transitory computer-readable medium of claim 1, wherein the acquisition cycle period for the target assay is dynamic.

7. determining the dynamic acquisition window width 10. The non-transitory computer-readable medium of claim 1, further comprising determining the acquisition cycle duration for the target assay based on a sampling rate requirement for the target assay and a fixed or time-varying elution peak width of the target assay.

8. determining the dynamic acquisition window width based on the acquisition cycle duration of the target assay, determining, for each subset of one or more target analytes included in the set of target analytes, a default acquisition window width value such that a maximum instrument time ratio for the target assay is less than or equal to a threshold ratio value; the maximum instrument time ratio for the target assay is the maximum ratio of instrument time performing an acquisition cycle to the acquisition cycle period for the acquisition cycle; and determining an acquisition window width default value, the threshold ratio value being less than or equal to 1; increasing an acquisition window width for a subset of one or more target analytes, such that after increasing the acquisition window width for the subset of one or more target analytes, the maximum instrument time ratio for the target assay is less than or equal to the threshold ratio value.

9. determining the dynamic acquisition window width based on the acquisition cycle duration for the target assay further comprises sorting the set of target analytes based on an estimated elution time of each target analyte included in the set of target analytes; Increasing the acquisition window width of the subset of one or more target analytes comprises:

10. The non-transitory computer-readable medium of claim 8, comprising an iterative process comprising increasing the acquisition window width for each subset of one or more target analytes included in the plurality of subsets of one or more target analytes in order of increasing estimated elution time based on the acquisition window width increment.

10. The non-transitory computer-readable medium of claim 9 , wherein the acquisition window width increment decreases with successive iterations.

11. The non-transitory computer-readable medium of claim 1 , wherein determining the dynamic acquisition window width is further based on a maximum acquisition window width value.

12. 1. A method for performing targeted mass spectrometry, comprising: generating an acquisition schedule for a target assay of a sample, the acquisition schedule scheduling acquisition by a mass spectrometer of a set of mass spectra of each target analyte in the set of target analytes contained in the sample as the target analytes elute from the separation system in an acquisition window having a dynamic acquisition window width, wherein generating the acquisition schedule includes determining the dynamic acquisition window width based on an acquisition cycle duration for the target assay; instructing the mass spectrometer to acquire each set of mass spectra according to the acquisition schedule; A method comprising:

13. determining the dynamic acquisition window width 13. The method of claim 12, further comprising determining the acquisition cycle duration for the target assay based on a sampling rate requirement for the target assay and a fixed or time-varying elution peak width of the target assay.

14. determining the dynamic acquisition window width based on the acquisition cycle duration of the target assay; determining, for each subset of one or more target analytes included in the set of target analytes, a default acquisition window width value such that a maximum instrument time ratio for the target assay is less than or equal to a threshold ratio value; the maximum instrument time ratio for the target assay is the maximum ratio of instrument time performing an acquisition cycle to the acquisition cycle period for the acquisition cycle; and determining an acquisition window width default value, the threshold ratio value being less than or equal to 1; 13. The method of claim 12, comprising increasing an acquisition window width for a subset of one or more target analytes, such that after increasing the acquisition window width for the subset of one or more target analytes, the maximum instrument time ratio for the target assay is less than or equal to the threshold ratio value.

15. determining the dynamic acquisition window width based on the acquisition cycle duration for the target assay further comprises sorting the set of target analytes based on an estimated elution time of each target analyte included in the set of target analytes; Increasing the acquisition window width for a subset of one or more target analytes comprises:

15. The method of claim 14, comprising an iterative process comprising increasing the acquisition window width for each subset of one or more target analytes included in a plurality of subsets of one or more target analytes in order of increasing estimated elution time based on the acquisition window width increment.

16. The method of claim 15 , wherein the acquisition window width increment decreases with successive iterations.

17. The method of claim 12 , wherein determining the dynamic acquisition window width is further based on a maximum acquisition window width value.

18. 1. A system for mass spectrometry, comprising: one or more processors; and a memory that stores executable instructions that, when executed by the one or more processors, cause the computing device to: generating an acquisition schedule for a target assay of a sample, the acquisition schedule scheduling acquisition by a mass spectrometer of a set of mass spectra for each target analyte in the set of target analytes contained in the sample as the target analytes elute from a separation system within an acquisition window having a dynamic acquisition window width, wherein generating the acquisition schedule includes determining the dynamic acquisition window width based on an acquisition cycle duration for the target assay; instructing the mass spectrometer to acquire each set of mass spectra according to the acquisition schedule.

19. determining the dynamic acquisition window width based on the acquisition cycle duration of the target assay; determining, for each subset of one or more target analytes included in the set of target analytes, a default acquisition window width value such that a maximum instrument time ratio for the target assay is less than or equal to a threshold ratio value; the maximum instrument time ratio for the target assay is the maximum ratio of instrument time performing an acquisition cycle to the acquisition cycle period for the acquisition cycle; and determining an acquisition window width default value, the threshold ratio value being less than or equal to 1; 20. The system of claim 18, further comprising: increasing an acquisition window width for a subset of one or more target analytes such that after increasing the acquisition window width for the subset of one or more target analytes, the maximum instrument time ratio for the target assay is less than or equal to the threshold ratio value.

20. The system of claim 18 further comprising the mass spectrometer.

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