Multiplexed ion pre-separation for mass spectrometry

Abstract

To provide a system for performing multiplexed ion pre-separation for mass spectrometry.SOLUTION: The system includes a pre-separation device for separating the precursor ions into a set of discrete fractions of precursor ions based on a physical property of the precursor ions and for sequentially transmitting a first subset of the discrete fractions of precursor ions included in the set of discrete fractions of precursor ions. The system further includes a mass spectrometer positioned downstream of the pre-separation device for receiving the first subset of the discrete fraction of precursor ions. The mass spectrometer includes an ion storage for accumulating a first population of product ions generated from each discrete fraction of precursor ions included in the first subset of discrete fractions of precursor ions and a mass analyzer for performing mass analysis of the first population of product ions.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to multiplexed ion pre-separation for mass spectrometry. [Background technology]

[0002] A mass spectrometer is an instrument that 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. A mass spectrometer generally includes 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. The mass spectrometer 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.

[0003] In some mass spectrometry 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, often designated MS / MS or MS2), certain ions are isolated and then 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 mass analysis. 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, based on predetermined criteria for isolation and fragmentation of the selected ion species and subsequent mass analysis of the fragment ions (product ions). For example, a mass spectrometer can perform a full MS survey scan of precursor ions over a wide precursor m / z range and then select one or more precursor ion species from the resulting spectrum for subsequent MS / MS or MSn analysis. Precursor ion species selection criteria can include intensity, charge state, m / z, inclusion / exclusion list, or isotope pattern.

[0005] In contrast to DDA, data independent acquisition DIA (Distributed Inertial Analysis) is a technique in which all precursor ion species within a wide precursor m / z range (e.g., 500-900 m / z) are isolated and fragmented to produce product ions via sequentially advancing isolation windows of fixed m / z width (e.g., 10 m / z, 20 m / z, etc.). MS / MS or MSn analysis is then performed on the product ions in a systematic and unbiased manner. The acquisition of a set of mass spectra spanning the entire precursor m / z range constitutes one acquisition cycle, which is repeated to produce MS / MS or MSn mass spectra of the 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.

[0006] However, due to limitations in instrument speed and sensitivity, there is a tension between isolation width and precursor m / z range. Generally, a wider isolation width allows for a wider precursor m / z range and therefore allows for the analysis of a greater number of precursor ion species, but produces lower-quality data because a wide isolation window can cause simultaneous isolation and fragmentation of adjacent analytes, potentially resulting in complex, indistinguishable, or poorly scored spectra. In contrast, a narrower isolation window produces higher sensitivity and better-quality data at the expense of fewer precursor ion species that can be analyzed due to the narrower precursor m / z range. For example, at a very narrow isolation width, the data has the highest quality in terms of sensitivity and selectivity, but the precursor ion species are analyzed to a minimum extent. Such a narrow isolation width can reduce the duty cycle for MS analysis by filtering out many precursor ions outside the narrow isolation width. The duty cycle for MS analysis can refer to the amount (e.g., ratio, percentage, number, etc.) of precursor ions generated by the ion source that are effectively analyzed during MS analysis. Due to filtering out many precursor ions during MS analysis using a narrow isolation width, fewer precursor ions are analyzed, thus reducing the duty cycle. Summary of the Invention

[0007] 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 it 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.

[0008] In certain illustrative examples, a system comprises one or more processors and a memory storing executable instructions that, when executed by the one or more processors, cause the computing device to direct a pre-separation device to separate precursor ions into a set of distinct fractions of precursor ions based on a physical property of the precursor ions, direct the pre-separation device to sequentially transfer a first subset of the distinct fractions of precursor ions included within the set of distinct fractions of precursor ions to a mass analyzer, direct the mass analyzer to sequentially generate product ions from each distinct fraction of precursor ions included within the first subset of distinct fractions of precursor ions, direct the mass analyzer to accumulate a first population of product ions in an ion store over an accumulation time, the first population of product ions including product ions generated from each distinct fraction of precursor ions included within the first subset of distinct fractions of precursor ions, and direct the mass analyzer to transfer the first population of product ions to the mass analyzer for mass analysis of the first population of product ions.

[0009] In some illustrative examples, the system comprises: a pre-separation device configured to separate precursor ions into a set of distinct fractions of precursor ions based on physical properties of the precursor ions and to sequentially transfer a first subset of the distinct fractions of precursor ions contained within the set of distinct fractions of precursor ions; a mass spectrometer positioned downstream of the pre-separation device and configured to receive the first subset of distinct fractions of precursor ions; an ion store configured to accumulate a first population of product ions generated from each distinct fraction of precursor ions contained within the first subset of distinct fractions of precursor ions; and a mass analyzer configured to perform mass analysis of the first population of product ions.

[0010] In certain illustrative examples, a non-transitory computer-readable medium stores instructions that, when executed, direct at least one processor of a computing device for mass analysis to perform a process including: directing a pre-separation device to separate precursor ions into a set of distinct fractions of precursor ions based on a physical property of the precursor ions; directing the pre-separation device to sequentially transfer a first subset of the distinct fractions of precursor ions included within the set of distinct fractions of precursor ions to a mass analyzer; directing the mass analyzer to sequentially generate product ions from each distinct fraction of precursor ions included within the first subset of distinct fractions of precursor ions; directing the mass analyzer to accumulate a first population of product ions in an ion store over an accumulation time, the first population of product ions including product ions generated from each distinct fraction of precursor ions included within the first subset of distinct fractions of precursor ions; and directing the mass analyzer to transfer the first population of product ions to the mass analyzer for mass analysis of the first population of product ions. [Brief explanation of the drawings]

[0011] 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 multiplexed ion pre-separation MS / MS system. [Figure 2] FIG. 1 illustrates a functional diagram of an exemplary multiplexed ion pre-isolation control module. [Figure 3] 1 illustrates an exemplary method for performing multiplexed ion pre-separation for mass spectrometry. [Figure 4] 4 illustrates an exemplary implementation of the method of FIG. 3. [Figure 5A] 4 shows an example schematic diagram of a timing scheme for carrying out the method of FIG. 3. [Figure 5B]4 shows an example schematic diagram of a timing scheme for carrying out the method of FIG. 3. [Figure 6A] 4 shows an example schematic diagram of a timing scheme for carrying out the method of FIG. 3. [Figure 6B] 4 shows an example schematic diagram of a timing scheme for carrying out the method of FIG. 3. [Figure 7] 4 illustrates another exemplary implementation of the method of FIG. 3. [Figure 8] 1 illustrates an exemplary computing device that may be specifically configured to perform one or more of the processes described herein. DETAILED DESCRIPTION OF THE INVENTION

[0012] Systems, apparatus, and methods for performing multiplexed ion pre-separation for mass analysis are described herein. For example, a mass analysis system may include a pre-separation device configured to separate precursor ions into a set of distinct fractions of precursor ions based on physical properties of the precursor ions (e.g., ion mobility, m / z, etc.) and sequentially transfer a first subset of the distinct fractions of precursor ions contained within the set of distinct fractions of precursor ions. The mass analysis system may further include a mass analyzer positioned downstream of the pre-separation device and configured to receive the first subset of distinct fractions of precursor ions. The mass analyzer may include an ion store configured to accumulate a first population of product ions generated from each distinct fraction of precursor ions contained within the first subset of distinct fractions of precursor ions, and a mass analyzer configured to perform mass analysis of the first population of product ions.

[0013] The systems, devices, and methods described herein improve the duty cycle for MS analysis compared to conventional MS analysis techniques by providing multiplexed pre-separation of multiple distinct fractions of precursor ions. For example, pre-separation of precursor ions into subsets of precursor ions according to their physical properties preserves the precursor ions while they await transfer to a mass spectrometer for MS analysis. Furthermore, such multiplexed pre-separation of precursor ions from multiple distinct fractions of precursor ions may enable detection and / or quantification of several analyte ions without overloading the mass spectrometer, improving the efficiency of mass analysis and reducing the charge load of ion pre-separation compared to MS analysis techniques without multiplexed pre-separation. Performing MS analysis techniques without multiplexed pre-separation of multiple distinct fractions of precursor ions, in which precursor ions may be stored in a single space while a single fraction of precursor ions is slowly scanned from the full sample injection, requires a high charge capacity, limits the amount of sample injected, and / or discards precursor ions from the sample that cannot be stored. Alternatively, multiplexed pre-separation of multiple distinct fractions of precursor ions as described herein may reduce the charge capacity required to store the precursor ions, increase the amount of sample injected, and / or preserve precursor ions from the sample, such as by pre-separating the precursor ions and spreading their charge over space and / or time. Thus, multiplexed pre-separation of multiple distinct fractions of precursor ions improves the duty cycle of MS analysis compared to conventional MS analysis techniques.

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

[0015] FIG. 1 shows a functional diagram of an exemplary multiplexed ion pre-separation MS / MS system 100 ("system 100"). System 100 includes an ion source 102, a pre-separation device 104, a mass spectrometer 106, and a controller 108. Mass spectrometer 106 may be implemented by a multi-stage mass spectrometer configured to perform multi-stage mass spectrometry (also denoted MSn). In some examples, as shown in FIG. 1, mass spectrometer 106 is a tandem mass spectrometer configured to perform tandem mass spectrometry. Tandem mass spectrometry (MS / MS) is a form of multi-stage mass spectrometry (MSn) where the number of stages (n) is 2. As used herein, multi-stage mass spectrometry refers to MS / MS as well as MSn mass spectrometry, where n is 3 or greater.

[0016] The ion source 102 is configured to generate a stream of precursor ions 110 from components contained within the sample and deliver the precursor ions to the pre-separation device 104. The ion source 102 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, or inductively coupled plasma. The ion source 102 may include various components for generating precursor ions from components contained within the sample and delivering the stream of precursor ions 110 to the pre-separation device 104.

[0017] The pre-separation device 104 is configured to separate precursor ions received from the ion source 102 into a set of distinct fractions 112 of precursor ions based on physical properties of the precursor ions and sequentially transfer a subset of the distinct fractions 112 of precursor ions contained within the set of distinct fractions 112 to the mass spectrometer 106. The physical properties of the precursor ions may include, without limitation, the mobility of the precursor ions, the m / z of the precursor ions, or any other suitable property for separating precursor ions. By way of example, the pre-separation device 104 may use any suitable mobility separation technique, including, but not limited to, trapped ion mobility separation (TIMS), drift ion mobility separation (including, for example, drift tubes and / or structures for lossless ion manipulation (SLIM)-enabled folded path separation), and differential mobility separation (DMA). Alternatively, the pre-separation device 104 may use any suitable m / z separation device, including, without limitation, a mass filter, an ion accumulator, an ion selector, a toroidal ion trap, or a linear ion trap. The m / z separation may be based on different principles that provide mass-dependent displacement of ions, such as RF field-induced pseudopotentials, various types of traveling waves, resonant activation, and others. The pre-separation device 104 may include various components for separating precursor ions into a set of distinct fractions 112 of precursor ions based on their physical properties (e.g., a stream 110 of precursor ions) and sequentially transferring one or more subsets of the distinct fractions 112 of precursor ions from the pre-separation device 104 to the mass analyzer 106 (e.g., each distinct fraction 112 of precursor ions contained within each subset of distinct fractions 112 is sequentially released from the pre-separation device 104).

[0018] The mass spectrometer 106 is positioned downstream of the pre-separation device 104 and is configured to receive a subset of the distinct fractions 112 of precursor ions from the pre-separation device 104 and to perform mass analysis of product ions generated from each subset of the distinct fractions 112 of precursor ions. As shown, the mass spectrometer 106 is tandem in space (e.g., has multiple mass filters and / or mass analyzers) and has two stages for performing MS / MS. However, the mass spectrometer 106 is not limited to this configuration and may have any other suitable configuration. For example, the mass spectrometer 106 may be tandem in time. Additionally or alternatively, the mass spectrometer 106 may be a multi-stage mass spectrometer having three or more stages for performing multi-stage mass analysis (e.g., MS / MS / MS).

[0019] In the illustrated example, the mass spectrometer 106 includes a mass filter 114, a collision cell 116, an ion store 118, and a mass analyzer 120. The mass spectrometer 106 may further include any additional or alternative components not shown that may be compatible with a particular implementation (e.g., ion optics, filters, lenses, autosamplers, detectors, etc.). Although the ion source 102 and pre-separation device 104 are shown as being separate from or external to the mass spectrometer 106, in other examples, the ion source 102 and / or pre-separation device 104 are included within the mass spectrometer 106.

[0020] The mass filter 114 is configured to isolate or separate precursor ions within each distinct fraction 112 of precursor ions contained within each subset of distinct fractions 112 of precursor ions according to the m / z of the precursor ions. The mass filter 114 may be implemented by any suitable mass filter, such as a quadrupole mass filter or 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.). The mass filter 114 is configured to receive the distinct fractions 112 of precursor ions from the pre-separation device 104, isolate precursor ions of a selected m / z range (e.g., an m / z range of an isolation window) for each distinct fraction 112 of precursor ions, and deliver packets 122 of isolated precursor ions from each distinct fraction 112 to the collision cell 116. In some embodiments, the m / z range of the mass filter 114 is adjusted to, for example, isolate one or more target precursor ions from each distinct fraction 112 of precursor ions.

[0021] The collision cell 116 is configured to receive packets 122 of precursor ions isolated from each distinct fraction 112 of precursor ions contained within each subset of distinct fractions 112 and generate product ions (e.g., fragment ions) via a controlled dissociation process. The collision cell 116 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 employed for collision-activated dissociation. For example, the collision cell 116 may be implemented in a variety of different ways, including collision-induced dissociation (CID), electron transfer dissociation (ECD), and the like. transfer dissociation (ETD), electron capture dissociation dissociation (ECD), photoinduced dissociation (PID) (e.g., infrared multiphoton dissociation (IRMPD), blackbody infrared radiative dissociation (BIRD), surface induced dissociation (SID), negative electron transfer dissociation (NDC) electron-transfer dissociation (NETD), electron-detachment dissociation dissociation (EDD), high-energy C-trap dissociation The collision cell 116 may be configured to fragment the precursor ions using charge-dissociation (HCD), charge-remote fragmentation, or ion / molecule reactions. The collision cell 116 directs packets of product ions 124 generated from each distinct fraction 112 contained within each subset of distinct fractions 112 to the ion store 118.

[0022] The ion store 118 is a device configured to accumulate product ions contained within a packet 124 of product ions generated from a subset of the distinct fractions 112 of precursor ions over an accumulation time. Illustratively, for each subset of the distinct fractions 112 of precursor ions, the ion store 118 is configured to accumulate a packet 124 of product ions generated from each distinct fraction 112 of precursor ions contained within the subset of distinct fractions 112. As used herein, "accumulation time" refers to the duration that product ions generated by the collision cell 116 accumulate within the ion store 118 before being ejected and transported to the mass analyzer 120. The accumulation time may also be known as the ion injection time or the ion fill time. In some examples, the ion store 118 is an ion storage device configured to buffer downstream processes, such as mass analysis, thereby increasing acquisition speed and instrument sensitivity. In some examples, the ion store 118 is a beam-type or trapping device such as a multipole ion guide (e.g., a quadrupole ion guide, a hexapole ion guide, an octapole ion guide, etc.), a linear quadrupole ion trap, a three-dimensional quadrupole ion trap, a cylindrical ion trap, a toroidal ion trap, an orbital electrostatic trap, or a Kingdon trap. In some examples, the ion store 118 takes the form of a curved trap (also known as a C-trap) of the type used in orbital electrostatic trap mass spectrometers. In some other examples, the ion store 118 comprises a collision cell 116 (e.g., the collision cell 116 is configured to accumulate ions).

[0023] The accumulation of ions in the ion store 118 can be adjusted to achieve a target population 126 of product ions in the ion store 118. The accumulation of ions can be adjusted in any suitable manner. In some examples, the accumulation of ions in the ion store 118 is adjusted by a gating device (not shown) that either transmits or blocks the flow of product ions. The gate can be opened for a given time to meter out an appropriate number of ions, after which the gate is closed. For each subset of distinct fractions 112 of precursor ions, an accumulated population 126 of product ions is transferred from the ion store 118 to the mass analyzer 120. It will be appreciated that other techniques for adjusting ion accumulation can be used.

[0024] The mass analyzer 120 is configured to perform filtering and / or mass analysis of the product ions in each population of product ions 126. For example, the mass analyzer 120 is configured to isolate or separate ions according to their respective m / z. The mass analyzer 120 may be implemented by any suitable mass analyzer, such as a quadrupole mass filter, an ion trap (e.g., a three-dimensional quadrupole ion trap, a cylindrical ion trap, a linear quadrupole ion trap, a toroidal ion trap, etc.), a time-of-flight (TOF) mass analyzer, an electrostatic trap mass analyzer (e.g., an orbital electrostatic trap such as an Orbitrap™ mass analyzer, a Kingdon trap, etc.), or a Fourier transform ion cyclotron resonance (FT-ICR) mass analyzer.

[0025] The ion detector (not shown) is configured to detect ions at each of a variety of different m / z and, in response, generate an electrical signal representative of the ion intensity. The electrical signal is transmitted to the controller 108 for processing, such as constructing a mass spectrum of the sample. For example, the mass analyzer 120 may emit an ejected beam of separated ions to an ion detector, which is configured to detect the ions in the ejected beam and generate or provide data that the controller 108 can use to construct a mass spectrum of the sample. The ion detector may be implemented by any suitable detection device, such as an electron multiplier or a Faraday cup. In some examples, the detector is included within or implemented by the mass analyzer 120.

[0026] The pre-separation device 104 is synchronized with the mass spectrometer 106 such that one or more operating parameters of the mass spectrometer 106 are adjusted during successive transfers of the distinct fractions 112 of precursor ions from the pre-separation device 104 for mass analysis of the next distinct fractions 112 of precursor ions. Synchronization may be achieved using the controller 108, as described in more detail below. For example, the collision energy of the collision cell 116 and / or the m / z range of the mass filter 114 may be adjusted based on the distinct fractions 112 of precursor ions prior to accepting each distinct fraction 112 of precursor ions. As used herein, the term "m / z range" refers to the width of the range of precursor ion masses isolated for each distinct fraction 112 of precursor ions. The product ion population 126 may be accumulated in the ion store 118 based on multiple m / z ranges (e.g., 1-50 m / z, 10-30 m / z, 10-20 m / z, etc.) within a precursor m / z range (e.g., 50-1600 m / z, 200-1200 m / z, 400-1000 m / z, etc.) to accumulate product ions generated from multiple distinct fractions 112 of precursor ions during an accumulation event. As used herein, the term "precursor m / z range" refers to the full range of m / z of precursor ions across multiple distinct fractions 112 of precursor ions that are transferred to the mass spectrometer 106 for mass analysis.

[0027] In some examples, the pre-separation device 104 is synchronized with the mass filter 114 of the mass spectrometer 106 so that the m / z range of precursor ions contained within each distinct fraction 112 of precursor ions transmitted from the pre-separation device 104 corresponds to the m / z range of the mass filter 114. For example, the m / z range of precursor ions contained within each distinct fraction 112 may correspond to the m / z range of the mass filter 114 by having an m / z range that is the same as the m / z range of the mass filter 114, having an m / z range that is within the m / z range of the mass filter 114, or having an m / z range that overlaps the m / z range of the mass filter 114. The pre-separation device 104 is configured to selectively transmit one or more distinct fractions 112 of precursor ions associated with the corresponding m / z range of the mass filter 114, while retaining the remaining distinct fractions 112 of precursor ions for subsequent transmission.

[0028] The controller 108 may be communicatively coupled to and configured to control the operation of the system 100 (e.g., the ion source 102, the pre-separation device 104, and the mass spectrometer 106). The controller 108 may include any suitable hardware (e.g., processors, circuits, etc.) and / or software configured to control the operation of and / or interface with the various components of the system 100 (e.g., the ion source 102, the pre-separation device 104, and the mass spectrometer 106).

[0029] By way of example, the controller 108 may be configured to control the settings and operation of the ion source 102, the pre-separation device 104, the mass filter 114, the collision cell 116, the ion store 118, and / or the mass analyzer 120. For example, the controller 108 may control an oscillating voltage power supply and / or a direct current (DC) power supply to supply radio frequency (RF) voltage and / or direct current (DC) voltage to the pre-separation device 104, the mass filter 114, and / or the mass analyzer 120, adjust the collision cell energy of the collision cell 116 to select valid m / z (including a mass acceptance window) for analysis, adjust the values ​​of the RF and DC voltages, and adjust the sensitivity of the ion detector (e.g., by adjusting the detector gain).

[0030] The controller 108 may also include and / or provide a user interface configured to enable interaction between a user of the mass spectrometer 106 and the controller 108. The user may interact with the controller 108 via the user interface through 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 allow the user to provide input to the controller 108. In other examples, the display device and / or input device may be separate from the controller 108 but communicatively coupled to it. For example, the display device and input device may be included in a computer (e.g., a desktop computer, a laptop computer, etc.) communicatively connected to the controller 108 via a wired connection (e.g., via one or more cables) and / or a wireless connection.

[0031] Controller 108 may include any suitable hardware (e.g., processor, circuitry, etc.) and / or software as may be useful for a particular implementation. Figure 1 illustrates that controller 108 is implemented separately from mass spectrometer 106 (e.g., a computing device communicatively coupled to mass spectrometer 106 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.)). Controller 108 may alternatively be contained entirely or partially within mass spectrometer 106.

[0032] The pre-separation methods, systems, and devices described herein may operate as part of or in conjunction with the system 100 described herein and / or with any other suitable mass spectrometer or mass spectrometry system, including a combined separation-mass spectrometry system such as a liquid chromatography-mass spectrometry (LC-MS) system, 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, systems, and devices described herein may also operate with continuous flow sample sources, such as flow-injection mass spectrometry (FI-MS), in which analytes are injected into the mobile phase and enter the mass spectrometer without separation in a column.

[0033] 1 shows a single pre-separation device 104 as being positioned upstream of the mass analyzer 106, while in some other examples, additional pre-separation devices 104 may be positioned upstream of the mass analyzer 106. For example, the system 100 may include a first pre-separation device 104 configured to separate precursor ions according to mobility or m / z, and a second pre-separation device 104 configured to separate precursor ions according to mobility or m / z. In some examples, the system 100 is configured to separate precursor ions according to both mobility and m / z.

[0034] System 100 can be used in conjunction with a multiplexed ion pre-separation control module to perform multiplexed ion pre-separation of precursor ions. Figure 2 shows a functional diagram of an exemplary multiplexed ion pre-separation control module 200 ("control module 200"). Control module 200 can be implemented in whole or in part by system 100 (e.g., by controller 108). Alternatively, control module 200 can be implemented separately from system 100 (e.g., a remote computing system or server separate from but communicatively coupled to controller 108).

[0035] Control module 200 may include, but is not limited to, memory 202 and processor 204 selectably and communicatively coupled to each other. Memory 202 and processor 204 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.). In some examples, memory 202 and processor 204 may be distributed among multiple devices and / or multiple locations, as may be useful for a particular implementation.

[0036] Memory 202 may maintain (e.g., store) executable data used by processor 204 to perform any of the operations described herein. For example, memory 202 may store instructions 206 that may be executed by processor 204 to perform any of the operations described herein. Instructions 206 may be executed by any suitable application, software, code, and / or other executable data instance.

[0037] Memory 202 may also maintain any data acquired, received, generated, managed, used, and / or transmitted by processor 204. For example, memory 202 may maintain hybrid ion pre-separation MS / MS data (e.g., acquired mass spectral data) and / or ion pre-separation algorithms, as described below.

[0038] The processor 204 may be configured to perform (e.g., execute instructions 206 stored in memory 202 to perform) various processing operations described herein. For example, the ion pre-separation control module 200 may control the pre-separation device to synchronize with a mass analyzer so that the m / z range of precursor ions emitted from the pre-separation device corresponds to a precursor m / z isolation window of the mass analyzer. The ion pre-separation control module 200 may also control the mass analyzer to acquire mass spectra of product ions resulting from precursor ions isolated based on the precursor m / z isolation window.

[0039] It will be appreciated that the operations and examples described herein are merely illustrative of many different types of operations that may be performed by processor 204. In the description herein, any reference to an operation performed by control module 200 may be understood to be performed by processor 204 of control module 200. Furthermore, in the description herein, any operation performed by control module 200 may be understood to include control module 200 instructing or commanding another system (e.g., system 100) or device (e.g., any component of system 100) to perform the operation.

[0040] Figure 3 illustrates an exemplary method 300 for performing multiplexed ion pre-separation. While Figure 3 illustrates exemplary operations according to one embodiment, other embodiments may omit, add, reorder, and / or modify any of the operations illustrated in Figure 3. One or more of the operations illustrated in Figure 3 may be performed by system 100 and / or control module 200, any components contained therein, and / or any implementation thereof (e.g., mass spectrometer 106, one or more components of mass spectrometer 106, controller 108, and / or a remote computing system separate from but communicatively coupled to mass spectrometer 106).

[0041] The method 300 includes, in operation 302, directing a pre-separation device (e.g., the pre-separation device 104) to separate precursor ions (e.g., received from the ion source 102) into a set of distinct fractions of precursor ions (e.g., distinct fractions 112) based on physical properties of the precursor ions, such as ion mobility and / or m / z of the precursor ions. As an illustrative example, the physical properties of the precursor ions include the mobility of the precursor ions, such that the pre-separation device includes a differential mobility analyzer having a differential mobility separator configured to spatially separate the precursor ions according to their ion mobility within a gas flow region of the pre-separation device. The gas flow region includes a gas flow in a first direction and an electric field gradient in a second direction different from the first direction. As the precursor ions are carried downstream in the first direction by the gas flow, the electric field gradient directs the precursor ions in the second direction. The precursor ions travel through the gas flow region of the pre-separation device according to the ion mobility properties of the precursor ions and, during travel, become spatially separated from one another. For example, larger precursor ions (e.g., precursor ions with a larger cross section) may move more slowly in the second direction than smaller precursor ions (e.g., precursor ions with a smaller cross section), resulting in separation of ions along the first direction into distinct fractions of precursor ions. This separation allows each distinct fraction of precursor ions exiting the gas flow region of the pre-separation device to have a different range of ion mobility compared to other subsets of precursor ions exiting the gas flow region. For example, smaller precursor ions may be separated into one distinct fraction of precursor ions, while larger precursor ions may be separated into another distinct fraction of precursor ions. The precursor ions may be separated into any suitable number of distinct fractions.

[0042] In some examples, instructing the pre-separation device to separate precursor ions includes instructing the pre-separation device to provide a gas flow and / or an electric field gradient within a gas flow region of the pre-separation device. Furthermore, instructing the pre-separation device to separate precursor ions may include setting or controlling one or more parameters of the gas flow (e.g., gas flow rate, gas type, gas flow direction, etc.) and / or electric field gradient (e.g., electric field gradient amount, electric field gradient direction, electric field gradient type, etc.) of the pre-separation device. By way of example, the pre-separation device may be instructed to apply the gas flow and / or electric field gradient at a constant gas flow rate and / or electric field gradient. Additionally or alternatively, the pre-separation device may be instructed to vary the gas flow and / or electric field gradient over time.

[0043] In some examples, the pre-separation device includes multiple channels configured to receive and / or store precursor ions as they exit the gas flow region. The multiple channels may include an ion trap, an RF ion guide, a DC ion lens, or a combination thereof. In these examples, instructing the pre-separation device to separate the precursor ions includes instructing the pre-separation device to store multiple distinct fractions of the precursor ions in the multiple channels. Illustratively, instructing the pre-separation device to store multiple distinct fractions of the precursor ions includes instructing the pre-separation device to provide a potential at each channel to selectively stop the flow of precursor ions out of the channel (e.g., to accumulate precursor ions in the channel). Each channel of the multiple channels may be instructed to store a distinct fraction of precursor ions included in the set of distinct fractions of precursor ions.

[0044] In an alternative example, directing the pre-separation device to separate precursor ions includes directing the pre-separation device to continuously transport precursor ions through the pre-separation device without storing distinct fractions of precursor ions in channels of the pre-separation device. As an illustrative example, the pre-separation device includes a trap ion mobility separator configured to simultaneously provide a gas flow in a first direction and a variable electric field gradient in a second direction (e.g., opposite to the first direction). By varying the electric field gradient, precursor ions are separated according to mobility. As another illustrative example, the pre-separation device includes a drift ion mobility separator (e.g., including a drift tube and / or a SLIM-enabled folded path separator) configured to trap and pulse precursor ions for subsequent ion mobility separation along an ion separation path. As described below, each distinct fraction of precursor ions included within the set of distinct fractions of precursor ions exits the pre-separation device at a distinct time according to the ion mobility of the precursor ions included within the set of distinct fractions of precursor ions.

[0045] Alternatively, the physical property of the precursor ions is the m / z of the precursor ions, whereby the pre-separation device is directed to provide an electric field gradient to each distinct fraction of precursor ions according to the m / z of the precursor ions. The pre-separation device may use mass-dependent ejection of precursor ions (e.g., axially, radially, or in multiple directions) such that precursor ions within a selected m / z range are ejected from the pre-separation device as distinct fractions of precursor ions, while precursor ions outside the selected m / z range are not ejected from the pre-separation device and / or are discarded from the pre-separation device.

[0046] The pre-separation device can be instructed to vary the electric field gradient to separate precursor ions having different m / z ranges into a set of distinct fractions of precursor ions (e.g., each distinct fraction of precursor ions exiting the pre-separation device has a different m / z range than the other distinct fractions of precursor ions). For example, precursor ions having a smaller m / z are separated into one distinct fraction of precursor ions, while precursor ions having a larger m / z are separated into another distinct fraction of precursor ions. Furthermore, instructing the pre-separation device to separate precursor ions can include setting or controlling one or more parameters of the electric field gradient provided by the pre-separation device (e.g., amount of electric field gradient, direction of electric field gradient, type of electric field gradient, etc.). Thus, each distinct fraction of precursor ions comprises a distinct m / z range of precursor ions, whereby the m / z range of precursor ions contained in one distinct fraction of precursor ions is unique relative to another m / z range of precursor ions contained in another distinct fraction of precursor ions (e.g., at least a portion of the m / z range of precursor ions contained in one distinct fraction of precursor ions does not overlap with another m / z range of precursor ions contained in another distinct fraction of precursor ions).

[0047] The method 300 includes, in operation 304, instructing a pre-separation device to sequentially transfer a first subset of the distinct fractions of precursor ions contained within the set of distinct fractions of precursor ions to a mass spectrometer (e.g., mass spectrometer 106). The first subset of distinct fractions includes a plurality of distinct fractions of precursor ions contained within the set of distinct fractions. As an illustrative example, the pre-separation device may separate precursor ions into nine distinct sets of fractions of precursor ions (e.g., n=9). The pre-separation device then sequentially transfers a first subset of precursor ions, comprising three distinct fractions of the precursor ions within the set of distinct fractions, to the mass analyzer, such that a first distinct fraction of the precursor ions contained within the first subset is transferred to the mass analyzer, a second distinct fraction of the precursor ions contained within the first subset is transferred to the mass analyzer after the first distinct fraction is transferred, and a third distinct fraction of the precursor ions contained within the first subset is transferred to the mass analyzer after the second distinct fraction is transferred. The process is repeated for each successive subset of distinct fractions of precursor ions.

[0048] If the pre-separation device is configured to store a set of distinct fractions of precursor ions in multiple channels, instructing the pre-separation device to sequentially transfer a first subset of distinct fractions may include instructing the pre-separation device to sequentially transfer the first subset of distinct fractions from the multiple channels. As an illustrative example, instructing the pre-separation device to sequentially transfer the first subset of distinct fractions includes instructing each channel to provide a potential that allows a flow of precursor ions (e.g., releases precursor ions from the channel) at a specific, controlled time. Each channel may be separately controlled such that the pre-separation device may be instructed to sequentially release each distinct fraction included in the first subset of distinct fractions from the multiple channels, such as one distinct fraction at a time. In another example, a subset of the multiple channels may be instructed to release each associated distinct fraction of precursor ions simultaneously, e.g., two or three channels. The subset of multiple channels that emit simultaneously can be chosen according to the spatial separation between the channels (e.g., channels that are not physically close together can be instructed to emit simultaneously) or according to the known or predicted identity of the precursor ions (e.g., channels containing precursor ions of a wide variety of m / z values ​​can be instructed to emit simultaneously).

[0049] If the pre-separation device does not store a set of distinct fractions of precursor ions in multiple channels, directing the pre-separation device to sequentially transport a first subset of distinct fractions of precursor ions includes directing the pre-separation device to sequentially transport the first subset of distinct fractions of precursor ions as distinct fractions included within the first subset of distinct fractions are sequentially transported through the pre-separation device. Illustratively, smaller precursor ions may travel more rapidly through the pre-separation device than larger precursor ions, such that a distinct fraction of precursor ions comprising smaller precursor ions is released prior to another distinct fraction of precursor ions comprising larger precursor ions.

[0050] In some examples, instructing the pre-separation device to sequentially transfer the first subset of distinct fractions of precursor ions further includes instructing the pre-separation device to transfer the distinct fractions of precursor ions according to a timing scheme. In some examples, the timing scheme includes transferring precursor ions from the pre-separation device at predetermined intervals (e.g., a first distinct fraction of precursor ions is released from the pre-separation device prior to releasing a next distinct fraction of precursor ions). The predetermined intervals may be based on one or more characteristics of the precursor ions (e.g., the number of distinct fractions of precursor ions, the number of channels storing distinct fractions of precursor ions, the number of precursor ions contained within each distinct fraction of precursor ions, the duration of accumulation of precursor ions in the pre-separation device, etc.) and / or may be performed periodically (e.g., about every 250 milliseconds (ms), 100 ms, 50 ms, 25 ms, 12 ms, etc.).

[0051] In some examples, instructing the pre-separation device to sequentially transfer the first subset of distinct fractions of precursor ions further includes selecting one or more distinct fractions of precursor ions contained within the first subset of distinct fractions of precursor ions. For example, one or more distinct fractions of precursor ions may be selected for multiplexed pre-separation to minimize or prevent overlap in m / z ranges of precursor ions contained within the first subset of distinct fractions of precursor ions, which may facilitate precursor ion-based mass analysis. As an illustrative example, selecting one or more distinct fractions of precursor ions may include selecting one or more distinct fractions of precursor ions, wherein one or more precursor ions contained within the one or more distinct fractions of precursor ions have at least one unique product ion. If the number of selected distinct fractions of precursor ions having at least one unique product ion increases, the confidence level associated with precursor ion detection and / or precursor ion-based mass analysis may also increase. Still other suitable methods may be used to select one or more distinct fractions of precursor ions contained within the first subset of distinct fractions of precursor ions.

[0052] For example, one or more distinct fractions of precursor ions whose precursor ions have similar neutral losses (e.g., during the generation of product ions) can be selected, which can allow a particular class of compounds to be analyzed. Additionally or alternatively, if the pre-separation device is configured to store a set of distinct fractions of precursor ions in multiple channels, selecting one or more distinct fractions of precursor ions can include selecting one or more channels of the pre-separation device to sequentially transfer one or more distinct fractions of precursor ions stored in the selected one or more channels. Furthermore, if at least a portion of the m / z ranges of precursor ions contained in the selected one or more channels overlap, deconvolution (e.g., via a Hadamard transform) can be used to determine the m / z range associated with each distinct fraction of precursor ions. Illustratively, the sequence of distinct fractions of precursor ions transferred from the pre-separation device can be encoded, and by alternating the selection of distinct fractions of precursor ions transferred from the pre-separation device, the precursor ions contained in each distinct fraction of precursor ions can be identified.

[0053] The method 300 includes, in operation 306, instructing a mass spectrometer to sequentially generate product ions from each distinct fraction of precursor ions contained within the first subset of distinct fractions of precursor ions as each distinct fraction of precursor ions is transferred from the pre-separation device. As an illustrative example, the mass spectrometer (e.g., via the collision cell 116) is configured to fragment the precursor ions contained within each distinct fraction of precursor ions to generate product ions, such as by applying collision energy to collide the precursor ions with a collision gas (e.g., argon, nitrogen, helium, ammonia, methane, oxygen, hydrogen, etc.). The collision energy and / or the pressure of the collision gas can be adjusted based on the distinct fraction of precursor ions to be fragmented, such as before receiving each distinct fraction of precursor ions. In some examples, the mass spectrometer is further configured to isolate one or more precursor ions from each distinct fraction of precursor ions contained within the first subset of distinct fractions (e.g., via the mass filter 114) before generating product ions. Such isolation of one or more precursor ions from each distinct fraction of precursor ions may allow the m / z range of each distinct fraction of precursor ions to be different from and non-overlapping with the m / z range of another distinct fraction of precursor ions.

[0054] In operation 308, the method 300 includes instructing the mass spectrometer to accumulate a first population of product ions in the ion store over an accumulation time. The first population of product ions includes product ions generated from each distinct fraction of precursor ions included in the first subset of distinct fractions of precursor ions. For example, if the first subset of distinct fractions of precursor ions includes three distinct fractions of precursor ions, the first population of product ions includes product ions generated from each of the three distinct fractions of precursor ions included in the first subset of distinct fractions. In some examples, the mass spectrometer (e.g., by the ion store 118) is configured to accumulate product ions as they are generated from each distinct fraction. For example, the mass spectrometer accumulates the first population of product ions as product ions are sequentially generated from a first distinct fraction of precursor ions, a second distinct fraction of precursor ions, and a third distinct fraction of precursor ions. Thus, the first population of product ions includes product ions generated from each of the first, second, and third distinct fractions of precursor ions contained within the first subset of distinct fractions.

[0055] The mass spectrometer accumulates the first population of product ions for an accumulation time, such as from the generation of product ions from the first distinct fraction of precursor ions until the accumulation of product ions generated from the third distinct fraction of precursor ions. The accumulation time may include any suitable period (e.g., approximately 250 ms, 150 ms, 100 ms, 50 ms, 36 ms, 12 ms, etc.) and may depend on one or more factors, such as the amount of product ions generated from each distinct fraction of precursor ions and / or the number of distinct fractions included in the first subset of distinct fractions. In some examples, the accumulation time is determined by an ion population control algorithm, such as an automatic gain control (AGC) process. In some examples, the accumulation time is less than the acquisition time for mass analysis. Such accumulation of product ions in the ion store 118 allows product ions generated from multiple distinct fractions of precursor ions to be accumulated within a single population of product ions.

[0056] Method 300 includes, in operation 310, instructing a mass spectrometer to transfer the first population of product ions to a mass analyzer (e.g., mass analyzer 120) for mass analysis of the first population of product ions. For example, the mass analyzer is further configured to obtain a mass spectrum based on the first population of product ions.

[0057] Operations 304-310 may be performed sequentially for one or more additional distinct subsets of fractions of precursor ions. For example, method 300 may further include instructing the pre-separation device to sequentially transfer a second subset of the distinct fractions of precursor ions to a mass analyzer after transferring the first subset of the distinct fractions of precursor ions. The mass analyzer may be further instructed to sequentially generate product ions from each distinct fraction of precursor ions included in the second subset of distinct fractions of precursor ions and accumulate a second population of product ions in the ion store for another accumulation time. The second population of product ions includes product ions generated from each distinct fraction of precursor ions included in the second subset of distinct fractions of precursor ions. The mass analyzer may be further instructed to transfer the second population of product ions from the ion store to a mass analyzer for mass analysis of the second population of product ions.

[0058] The second subset of distinct fractions of precursor ions can be transferred from the pre-separation device to the mass analyzer simultaneously with the transfer of the first population of product ions to the mass analyzer. Additionally, one or more operating parameters of the mass analyzer (e.g., collision energy, m / z range, etc.) can be adjusted between the transfer of the first subset of distinct fractions of precursor ions and the transfer of the second subset of distinct fractions of precursor ions to the mass analyzer, such as to target selected precursor ions from the second subset of distinct fractions of precursor ions. For example, targeting selected precursor ions from the second subset of distinct fractions of precursor ions can be based on mass analysis of the first subset of distinct fractions or precursor ions.

[0059] Pre-separation of precursor ions into a subset of distinct fractions of precursor ions according to their physical characteristics preserves the precursor ions while they await transmission to a mass spectrometer for mass analysis. Furthermore, multiplexed mass spectral acquisition of precursor ions from multiple distinct fractions of precursor ions allows for detection and / or quantification of several analyte ions without overloading the mass spectrometer, improving the efficiency of mass analysis and reducing the charge load of ion pre-separation, compared to MS analysis techniques without multiplexed pre-separation. Thus, multiplexed mass spectral acquisition of multiple distinct fractions of precursor ions improves the duty cycle of MS analysis, compared to conventional MS analysis techniques.

[0060] Other suitable techniques for obtaining a mass spectrum for the accumulated population of product ions may also be used. As an illustrative example, the pre-separation device may be synchronized with the mass analyzer such that the ion mobility range of the precursor ions in each distinct fraction of precursor ions transferred from the pre-separation device to the mass analyzer includes precursor ions that are within the precursor m / z range of the mass analyzer. Furthermore, any suitable number of subsets of distinct fractions of precursor ions may be included within a set of distinct fractions of precursor ions, any suitable number of distinct fractions of precursor ions may be included in each subset of distinct fractions of precursor ions, and / or any suitable number of populations of product ions may be accumulated for mass analysis of the set of distinct fractions.

[0061] 4 shows an example implementation 400 of the system 100 for performing the method of FIG. 3. As shown, the pre-separation device 104 is positioned downstream of the ion source 102 and is implemented by a differential mobility analyzer including a differential mobility separator. The pre-separation device 104 includes an ion mobility cell 402 having a gas flow region in which gas flow (indicated by arrow 404) flows in a first direction from a gas inlet 406 at one end of the ion mobility cell 402 to a gas outlet 408 at the other (e.g., opposite) end of the ion mobility cell 402. Additionally, an electric field gradient (indicated by arrow 410) is applied in a second direction. In various examples, the first and second directions can form an angle between about 0 degrees and 180 degrees, such as between about 70 degrees and about 110 degrees, such as between about 45 degrees and 135 degrees. In certain examples, the first direction and the second direction may be substantially perpendicular to one another (with a small difference, e.g., within ±5 degrees of the perpendicular angle). The gas pressure within the ion mobility cell 402 may be between about 1 Torr and about 20 Torr, between about 3 Torr and about 6 Torr, or any other suitable range or value. In various examples, the gas velocity within the ion mobility cell 402 may be between about 100 m / s and about 300 m / s, between about 150 m / s and about 200 m / s, or any other suitable range or value.

[0062] Ions provided by the ion source 102 enter the ion mobility cell 402 through an ion inlet 412. The pre-separation device 104 further includes a plurality of ion channels 414 (e.g., channels 414-1 through 414-n) located proximate to a plurality of ion exit orifices 416. In some examples, the ion channels 414 and the ion exit orifices 416 are arranged in an array along the first direction. For example, the ion exit orifices 416 are located on opposite sides of the ion inlet 412 in the second direction, are at the same height as the ion inlet 412, and / or are offset (downstream) from the ion inlet 412, and are spaced apart from each other in the first direction. Precursor ions entering the ion mobility cell 402 from the ion source 102 are separated into distinct fractions 112 of precursor ions (e.g., distinct fractions 112-1 through 112-n, represented by arrows) based on their differential ion mobilities, exit through an ion exit orifice 416, and are directed into an array of ion channels 414.

[0063] Illustratively, precursor ions flow at substantially the same velocity along a first direction (due to the gas flow) and move differentially in a second direction according to their collision cross-sections. Precursor ions with larger collision cross-sections (e.g., precursor ions contained within distinct fraction 112-n) move more slowly in the second direction due to a greater number of collisions with molecules in the gas flow than precursor ions with smaller collision cross-sections (e.g., precursor ions contained within distinct fraction 112-1). Due to their slower movement in the second direction, precursor ions with larger collision cross-sections move further along the first direction while passing through the ion mobility cell 402. In this manner, precursor ions with successively larger collision cross-sections are sorted into distinct fractions 112 in the array of ion channels 414 such that precursor ions contained within a distinct fraction 112 of precursor ions in an ion channel have a different range of ion mobility than precursor ions contained within another distinct fraction 112 of precursor ions in an adjacent ion channel. For example, a first distinct fraction 112-1 of precursor ions having a first range of ion mobilities is separated into a first channel 414-1, a second distinct fraction 112-2 of precursor ions having a second range of ion mobilities is separated into a second channel 414-2, a third distinct fraction 112-3 of precursor ions having a third range of ion mobilities is separated into a third channel 414-3, and so on.

[0064] In various examples, the ion channel 414 is implemented by one or more ion traps, RF ion guides, DC ion lenses, or combinations thereof. In some examples, the ion channel 414 includes ion traps each defined by a plurality of rod electrodes (e.g., quadrupoles). Additionally, each ion trap may include one or more drag vanes. In one particular example, adjacent ion traps in an array of ion traps share a pair of rod electrodes.

[0065] In various examples, the plurality of ion channels 414 includes between about 3 ion channels and about 50 ion channels, between about 5 ion channels and about 20 ion channels, between about 7 ion channels and about 15 ion channels, or any other suitable number of ion channels. The ion channels 414 are shown as linear channels arranged in a linear array. In other examples, the ion channels 414 may have any other suitable shape (e.g., curved, bent, non-linear, etc.) and / or orientation, and the arrangement of the plurality of ion channels may have any suitable configuration, such as a circular array or a curved array.

[0066] In various examples, a lens array (not shown) may be positioned between the ion exit orifice 416 and the ion channels 414. The lens array may be configured to direct the distinct fractions 112 of precursor ions into respective ion channels 414, such as by focusing the distinct fractions 112 of precursor ions toward the centerline of the ion channels 414.

[0067] The control module 200 may be configured to instruct the pre-separation device 104 to spatially separate the precursor ions received from the ion source 102 into a set of distinct fractions 112 of precursor ions, such as by instructing the pre-separation device 104 to accept precursor ions from the ion source 102, and to provide a gas flow and / or an electric field gradient within the ion mobility cell 402 of the pre-separation device 104. Further, the control module 200 may instruct the pre-separation device 104 to spatially separate the precursor ions by setting one or more parameters of the gas flow (e.g., gas flow rate, gas type, gas flow direction, etc.) and / or electric field gradient (e.g., electric field gradient amount, electric field gradient direction, electric field gradient type, etc.) of the pre-separation device 104.

[0068] 4 , the pre-separation device 104 is configured (e.g., in response to control signals received from the control module 200) to sequentially transfer the distinct fractions 112 of precursor ions contained within a subset of the distinct fractions 112 from the channels 414. Illustratively, the control module 200 is configured to direct the channels 414 of the pre-separation device 104 at certain times to provide a potential to stop the flow of precursor ions in the channels 414, and to direct one or more selected channels 414 of the pre-separation device 104 at certain other times to provide a potential (e.g., a reduced potential) to allow the flow of precursor ions from the one or more selected channels 414. The one or more selected channels 414 are controlled to sequentially release the distinct fractions 112 of precursor ions contained within the subset of the distinct fractions 112. For example, the first subset of distinct fractions 112 of precursor ions may include a first distinct fraction 112-1 and a second distinct fraction 112-2, such that a first distinct fraction 112-1 of precursor ions is released from a first channel 414-1 and a second distinct fraction 112-2 of precursor ions is released from a second channel 414-2 (e.g., after the first distinct fraction 112-1 of precursor ions is released). Such sequential transfer of distinct fractions 112 of precursor ions is continued from the pre-separation device 104 until a desired number of subsets of distinct fractions 112 of precursor ions are released from the pre-separation device 104. The channels 414 may be sequentially controlled to permit the flow of precursor ions (e.g., to release distinct fractions 112 of precursor ions in any sequence) in any order. In some examples, the channels 414 are controlled to sequentially release distinct fractions 112 of precursor ions in an order of increasing (or decreasing) ion mobility.

[0069] Ion optics 418 (e.g., cooling / transport guides) are located adjacent to the multiple ion channels 414 between the pre-separation device 104 and the mass analyzer 106. The ion optics 418 are configured to guide the distinct fractions 112 of precursor ions emitted from the pre-separation device 104 to the mass analyzer 106. For example, the ion optics 418 may direct precursor ions contained within each distinct fraction 112 of precursor ions emitted from the pre-separation device 104 toward the central axis of the mass filter 114 of the mass analyzer 106. In the example of FIG. 4 , the ion optics 420 are illustrated as a funnel. However, the funnel is merely optional, as any one or more additional and / or alternative devices and / or ion optics may be used to guide ions from the ion channel 414 to the mass analyzer 106.

[0070] The mass filter 114 of the mass spectrometer 106 is positioned downstream of the pre-separation device 104 and the collector funnel 418 such that as the distinct fractions 112 of precursor ions are sequentially transmitted from the ion channel 414, the mass filter 114 is configured to receive each distinct fraction 112 of precursor ions contained within a subset of the distinct fractions 112. For each distinct fraction 112 of precursor ions contained within a subset of the distinct fractions 112, the mass filter 114 isolates precursor ions of a selected m / z range (e.g., the m / z range of an isolation window) and transmits the isolated precursor ions as packets 122 of precursor ions. Illustratively, the mass filter 114 receives a first distinct fraction 112-1, isolates one or more precursor ions contained within the first distinct fraction 112-1 based on m / z, and transfers the isolated one or more precursor ions from the mass filter 114 to the collision cell 116 as a first packet of precursor ions 122-1. After isolating the precursor ions contained within the first packet 122-1 and transferring the first packet 122-1 to the collision cell 116, the mass filter 114 receives a second distinct fraction 112-2 contained within the first subset of distinct fractions 112 from the pre-separation device 104, isolates one or more precursor ions contained within the second distinct fraction 112-2 based on m / z, and transfers the isolated one or more precursor ions from the mass filter 114 to the collision cell 116 as a second packet of precursor ions 122-2.

[0071] As shown, the mass filter 114 is an m / z separator that includes a linear ion trap defined by a plurality of rod electrodes 420 (e.g., quadrupole, hexapole, octopole, etc.). In some examples, the rod electrodes 420 are configured to provide an electric field gradient (e.g., an RF field pseudopotential) that is m / z dependent such that precursor ions within an m / z range that are stable within the electric field gradient accumulate within the mass filter 114 as packets 122 of precursor ions, while precursor ions outside the m / z range that are unstable within the electric field gradient do not accumulate within the mass filter 114 and / or are discarded from the mass filter 114. In the illustrated example, the rod electrodes 420 of the mass filter 114 are configured to provide the electric field gradient in response to control signals received from the control module 200. Furthermore, the control module 200 may set or control one or more parameters of the electric field gradient provided by the mass filter 114 (e.g., the amount of the electric field gradient, the direction of the electric field gradient, the type of electric field gradient, etc.).

[0072] The electric field gradient provided by the rod electrodes 420 can be varied for each distinct fraction 112 of precursor ions such that precursor ions having different m / z ranges are sequentially emitted from the mass filter 114 as packets 122 of precursor ions (e.g., each distinct packet 122 of precursor ions has a different m / z range compared to the other packets 122 of precursor ions). As an illustrative example, the electric field gradient provided by the rod electrodes 420 is adjusted for each distinct fraction 112 of precursor ions, such as to order the packets 122 of precursor ions in an m / z-dependent manner (e.g., in order of increasing or decreasing m / z value).

[0073] The mass filter 114 includes an aperture 422 through which, for each distinct fraction 112 of precursor ions contained within a subset of the distinct fractions 112, a packet 122 of precursor ions is transferred from the mass filter 114 to the collision cell 116. For example, the mass filter 114 transfers a first packet 122-1 of precursor ions isolated from a first distinct fraction 112-1 to the collision cell 116, and then transfers a second packet 122-2 of precursor ions isolated from a second distinct fraction 112-2 to the collision cell 116. The collision cell 116 is configured to fragment the precursor ions contained within each packet 122 of precursor ions to generate a packet 124 of product ions for each packet 122 of precursor ions. For example, the collision cell 116 fragments precursor ions contained within a first packet of precursor ions 122-1 to generate a first packet of product ions 124-1, and then fragments precursor ions contained within a second packet of precursor ions 122-2 to generate a second packet of product ions 124-2.

[0074] The collision cell 116 uses collision-induced dissociation to fragment precursor ions by applying collision energy to cause the precursor ions to collide with a collision gas. In some examples, the collision energy may be applied at about 5 volts (V) to about 50 V, about 5 V to about 25 V, or any other suitable range or value. The collision gas may have a gas pressure of about 0.1 mTorr to about 10 mTorr, or any other suitable range or value. In the illustrated example, the collision cell 116 is configured to provide the collision energy in response to a control signal received from the control module 200. Furthermore, the control module 200 may set or control one or more parameters of the collision energy provided by the collision cell 116 (e.g., the amount of collision energy, the direction of the collision energy, the type of collision energy, etc.). By way of example, the collision energy may be adjusted for each packet 122 of precursor ions received from the mass filter 114 to produce a packet 124 of product ions (e.g., the collision energy may be adjusted before receiving the first packet of precursor ions 122-1 and / or before receiving the second packet of precursor ions 122-2).

[0075] The collision cell 116 includes an opening 424 through which a packet of product ions 124 is transferred from the collision cell 116 to the ion store 118 for each distinct fraction 112 of precursor ions contained within the subset of distinct fractions 112. As shown, the ion store 118 includes a linear ion trap defined by a plurality of rod electrodes 426 (e.g., a quadrupole, hexapole, octapole, etc.) and an end electrode 428 positioned at the downstream end of the rod electrodes 426 such that the ion store 118 is configured to accumulate, over an accumulation time, product ions contained within the packet of product ions 124 generated from each distinct fraction 112 of precursor ions contained within the subset of distinct fractions 112. For example, the ion store 118 accumulates a first packet of product ions 124-1 and a second packet of product ions 124-2 within the ion store 118 over an accumulation time to accumulate a first population of product ions 126-1.

[0076] In some examples, the rod electrodes 426 are configured to provide an electric field gradient (e.g., an RF field pseudopotential) such that product ions within an m / z range stable within the electric field gradient accumulate in the ion store 118 as a population 126 of product ions generated from each distinct fraction 112 of precursor ions contained within the subset of distinct fractions 112. In the illustrated example, the rod electrodes 426 of the ion store 118 are configured to provide the electric field gradient in response to a control signal received from the control module 200. Furthermore, the control module 200 may set or control one or more parameters of the electric field gradient provided by the ion store 118 (e.g., the amount of the electric field gradient, the direction of the electric field gradient, the type of electric field gradient, etc.). The product ions are accumulated in the ion store 118 over an accumulation time (e.g., until all packets 124 of product ions generated from the subset of distinct fractions 112 of precursor ions have been accumulated).

[0077] The end electrode 428 of the ion store 118 includes an aperture 430 through which the first population of product ions 126-1 are transferred from the ion store 118 to the mass analyzer 120. For example, the end electrode 428 is configured to provide a blocking potential (e.g., a DC blocking potential) configured to stop the flow of product ions in the ion store 118 at certain times (e.g., during an accumulation time for accumulating the first population of product ions 126-1) and to allow the flow of product ions through the aperture 430 at certain other times (e.g., the blocking potential may be reduced at certain times to allow the first population of precursor ions 126-1 to flow through the aperture 430). In the illustrated example, the end electrode 428 is configured to provide the blocking potential in response to a control signal received from the control module 200. Furthermore, the control module 200 may control one or more parameters of the blocking potential provided by the ion store 118 (e.g., the amount of blocking potential, the direction of the blocking potential, the type of blocking potential, etc.).

[0078] The mass analyzer 120 is positioned downstream of the ion store 118 and is configured to receive the first population of product ions 126-1 transferred from the ion store 118 and acquire a mass spectrum based on the first population of product ions 126-1. For example, the mass analyzer 120 generates signals based on product ions contained within the first population of product ions 126-1 having a variety of different m / z. The signals may include electrical signals representing ion intensities based on product ions contained within the first population of product ions 126-1. In some examples, the control module 200 may be configured to instruct the mass analyzer 120 to generate signals and / or acquire signals generated by the mass analyzer 120.

[0079] The pre-separation device 104 may be configured to transfer the distinct fractions 112 of precursor ions and / or the ion store 118 may be configured to eject the population of product ions 126 according to a timing scheme. Figures 5A and 5B show schematic diagrams of exemplary timing schemes 500 and 502, respectively. Each timing scheme 500 and 502 includes transferring an initial subset of the distinct fractions 112 of precursor ions from the pre-separation device 104 to the mass filter 114, accumulating a population of product ions 126 generated from the initial subset of the distinct fractions 112 of precursor ions in an ion store, and transferring the population of product ions 126 from the ion store 118 concurrently with the transfer of the next subset of the distinct fractions 112 of precursor ions from the pre-separation device 104 to the mass filter 114.

[0080] 5A, the pre-separation device 104 is configured to accumulate precursor ions (e.g., in channel 414) over a first time period (e.g., from time t0 to time t1) of the timing scheme 500. The pre-separation device 104 then sequentially transfers the distinct fractions 112 of precursor ions (e.g., distinct fractions F1-F3) contained within a first subset of the accumulated distinct fractions 112 of precursor ions (e.g., a subset including distinct fractions F1-F3) to the mass filter 114, and the collision cell 116 fragments the precursor ions contained within the first subset of distinct fractions into packets 124 of product ions (e.g., packets P1-P3 such that packet P1 is generated from distinct fraction F1, then packet P2 is generated from distinct fraction F2, and then packet P3 is generated from distinct fraction F3) over a second time period (e.g., from time t1 to time t2) of the timing scheme 500. 5B, the pre-separation device 104 is alternatively configured to continuously accumulate precursor ions (e.g., in channel 414) during and after a first period of time, such as during which the pre-separation device sequentially transfers distinct fractions 112 of precursor ions. Illustratively, the pre-separation device 104 may accumulate precursor ions contained in distinct fraction F2 while the pre-separation device transfers precursor ions contained in distinct fraction F1, and / or the pre-separation device 104 may accumulate precursor ions contained in distinct fraction F3 while the pre-separation device transfers precursor ions contained in distinct fraction F2. The pre-separation device 104 may be configured to continuously and / or periodically accumulate precursor ions through a timing scheme 502.

[0081] The ion store 118 accumulates the first population of product ions 126-1 (e.g., product ions contained within packets P1-P3) over a third time period (accumulation time, e.g., time t2-time t3) of timing schemes 500 and 502. In some examples, the third time period may overlap with the second time period such that the ion store 118 accumulates the first population of product ions 126-1 as the product ions are generated by the collision cell 116. In some other examples, each packet (e.g., packets P1-P3) contained within the first population of product ions 126-1 is transferred to the ion store 118 simultaneously (e.g., after the last packet of product ions contained within the first population of product ions 126-1 is generated). The ion store 118 transfers the first population of product ions 126-1 to the mass analyzer 120, which performs mass analysis of the first population of product ions 126-1 over a fourth period of timing schemes 500 and 502 (e.g., from time t3 to time t4).

[0082] Concurrent with mass analysis of the first population of product ions 126-1 over a fourth time period, the pre-separation device 104 sequentially transports the distinct fractions 112 of precursor ions (e.g., distinct fractions F4-F6) contained within a second subset of the accumulated distinct fractions 112 of precursor ions (e.g., a subset including distinct fractions F4-F6), and the collision cell 116 fragments the precursor ions contained within the second subset of distinct fractions into packets 124 of product ions (e.g., packets P4-P6, such that packet P4 is generated from distinct fraction F4, then packet P5 is generated from distinct fraction F5, and then packet P6 is generated from distinct fraction F6) over a fourth time period (e.g., from time t3 to time t4). The ion store 118 accumulates the second population of product ions 126-2 (e.g., product ions contained in packets P4-P6) over a fifth time period (accumulation time, e.g., from time t4 to time t5) of the timing scheme 500. In some examples, the fifth time period may overlap with the fourth time period, such that the ion store 118 accumulates the second population of product ions 126-2 as the product ions are generated by the collision cell 116. In some other examples, each packet (e.g., packets P4-P6) contained in the second population of product ions 126-2 is transferred to the ion store 118 simultaneously (e.g., after the last packet of product ions contained in the second population of product ions 126-2 is generated). The ion store 118 transfers the second population of product ions 126-2 to the mass analyzer 120, which performs mass analysis of the second population of product ions 126-2 over a sixth time period (e.g., from time t5 to time t6) of timing schemes 500 and 502. The pre-separation device 104 may continue to sequentially eject subsets of the distinct fractions 112 of precursor ions until each distinct fraction 112 of precursor ions has been ejected from each channel 414.

[0083] As an illustrative example, the pre-separation device 104 may include nine channels 414 (e.g., n=9), such that during a first time period (e.g., about 250 ms), the pre-separation device 104 accumulates a set of distinct fractions 112 of precursor ions in the nine channels 414. The set of nine distinct fractions 112 includes three subsets of distinct fractions 112 of precursor ions, such that each subset of distinct fractions 112 includes three distinct fractions 112. Each subset of three distinct fractions 112 of precursor ions is sequentially transferred from the pre-separation device 104 for mass analysis over the remainder of the time period (e.g., about 36 ms, such as about 12 ms per subset of distinct fractions 112).

[0084] Prior to transferring a first distinct fraction 112-1 of precursor ions contained within a first subset of distinct fractions 112 from the pre-separation device 104, one or more operating parameters of the mass filter 114 and / or collision cell 116 may be adjusted (e.g., over a period of about 1-2 ms) to accommodate the precursor ions contained in the first distinct fraction 112-1. The first distinct fraction 112-1 is transferred (e.g., over a period of about 2-3 ms) from the first channel 414-1 of the pre-separation device 104 to the mass filter 114, collision cell 116, and ion store 118. One or more operating parameters of the mass filter 114 and / or collision cell 116 may be further adjusted (e.g., over a period of about 1-2 ms) to accommodate the precursor ions contained within the second distinct fraction 112-2. The second distinct fraction 112-2 is transferred (e.g., over a period of about 2-3 ms) from the second channel 414-2 of the pre-separation device 104 to the mass filter 114, the collision cell 116, and the ion store 118. One or more operating parameters of the mass filter 114 and / or the collision cell 116 may be further adjusted (e.g., over a period of about 1-2 ms) to accommodate precursor ions contained within the third distinct fraction 112-3, and the third distinct fraction 112-3 is transferred (e.g., over a period of about 2-3 ms) from the third channel 414-3 of the pre-separation device 104 to the mass filter 114, the collision cell 116, and the ion store 118. The ion store 118 accumulates a first population 126-1 of product ions generated from precursor ions contained in the first distinct fraction 112-1, the second distinct fraction 112-2, and the third distinct fraction 112-3 over an accumulation time and transfers the first population 126-1 to the mass analyzer 120 for mass analysis of the first population 126-1. After the first population 126-1 is transferred from the ion store 118, the process is repeated for the second and third subsets of distinct fractions 112.While the illustrated example includes nine channels 414, nine distinct fractions 112 of precursor ions, and three distinct fractions 112 within each subset of distinct fractions, any suitable number of channels 414, distinct fractions 112, and subsets of distinct fractions 112 may be used for multiplexing separation over any suitable length of time.

[0085] Such pre-separation of precursor ions according to timing schemes 500 and 502 improves the duty cycle for mass analysis. For example, accumulating precursor ions according to mobility in pre-separation device 104 during a first period and pre-separating them into distinct fractions 112 of precursor ions stores the precursor ions while they wait to be transferred to mass analyzer 106 for mass analysis. Furthermore, accumulating and analyzing product ions generated from precursor ions contained within a subset of distinct fractions 112 during a remaining period allows mass analyzer 106 to simultaneously analyze product ions generated from each subset of distinct fractions 112 without overloading mass analyzer 106. Mobility-based pre-separation may further reduce charge loading that would otherwise hinder m / z-based separation, provide charge state separation of interfering ions, and increase the efficiency of m / z-based separation.

[0086] 6A and 6B show schematic diagrams of other exemplary timing schemes 600 and 602, respectively. Each timing scheme 600 and 602 includes ejecting a first distinct fraction 112 of precursor ions from the pre-separation device 104 and ejecting a next distinct fraction 112 of precursor ions from the pre-separation device 104 simultaneously with generating product ions from the first distinct fraction 112 of precursor ions.

[0087] 6A, the pre-separation device 104 accumulates precursor ions (e.g., in channel 414) over a first time period (e.g., from time t0 to time t1) of a timing scheme 600. The pre-separation device 104 then sequentially transfers distinct fractions 112 of precursor ions (e.g., distinct fractions F1-F3) contained within a first subset of the accumulated distinct fractions 112 of precursor ions to the mass filter 114 and collision cell 116 over a second time period (e.g., from time t1 to time t2) of the timing scheme 600. For example, a first distinct fraction F1 is transferred from the first channel 414-1, and a first packet of product ions P1 is generated from the first distinct fraction F1 (e.g., by the collision cell 116). While a first packet of product ions P1 is generated and / or transferred from the collision cell 116, the pre-separation device 104 transfers a second distinct fraction F2 from the second channel 414-2, such that a second packet of product ions P2 is generated from the second distinct fraction F2. Then, while the second packet of product ions P2 is generated and / or transferred from the collision cell 116, the pre-separation device 104 transfers a third distinct fraction F3 from the third channel 414-3, such that a third packet of product ions P3 is generated in the collision cell 116 from the third distinct fraction F3.

[0088] 6B, the pre-separation device 104 is alternatively configured to continuously accumulate precursor ions (e.g., in channel 414) during and after a first period of time, such as during which the pre-separation device 104 sequentially transfers distinct fractions 112 of precursor ions. Illustratively, the pre-separation device 104 may accumulate precursor ions contained in distinct fraction F2 while the pre-separation device transfers precursor ions contained in distinct fraction F1, and / or the pre-separation device 104 may accumulate precursor ions contained in distinct fraction F3 while the pre-separation device transfers precursor ions contained in distinct fraction F2. The pre-separation device 104 may be configured to continuously and / or periodically accumulate precursor ions through a timing scheme 602.

[0089] The ion store 118 accumulates the first population of product ions 126-1 (e.g., product ions contained in packets P1-P3) over a third time period (e.g., from time t2 to time t3) of timing schemes 600 and 602. In some examples, the third time period may overlap with the second time period such that the ion store 118 accumulates the first population of product ions 126-1 as the product ions are generated by the collision cell 116. In some other examples, each packet (e.g., packets P1-P3) contained in the first population of product ions 126-1 is transferred to the ion store 118 simultaneously (e.g., after the last packet of product ions contained in the first population of product ions 126-1 is generated). The ion store 118 then transfers the first population of product ions 126-1 to the mass analyzer 120, which performs mass analysis of the first population of product ions 126-1 over a fourth period of timing schemes 600 and 602 (e.g., from time t3 to time t4).

[0090] Concurrent with mass analysis of the first population of product ions 126-1 over a fourth time period, the pre-separation device 104 sequentially transfers distinct fractions 112 of precursor ions (e.g., distinct fractions F4 to F6) contained within a second subset of the accumulated distinct fractions 112 of precursor ions to the mass filter 114 and collision cell 116 (e.g., distinct fraction F4 from the fourth channel 414-4, then distinct fraction F5 from the fifth channel 414-5, then distinct fraction F6 from the sixth channel 414-6), and the collision cell 116 fragments the precursor ions contained within the second subset into packets 124 of product ions over the fourth time period (e.g., packet P4 from distinct fraction F4, then packet P5 from distinct fraction F5, then packet P6 from distinct fraction F6). For example, a subsequent fraction of precursor ions is transferred from the pre-separation device 104 after generating product ions from a previous fraction of precursor ions in the collision cell 116. The ion store 118 accumulates the second population of product ions 126-2 (e.g., product ions included in packets P4-P6) over a fifth time period (e.g., from time t4 to time t5) of the timing scheme 600. In some examples, the fifth time period may overlap with the fourth time period such that the ion store 118 accumulates the second population of product ions 126-2 as product ions are generated by the collision cell 116. In some other examples, each packet included in the second population of product ions 126-2 (e.g., packets P4-P6) is transferred to the ion store 118 simultaneously (e.g., after the last packet of product ions included in the second population of product ions 126-2 is generated). The ion store 118 transfers the second population of product ions 126-2 to the mass analyzer 120, which performs mass analysis of the second population of product ions 126-2 over a sixth time period (e.g., from time t5 to time t6) of the timing scheme 600. The pre-separation device 104 may continue to sequentially eject subsets of the distinct fractions 112 of precursor ions until each distinct fraction 112 of precursor ions has been ejected from each channel 414.Such transfer of distinct fractions 112 of precursor ions while product ions are being generated further reduces the amount of time for mass analysis of a set of distinct fractions 112 .

[0091] 7 shows another exemplary implementation 700 of the system 100 in which precursor ions are transported sequentially through a pre-separation device 104 and a mass spectrometer 106. As shown, the pre-separation device 104 is positioned downstream of the ion source 102 and includes a trap ion mobility separator, where precursor ions are spatially separated within the separation region 702 of the pre-separation device 104 based on a simultaneous acting gas flow (indicated by arrow 704) and a variable electric field gradient (indicated by arrow 706) in the separation region 702. The gas flow is in a first direction from an inlet 708 at one end of the separation region 702 to an outlet 710 at the other end (e.g., the opposite end) of the separation region 702. Additionally, an electric field gradient is applied in a second direction (e.g., opposite the first direction). Thus, the gas flow transports precursor ions in a first direction against the second direction of the electric field gradient to spatially separate the precursor ions according to their mobility. The electric field gradient is varied over time to sequentially transfer the distinct fractions 112 of precursor ions into the mass spectrometer 106. Illustratively, the electric field gradient is gradually decreased to sequentially transfer the distinct fractions 112 of precursor ions with increasing mobility. As shown, the distinct fractions 112 of precursor ions are continuously transferred from the pre-separation device 104. Alternatively, the electric field gradient can be stepped such that the distinct fractions 112 of precursor ions are transferred from the pre-separation device 104 in stages. In some examples, the distinct fractions 112 of precursor ions can be directed to different locations (e.g., within a DMA separator based on arrival time).

[0092] The mass filter 114 of the mass spectrometer 106 is positioned downstream of the pre-separation device 104 such that the mass filter 114 is configured to receive the distinct fractions 112 of precursor ions (e.g., through the outlet 710). For each distinct fraction 112 of precursor ions, the mass filter 114 isolates precursor ions of a selected m / z range of precursor ions (e.g., the m / z range of an isolation window). The mass filter 114 transfers the isolated precursor ions for each distinct fraction 112 of precursor ions to the collision cell 116. The collision cell 116 fragments the isolated precursor ions received from the mass filter 114 into product ions. For example, the collision cell 116 uses collision-induced dissociation to fragment the precursor ions by applying collision energy to collide the precursor ions with a collision gas. One or more operating parameters of the mass filter 114 and / or collision cell 116 may be adjusted prior to receiving each distinct fraction 112 of precursor ions, such as to isolate, fragment, and / or accumulate ions within each distinct fraction 112.

[0093] In the illustrated example, the collision cell 116 includes an ion store (e.g., ion store 118) such that the collision cell 116 is configured to accumulate a population of product ions 126 generated from each subset of distinct fractions 112 of precursor ions within the collision cell 116. The product ions are accumulated within the collision cell 116 for an accumulation time (e.g., until a product ion is generated from each distinct fraction of precursor ions included within the subset of distinct fractions 112). The collision cell 116 then transfers the population of product ions 126 to the mass analyzer 120 for mass analysis. For example, the collision cell 116 may include end electrodes configured to provide a blocking potential (e.g., a DC blocking potential) configured to stop the flow of product ions within the collision cell 116 at certain times (e.g., during the accumulation time) and allow the flow of product ions out of the collision cell 116 at certain other times (e.g., the blocking potential may be reduced at certain times to allow the population of precursor ions 126 to flow).

[0094] The mass analyzer 120 of the mass spectrometer 106 is positioned downstream from the collision cell 116 and is configured to receive the population of product ions 126 transferred from the collision cell 116 and acquire a mass spectrum of the population of product ions 126. For example, the mass analyzer 120 generates a signal based on product ions generated from precursor ions contained within each distinct fraction 112 of precursor ions transferred from the pre-separation device 104 as a subset of the distinct fractions 112. The signal may include an electrical signal representing ion intensities based on the product ions contained within the population of product ions 126. In some examples, the control module 200 may be configured to instruct the mass analyzer 120 to generate a signal and / or acquire the signal generated by the mass analyzer 120. The mass analyzer 120 may further generate a mass spectrum for an additional population of product ions 126 accumulated in the collision cell 116 generated from an additional subset of the distinct fractions 112 of precursor ions transferred from the pre-separation device 104.

[0095] The systems and methods described herein can be applied to other types of instruments for multiplexed pre-separation. For example, the pre-separation device 104 can implement any suitable technique for spatially separating precursor ions according to mobility, such as DMA separation, drift ion mobility separation, traveling wave ion mobility separation, and trapped ion mobility separation. Additionally or alternatively, the pre-separation device 104 can implement any suitable technique for separating precursor ions based on m / z, such as separation based on competing pseudopotentials resulting from RF stacked ring ion guides, traveling waves, and DC gradients. Such systems and techniques can be used for DDA and / or DIA MS analysis.

[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 systems and / or components described above may include or be realized by any computer hardware and / or computer-executable 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.

[0097] In certain embodiments, one or more of the processes described herein may be executed, 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.

[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 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.

[0099] FIG. 8 illustrates an exemplary computing device 800 that may be specifically configured to perform one or more of the processes described herein. As shown in FIG. 8, computing device 800 may include a communication interface 802, a processor 804, a storage device 806, and an input / output ("I / O") module 808 that are communicatively coupled to each other via a communication infrastructure 810. While FIG. 8 illustrates an exemplary computing device 800, the components illustrated in FIG. 8 are not intended to be limiting. In other embodiments, additional or alternative components may be used. The components of computing device 800 illustrated in FIG. 8 will now be described in further detail.

[0100] The communication interface 802 may be configured to communicate with one or more computing devices. Examples of the communication interface 802 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 804 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. The processor 804 may perform operations by executing computer-executable instructions 812 (e.g., applications, software, code, and / or other executable data instances) stored in the storage device 806.

[0102] Storage device 806 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 806 may include, without limitation, any combination of non-volatile and / or volatile media described herein. Electronic data, including the data described herein, may be temporarily and / or permanently stored in storage device 806. For example, data representing computer-executable instructions 812 configured to instruct processor 804 to perform any of the operations described herein may be stored in storage device 806. In some examples, data may be located in one or more databases residing in storage device 806.

[0103] I / O module 808 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 808 may include any hardware, firmware, software, or combination thereof that supports input and output capabilities. For example, I / O module 808 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.

[0104] I / O module 808 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 a particular embodiment, I / O module 808 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 for 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 800. For example, memory 202 may be implemented by storage device 806, and processor 204 may be implemented by processor 804.

[0106] 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 realized, 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.

[0107] The advantages and features of the present disclosure can be further illustrated by the following examples.

[0108] Example 1. A system comprising: one or more processors; and a memory storing executable instructions, the executable instructions, when executed by the one or more processors, causing the computing device to direct a pre-separation device to separate precursor ions into a set of distinct fractions of precursor ions based on a physical property of the precursor ions; direct the pre-separation device to sequentially transfer a first subset of the distinct fractions of precursor ions included within the set of distinct fractions of precursor ions to a mass analyzer; direct the mass analyzer to sequentially generate product ions from each distinct fraction of precursor ions included within the first subset of distinct fractions of precursor ions; direct the mass analyzer to accumulate a first population of product ions in an ion store over an accumulation time, the first population of product ions including product ions generated from each distinct fraction of precursor ions included within the first subset of distinct fractions of precursor ions; and direct the mass analyzer to transfer the first population of product ions to the mass analyzer for mass analysis of the first population of product ions.

[0109] Example 2. The system of Example 1, wherein the pre-separation device is configured to spatially separate precursor ions into a set of distinct fractions of precursor ions according to the mobilities of the precursor ions.

[0110] Example 3. The system of example 2, wherein the pre-separation device comprises a trapped ion mobility separator, a drift ion mobility separator, or a differential mobility separator.

[0111] Example 4. The system of Example 1, wherein the pre-separation device is configured to separate precursor ions into a set of distinct fractions of precursor ions based on the mass-to-charge ratio (m / z) of the precursor ions.

[0112] Example 5. The system of example 4, wherein the pre-separation device comprises a mass filter, an ion accumulator, an ion selector, a toroidal ion trap, or a linear ion trap.

[0113] Example 6. The system of Example 1, wherein the pre-separation device comprises a plurality of channels configured to store a plurality of sets of precursor ions within the plurality of channels, and the pre-separation device is configured to sequentially transfer a first subset of distinct fractions of precursor ions from a first subset of channels included in the plurality of channels.

[0114] Example 7. The system of Example 6, wherein each channel of the plurality of channels is configured to store a distinct fraction of precursor ions contained within a set of distinct fractions of precursor ions, and to sequentially transfer each distinct fraction of precursor ions from the plurality of channels.

[0115] Example 8. The system of Example 1, wherein the pre-separation device is configured to continuously transport precursor ions through the pre-separation device to spatially separate the precursor ions into a set of distinct fractions of precursor ions.

[0116] Example 9. The system of Example 1, wherein each distinct fraction of precursor ions comprises a distinct m / z range of precursor ions such that an m / z range of precursor ions contained in one distinct fraction of precursor ions does not overlap with another m / z range of precursor ions contained in another distinct fraction of precursor ions.

[0117] Example 10. The system of example 1, wherein the product ions are generated in a collision cell of the mass spectrometer, and the ion store comprises the collision cell.

[0118] Example 11. The system of example 1, wherein the ion store is positioned downstream of a collision cell of the mass spectrometer, the collision cell configured to generate product ions and sequentially transfer the product ions to the ion store for accumulation of a first population of product ions.

[0119] Example 12. The system of example 11, wherein the ion store comprises an ion trap or a C-trap.

[0120] Example 13. The system of example 1, wherein the instructions, when executed by the one or more processors, further cause the computing device to adjust one or more operating parameters of the mass spectrometer during successive transfers of the distinct fractions of precursor ions from the pre-separation device to the mass spectrometer for processing of the next distinct fraction of precursor ions.

[0121] Example 14. The system of example 13, wherein the one or more operating parameters include a collision energy of a collision cell included in the mass spectrometer and configured to generate product ions.

[0122] Example 15. The system of example 13, wherein the one or more operating parameters are included within the mass spectrometer and include an m / z isolation window of a mass filter configured to filter a set of distinct fractions of precursor ions.

[0123] Example 16. The system of example 1, wherein the instructions, when executed by the one or more processors, further cause the computing device to direct the mass spectrometer to acquire a mass spectrum based on the first population of product ions.

[0124] Example 17. The system of Example 1, wherein the accumulation time is less than the acquisition time for mass analysis.

[0125] Example 18. The system of example 1, wherein the instructions, when executed by the one or more processors, further cause the computing device to direct the pre-separation device to sequentially transfer a second subset of the distinct fractions of precursor ions to the mass analyzer after the transfer of the first subset of the distinct fractions of precursor ions; direct the mass analyzer to sequentially generate product ions from each distinct fraction of precursor ions included in the second subset of the distinct fractions of precursor ions; direct the mass analyzer to accumulate a second population of product ions in the ion store for another accumulation time, the second population of product ions including product ions generated from each distinct fraction of precursor ions included in the second subset of the distinct fractions of precursor ions; and direct the mass analyzer to transfer the second population of product ions to the mass analyzer for mass analysis of the second population of product ions.

[0126] Example 19. The system of example 18, wherein a second subset of the distinct fraction of precursor ions is transferred from the pre-separation device to the mass analyzer simultaneously with the transfer of the first population of product ions to the mass analyzer.

[0127] Example 20. The system of Example 18, wherein the instructions, when executed by the one or more processors, further cause the computing device to adjust one or more operating parameters of the mass spectrometer between the transfer of the first subset of the distinct fractions of precursor ions to the mass spectrometer and the transfer of the second subset of the distinct fractions of precursor ions to the mass spectrometer to target selected precursor ions from the second distinct fraction of precursor ions.

[0128] Example 21. The system of example 20, wherein targeting selected precursor ions from the second subset of distinct fractions of precursor ions is based on mass analysis of the first subset of distinct fractions or precursor ions.

[0129] Example 22. A system comprising: a pre-separation device configured to separate precursor ions into a set of distinct fractions of precursor ions based on a physical property of the precursor ions and to sequentially transfer a first subset of the distinct fractions of precursor ions contained within the set of distinct fractions of precursor ions; and a mass spectrometer positioned downstream of the pre-separation device and configured to receive the first subset of distinct fractions of precursor ions, wherein the mass spectrometer comprises an ion store configured to accumulate a first population of product ions generated from each distinct fraction of precursor ions contained within the first subset of distinct fractions of precursor ions; and a mass analyzer configured to perform mass analysis of the first population of product ions.

[0130] Example 23. The system of example 22, wherein the product ions are generated in a collision cell of the mass spectrometer, and the ion store comprises the collision cell.

[0131] Example 24. The system of Example 22, wherein the ion store is positioned downstream of a collision cell of the mass spectrometer, and the collision cell is configured to generate product ions and sequentially transfer the product ions to the ion store for accumulation of a first population of product ions.

[0132] Example 25. A non-transitory computer-readable medium storing instructions that, when executed, direct at least one processor of a computing device for mass spectrometry to: direct a pre-separation device to separate precursor ions into a set of distinct fractions of precursor ions based on a physical property of the precursor ions; direct the pre-separation device to sequentially transfer a first subset of the distinct fractions of precursor ions contained within the set of distinct fractions of precursor ions to a mass spectrometer; and direct the mass spectrometer to sequentially transfer a first subset of the distinct fractions of precursor ions contained within the first subset of distinct fractions of precursor ions. a first subset of the distinct fractions of precursor ions; directing the mass spectrometer to accumulate a first population of product ions in an ion store over an accumulation time, the first population of product ions including product ions generated from each distinct fraction of precursor ions contained in the first subset of the distinct fractions of precursor ions; and directing the mass spectrometer to transfer the first population of product ions to a mass analyzer for mass analysis of the first population of product ions.

[0133] Example 26. The non-transitory computer-readable medium of Example 25, wherein the instructions, when executed by at least one processor, further cause the computing device to adjust one or more operating parameters of the mass spectrometer during successive transfers of the distinct fractions of precursor ions from the pre-separation device to the mass spectrometer for processing of a next distinct fraction of precursor ions.

[0134] Example 27. The non-transitory computer-readable medium of Example 26, wherein the one or more operating parameters include a collision energy of a collision cell included in the mass spectrometer and configured to generate product ions.

[0135] Example 28. The non-transitory computer-readable medium of Example 26, wherein the one or more operating parameters are included within the mass spectrometer and include an m / z isolation window of a mass filter configured to filter a set of distinct fractions of precursor ions.

Claims

1. 1. A system 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: directing the pre-separation device to separate the precursor ions into a set of distinct fractions of precursor ions based on physical properties of said precursor ions; directing the pre-separation device to sequentially transfer a first subset of distinct fractions of precursor ions contained within the set of distinct fractions of precursor ions to a mass spectrometer; directing the mass spectrometer to sequentially generate product ions from each distinct fraction of precursor ions contained within the first subset of distinct fractions of precursor ions; directing the mass spectrometer to accumulate in an ion store over an accumulation time a first population of product ions, the first population of product ions including product ions generated from each distinct fraction of precursor ions included within the first subset of distinct fractions of precursor ions; directing the mass spectrometer to transmit the first population of product ions to a mass analyzer for mass analysis of the first population of product ions.

2. 10. The system of claim 1, wherein the pre-separation device is configured to spatially separate precursor ions into a set of distinct fractions of the precursor ions according to the mobilities of the precursor ions.

3. The system of claim 2 , wherein the pre-separation device comprises a trapped ion mobility separator, a drift ion mobility separator, or a differential mobility separator.

4. 2. The system of claim 1, wherein the pre-separation device is configured to separate precursor ions into a set of distinct fractions of the precursor ions based on the mass-to-charge ratio (m / z) of the precursor ions.

5. The system of claim 4 , wherein the pre-separation device comprises a mass filter, an ion accumulator, an ion selector, a toroidal ion trap, or a linear ion trap.

6. 2. The system of claim 1, wherein the pre-separation device comprises a plurality of channels configured to store a set of distinct fractions of the precursor ions within the plurality of channels, and the pre-separation device is configured to sequentially transfer a first subset of the distinct fractions of the precursor ions from a first subset of channels included within the plurality of channels.

7. 7. The system of claim 6, wherein each channel of the plurality of channels is configured to store a distinct fraction of precursor ions included in the set of distinct fractions of precursor ions and to sequentially transfer each distinct fraction of precursor ions from the plurality of channels.

8. 2. The system of claim 1, wherein the pre-separation device is configured to sequentially transport the precursor ions through the pre-separation device to spatially separate the precursor ions into a set of distinct fractions of the precursor ions.

9. 2. The system of claim 1, wherein each distinct fraction of precursor ions comprises a distinct m / z range of precursor ions such that an m / z range of precursor ions contained in one distinct fraction of precursor ions does not overlap with another m / z range of precursor ions contained in another distinct fraction of precursor ions.

10. The system of claim 1 , wherein the product ions are generated in a collision cell of the mass spectrometer, and the ion store comprises the collision cell.

11. 2. The system of claim 1, wherein the ion store is positioned downstream of a collision cell of the mass spectrometer, the collision cell configured to generate the product ions and sequentially transfer the product ions to the ion store for accumulation of the first population of product ions.

12. The system of claim 11 , wherein the ion store comprises an ion trap or a C-trap.

13. 2. The system of claim 1, wherein the executable instructions, when executed by the one or more processors, further cause the computing device to adjust one or more operating parameters of the mass spectrometer during successive transfers of distinct fractions of precursor ions from the pre-separation device to the mass spectrometer for processing of a next distinct fraction of precursor ions.

14. 14. The system of claim 13, wherein the one or more operating parameters include a collision energy of a collision cell contained within the mass spectrometer and configured to produce the product ions.

15. 14. The system of claim 13, wherein the one or more operating parameters are comprised within the mass spectrometer and include an m / z isolation window of a mass filter configured to filter a set of distinct fractions of the precursor ions.

16. 2. The system of claim 1, wherein the executable instructions, when executed by the one or more processors, further cause the computing device to instruct the mass spectrometer to acquire a mass spectrum based on the first population of product ions.

17. The system of claim 1 , wherein the accumulation time is less than an acquisition time for the mass analysis.

18. The executable instructions, when executed by the one or more processors, further cause the computing device to: directing the pre-separation device to sequentially transmit a second subset of distinct fractions of precursor ions to the mass spectrometer after the transmission of a first subset of distinct fractions of precursor ions; directing the mass spectrometer to sequentially generate product ions from each distinct fraction of precursor ions contained within the second subset of distinct fractions of precursor ions; directing the mass spectrometer to accumulate in the ion store for another accumulation time a second population of product ions, the second population of product ions including product ions generated from each distinct fraction of precursor ions included in a second subset of the distinct fractions of precursor ions; The system of claim 1 , wherein the mass spectrometer is directed to transfer the second population of product ions to a mass analyzer for mass analysis of the second population of product ions.

19. 20. The system of claim 18, wherein the executable instructions, when executed by the one or more processors, further cause the computing device to adjust one or more operating parameters of the mass spectrometer between the transfer of a first subset of the distinct fractions of precursor ions to the mass spectrometer and the transfer of a second subset of the distinct fractions of precursor ions to the mass spectrometer to target selected precursor ions from a second distinct fraction of precursor ions.

20. 1. A system comprising: a pre-separation device configured to separate precursor ions into a set of distinct fractions of precursor ions based on a physical property of the precursor ions and to sequentially transfer a first subset of the distinct fractions of precursor ions contained within the set of distinct fractions of precursor ions; a mass spectrometer positioned downstream of the pre-separation device and configured to receive a first subset of the distinct fractions of precursor ions, the mass spectrometer comprising: an ion store configured to accumulate a first population of product ions generated from each distinct fraction of precursor ions included within the first subset of distinct fractions of precursor ions; a mass analyzer configured to perform mass analysis of the first population of product ions.

21. A non-transitory computer-readable medium storing instructions that, when executed, direct at least one processor of a computing device for mass spectrometry to: directing a pre-separation device to separate the precursor ions into a set of distinct fractions of precursor ions based on physical properties of said precursor ions; directing the pre-separation device to sequentially transfer a first subset of the distinct fractions of precursor ions contained within the set of distinct fractions of precursor ions to a mass spectrometer; directing the mass spectrometer to sequentially generate product ions from each distinct fraction of precursor ions contained within the first subset of distinct fractions of precursor ions; directing the mass spectrometer to accumulate in an ion store over an accumulation time a first population of product ions, the first population of product ions including product ions generated from each distinct fraction of precursor ions included within a first subset of the distinct fractions of precursor ions; and instructing the mass spectrometer to transfer the first population of product ions to a mass analyzer for mass analysis of the first population of product ions.

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