Data independent acquisition with parallel isolation multiplexing

Abstract

The invention relates to data independent acquisition with parallel isolation multiplexing. The system may control a mass spectrometer to acquire a set of mass spectra of product ions derived from precursor ions isolated based on parallel isolation windows continuously positioned throughout the precursor mass-to-charge ratio (m / z) range during a plurality of acquisitions constituting an acquisition cycle. The precursor m / z range is divided into a plurality of isolation window cells. For each acquisition of the acquisition cycle, the parallel isolation window includes a set of isolation sub-windows corresponding to a different set of isolation window cells of the precursor m / z range. At least two adjacent isolation sub-windows of the parallel isolation window are discontinuous. Each isolation window cell of the precursor m / z range is analyzed at least twice during the acquisition cycle. A mass spectrum for the precursor m / z range may be generated based on the set of mass spectra acquired during the acquisition cycle.

Description

[0001] Government Support Statement

[0002] This invention was made with government support under Grant Nos. P41 GM103533 and R21 CA192983 awarded by the National Institute of Health. The government has certain rights in this invention. Background Art

[0003] A mass spectrometer is a sensitive instrument that can be used to detect, identify and / or quantify molecules based on the mass-to-charge ratio (m / z) of the ions generated by the molecules. A mass spectrometer generally includes an ion source for generating ions from the molecules contained in the sample, a mass analyzer for separating ions based on its m / z, and an ion detector for detecting separated ions. A mass spectrometer may include or be connected to a computer-based software platform that uses data from the ion detector to construct a mass spectrogram that shows the relative abundance of each ion in the detected ions that varies with m / z. A mass spectrogram may be used to detect and quantify molecules in simple and complex mixtures. Separation devices such as liquid chromatographs (LC), gas chromatographs (GC), or capillary electrophoresis (CE) systems may be coupled to a mass spectrometer in a combined system (e.g., LC-MS, GC-MS, or CE-MS systems) to separate components in a sample before the components are introduced into the mass spectrometer.

[0004] One application of mass spectrometry is the identification, quantification and structural determination of peptides, proteins and related molecules in complex biological samples. In some such experiments, such as multistage mass spectrometry (MSn, where n is 2 or greater) or tandem mass spectrometry (a form of multistage mass spectrometry where n is 2, usually denoted as MS / MS or MS2), certain ions are isolated in a controlled manner and then fragmented to produce product ions. The product ions are then mass analyzed 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 identification results, determine the quantity and / or derive structural details about the analyte of interest.

[0005] Various techniques can be used to collect mass spectra using multi-stage mass spectrometry. A common technique is data-dependent acquisition (DDA), which uses the data collected in a certain mass analysis to select one or more ion species or narrow m / z ranges based on predetermined criteria for isolation and fragmentation of the selected ion species and subsequent mass analysis of fragmented ions (product ions). For example, a mass spectrometer can perform a full MS survey scan of precursor ions within a wide precursor m / z range, and then select one or more precursor ion species from the resulting spectra for subsequent MS / MS or MSn analysis. The selection criteria for precursor ion species can include intensity, charge state, m / z, inclusion / exclusion lists, or isotope patterns. The main disadvantage of DDA technology is the inherent random nature of the results. When performing technical repetitions of the same sample or comparative analysis of other samples, some analytes can be measured in one experiment, but not in other experiments. This hinders attempts to perform repeatable analysis and is known as the "missing value problem."

[0006] In contrast to DDA, data-independent acquisition (DIA) is a technique in which all precursor ion species within a wide precursor m / z range (e.g., 500m / z-900m / z) are isolated and fragmented to generate product ions via an isolation window with a fixed m / z width (e.g., 20m / z) and sequential advancement. MS or MSn analysis is then performed on the product ions in an organized and unbiased manner. The acquisition of a set of mass spectra across the entire precursor m / z range constitutes an acquisition cycle, which is repeated to generate MS / MS or MSn mass spectra of these product ions. In the DIA technique, the separation and fragmentation of one or more precursor ion species does not rely on the data acquired in the survey mass analysis (as in DDA), and is more suitable for comparing results for different samples than DDA. The advantage of DIA is repeatable sampling, which eliminates the missing value problem inherent in the DDA technique.

[0007] However, due to instrument speed and sensitivity limitations, there is a tension between isolation width, precursor m / z range, and acquisition cycle time. The acquisition cycle time is typically chosen to be less than or equal to a typical LC peak width divided by about 6, where the m / z range will be fully sampled during the acquisition cycle time, allowing accurate integration of the area of ​​the eluting compound. In general, the wider the isolation width, the wider the precursor m / z range that can be achieved, and therefore a greater number of precursor ion species can be analyzed, but lower quality data is produced because the wide isolation window may result in co-isolation and co-fragmentation of adjacent analytes, resulting in complex, unrecognizable, or low-scoring spectra. On the other hand, smaller isolation widths produce better quality data with increased sensitivity, but at the expense of fewer precursor ion species available for analysis due to the narrower precursor m / z range. For example, in the extreme case of very narrow isolation widths, the data is of the highest quality in terms of sensitivity and selectivity, but the range of precursor ion species analyzed is the smallest. Conversely, in the extreme case of very wide isolation widths, a wider range of precursor ion species can be analyzed, but the quality of the resulting data is compromised because the dynamic range of quantification may be compromised and the spectrum may have mixed signals arising from many different analytes. As a result, the spectrum is difficult to interpret. Summary of the invention

[0008] The following description presents a simplified overview 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 covered aspects, and is neither intended to identify key or critical elements of all aspects, nor 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 presented below.

[0009] In some illustrative examples, a system includes: one or more processors; and a memory storing executable instructions that, when executed by the one or more processors, cause a computing device to perform a process including the following: controlling a mass spectrometer to acquire a set of mass spectra of product ions during multiple acquisitions constituting an acquisition cycle, the product ions being derived from precursor ions isolated based on parallel isolation windows positioned continuously across a precursor mass-to-charge ratio (m / z) range; wherein: the precursor m / z range is divided into a plurality of isolation window units; for each acquisition of the acquisition cycle, the parallel isolation windows include a set of isolation sub-windows corresponding to a different set of isolation window units of the precursor m / z range; at least two adjacent isolation sub-windows of the parallel isolation windows are discontinuous; and each isolation window unit of the precursor m / z range is analyzed at least twice during the acquisition cycle.

[0010] In some illustrative examples, a system includes: a mass spectrometer; and a controller configured to control the mass spectrometer to acquire a set of mass spectra of product ions during multiple acquisitions constituting an acquisition cycle, the product ions being derived from precursor ions isolated based on parallel isolation windows positioned continuously across a precursor mass-to-charge ratio (m / z) range; wherein: the precursor m / z range is divided into a plurality of isolation window units; for each acquisition of the acquisition cycle, the parallel isolation windows include a set of isolation sub-windows corresponding to a different set of isolation window units of the precursor m / z range; at least two adjacent isolation sub-windows of the parallel isolation windows are discontinuous; and each isolation window unit of the precursor m / z range is analyzed at least twice during the acquisition cycle.

[0011] In some illustrative examples, a non-transitory computer-readable medium stores instructions that, when executed, direct at least one processor of a computing device for mass spectrometry to perform a process comprising: controlling a mass spectrometer to acquire a set of mass spectra of product ions during multiple acquisitions constituting an acquisition cycle, the product ions being derived from precursor ions isolated based on parallel isolation windows positioned continuously across a precursor mass-to-charge ratio (m / z) range; wherein: the precursor m / z range is divided into a plurality of isolation window units; for each acquisition of the acquisition cycle, the parallel isolation windows include a set of isolation sub-windows corresponding to a different set of isolation window units of the precursor m / z range; at least two adjacent isolation sub-windows of the parallel isolation windows are discontinuous; and each isolation window unit of the precursor m / z range is analyzed at least twice during the acquisition cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings illustrate various embodiments and are part of the specification. The embodiments shown are examples only and do not limit the scope of the present disclosure. Throughout the drawings, the same or similar reference numerals represent the same or similar elements.

[0013] Figure 1 A functional diagram of an exemplary LC-MS system is shown.

[0014] Figure 2 shows that it may be included in Figure 1 Functional diagram of an exemplary implementation of a mass spectrometer in an LC-MS system.

[0015] Figure 3 A functional diagram of an exemplary mass spectrometry control system is shown.

[0016] Figure 4 An exemplary method for performing a DIA experiment using parallel isolation multiplexing is shown.

[0017] Figure 5 An exemplary acquisition scheme using an acquisition cycle of parallel isolation windows is shown.

[0018] FIG. 6A to FIG. 6D Shows Figure 4 Various exemplary instrument configurations and specific implementations of the methods.

[0019] 7A to 7C Shows Figure 4 Various additional illustrative instrument configurations and specific implementations of the methods.

[0020] Figures 8 to 12 Various illustrative acquisition schemes using acquisition cycles of parallel isolation windows are shown.

[0021] Fig.13 An illustrative computing device is shown that may be specifically configured to perform one or more of the processes described herein. DETAILED DESCRIPTION

[0022] This article describes systems, devices and methods for performing data-independent acquisition (DIA) using parallel isolation multiplexing. For example, the system can control a mass spectrometer to collect a set of mass spectra of product ions during multiple acquisitions that constitute an acquisition cycle, and the product ions are derived from precursor ions isolated based on parallel isolation windows that are continuously positioned throughout the precursor mass-to-charge ratio (m / z) range. The precursor m / z range is divided into multiple isolation window units. For each acquisition of the acquisition cycle, the parallel isolation window includes a set of isolation sub-windows corresponding to different sets of isolation window units of the precursor m / z range. At least two adjacent isolation sub-windows of the parallel isolation window are discontinuous. Each isolation window unit of the precursor m / z range is analyzed at least twice during the acquisition cycle. A mass spectrum for the precursor m / z range can be generated based on a set of mass spectra collected during the acquisition cycle. For example, a set of mass spectra can be demultiplexed to assign one or more signals in the set of mass spectra to the appropriate isolation window units of its precursor m / z range.

[0023] Compared to conventional DIA techniques, the systems, devices, and methods described herein improve the sensitivity of DIA analysis by using a relatively wide parallel isolation width. At the same time, compared to conventional DIA techniques, selectivity is improved by utilizing one or more m / z gaps between the isolation sub-windows, which increases the diversity of analysis in a set of mass spectra collected during an acquisition cycle by the isolation window unit of the precursor m / z range. For example, the selectivity of the demultiplexed mass spectrum can be measured as the isolation width of the parallel isolation window divided by the number of different overlaps of each isolation window unit of each acquisition cycle. Therefore, a demultiplexed mass spectrum generated based on a parallel isolation window with a parallel isolation width of 8m / z will have a selectivity of less than 8m / z (e.g., 4m / z or 2m / z). In addition, the use of a wide parallel isolation width enables coverage of a wider precursor m / z range, thereby increasing throughput, compared to conventional DIA techniques.

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

[0025] In some specific implementations, the method and system for performing DIA with parallel isolation can be used in combination with a combined separation-mass spectrometry system, such as an LC-MS system. Thus, the LC-MS system will now be described. The LC-MS system described is illustrative and not restrictive. The method and system described herein can be used as a part of the LC-MS system described herein or run with it, and / or run with any other suitable separation mass spectrometry system, which includes 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 method and system described herein can also be run with any other continuous flow sample source, such as a flow injection mass spectrometry system (FI-MS), wherein analyte is injected into the mobile phase (not separated in a column), and enters a mass spectrometer in a manner that intensity varies over time (e.g., a Gaussian peak).

[0026] Figure 1 A functional diagram of an exemplary LC-MS system 100 is shown. 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 components (e.g., analytes) within a sample 108 injected into the liquid chromatograph 102 over time. The sample 108 may include, for example, chemical components (e.g., molecules, ions, etc.) and / or biological components (e.g., metabolites, proteins, peptides, lipids, etc.) for detection and analysis by the LC-MS system 100. The liquid chromatograph 102 may be implemented by, for example, any liquid chromatograph that may conform to a particular implementation. In the liquid chromatograph 102, the sample 108 may be 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, components within the sample 108 elute from the column 110 at different times based on, for example, their size, their affinity for the stationary phase, their polarity, and / or their hydrophobicity.

[0027] Detector (e.g., ion detector assembly, ion-electron converter and electron multiplier of mass spectrometer 104, etc.) can measure the relative intensity of the signal modulated by each separated component in the eluate 112 from column 110. The data generated by the detector can be represented as a chromatogram, which plots retention time on the x-axis and plots the signal representing relative intensity on the y-axis. The retention time of the component is usually measured as the time period between the injection of sample 108 into the mobile phase and the appearance of the maximum peak of relative intensity after chromatographic separation. In some examples, relative intensity can be related to the relative abundance of the separated components or represent the relative abundance of the separated components. The data generated by liquid chromatograph 102 can be output to controller 106.

[0028] In some cases, particularly in the analysis of complex mixtures, multiple different components in sample 108 co-elute from column 110 at approximately the same time and may therefore have the same or similar retention times. Therefore, determining the relative intensities of the individual components within sample 108 requires further separation of the individual components. To this end, liquid chromatograph 102 directs the components contained in eluate 112 to mass spectrometer 104 for identification and / or quantification of one or more components.

[0029] The mass spectrometer 104 is configured to generate ions from the components received by the liquid chromatograph 102 and to classify or separate the generated ions based on the m / z of the ions. The detector in the mass spectrometer 104 measures the intensity of the signal generated by the ions. As used herein, "intensity" or "signal intensity" refers to the response of the detector and can represent absolute abundance, relative abundance, ion count, intensity, relative intensity, ion current, or any other suitable ion detection measure. The data generated by the detector can be represented by a mass spectrogram, which plots the intensity of the observed signal as a function of the m / z of the detected ions. The data collected by the mass spectrometer 104 can be output to the controller 106.

[0030] The mass spectrometer 104 can be implemented by a multistage mass spectrometer configured to perform multistage mass spectrometry (also indicated as MSn). In some examples, the mass spectrometer is a tandem mass spectrometer configured to perform tandem mass spectrometry. Tandem mass spectrometry (MS / MS) is a form of multistage mass spectrometry (MSn) in which the number of stages (n) is 2. As used herein, multistage mass spectrometry refers to MS / MS and MSn mass spectrometry (where n is greater than two).

[0031] The controller 106 can be communicatively coupled to the LC-MS system 100 (e.g., the liquid chromatograph 102 and the mass spectrometer 104) and configured to control the operation of the LC-MS system. The controller 106 can include any suitable hardware (e.g., a processor, circuitry, etc.) and / or software configured to control the operation of and / or interface with various components of the LC-MS system 100 (e.g., the liquid chromatograph 102 or the mass spectrometer 104).

[0032] Figure 2 A functional diagram of an exemplary implementation of a mass spectrometer 104 is shown. As shown, the mass spectrometer 104 is spatially tandem (e.g., having multiple mass filters and / or mass analyzers) and has two stages for performing MS / MS. However, the mass spectrometer 104 is not limited to this configuration, but may have any other suitable configuration. For example, the mass spectrometer 104 may be temporally tandem. Additionally or alternatively, the mass spectrometer 104 may be a multistage mass spectrometer having three or more stages for performing multistage tandem mass spectrometry (e.g., MS / MS / MS).

[0033] As shown, the 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. The mass spectrometer 104 may also include any additional or alternative components that may be suitable for a particular implementation (e.g., ion optics, filters, lenses, ion sources, autosamplers, detectors, etc.) and are not shown.

[0034] The ion source 202 is configured to generate an ion flow 208 from the components and deliver the ions to the first mass analyzer 204-1. The ion source 202 can 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 can include various components for generating ions from the components contained in the sample 108 and delivering the ions to the first mass analyzer 204-1.

[0035] The first mass analyzer 204-1 is configured to receive the ion flow 208, isolate precursor ions of a selected m / z range (e.g., the m / z range of the parallel isolation window), and deliver the precursor ion beam 210 to the collision cell 204-2. The collision cell 204-2 is configured to receive the precursor ion beam 210 and generate product ions (e.g., fragment ions) via a controlled dissociation process. The collision cell 204-2 directs the product ion beam 212 to the second mass analyzer 204-3. The second mass analyzer 204-3 is configured to filter and / or perform mass analysis of the product ions.

[0036] The mass analyzers 204-1 and 204-3 are configured to isolate or separate ions according to the m / z of each ion. The 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 circular ion trap, etc.), a time-of-flight (TOF) mass analyzer, an electrostatic trap mass analyzer (e.g., an orbital electrostatic trap, such as an orbital trap (Orbitrap) mass analyzer, a Kingdon trap, etc.), a Fourier transform ion cyclotron resonance (FT-ICR) mass analyzer, etc. The mass analyzers 204-1 and 204-3 do not need to be implemented by the same type of mass analyzer.

[0037] The collision cell 204-2 can be implemented by any suitable collision cell. As used herein, the term "collision cell" can encompass any structure or device configured to produce product ions via a controlled dissociation process, and is not limited to devices for collision-activated dissociation. For example, the collision cell 204-2 can be configured to fragment precursor ions using collision-induced dissociation (CID), electron transfer dissociation (ETD), electron capture dissociation (ECD), photoinduced dissociation (PID) (e.g., infrared multiphoton dissociation (IRMPD), blackbody infrared radiation dissociation (BIRD)), surface-induced dissociation (SID), negative electron transfer dissociation (NETD), electron detachment dissociation (EDD), higher energy C-trap dissociation (HCD), charge remote fragmentation, ion / molecule reactions, etc.

[0038] An ion detector (not shown) is configured to detect ions at each of the various m / z and responsively generate an electrical signal representing the ion intensity. The electrical signal is sent to the controller 206 for processing, such as constructing a mass spectrum of the sample. For example, the mass analyzer 204-3 can emit an emission beam of separated ions to the ion detector, which is configured to detect ions in the emission beam and generate or provide data that can be used by the controller 206 to construct a sample mass spectrum. The ion detector can be implemented by any suitable detection device including, but not limited to, an electron multiplier, a Faraday cup, etc.

[0039] The controller 206 may be 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. For illustration, 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 a radio frequency (RF) voltage and / or a direct current (DC) voltage to the mass analyzer 204, adjust the values ​​of the RF voltage and the DC voltage to select an effective m / z (including a mass tolerance window) for analysis, and adjust the sensitivity of the ion detector (e.g., by adjusting the detector gain).

[0040] The controller 206 may also include and / or provide a user interface that is configured to enable interaction between a user of the mass spectrometer 104 and the controller 206. The user may interact with the controller 206 via the user interface through tactile, visual, auditory, and / or other sensory types of communication. For example, the user interface may include a display device (e.g., a liquid crystal display (LCD) display screen, a touch screen, etc.) for displaying information (e.g., a mass spectrogram, a notification, etc.) to the user. The user interface may also include an input device (e.g., a keyboard, a mouse, a touch screen device, etc.) that allows the user to provide input to the controller 206. In other examples, the display device and / or the input device may be separate from the controller 206 but communicatively coupled to the controller. For example, the display device and the input device may be included in a computer (e.g., a desktop computer, a laptop computer, etc.) that is communicatively connected to the controller 206 via a wired connection (e.g., via one or more cables) and / or a wireless connection.

[0041] Controller 206 may include any suitable hardware (eg, processor, circuit, etc.) and / or software as may serve a particular implementation. Figure 2 Controller 206 is shown to be included in mass spectrometer 104, but controller 206 may alternatively be implemented in a manner that is completely or partially separate from mass spectrometer 104, such as by a computing device that is communicatively coupled to mass spectrometer 104 via a wired connection (e.g., a cable) and / or a network (e.g., a local area network, a wireless network (e.g., Wi-Fi), a wide area network, the Internet, a cellular data network, etc.). In some examples, controller 206 may be implemented in whole or in part by controller 106.

[0042] The LC-MS system 100 can be used in conjunction with a mass spectrometry control system to perform DIA experiments using parallel isolation multiplexing. Figure 3A functional diagram of an exemplary mass spectrometry control system 300 ("system 300") is shown. System 300 may be implemented in whole or in part by LC-MS system 100 (e.g., by controller 106 and / or controller 206). Alternatively, system 300 may be implemented separately from LC-MS system 100 (e.g., a remote computing system or server that is separate from but communicatively coupled to controller 106 and / or controller 206).

[0043] System 300 may include, but is not limited to, memory 302 and processor 304 selectively and communicatively coupled to each other. Memory 302 and processor 304 may each include or be implemented by hardware and / or software components (e.g., processor, memory, communication interface, instructions stored in memory for execution by processor, etc.). In some examples, memory 302 and processor 304 may be distributed among multiple devices and / or multiple locations that may serve a particular implementation.

[0044] The memory 302 may maintain (e.g., store) executable data used by the processor 304 to perform any of the operations described herein. For example, the memory 302 may store instructions 306 that may be executed by the processor 304 to perform any of the operations described herein. The instructions 306 may be implemented by any suitable application, software, code, and / or other executable data instance.

[0045] The memory 302 may also maintain any data acquired, received, generated, managed, used, and / or sent by the processor 304. For example, the memory 302 may maintain LC-MS data (e.g., acquired chromatogram data and / or mass spectrometer data) and / or a demultiplexing algorithm, as described below.

[0046] The processor 304 can be configured to perform (e.g., execute instructions 306 stored in the memory 302 to perform) various processing operations described herein. For example, the system 300 can control the mass spectrometer to acquire a set of mass spectra of product ions through multiple acquisitions constituting an acquisition cycle, the product ions originating from precursor ions isolated based on parallel isolation windows positioned continuously across the precursor mass-to-charge ratio (m / z) range. The system 300 can also generate a mass spectrum for a precursor m / z range based on the set of mass spectra acquired at operation 402. In some examples, the system 300 demultiplexes the set of mass spectra to determine a signal for each isolation window unit in a plurality of isolation window units.

[0047] It will be appreciated that the operations and examples described herein are merely illustrative of the many different types of operations that may be performed by the processor 304. In the following description, any reference to operations performed by the system 300 may be understood to be performed by the processor 304 of the system 300. Furthermore, in the description herein, any operations performed by the system 300 may be understood to include the system 300 directing or instructing another system or device to perform an operation.

[0048] Figure 4 An exemplary method 400 for performing a DIA experiment using parallel isolation multiplexing is shown. Figure 4 Exemplary operations according to one embodiment are shown, but other embodiments may omit, add to, reorder, and / or modify Figure 4 Any of the operations shown. Figure 4 One or more of the operations illustrated may be performed by LC-MS system 100 and / or system 300, any components included therein, and / or any specific 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).

[0049] At operation 402, parallel isolation is used to isolate a first population of precursor ions having an m / z within an m / z range of a parallel isolation window. Parallel isolation using a parallel isolation window will be described in more detail below.

[0050] At operation 404, the isolated precursor ions are fragmented to produce product ions. The isolated precursor ions may be fragmented in any suitable manner.

[0051] At operation 406, the product ions are mass analyzed to acquire a mass spectrum for the m / z range corresponding to the parallel isolation window.

[0052] Operations 402 to 406 constitute one acquisition of an acquisition cycle, and the mass spectrum acquired at operation 406 is included in a set of mass spectra acquired during the acquisition cycle.

[0053] At operation 408, the system 300 determines whether the acquisition cycle is complete. In some examples, the acquisition cycle is complete when each isolation window cell of the precursor m / z range has been mass analyzed a threshold number of times (e.g., at least twice). If the system 300 determines that the acquisition cycle is not complete, the method 400 proceeds to operation 410.

[0054] At operation 410, the parallel isolation window is moved to the next position within the precursor m / z range. For example, the control voltage that sets the m / z range of the parallel isolation window may be adjusted to perform the next acquisition. The next position of the parallel isolation window may be determined in any suitable manner, such as based on a preconfigured acquisition cycle schedule or randomly. Processing then returns to operations 402 to 406 to perform another acquisition using the parallel isolation window at the new position. This cycle (operations 402 to 410) is repeated until the system 300 determines at operation 408 that the acquisition cycle is complete, at which point processing proceeds to operation 412.

[0055] At operation 412, a mass spectrum corresponding to the precursor m / z range is generated based on a set of mass spectra acquired during the acquisition cycle (e.g., a set of mass spectra acquired at each operation 408). For example, a set of mass spectra can be demultiplexed to assign one or more signals in the set of mass spectra to an appropriate m / z window. Demultiplexing of a set of mass spectra will be described in more detail below.

[0056] Figure 5 An exemplary acquisition scheme 500 for an acquisition cycle using parallel isolation based on a parallel isolation window is shown. The acquisition scheme 500 may be used to implement the method 400. However, the acquisition scheme 500 is merely exemplary, and other acquisition schemes may be used to implement the method 400. Figure 5 As shown, acquisition cycle 502 includes multiple acquisitions 504 (e.g., acquisitions 504-1, 504-2, 504-3, ... 504-N). Each acquisition 504 can be performed as described above for operations 402, 404, and 406 of method 400. During acquisition cycle 502, parallel isolation windows 506 are continuously positioned (e.g., at operation 410 of method 400) across the precursor m / z range 508 over time (e.g., at different locations within the precursor m / z range 508 for each acquisition 504) to analyze the entire precursor m / z range 508. Figure 5 In the example of , the precursor m / z range 508 spans 400 m / z to 900 m / z. However, the precursor m / z range 508 may span any other range that may suit a particular implementation. The precursor m / z range 508 is divided into a plurality of isolation window cells 510 of fixed cell width. Figure 5In the example of , the isolation window cell 510 has a cell width of 20m / z. However, the isolation window cell 510 may have any other cell width that may conform to a particular implementation. In some examples, the isolation window cell 510 has a cell width between 1m / z and 20m / z (inclusive). In another example, the isolation window cell 510 has a cell width between 2m / z and 10m / z (inclusive). In yet another example, the isolation window cell 510 has a cell width between 2m / z and 5m / z (inclusive). In some examples, the isolation window cell 510 has a cell width between 2m / z and 4m / z (inclusive).

[0057] For each acquisition 504, the parallel isolation window 506 includes a group of two or more isolation sub-windows 512 corresponding to a different group of isolation window cells 510, wherein at least two adjacent isolation sub-windows 512 of the parallel isolation window 506 are discontinuous. As used herein, "adjacent" isolation sub-windows 512 mean isolation sub-windows 512 that are adjacent in the positive or negative m / z direction without any intervening isolation sub-windows 512. Adjacent isolation sub-windows 512 do not need to be continuous. As used herein, "continuous" means adjacent to each other without an m / z gap therebetween. As used herein, "discontinuous" means not adjacent to each other so that there is an m / z gap between the isolation sub-windows 512. In some examples, the m / z gap has a width that is an integer multiple of the cell width, so that the isolation sub-windows 512 of different acquisitions are aligned with each other along the precursor m / z range 508.

[0058] exist Figure 5 In the example of , the parallel isolation window 506 includes a set of four isolation sub-windows 512 (e.g., isolation sub-windows 512-1, 512-2, 512-3, and 512-4). Isolation sub-window 512-1 and isolation sub-window 512-2 are adjacent to each other and continuous with each other. Isolation sub-window 512-2 and isolation sub-window 512-3 are adjacent to each other but not continuous (e.g., an m / z gap 514-1 equal to one unit width is located between isolation sub-window 512-2 and isolation sub-window 512-3). Similarly, isolation sub-window 512-3 and isolation sub-window 512-4 are adjacent to each other but not continuous with each other (e.g., an m / z gap 514-2 equal to one unit width is located between isolation sub-window 512-3 and isolation sub-window 512-4). Isolation sub-window 512-1 is not adjacent to isolation sub-window 512-3 or isolation sub-window 512-4, isolation sub-window 512-2 is not adjacent to isolation sub-window 512-4, isolation sub-window 512-3 is not adjacent to isolation sub-window 512-1, and isolation sub-window 512-4 is not adjacent to isolation sub-window 512-1 or isolation sub-window 512-2.

[0059] As used herein, the m / z range of a parallel isolation window 506 refers to the total m / z range of only one set of isolation sub-windows 512 of the parallel isolation window 506 for a given acquisition 504 and does not include any m / z gaps 514 between the isolation sub-windows 512. The isolation width of a parallel isolation window 506 is the width of the m / z range of the parallel isolation window. Therefore, the isolation width of the parallel isolation window 506 is 80m / z (four isolation sub-windows 512 of a cell width of 20m / z each). On the other hand, the total m / z span of a parallel isolation window 506 refers to the total span of all isolation sub-windows 512 for a given acquisition 504 and any m / z gaps 514 between the isolation sub-windows 512. Therefore, the width of the total m / z span of a parallel isolation window 506 is 120m / z.

[0060] The parallel isolation window 506 is not limited to Figure 5 , but may have any other suitable configuration (e.g., number and / or position of isolation sub-windows 512, parallel isolation width, m / z range, total m / z span). In addition, m / z gap 514 may have any other suitable width (e.g., two or more unit widths). Various other exemplary configurations of parallel isolation windows 506 will be described in more detail below.

[0061] During the acquisition cycle 502, the parallel isolation windows 506 are positioned continuously throughout the precursor m / z range 508, such that each isolation window cell 510 of the precursor m / z range 508 is analyzed multiple times (e.g., by at least two separate acquisitions 504). In some examples, the portion of the parallel isolation windows 506 that are cut off at the tail end of the precursor m / z range 508 (e.g., the group 516 of isolation sub-windows shown as dashed lines and gradient shading beyond 900 m / z) is flipped to the beginning of the precursor m / z range 508 (starting at 400 m / z). Figure 5As shown, the group 516 of the isolation sub-windows is flipped to the subsequent acquisition 504 instead of in its original acquisition 504, because the m / z gap between the flipped isolation sub-window 512-4 and the unflipped isolation sub-window 512-1 may be too large for parallel isolation. Generally, parallel isolation becomes difficult when the m / z gap is greater than about 100m / z. In other examples (not shown), the group 516 of the isolation sub-windows is flipped in the same acquisition 504. In another example, the group 516 of the isolation sub-windows that exceeds the upper limit of the precursor m / z range 508 is not flipped, but included in the acquisition, and the collected data is discarded. In an additional or alternative example, the portion of the parallel isolation window 506 cut off at the minimum end of the precursor m / z range 508 can be processed in a similar but opposite manner to the group 516 of the isolation sub-windows. For example, acquisition cycle 502 may include one or more acquisitions 504 prior to acquisition 504-1, and a set of isolation subwindows that were cut off before 400 m / z may be flipped to the upper limit of the precursor m / z range 508, or a set of isolation subwindows may be included and the data discarded.

[0062] During each acquisition 504, precursor ions having an m / z within the m / z range of the parallel isolation window 506 are isolated by parallel isolation (e.g., operation 402) for fragmentation into product ions (e.g., operation 404) to be mass analyzed (e.g., operation 406). Parallel isolation is a technique for simultaneously isolating precursor ions over a discrete m / z range (e.g., an m / z range having one or more m / z gaps in which ions are not isolated, such as the m / z range of the parallel isolation window 506). Any suitable parallel isolation technique and instrument configuration may be used for parallel isolation.

[0063] In some examples, precursor ions are isolated by parallel isolation using parallel waveform isolation technology. Parallel waveform isolation can be performed using an ion trap (e.g., a linear quadrupole ion trap) and / or using a mass filter (e.g., a quadrupole mass filter). For example, in a linear ion trap and a mass filter, ions are radially captured by an RF quadrupole field. In a linear ion trap, ions are also axially captured by a static DC potential at an end (e.g., at the end of a quadrupole array). Parallel waveform isolation is a technique that applies a broadband auxiliary alternating current (AC) waveform to remove unwanted ions from an ion population captured in an ion trap or passing through a mass filter. The broadband auxiliary AC waveform has a frequency distribution that contains energy at the swing frequency of the unwanted ions and does not contain energy at the swing frequency of the precursor ions of interest, thereby forming one or more "notches" corresponding to the isolated (retained) precursor ions of interest. Various exemplary methods of parallel waveform isolation are described in detail in US Pat. No. 10,170,290, US Pat. No. 9,875,885, and US Pat. No. 9,048,074, each of which is incorporated herein by reference in its entirety.

[0064] In other examples, precursor ions are isolated using the mass selective ejection of trapped ions. Mass selective ejection includes mass selective radial ejection and mass selective axial ejection. When the RF voltage for the radial confinement RF quadrupole field is increased in the presence of a radially applied auxiliary AC resonant ejection voltage, mass selective radial ejection occurs, which causes the trapped ions to be ejected from the ion trap through the slots in the quadrupole. When a degree of radial excitation is given to the trapped ions via a resonant excitation process, mass selective axial ejection of the ions occurs, which gives the ions enough axial kinetic energy to overcome the static DC exit barrier.

[0065] A variety of different instrument configurations may be used to implement method 400, as will now be referenced. FIG. 6A to FIG. 6D and 7A to 7C Described.

[0066] Fig. 6A An exemplary implementation of method 400 using an exemplary instrument configuration 600A is shown. As shown, instrument configuration 600A includes an ion source 602 and an ion trap 604. Ion source 602 can be implemented by any ion source described herein (e.g., by ion source 202). Ion trap 604 can be implemented by any suitable ion trap, such as a linear ion trap (e.g., a linear quadrupole ion trap, a linear hexapole ion trap, a linear octopole ion trap, etc.), a spherical ion trap, a toroidal ion trap, or any other suitable ion trap capable of performing parallel isolation. With this configuration, parallel isolation, fragmentation, and mass analysis are all performed sequentially in ion trap 604. Parallel isolation can be performed using parallel waveform isolation or mass selective ejection of ions.

[0067] Figure 6B Another exemplary embodiment of the method 400 using an exemplary instrument configuration 600B is shown. As shown, the instrument configuration 600B (e.g., triple quadrupole instrument) includes an ion source 602, a mass filter 606 (e.g., a quadrupole mass filter), a collision cell 608 located downstream of the mass filter 606, and a mass analyzer 610 located downstream of the collision cell 608. The mass analyzer 610 can be implemented by any suitable mass analyzer, including any mass analyzer described herein (e.g., mass analyzer 204-3). When ions pass through the mass filter 606, the mass filter 606 uses parallel waveform isolation to perform parallel isolation. The isolated precursor ions are accumulated and fragmented in the collision cell 608 subsequently. The resulting product ions are subsequently transferred to the mass analyzer 610 to perform mass analysis. In some examples, a first population of ions (e.g., product ions) is mass analyzed within mass analyzer 610 during a first acquisition (e.g., acquisition 504-1), while a second population of ions (e.g., isolated precursor ions) is isolated by mass filter 606 and fragmented in collision chamber 608 during a second acquisition (e.g., acquisition 504-2), thereby achieving increased experimental throughput compared to instrument configuration 600A.

[0068] Figure 6C Another exemplary implementation of the method 400 using an exemplary instrument configuration 600C is shown. As shown, the instrument configuration 600C (e.g., a triple quadrupole instrument) includes an ion source 602, a mass filter 606, a collision cell 608, and an ion trap 612 positioned downstream of the collision cell 608. The mass filter 606 and the collision cell 608 are controlled to pass all ions to the ion trap 612. Alternatively, the collision cell 608 is controlled to accumulate ions before ejecting the accumulated ions into the ion trap 612. The ion trap 612 then performs parallel isolation of precursor ions (e.g., using parallel waveform isolation or mass selective ejection), fragmentation of the isolated precursor ions, and mass analysis of the product ions. In some examples, a first population of ions may be processed (e.g., parallel isolation, fragmentation, and mass analysis) within ion trap 612 during a first acquisition (e.g., acquisition 504-1) while a second population of ions is simultaneously accumulated within collision chamber 608 during a second acquisition (e.g., acquisition 504-2) until the second population of ions can be transferred to ion trap 612 for parallel isolation, fragmentation, and mass analysis.

[0069] Fig.6D Another exemplary implementation of method 400 using instrument configuration 600C is shown. Fig.6DIn the example of , the mass filter 606 and the collision cell 608 are controlled to pass all ions to the ion trap 612. The ion trap 612 then performs parallel isolation of the precursor ions (e.g., using parallel waveform isolation or mass selective ejection). The isolated precursor ions are then transferred back to the collision cell 608 for fragmentation. The resulting product ions are then returned to the ion trap 612 for mass analysis.

[0070] exist Figure 6C and Fig.6D In the example of FIG. 6 , the ions are transferred to the ion trap 612 for parallel isolation in the form of a beam rather than an extended stream. However, the precursor ion beam may suffer from space charge problems in the ion trap 612, which prevents parallel waveform isolation techniques for parallel isolation. To prevent space charge problems, the precursor ions may be pre-isolated (e.g., pre-filtered) before being transferred to the ion trap 612 for parallel isolation, as will now be referred to. 7A to 7C Described.

[0071] Fig. 7A An exemplary implementation of method 400 using instrument configuration 600C is shown. Fig. 7A In the example of , the mass filter 606 is controlled to pre-isolate the ion population having an m / z within the total m / z span of the parallel isolation window. For example, in scheme 500 ( Figure 5 ), the total m / z span of the parallel isolation window 506 during the first acquisition 504-1 ranges from 400 m / z to 520 m / z. Therefore, the mass filter 606 is controlled to pre-isolate ions having m / z using an isolation window ranging from 400 m / z to 520 m / z (or any other larger isolation window covering the range of 400 m / z to 520 m / z, such as 400 m / z to 540 m / z). The collision cell 608 is controlled to accumulate and / or pass the population of pre-isolated ions to the ion trap 612. The ion trap 612 performs parallel isolation (e.g., using parallel waveform isolation or mass selective ejection) on the population of pre-isolated ions to isolate a population of precursor ions having m / z within the m / z range of the parallel isolation window 506. Therefore, the population of precursor ions is derived from the population of pre-isolated ions. By performing pre-isolation at mass filter 606, the number of ions transferred to ion trap 612 can be reduced to reduce or prevent space charge problems in ion trap 612. Ion trap 612 then performs fragmentation of the population of precursor ions and mass analysis of the resulting population of product ions.

[0072] Figure 7B Another exemplary implementation of method 400 using instrument configuration 600C is shown. Figure 7B Similar to Fig. 7A , the difference is that in Figure 7BIn FIG. 6 , after the ion trap 612 performs parallel isolation of the population of precursor ions, the population of precursor ions is transferred back to the collision cell 608 for fragmentation. The resulting population of product ions is then returned to the ion trap 612 for mass analysis.

[0073] Figure 7C Another exemplary implementation of method 400 using instrument configuration 600C is shown. Figure 7C Similar to Fig. 7A , the difference is that in Figure 7C In the example of , two ion populations are processed substantially simultaneously. For example, a first ion population is processed (e.g., isolated, fragmented, and mass analyzed in parallel) within ion trap 612 during a first acquisition (e.g., acquisition 504-1), while a second ion population is pre-isolated in mass filter 606 and accumulated in collision cell 608 during a second acquisition (e.g., acquisition 504-2). Once processing of the first ion population is completed in ion trap 612, the second ion population is transferred to ion trap 612 for processing, while a third ion population is processed in mass filter 606 and collision cell 608 during a third acquisition (e.g., acquisition 504-3).

[0074] The specific implementation of method 400 is not limited to reference FIG. 6A to FIG. 6D and Figures 7A to 7C The examples shown and described are not exhaustive, as other implementations and other instrument configurations may be used. For example, other instrument configurations may include a multi-stage mass spectrometer with more than two stages.

[0075] Demultiplexing a set of mass spectra acquired during an acquisition cycle to generate a mass spectrum for a precursor m / z range will now be described. Figure 5 , each mass spectrum acquired during acquisition 504 includes signals representing ions derived from precursor ions having an m / z within the m / z range of parallel isolation window 506. However, the signals in the mass spectrum cannot be accurately assigned to the appropriate isolation window cell 510 (e.g., the isolation window cell 510 corresponding to the precursor m / z from which the signal was derived). Therefore, the mass spectra have an m / z range equal to the parallel isolation window 506 (e.g., Figure 5 ) in the example of four isolation sub-windows 512). However, each isolation window unit 510 of the precursor m / z range 508 is analyzed multiple times via multiple unique acquisitions 504, which enables demultiplexing of signals in a set of mass spectra to generate a demultiplexed mass spectrum with improved selectivity. The selectivity measure of the demultiplexed mass spectrum is equal to the m / z range of the parallel isolation windows 506 divided by the number of overlaps of each isolation window unit 510 per acquisition cycle 502. Therefore, the selectivity measure of the demultiplexed mass spectrum is less than the m / z range of the parallel isolation windows 506. Figure 5In the example of , the m / z range of the parallel isolation window 506 is four isolation sub-windows 512 (e.g., 80 m / z) and each isolation window unit 510 has four overlaps (e.g., analyzed in four different acquisitions 504), so the selectivity of the demultiplexing is one isolation window unit (20 m / z).

[0076] Demultiplex a set of mass spectra using Figure 5 The system 300 may use any suitable demultiplexing algorithm to demultiplex a set of mass spectra. In some examples, the demultiplexing algorithm uses acquisition 504 in acquisition cycle 502 to generate a system of linear equations and solve the system of linear equations, such as by using a non-negative least squares technique.

[0077] To illustrate, the demultiplexing algorithm can approximate each acquisition 504 in the acquisition cycle 502 as a linear superposition of data from each isolation sub-window 512 of the parallel isolation window 506. Each acquisition 504 contains a different set of isolation sub-windows 512 (e.g., a set of isolation sub-windows 512 corresponding to a different set of isolation window cells 510), enabling the signal to be localized to the appropriate isolation window cell 510 precursor m / z range 508. The demultiplexing algorithm localizes the signal from the parallel isolation windows 506 to its appropriate isolation window cell 510 by solving a system of linear equations. For example, for each acquisition of interest and each transition of interest, a set of acquisitions surrounding the acquisition of interest is used to form a system of linear equations that relates the measured intensity of each acquisition to the combination of isolation window cells analyzed by the acquisition of interest. The matrix equation (1) is then solved:

[0078] X·A=Y ​​(1)

[0079] Where X is an n×n "design matrix" whose rows represent different acquisitions (plus one regular row) and whose columns represent isolation window cells in the precursor m / z range (n is the number of isolation window cells in the precursor m / z range), and whose entries contain one (1) if the acquisition includes a given isolation window cell and zero (0) if the acquisition does not include the given isolation window cell; Y is the "observation data", an n×m matrix whose rows represent different acquisitions (plus one regular row) and whose columns represent product ions (m is the number of product ions of interest), and whose entries represent the observed intensity of each transition for each acquisition; and A is an n×m matrix whose rows represent isolation window cells and whose columns represent transitions, and whose entries represent the unknown isolation window cell intensities being solved for. Because mass spectrometry signal intensities are required to be positive, the matrix equation (1) can be solved using non-negative least squares techniques, such as the Lawson-Hansen algorithm. Exemplary demultiplexing algorithms that can be used are described in more detail in Amodei et al., Improving Precursor Selectivity in Data-Independent Acquisition Using Overlapping Windows. Mass Spectrom. 2019, 30(4): 669–684, which is incorporated herein by reference in its entirety.

[0080] After completing acquisition cycle 502, another acquisition cycle may be performed in the same or similar manner, and the process may be continuously repeated as the analytes elute from the separation system.

[0081] As described above, method 400 may be implemented by other acquisition schemes than acquisition scheme 500. Illustrative examples of alternative acquisition schemes will now be described.

[0082] Figure 8 Another exemplary acquisition scheme 800 is shown. The acquisition scheme 800 is similar to Figure 5800, except that in the acquisition scheme 800, the acquisition cycle 502 includes a first sub-cycle 802-1 and a second sub-cycle 802-2, the first sub-cycle including a first subset of acquisitions 504 (e.g., acquisitions 504-1, 504-2, 504-3 to 504-M), and the second sub-cycle including a second subset of acquisitions 504 (e.g., acquisitions 504-M+1, 504-M+2 to 504-N). The second sub-cycle 802-2 is performed after the first sub-cycle 802-1. The parallel isolation windows 506 are continuously positioned throughout the precursor m / z range 508, so that each isolation window cell 510 of the precursor m / z range 508 is analyzed at least once during the first sub-cycle 802-1. Similarly, the parallel isolation windows 506 are continuously positioned throughout the precursor m / z range 508, so that each isolation window cell 510 of the precursor m / z range 508 is analyzed at least once during the second sub-cycle 802-2.

[0083] With the dual sub-cycle ordering of acquisition 504 of acquisition scheme 800, the entire precursor m / z range 508 is analyzed more frequently (e.g., at twice the sampling frequency of acquisition scheme 500) than acquisition scheme 500. This higher frequency sampling can be configured to achieve sampling at or above the Nyquist limit or any other desired or required frequency.

[0084] Fig. 9 Another exemplary acquisition scheme 900 is shown. The acquisition scheme 900 is similar to Figure 5 The acquisition scheme 500 is different from the acquisition scheme 500, except that in the acquisition scheme 900, the parallel isolation window 506 has a different configuration than that in the acquisition scheme 500. In the acquisition scheme 900, the parallel isolation window 506 has only one m / z gap 514-1 (between the isolation sub-window 512-2 and the isolation sub-window 512-3), and the isolation sub-window 512-3 is adjacent to and continuous with the isolation sub-window 512-4.

[0085] Fig.10 Another exemplary acquisition scheme 1000 is shown. The acquisition scheme 1000 is similar to Fig. 9900 of the acquisition scheme 1000, except that in the acquisition scheme 1000, the acquisition cycle 502 includes a first sub-cycle 1002-1 of the acquisition 504 (e.g., acquisitions 504-1, 504-2, 504-3 to 504-M) and a second sub-cycle 1002-2 of the acquisition 504 (e.g., acquisitions 504-M+1, 504-M+2 to 504-N). The second sub-cycle 1002-2 is performed after the first sub-cycle 1002-1. The parallel isolation windows 506 are continuously positioned throughout the precursor m / z range 508, so that each isolation window cell 510 of the precursor m / z range 508 is analyzed at least once during the first sub-cycle 1002-1. Similarly, the parallel isolation windows 506 are continuously positioned throughout the precursor m / z range 508, so that each isolation window cell 510 of the precursor m / z range 508 is analyzed at least once during the second sub-cycle 1002-2.

[0086] With the dual sub-cycle ordering of acquisitions 504 of acquisition scheme 1000, the entire precursor m / z range 508 is analyzed more frequently (e.g., at twice the sampling frequency of acquisition scheme 500) than acquisition scheme 900. This higher frequency sampling can be configured to achieve sampling at or above the Nyquist limit or any other desired or required frequency.

[0087] Fig.11 Another exemplary acquisition scheme 1100 is shown. Acquisition scheme 1100 is similar to acquisition scheme 500, except that, in acquisition scheme 1100, parallel isolation window 506 has a different configuration than in acquisition scheme 500. In acquisition scheme 1100, parallel isolation window 506 has three isolation sub-windows 512 (e.g., isolation sub-windows 512-1, 512-2, and 512-3; isolation sub-window 512-4 of acquisition scheme 500 is omitted).

[0088] Fig.12Another exemplary acquisition scheme 1200 is shown. In the acquisition scheme 1200, the parallel isolation window 506 has three adjacent and continuous isolation sub-windows 512 (e.g., isolation sub-windows 512-1, 512-2, and 512-3) and an isolation sub-window 512-4 that is adjacent to but not continuous with the isolation sub-window 512-3. The m / z gap 514-1 is located between the isolation sub-window 512-3 and the isolation sub-window 512-4. In the acquisition scheme 1200, the acquisition cycle 502 includes: a first sub-cycle 1202-1, a second sub-cycle 1202-2, and a third sub-cycle 1202-3, wherein the first sub-cycle includes a first sub-group of acquisitions 504 (e.g., acquisitions 504-1, 504-2, 504-3 to 504-L); the second sub-cycle includes a second sub-group of acquisitions 504 (e.g., acquisitions 504-L+1, 504-L+2 to 504-M), and the third sub-cycle includes a third sub-group of acquisitions 504 (e.g., acquisitions 504-M+1, 504-M+2 to 504-N). The second sub-cycle 1202-2 is executed after the first sub-cycle 1202-1, and the third sub-cycle 1202-3 is executed after the second sub-cycle 1202-2. The parallel isolation windows 506 are positioned continuously throughout the precursor m / z range 508 such that each isolation window cell 510 of the precursor m / z range 508 is analyzed at least once during the first sub-cycle 1202-1. Similarly, the parallel isolation windows 506 are positioned continuously throughout the precursor m / z range 508 such that each isolation window cell 510 of the precursor m / z range 508 is analyzed at least once during the second sub-cycle 1202-2. Similarly, the parallel isolation windows 506 are positioned continuously throughout the precursor m / z range 508 such that each isolation window cell 510 of the precursor m / z range 508 is analyzed at least once during the third sub-cycle 1202-3.

[0089] Utilizing the triplet cycle ordering of acquisition 504 of acquisition scheme 1200, the entire precursor m / z range 508 is analyzed more frequently than acquisition scheme 500 (e.g., at three times the sampling frequency of acquisition scheme 500), acquisition scheme 900, and acquisition scheme 1100, even though the total acquisition cycle time is the same.

[0090] Various modifications may be made to the above examples and embodiments. In some examples, the cell width of the isolation window cell 510, the isolation width of the parallel isolation window 506, and / or the total m / z span of the parallel isolation window 506 are not fixed within the acquisition cycle 502, but vary based on m / z. For example, the cell width of the isolation window cell 510 at the edge interval of the precursor m / z range 508 (e.g., from 400m / z-500m / z to 800m / z-900m / z) may be greater than (or less than) the cell width at the center interval of the precursor m / z range 508 (e.g., from 500m / z to 800m / z). In additional or alternative examples, the configuration of the parallel isolation window 506 may vary throughout the acquisition cycle as long as each isolation window cell 510 of the precursor m / z range 508 is analyzed at least twice.

[0091] In yet another example, the acquisition scheme used to implement method 400 during a DIA experiment can vary with the elution gradient. For example, different acquisition schemes can be used at different times during the elution gradient. For example, acquisition scheme 500 can be used during a first time period during which a component is eluted from liquid chromatograph 102, and acquisition scheme 900 or acquisition scheme 1100 can be used during a second time period during which a component is eluted from liquid chromatograph 102.

[0092] like Figure 5 , FIG. 6A to FIG. 6D and 7A to 7C As shown in the example of , the parallel isolation windows 506 are sequentially positioned within the precursor m / z range 508 in order of increasing m / z within the acquisition cycle 502 or within the acquisition sub-cycles 802, 1002, and 1202. However, any other ordering of the parallel isolation windows 506 within the acquisition cycle 502 and / or within the sub-cycles 802, 1002, and 1202 may be used, including sequential ordering in order of decreasing m / z, non-sequential ordering along the m / z domain (e.g., staggered, alternating, or some other pattern), or randomly ordering.

[0093] In the acquisition scheme described above, the parallel isolation window 506 has a "square" profile limited only by zero and one, where one indicates an isolation sub-window 512 for the isolation of precursor ions and zero indicates an m / z gap. However, the parallel isolation window 506 can have any other suitable profile. In some examples, the parallel isolation window 506 is formed by a parallel isolation waveform such as a square wave, which decreases from one to zero at the minimum and maximum ends of the parallel isolation window 506 and at the edge near the m / z gap. For example, the edge of the parallel isolation window 506 may have a value between zero and one (e.g., 0.80, 0.65, 0.50, 0.35, etc.). In other examples, the parallel isolation window 506 has a gradual continuous transition between zero and one at the edge. For example, the parallel isolation window 506 may have a non-square profile that smoothly changes between zero and one at the edge, such as a sine waveform. These modified profiles of the parallel isolation window 506 can better promote demultiplexing than square profiles with only zero and one.

[0094] The systems and methods described herein can be applied to other types of instruments, wherein the concept of parallel isolation is extended to include any group ion isolation that can be performed without losing duty cycle. For example, an instrument with an upstream ion accumulator (e.g., a collision chamber or ion storage device) and a downstream ion separator (e.g., a quadrupole mass filter, an ion trap, etc.) can be controlled so that when ions accumulate in the accumulator, a group of one or more ion separations occur continuously in the downstream ion separator to produce a population of precursor ions. Continuous isolation can be performed, for example, using mass selective injection of ions, as described above. The isolated precursor ions are then fragmented, and the total population of ion fragments is analyzed in a single MS / MS acquisition. Such instruments will enjoy the benefits of DIA acquisition using parallel isolation multiplexing without the need for parallel isolation techniques, such as waveform isolation.

[0095] The concept of parallel isolation can also be extended to ion mobility mass spectrometry (IM-MS) instruments. For example, by using a parallel mobility window positioned continuously within the ion mobility range during acquisition, different ion packets can be continuously released from the ion mobility separator. The parallel mobility window can be configured similar to the parallel isolation window described herein. For example, for each release of ions, the parallel mobility window can include a different set of ion mobility sub-windows, wherein at least two adjacent ion mobility sub-windows are discontinuous along the ion mobility domain. Each different ion packet released from the ion mobility separator is transferred to a fragmentor (e.g., a collision chamber) for generating product ions. Product ions from all ion packets for acquisition can be accumulated in an accumulator, and all accumulated ions can be subsequently mass analyzed in a mass analyzer in a single mass spectrum (e.g., a single MS / MS spectrum).

[0096] 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 above-mentioned systems and / or components may include or be implemented by any computer hardware and / or computer implementation instructions (e.g., software) embodied on at least one non-transient computer-readable medium configured to perform one or more processes in the processes described above. Specifically, the system component may be implemented on a physical computing device, or may be implemented on more than one physical computing device. Therefore, the system component may include any number of computing devices, and any number of computer operating systems may be used.

[0097] In certain embodiments, one or more of the processes described herein may be implemented at least in part as instructions embodied in a non-transitory computer-readable medium and executable by one or more computing devices. Typically, a processor (e.g., a microprocessor) receives instructions from a non-transitory computer-readable medium (e.g., a memory, etc.) and executes those instructions, thereby performing one or more processes, including one or more of the processes described herein. Any of a variety of known computer-readable media may be used to store and / or send such instructions.

[0098] Computer-readable media (also referred to as processor-readable media) include any non-transitory media that participate in providing data (e.g., instructions) that can be read by a computer (e.g., by a processor of a computer). Such media can take many forms, including but not limited to non-volatile media and / or volatile media. Non-volatile media can include, for example, optical or magnetic disks and other permanent memories. Volatile media can include, for example, dynamic random access memory ("DRAM") that typically constitutes main memory. Common forms of computer-readable media include, for example, disks, hard disks, tapes, any other magnetic media, compact disk read-only memory ("CD-ROM"), digital video disks ("DVD"), any other optical media, random access memory ("RAM"), programmable read-only memory ("PROM"), electrically erasable programmable read-only memory ("EPROM"), FLASH-EEPROM, any other memory chip or cartridge, or any other tangible medium that a computer can read.

[0099] Fig.13 An exemplary computing device 1300 is shown, which may be specifically configured to perform one or more of the processes described herein. Fig.13 As shown, computing device 1300 may include a communication interface 1302, a processor 1304, a storage device 1306, and an input / output ("I / O") module 1308 communicatively coupled to one another via a communication infrastructure 1310. Fig.13 An exemplary computing device 1300 is shown, but Fig.13 The components shown are not intended to be limiting. In other embodiments, additional or alternative components may be used. Fig.13 Components of computing device 1300 are shown.

[0100] Communication interface 1302 may be configured to communicate with one or more computing devices. Examples of communication interface 1302 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.

[0101] The processor 1304 generally represents any type or form of processing unit capable of processing data and / or interpreting, executing and / or directing one or more of the instructions, processes and / or operations described herein. The processor 1304 may perform operations by executing computer-executable instructions 1312 (e.g., applications, software, code and / or other executable data instances) stored in the storage device 1306.

[0102] Storage device 1306 may include one or more data storage media, devices, or configurations, and may employ any type, form, and combination of data storage media and / or devices. For example, storage device 1306 may include, but is not limited to, any combination of non-volatile media and / or volatile media described herein. Electronic data, including data described herein, may be temporarily and / or permanently stored in storage device 1306. For example, data representing computer executable instructions 1312 configured to direct processor 1304 to perform any of the operations described herein may be stored within storage device 1306. In some examples, the data may be arranged in one or more databases residing within storage device 1306.

[0103] The I / O module 1308 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. The I / O module 1308 may include any hardware, firmware, software, or combination thereof that supports input capabilities and output capabilities. For example, the I / O module 1308 may include hardware and / or software for capturing user input, including but not limited to a keyboard or keypad, a touch screen component (e.g., a touch screen display), a receiver (e.g., an RF or infrared receiver), a motion sensor, and / or one or more input buttons.

[0104] I / O module 1308 may include one or more devices for presenting output to a user, including but not limited to a graphics engine, a display (e.g., a display screen), one or more output drivers (e.g., a display driver), one or more audio speakers, and one or more audio drivers. In certain embodiments, I / O module 1308 is configured to provide graphical data to the display for presentation to the user. Graphics data may represent one or more graphical user interfaces and / or any other graphical content such as may serve a particular implementation.

[0105] In some examples, any of the systems, computing devices, and / or other components described herein may be implemented by computing device 1300. For example, memory 302 may be implemented by storage device 1306, and processor 304 may be implemented by processor 1304.

[0106] Those of ordinary skill in the art will recognize that, although 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 alterations may be made thereto, and additional embodiments may be implemented, without departing from the scope of the invention as set forth in the appended 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 description and drawings should be regarded as illustrative rather than restrictive.

Claims

1. A system, comprising: one or more processors; and A memory storing executable instructions that, when executed by the one or more processors, cause a computing device to perform a process comprising: controlling the mass spectrometer to acquire, during a plurality of acquisitions constituting an acquisition cycle, a set of mass spectra of product ions derived from precursor ions isolated based on parallel isolation windows positioned sequentially across a range of precursor mass-to-charge ratios (m / z); in: dividing the precursor m / z range into a plurality of isolation window units; For each acquisition of the acquisition cycle, the parallel isolation window includes a set of isolation sub-windows corresponding to a different set of isolation window cells for the precursor m / z range; At least two adjacent isolation sub-windows of the parallel isolation window are discontinuous; and Each isolation window cell of the precursor m / z range is analyzed at least twice during the acquisition cycle.

2. The system of claim 1, wherein the process further comprises: A mass spectrum is generated for the precursor m / z range based on the set of mass spectra acquired during the acquisition cycle.

3. The system of claim 2, wherein generating the mass spectrum comprises demultiplexing the set of mass spectra to assign signals representing product ions to appropriate isolation window cells for the precursor m / z range.

4. The system of claim 2, wherein the selectivity measure of the mass spectrum is less than the m / z range of the parallel isolation window.

5. The system of claim 1, wherein: The acquisition cycle includes a first sub-cycle and a second sub-cycle, the first sub-cycle includes a first subset of acquisitions, the second sub-cycle includes a second subset of acquisitions, wherein the second sub-cycle is executed after the first sub-cycle; Each isolation window cell of the precursor m / z range is analyzed at least once during the first sub-cycle; and Each isolation window cell of the precursor m / z range is analyzed at least once during the second sub-cycle.

6. The system of claim 1 , wherein for each acquisition of the acquisition cycle, acquiring the set of mass spectra comprises: isolating a population of said precursor ions having an m / z within an m / z range of said parallel isolation window; fragmenting the population of precursor ions to produce the population of product ions; as well as The population of the product ions is mass analyzed.

7. The system of claim 6, wherein the isolation, the fragmentation, and the mass analysis are performed in an ion trap.

8. The system of claim 6, wherein: The isolation is performed in a mass filter; The fragmentation is performed in a collision cell positioned downstream of the mass filter; and The mass analysis is performed in a mass analyser positioned downstream of the collision cell.

9. The system of claim 6, wherein the isolation is performed using parallel waveform isolation.

10. The system of claim 6, wherein the isolating is performed using mass selective ejection of ions.

11. The system of claim 6, wherein for each acquisition of the acquisition cycle, acquiring the set of mass spectra further comprises: prior to said isolating said population of said precursor ions, isolating a population of pre-isolated ions having an m / z within a total m / z span of said parallel isolation windows; wherein the population of the precursor ions is derived from the population of the pre-isolated ions.

12. The system of claim 11, wherein: said isolating said population of pre-isolated ions is performed in a mass filter; and Said isolating said population of said precursor ions is performed in an ion trap positioned downstream of said mass filter.

13. The system of claim 12, wherein the fragmentation is performed in the ion trap or in a collision cell positioned between the mass filter and the ion trap.

14. The system of claim 1, wherein the parallel isolation window has a waveform that varies between zero and one at an edge of the parallel isolation window.

15. A system, comprising: Mass spectrometer; and a controller configured to control the mass spectrometer to acquire a set of mass spectra of product ions derived from precursor ions isolated based on parallel isolation windows positioned sequentially across a precursor mass-to-charge ratio (m / z) range during a plurality of acquisitions constituting an acquisition cycle; in: dividing the precursor m / z range into a plurality of isolation window units; For each acquisition of the acquisition cycle, the parallel isolation window includes a set of isolation sub-windows corresponding to a different set of isolation window cells for the precursor m / z range; At least two adjacent isolation sub-windows of the parallel isolation window are discontinuous; and Each isolation window cell of the precursor m / z range is analyzed at least twice during the acquisition cycle.

16. The system of claim 15, wherein for each acquisition of the acquisition cycle, acquiring the set of mass spectra comprises: isolating a population of said precursor ions having an m / z within an m / z range of said parallel isolation window; fragmenting the population of the precursor ions to produce the population of product ions; as well as The population of the product ions is mass analyzed.

17. The system of claim 16, wherein: The mass spectrometer comprises an ion trap; and The isolation and the mass analysis are performed in the ion trap.

18. The system of claim 16, wherein: The mass spectrometer includes a mass filter, a collision cell positioned downstream of the mass filter, and a mass analyzer positioned downstream of the collision cell; The isolation is performed in the mass filter; said fragmentation is performed in said collision cell; and The mass analysis is performed in the mass analyzer.

19. The system of claim 16, wherein for each acquisition of the acquisition cycle, the acquiring the set of mass spectra further comprises: prior to said isolating said population of said precursor ions, isolating a population of pre-isolated ions having an m / z within a total m / z span of said parallel isolation windows; wherein the population of the precursor ions is derived from the population of the pre-isolated ions.

20. The system of claim 19, wherein: The mass spectrometer includes a mass filter and an ion trap positioned downstream of the mass filter; said isolating said population of pre-isolated ions is performed in said mass filter; and The isolating the population of the precursor ions is performed in the ion trap.

21. The system of claim 20, wherein: The mass spectrometer also includes a collision cell positioned downstream of the mass filter and upstream of the ion trap; and The fragmentation is performed in the ion trap.

22. The system of claim 21, wherein a first population of ions is processed in the ion trap during a first acquisition and a second population of ions is processed in the mass filter and the collision cell during a second acquisition.

23. The system of claim 15, wherein the controller is further configured to generate a mass spectrum for the precursor m / z range based on the set of mass spectra.

24. A non-transitory computer-readable medium storing instructions that, when executed, direct at least one processor of a computing device for performing mass spectrometry to perform a process comprising: controlling the mass spectrometer to acquire, during a plurality of acquisitions constituting an acquisition cycle, a set of mass spectra of product ions derived from precursor ions isolated based on parallel isolation windows positioned sequentially across a range of precursor mass-to-charge ratios (m / z); in: dividing the precursor m / z range into a plurality of isolation window units; For each acquisition of the acquisition cycle, the parallel isolation window includes a set of isolation sub-windows corresponding to a different set of isolation window cells for the precursor m / z range; At least two adjacent isolation sub-windows of the parallel isolation window are discontinuous; and Each isolation window cell of the precursor m / z range is analyzed at least twice during the acquisition cycle.

25. The computer readable medium of claim 24, wherein the process further comprises: A mass spectrum is generated for the precursor m / z range based on the set of mass spectra acquired during the acquisition cycle.

26. The computer readable medium of claim 25, wherein said generating said mass spectrum comprises demultiplexing said set of mass spectra to assign signals representing product ions to appropriate isolation window cells for said precursor m / z range.

27. The computer-readable medium of claim 24, wherein: The acquisition cycle includes a first sub-cycle and a second sub-cycle, the first sub-cycle includes a first subset of acquisitions, the second sub-cycle includes a second subset of acquisitions, wherein the second sub-cycle is executed after the first sub-cycle; Each isolation window cell of the precursor m / z range is analyzed at least once during the first sub-cycle; and Each isolation window cell of the precursor m / z range is analyzed at least once during the second sub-cycle.

28. The computer readable medium of claim 24, wherein for each acquisition of the acquisition cycle, acquiring the set of mass spectra comprises: isolating a population of said precursor ions having an m / z within an m / z range of said parallel isolation window; fragmenting the population of precursor ions to produce the population of product ions; as well as The population of the product ions is mass analyzed.

29. The computer readable medium of claim 28, wherein for each acquisition of the acquisition cycle, acquiring the set of mass spectra further comprises: prior to said isolating said population of said precursor ions, isolating a population of pre-isolated ions having an m / z within a total m / z span of said parallel isolation windows; wherein the population of the precursor ions is derived from the population of the pre-isolated ions.

Images