Ion analysis apparatus and method

Abstract

An ion analysis apparatus comprising: an ionization source configured to generate an ion beam; a trapping device configured to receive the ion beam and to transfer ions axially downstream; a first quadrupole mass filter configured to receive ions transferred from the trapping device and to transfer at least a first subset of the received ions; a segmented linear quadrupole ion trap configured to receive at least the first subset of ions transferred from the first quadrupole mass filter, to perform a first processing step on at least the first subset of the received ions, and to transfer the processed ions; a second quadrupole mass filter configured to receive the processed ions transferred from the segmented linear quadrupole ion trap and to transfer at least a second subset of the processed ions; a collision cell configured to receive at least the second subset of ions transferred from the second quadrupole mass filter, to perform a second processing step on at least the second subset of the received ions, and to transfer the processed ions; a mass analyzer configured to receive the processed ions from the collision cell and to mass analyze the received processed ions.

Description

Technical Field

[0001] This invention relates to ion spectrometers, such as mass spectrometers.

[0002] introduction

[0003] Conventional mass spectrometers are typically configured for bottom-up workflows, such as for complex analytes, like biological molecules (also known as biomolecules), based on MS2-type experiments, whereby a single fragmentation step is performed on one or more precursor ions selected by a first mass analyzer (usually a quadrupole mass filter), while the mass-to-charge ratio of the product (also known as fragment) ions is measured by a second mass analyzer. Summary of the Invention

[0004] The first aspect provides ion analysis equipment, which includes:

[0005] The ionization source is configured to generate an ion beam;

[0006] The capture device is configured to receive an ion beam and transfer ions axially upward and downstream.

[0007] A first quadrupole mass filter is configured to receive ions transferred from a capture device and to transfer at least a first subset of the received ions;

[0008] A piecewise linear quadrupole ion trap is configured to receive at least a first subset of ions transferred from a first quadrupole mass filter, perform a first processing step on the received at least a first subset of ions, and transfer the processed ions.

[0009] The second quadrupole mass filter is configured to receive processed ions transferred from the piecewise linear quadrupole ion trap, and to transfer at least a second subset of the processed ions;

[0010] The collision cell is configured to receive at least a second subset of ions transferred from the second quadrupole mass filter, perform a second processing step on the received at least a second subset of ions, and transfer the processed ions.

[0011] A mass analyzer is configured to receive processed ions from a collision cell and perform mass analysis on the received processed ions.

[0012] The second aspect provides ion analysis methods, which include:

[0013] Generate an ion beam;

[0014] The ion beam is received by a capture device and the ions are transferred axially upwards and downstreams.

[0015] At least a first subset of the transferred ions are mass filtered by a first quadrupole mass filter.

[0016] The first processing step is performed on at least a first subset of mass-filtered ions using a piecewise linear quadrupole ion trap.

[0017] At least a second subset of the treated ions are mass filtered by a second quadrupole mass filter;

[0018] A second processing step is performed on at least a second subset of the mass-filtered ions using a collision cell; and

[0019] Mass analysis is performed on at least a second subset of the processed ions using a mass analyzer.

[0020] Detailed description of the invention

[0021] The first aspect provides ion analysis equipment, which includes:

[0022] The ionization source is configured to generate an ion beam;

[0023] The capture device is configured to receive an ion beam and transfer ions axially upward and downstream.

[0024] A first quadrupole mass filter is configured to receive ions transferred from a capture device and to transfer at least a first subset of the received ions;

[0025] A piecewise linear quadrupole ion trap is configured to receive at least a first subset of ions transferred from a first quadrupole mass filter, perform a first processing step on the received at least a first subset of ions, and transfer the processed ions.

[0026] The second quadrupole mass filter is configured to receive processed ions transferred from the piecewise linear quadrupole ion trap, and to transfer at least a second subset of the processed ions;

[0027] The collision cell is configured to receive at least a second subset of ions transferred from the second quadrupole mass filter, perform a second processing step on the received at least a second subset of ions, and transfer the processed ions.

[0028] A mass analyzer is configured to receive processed ions from a collision cell and perform mass analysis on the received processed ions.

[0029] Additionally and / or alternatively, the first aspect provides a mass spectrometer comprising:

[0030] Ionization source;

[0031] Capture device;

[0032] First four-stage quality filter;

[0033] Piecewise linear ion trap;

[0034] Second-stage four-stage quality filter;

[0035] Collision pool;

[0036] Quality analyzer; and

[0037] Controller;

[0038] The ionization source, trapping device, first quadrupole mass filter, segmented linear ion trap, second quadrupole mass filter, collision cell, and mass analyzer are fluidly coupled in series; and

[0039] The controller is configured to control (e.g., synchronously and / or simultaneously) the processing of corresponding ions in the capture device, the first quadrupole mass filter, the segmented linear ion trap, the second quadrupole mass filter, and the collision cell.

[0040] New design architectures for mass spectrometers are desired to increase analytical depth across different classes of molecules, characterizing their structure and topology, maximizing sequence coverage, identifying binding domains in conjugates, and studying molecular complexes and their stoichiometry. Achieving such in-depth characterization of biomolecular ions requires tandem mass spectrometry performed using multiple mass-selective steps, each followed by a customized approach for ion activation and / or dissociation selection. Such a multi-stage, multi-dimensional workflow is preferably performed at the highest possible scan rate to enable efficient coupling with front-end online separation methods (such as liquid chromatography (LC) or capillary electrophoresis (CE)) widely used for separating complex samples prior to mass analysis. Furthermore, high scan rates will allow for multiple scans to improve the signal-to-noise level of product ions through extended averaging, thus addressing limitations in analyzing highly crowded mass spectrometers by increasing the confidence of annotation (i.e., ion recognition). Top-down approaches or analyses of more complex analytes require new instruments that offer a wide range of ion processing options and the ability to orchestrate multifaceted data-dependent acquisition (DDA) workflows to increase analytical depth.

[0041] The novel design architecture disclosed herein enhances identification and achieves new levels of ion molecular structure characterization and sequence coverage. The novel geometry (i.e., the design architecture) incorporates a series of ion optical elements, including a high-capacity trapping region (e.g., an ion trap or first ion trap) configured with one or more adjacent ion-activated dissociation regions; a first quadrupole mass filter for mass-selecting analyte ions; a linear ion trap (i.e., a second ion trap) for activating and / or dissociating the mass-selected ions (MS2 / MS3) by various means (including electrons, photons, reagent ions, activated neutrals, collisions with buffer gas molecules, and combinations thereof), preferably a segmented linear quadrupole ion trap; a second quadrupole mass filter for selectively delivering one or more of the activated analyte ions and / or dissociation products of the analyte ions; an additional collision cell for dissociating the ions (MS3 / MS4 / MS5) to form the final product ion species; and finally, a mass analyzer equipped with at least one reflective unit for ion detection of the final product ion species, preferably a time-of-flight analyzer. This new geometry can support various MSn (e.g., n=1, 2, 3 and / or 4; e.g., n=1, 2, 3, 4, 5) operating modes or workflows, combined with standard MS2 experiments known in the art, such as fragmenting mass-selected analyte ions using a piecewise linear quadrupole ion trap alone or using a collision cell alone, and customizing the desired MS2 ion activation-dissociation method for specific classes of analytes. That is, in one example, the ion analysis device can be configured to operate in a set of operating modes (e.g., including MS operating modes (also known as Level 1 full mode), MS2 operating mode, MS3 operating mode, MS4 operating mode, and / or MS5 operating mode).

[0042] In one example, the ion analysis device is configured to switch between its set of operating modes between scans. In this way, the ion analysis device can operate in different operating modes during different scans, as described herein. In this way, continuous portions of ions can be analyzed in continuous scans during different operating modes, as described herein. For example, continuous portions of ions can be analyzed in MS1 ​​(i.e., first-level full scan) followed by continuous scans of the MSn ion processing workflow, where n = 2, 3, 4, and / or 5, respectively.

[0043] As described in this article, each operating mode can be implemented through a set of configurations for the ion analysis device (e.g., alternative configurations).

[0044] In one example, the ion analysis device is configured to change between configurations of its operating modes (e.g., a specific operating mode) between scans. In this way, the ion analysis device can implement different configurations of the operating modes (e.g., a specific operating mode) in different scans, as described herein. In this way, continuous portions of ions can be analyzed in continuous scans under different configurations of the operating modes (e.g., a specific operating mode), as described herein. For example, continuous portions of ions can be analyzed in MS2 operating mode by activation-dissociation in a piecewise linear quadrupole ion trap and a collision cell, respectively, in continuous scans.

[0045] For example, in MS2 operating mode, activation-dissociation of the received ions or subsets thereof can be performed in any of the ionization source, activation-dissociation region, piecewise linear ion trap, and collision cell.

[0046] In one example, the ion analysis device can be configured to operate in MS2 mode, wherein the ion analysis device can be configured to implement MS2 operation mode through a configuration set of the ion analysis device, the configuration set including:

[0047] In the first configuration, the ionization source is configured to perform activation-dissociation of the received ions or a subset thereof;

[0048] The second configuration, wherein the capture device includes an activation-dissociation region and / or is coupled to the activation-dissociation region, the activation-dissociation region being configured to perform activation-dissociation of the received ions or a subset thereof;

[0049] A third configuration, wherein the piecewise linear quadrupole ion trap is configured to perform a first processing step, wherein the first processing step includes activating-dissociating the received ions or a subset thereof and / or activating-dissociating the received ions or a subset thereof; and

[0050] The fourth configuration, wherein the collision pool is configured to perform a second processing step, wherein the second processing step includes collision activation of the received ions or a subset thereof and / or collision activation of the received ions or a subset thereof.

[0051] It should be understood that these configurations are alternative configurations to the MS2 operating mode for the received ions or subsets thereof. That is, in MS2 operating mode, activation-dissociation of the received ions or subsets thereof is performed in one (e.g., only one) of the ion source, activation-dissociation region, piecewise linear quadrupole ion trap, and collision cell. In other words, when the ion analyzer is configured in a certain configuration, it will not be simultaneously configured in any other configuration for the same received ions or subsets thereof (i.e., parallel processing of different received ions or corresponding subsets thereof can still be performed). It should be understood that in each configuration of MS2 operating mode, one ion optics is configured to perform activation-dissociation of the received ions or subsets thereof, and other ion optics are configured to perform any of their functions, excluding activation-dissociation of the received ions or subsets thereof.

[0052] For example, in MS3 operating mode, activation-dissociation of the received ions or subsets thereof can be performed in any one or two of the ionization source, activation-dissociation region, piecewise linear ion trap, and collision cell. It should be understood that in MS3 operating mode, activation-dissociation of the received ions or subsets thereof can be repeatedly performed in any one of the ionization source, activation-dissociation region, piecewise linear ion trap, and collision cell (e.g., by capturing or accumulating MS2 product ions in one of the ion optics devices and transferring the MS2 product ions back to the ionization source, activation-dissociation region, piecewise linear ion trap, and collision cell).

[0053] In one example, the ion analysis device can be configured to operate in MS3 mode, wherein the ion analysis device can be configured to implement MS3 operation mode through a configuration set of the ion analysis device, the configuration set including:

[0054] In a first configuration, an ionization source is configured to perform activation-dissociation of received ions or subsets thereof, and a trapping device includes an activation-dissociation region and / or is coupled to the activation-dissociation region, the activation-dissociation region being configured to perform activation-dissociation of received ions or subsets thereof; or a piecewise linear quadrupole ion trap is configured to perform a first processing step, the first processing step including activation-dissociation of received ions or subsets thereof and / or activation-dissociation of received ions or subsets thereof; or a collision cell is configured to perform a second processing step, the second processing step including collision activation of received ions or subsets thereof and / or collision activation of received ions or subsets thereof.

[0055] A second configuration, wherein the capture device includes an activation-dissociation region and / or is coupled to the activation-dissociation region, the activation-dissociation region being configured to perform activation-dissociation of received ions or subsets thereof, and wherein a piecewise linear quadrupole ion trap is configured to perform a first processing step, wherein the first processing step includes activation-dissociation of received ions or subsets thereof and / or activation-dissociation of received ions or subsets thereof, or wherein a collision cell is configured to perform a second processing step, wherein the second processing step includes collision activation of received ions or subsets thereof and / or collision activation of received ions or subsets thereof;

[0056] A third configuration, wherein a piecewise linear quadrupole ion trap is configured to perform a first processing step, wherein the first processing step includes activation-dissociation of the received ions or a subset thereof and / or activation-dissociation of the received ions or a subset thereof, and wherein a collision cell is configured to perform a second processing step, wherein the second processing step includes collision activation of the received ions or a subset thereof and / or collision activation of the received ions or a subset thereof; and

[0057] The fourth configuration, wherein the piecewise linear quadrupole ion trap is configured to repeatedly perform a first processing step, wherein the first processing step includes activating-dissociating the received ions and / or activating-dissociating the received ions.

[0058] It should be understood that these configurations are alternative configurations to the MS3 operating mode for the received ions or subsets thereof. That is, in MS3 operating mode, activation-dissociation of the received ions or subsets thereof is performed in any one or two of the ionization source, activation-dissociation region, piecewise linear quadrupole ion trap, and collision cell. In other words, when the ion analyzer is configured in a certain configuration, it cannot be simultaneously configured in any other configuration for the same received ions or subsets thereof (i.e., parallel processing of different received ions or corresponding subsets thereof can still be performed). It should be understood that in each configuration in MS3 operating mode, one or two ion optics are configured to perform activation-dissociation of the received ions or subsets thereof, and other ion optics are configured to perform any of their functions, excluding activation-dissociation of the received ions or subsets thereof.

[0059] For example, in MS4 operating mode, activation-dissociation of the received ions or a subset thereof can be performed in any two or three of the ionization source, activation-dissociation region, piecewise linear ion trap, and collision cell. It should be understood that in MS4 operating mode, activation-dissociation of the received ions or a subset thereof can be performed repeatedly in any one of the ionization source, activation-dissociation region, piecewise linear ion trap, and collision cell (e.g., by capturing or accumulating MS2 product ions in one of the ion optics devices and transferring the MS2 product ions back to one of the ionization source, activation-dissociation region, piecewise linear ion trap, and collision cell), or activation-dissociation of the received ions or a subset thereof in any two of the ionization source, activation-dissociation region, piecewise linear ion trap, and collision cell.

[0060] In one example, the ion analysis device can be configured to operate in MS4 mode, wherein the ion analysis device can be configured to implement MS4 operation mode through a configuration set of the ion analysis device, the configuration set including:

[0061] In a first configuration, an ionization source is configured to perform activation-dissociation of received ions or subsets thereof, and a piecewise linear quadrupole ion trap is configured to perform a first processing step, wherein the first processing step includes activation-dissociation of received ions or subsets thereof and / or activation-dissociation of received ions or subsets thereof, and wherein a collision cell is configured to perform a second processing step, wherein the second processing step includes collision activation of received ions or subsets thereof and / or collision activation of received ions or subsets thereof;

[0062] A second configuration, wherein the capture device includes an activation-dissociation region and / or is coupled to the activation-dissociation region, the activation-dissociation region being configured to perform activation-dissociation of received ions or subsets thereof, and wherein a piecewise linear quadrupole ion trap is configured to perform a first processing step, wherein the first processing step includes activation-dissociation of received ions or subsets thereof and / or activation-dissociation of received ions or subsets thereof, and wherein a collision cell is configured to perform a second processing step, wherein the second processing step includes collision activation of received ions or subsets thereof and / or collision activation of received ions or subsets thereof;

[0063] A third configuration, wherein an ionization source is configured to perform activation-dissociation of received ions or subsets thereof, and wherein a piecewise linear quadrupole ion trap is configured to repeatedly perform a first processing step, wherein the first processing step includes activation-dissociation of received ions or subsets thereof and / or activation-dissociation of received ions or subsets thereof, and wherein a collision cell is configured to perform a second processing step, wherein the second processing step includes collision activation of received ions or subsets thereof and / or collision activation of received ions or subsets thereof; and

[0064] The fourth configuration includes an activation-dissociation region and / or a region coupled to the activation-dissociation region configured to perform activation-dissociation of received ions or a subset thereof, and wherein the piecewise linear quadrupole ion trap is configured to repeatedly perform a first processing step, wherein the first processing step includes activation-dissociation of received ions and / or activation-dissociation of received ions.

[0065] It should be understood that these configurations are alternative configurations to the MS4 operating mode for the received ions or subsets thereof. That is, in MS4 operating mode, activation-dissociation of the received ions or subsets thereof is performed in any one or two of the ionization source, activation-dissociation region, piecewise linear quadrupole ion trap, and collision cell. In other words, when the ion analyzer is configured in a particular configuration, it cannot be simultaneously configured in any other configuration for the same received ions or subsets thereof (i.e., parallel processing of different received ions or corresponding subsets thereof can still be performed). It should be understood that in each configuration of MS4 operating mode, one or two ion optics are configured to perform activation-dissociation of the received ions or subsets thereof, and other ion optics are configured to perform any of their functions, excluding activation-dissociation of the received ions or subsets thereof.

[0066] In an example operating mode (referred to herein as Level 1 Full Mode), both the piecewise linear quadrupole ion trap and the collision cell operate without applying activation or fragmentation to the analyte ions, while both the first and second quadrupole mass filters operate in transport (also referred to as through) mode to provide a panoramic view of the analytes present in the sample (i.e., information about the analytes at the MS level). Such a panoramic view of the analytes across a wide range of mass-to-charge ratios and at any point in time during LC / CE analysis is referred to as Level 1 Full Scan. Typically, Level 1 Full Scan information (i.e., information at the MS level) is used automatically to drive a series of subsequent MS2 experiments (i.e., MS2 scans), where different analytes are batch-selected and sequentially fragmented based on the information at the MS level. This, in turn, provides information at the MS2 level. For example, one or more analyte ions detected from the information at the MS level can be sequentially (i.e., in turn) mass-selected, fragmented, and their corresponding product ions can be detected, thus providing information about one or more analyte ions at the MS2 level. A first-level full scan can be performed with the capture region (i.e., the first ion trap) operating in accumulation mode and by dynamically controlling the amount of accumulated charge. Most preferably, a first-level full scan is performed with the capture region operating in pass-through mode, wherein analyte ions are simply transported through the capture region without being stored and accumulated, thus eliminating the mass discrimination effect typically observed under high space charge loads, such as for complex samples.

[0067] In one example, in the first scan, the precursor ions fragment in a collision cell, generating MS2 product ions from the precursor ions, and a first mass spectrum of the MS2 product ions is acquired by a mass analyzer, thereby allowing identification of the MS2 product ions from the first mass spectrum. In one example, in the second scan (e.g., following the first scan), the precursor ions fragment in an activation-dissociation region, generating MS2 product ions from the precursor ions, and one or more MS2 product ions are identified from a first mass spectrum selected using a first quadrupole mass filter (i.e., mass-filtered) for processing in a linear quadrupole ion trap, a second quadrupole mass filter, and / or a collision cell, followed by mass analysis of the final product ions. In this way, information provided at the MS2 level from fragmentation relatively downstream in the first scan is used for fragmentation relatively upstream in the second scan, thereby achieving complete processing up to these product ions and thus providing information at the MS3, MS4, and / or MS5 levels in real time. Alternatively and / or additionally, in the second scan, the precursor ions may be fragmented in a linear quadrupole ion trap, and one or more of the MS2 product ions may be identified from a first mass spectrometer selected using a second quadrupole mass filter (i.e., mass-filtered) for processing in a collision cell, followed by mass analysis of the final product ions.

[0068] In one example operating mode (referred to herein as the first MS3 mode), the novel design architecture disclosed in this invention further enables MS3 experiments to be performed at the highest possible scan rate. This is achieved by processing information at the MS2 level and further utilizing that information at the MS2 level, subsequently driving a series of MS3 and / or MS4 experiments (i.e., MS3 and / or MS4 scans) by automatically selecting MS2 product ions. This is preferably performed in real time during the same LC / CE run, thereby identifying MS2 product ions based on information at the MS2 and MS3 levels respectively and subsequently mass-selecting them for further processing at the MS3 and MS4 levels. It should be understood that real time during the same LC / CE run means analyzing the same analyte of the same sample sufficiently quickly during the same LC / CE separation, such as within the same chromatographic peak, and therefore the scan rates are chromatographically compatible. Conventional mass spectrometers cannot achieve such a real-time MS / MS2 / MS3 / MS4 workflow, which is achieved in the new design architecture through the arrangement of its ion optics and its corresponding control. Alternatively, a standalone target MS3 LC / CE run can be designed offline, thereby defining the product ion list based on post-processing of the first DDA MS2 LC / CE dataset.

[0069] In one example, the trapping device can be configured to operate in an ion transport mode, where, for example, ions received in the trapping device are axially transferred from the trapping device without being trapped or accumulated (i.e., by). In another example, the trapping device can be configured to operate in an ion accumulation mode, where, for example, ions continue to be received and accumulated in the trapping device, while the accumulated ions remain retained in the trapping device. The axial transfer of ions accumulated from the trapping device can be gated, for example, as packets. In one example, the trapping device (i.e., a first ion trap) is configured with (i.e., includes) an ion accumulation region that receives an incident ion beam (continuous or pulsed), accumulates the incident ions, and sequentially axially transfers (also referred to as jetting) packets of accumulated ions to a downstream ion optics element, i.e., the packets of accumulated ions are transferred sequentially. In particular, by axially (relative to the accumulation direction) transferring packets of ions, the temporal order of the packets is preserved, without mixing packets of ions to be transferred with the accumulated ions. In this way, incident ions can be accumulated simultaneously with the transfer of packets of previously accumulated ions (i.e., accumulation and transfer of different ions are concurrent or simultaneous) without ion mixing. Furthermore, such accumulation and transfer are compatible with ion mobility separation, which can optionally be performed in the trapping device, as described below. In contrast, transferring packets of ions orthogonally (relative to the accumulation direction) (also known as jetting) requires radial excitation of the ions, and controlling radial jetting becomes particularly problematic, especially because the intensity of the incident ion beam varies over time (typically rapidly) in typical LC / MS experiments, and long-term space charge frequency shifts occur across the entire m / z range. Incomplete radial jetting can lead to mixing of packets of ions to be transferred, which may result in suboptimal DDA selection of precursor ions and / or information loss. Axial jetting does not require ion excitation and is therefore less susceptible to such intensity variations and / or mixing problems. Furthermore, the axial space charge capacity is much greater than the radial capacity of RF trapping devices. Therefore, axial jetting is superior to orthogonal jetting. Therefore, axial injection mitigates or eliminates the problems of orthogonal injection, while having a larger space charge capacity, thus enabling at least partially the real-time MS / MS2 / MS3 / MS4 / MS5 workflow described herein.

[0070] Piecewise linear quadrupole ion trap

[0071] In one example, a piecewise linear quadrupole ion trap is as described in WO 2017 / 134436 A1, the subject of which is incorporated herein by reference in its entirety.

[0072] In one example, a piecewise linear quadrupole ion trap includes:

[0073] At least two discrete trapping regions for processing ions;

[0074] An RF potential generator is used to generate two RF waveforms and is arranged to apply each RF waveform to a pair of electrodes of a linear ion trap to form an RF trapping field component to trap ions radially.

[0075] A multi-output DC potential generator is used to generate multiple DC field components superimposed on the RF field components and distributed across the length of the linear ion trap to control ions in the axial direction.

[0076] A control unit configured to: switch a DC potential and a corresponding DC field component to jointly form a first trapping region filled with ions in at least two discrete trapping regions to change the ion potential energy from a first level to a second level, and to enable a first ion processing step at at least one of the first and second levels; and

[0077] The control unit is configured to switch the DC potential at a second level on one side of the first capture region to a value less than the minimum DC potential of the first capture region, thereby allowing the release of ions confined in the first capture region to move away from the first capture region along the axis of the linear ion trap by acceleration.

[0078] In one example, a piecewise linear quadrupole ion trap includes:

[0079] At least two discrete trapping regions for processing ions;

[0080] An RF potential generator is used to generate two RF waveforms and is arranged to apply each RF waveform to a pair of electrodes of a linear ion trap to form an RF trapping field component to radially confine ions.

[0081] A multi-output DC potential generator (103, 300) is used to generate multiple DC field components superimposed on the RF field components and distributed across the linear ion trap length for axial ion control; and

[0082] A control unit is configured to: switch a DC potential and a corresponding DC field component to jointly form a first capture region in at least two discrete capture regions, the first capture region being filled with ions axially confined therein, to change the ion potential energy from a first ion potential energy level to a second ion potential energy level by simultaneously changing the DC potential of the first capture region between a first DC potential energy level and a second DC potential energy level; and to enable a first ion processing step at at least one of the first ion potential energy level and the second ion potential energy level.

[0083] In one example, a piecewise linear quadrupole ion trap includes:

[0084] At least two discrete trapping regions for processing ions;

[0085] An RF potential generator is used to generate two RF waveforms and is arranged to apply each RF waveform to a pair of electrodes of a linear ion trap to form an RF trapping field component to radially confine ions.

[0086] A multi-output DC potential generator is used to generate multiple DC field components superimposed on the RF field components and distributed across the length of the linear ion trap to control ions axially.

[0087] A control unit configured to switch a DC potential and a corresponding DC field component to jointly form a first capture region within at least two discrete capture regions, the first capture region being filled with ions axially confined therein, to change the ion potential energy from a first ion potential energy level to a second ion potential energy level by simultaneously changing the DC potential of the first capture region between a first DC potential energy level and a second DC potential energy level; and to enable a first ion processing step at at least one of the first and second ion potential energy levels; and

[0088] The control unit is configured to switch one or more of the DC potentials at the second ion potential level on one side of the first capture region to a value less than the minimum DC potential of the first capture region, thereby allowing the release of ions confined in the first capture region to move away from the first capture region along the axis of the linear ion trap by acceleration.

[0089] In one example, the control unit is also configured to switch at least one of the multiple DC field components that together form the first capture region between three different DC potential levels.

[0090] In one example, the control unit is also configured to switch at least one of a plurality of DC field components to transfer ions from a first capture region to a second capture region in at least two discrete capture regions to implement a second processing step.

[0091] In one example, the control unit is also configured to switch multiple DC field components from multiple DC field components that together form a second trapping region in at least two discrete trapping regions, so as to change the potential energy of ions stored in the second trapping region from a first level to a second level.

[0092] In one example, the control unit is also configured to switch at least one of the multiple DC field components that together form the second capture region between three different DC potential levels.

[0093] In one example, the RF waveform consists of a generally rectangular train of voltage pulses.

[0094] In one example, the piecewise linear quadrupole ion trap includes a pair of electrodes configured to receive at least one injected particle beam passing through two discrete trap regions filled with ions at a first potential level.

[0095] In one example, the particles are injected in at least one of the two discrete capture regions without any colliding gas.

[0096] In one example, the control unit is also configured to switch multiple DC field components to release ions from the first capture region toward the second capture region with sufficient kinetic energy to perform collision-induced dissociation.

[0097] In one example, the control unit is also configured to switch at least one DC field component to spray the treated ions toward a mass analyzer for measuring the mass-to-charge ratio.

[0098] In one example, the piecewise linear quadrupole ion trap is configured with at least two trapping regions and is configured to perform ion processing in at least one of the two trapping regions. In one example, one of the at least two trapping regions is configured to receive electrons from an external electron source to activate the ions trapped therein. In one example, the ion analysis device includes a power supply configured to drive the piecewise linear quadrupole ion trap using a pair of inverted rectangular waveforms.

[0099] In one example, the processing of a first packet of ions ejected from the capture device, or a mass selection portion of the first packet of ions (using a first quadrupole mass filter) is performed downstream of the capture device in a piecewise linear quadrupole ion trap and / or in a collision cell. In one example, in parallel with this processing (i.e., concurrently, synchronously), the capture device accumulates a second packet of ions, thus enhancing the duty cycle of the mass spectrometer.

[0100] In one example, the piecewise linear quadrupole ion trap includes at least three trapping regions for processing ions. Most preferably, each trapping region is designed to support (e.g., each supports) unique and independent processing functions (e.g., at least two of the processing functions). For example, a first processing step in a first trapping region of the three trapping regions may involve ion isolation using an AC waveform applied in bipolar mode or by applying a resolved DC, while a second processing step may involve slowly heating the CID using an AC excitation waveform. A first processing step in a second trapping region may involve injecting electrons externally through an aperture on one electrode, while a second processing step may involve injecting radical species externally from the opposite electrode. Finally, a first processing step in a third trapping region may involve ion accumulation, while a second processing step may involve irradiating ions with photons. The ability to raise, lower, and modulate the ion potential energy by controlling the DC potential and corresponding DC field components distributed across the linear ion trap is crucial for optimizing activation dissociation experiments (e.g., tandem execution) and any other processing steps performed sequentially or simultaneously. Switching the DC potential between at least three potential levels (e.g., applied to a single segment) to receive and eject ions is desirable, facilitating transport between the trapping regions.

[0101] In one example, a piecewise linear quadrupole ion trap is designed with nine segments, each supplied with a switchable DC potential to form three trap regions within the segment. Processing in the first trap region involves ion collection and storage, ion accumulation, ion motion excitation for isolation of a single mass-to-charge ratio or multiple precursor ions, slow-heating collision-induced dissociation using dipole excitation, broadband excitation or DC dipole excitation methods, activation without driving ions to dissociate, and combinations thereof. The third trap region is designed to store and accumulate product ions before being ejected at appropriate energy toward an oTOF pulser to meet the demands imposed by downstream optics and a mass analyzer. In a simple operating mode, the potential energy of the ions stored in the segment is increased above the potential applied to adjacent segments, and then switching the appropriate DC field component is performed to efficiently transfer ions between the trap regions.

[0102] MS2

[0103] In one example, the mass spectrometer is configured to activate and / or dissociate incident ions and / or accumulated ions. For example, the trapping device may include an activation-dissociation region and / or be coupled to it, for example, operating at approximately the same pressure, whereby ions can be activated and / or dissociated via collisions with buffer gas molecules and / or other means (e.g., UV photons). In one example, the activation-dissociation region for activating and dissociating at least a portion of the ions generated in the ionization source is positioned before the trapping device (i.e., between the ionization source and the trapping device, and therefore upstream of the trapping device and upstream of the first quadrupole mass filter), within the trapping device, and / or after the trapping device (i.e., between the trapping device and the first quadrupole mass filter, and therefore downstream of the trapping device and upstream of the first quadrupole mass filter), thus enabling high scan rate multi-stage tandem MSn experiments by utilizing downstream ion optics (particularly the first and second quadrupole mass filters) for mass selection of activated and / or dissociated ions. In one example, the ion analysis apparatus includes an ion activation-dissociation region adjacent to the trapping device and upstream of the first quadrupole mass filter. In a preferred embodiment, the activation-dissociation region is positioned before or after the capture device. If the capture device includes an IMS and / or an IMS, activation-dissociation of the precursor ions in the upstream activation-dissociation region enables ion mobility separation of the MS2 product ions generated from the precursor ions in the activation-dissociation region, thereby transferring the ion mobility-separated MS2 product ions downstream to the first quadrupole mass filter. That is, the precursor ions are first activated-dissociated, and the subsequently generated MS2 ions are separated by ion mobility, thereby maintaining the IMS separation of the MS2 product ions. If the capture device includes an IMS and / or an IMS, activation-dissociation of the precursor ions in the downstream activation-dissociation region enables activation-dissociation of the ion mobility-separated precursor ions, thereby generating MS2 product ions from the ion mobility-separated precursor ions, thereby transferring the ion mobility-separated MS2 product ions of the precursor ions downstream to the first quadrupole mass filter. In other words, the precursor ions are first separated by ion mobility, and these ion mobility-separated ions are then activated-dissociated, thereby maintaining the IMS separation of the precursor ions in the MS2 product ions. In one example, no activation-dissociation region is provided within the capture device.

[0104] MS3

[0105] By enabling ion activation and / or dissociation before or after the trapping device configured with an ion accumulation region, the new design architecture enables subsequent mass selection of product ions at the MS2 level using a first quadrupole mass filter, MS3 activation-dissociation of at least a portion of the MS2 product ions in a downstream piecewise linear quadrupole ion trap, followed by mass selection of the MS3 product ions using a second quadrupole mass filter to selectively deliver at least a portion of these MS3 product ions (i.e., fragments and / or activated ions), and finally, MS4 collision-induced dissociation (CID) in a collision cell to form a final product species, which is then preferably mass-analyzed by a time-of-flight mass analyzer. This new design architecture can support different operating modes or workflows, such as (a) using the activation-dissociation region coupled to the trapping device configured with the accumulation region alone, (b) using the piecewise linear quadrupole ion trap alone, or (c) using the collision cell alone to facilitate MS2 experiments including a single ion activation-dissociation step. Therefore, different sequences and / or alternating MS-1 full scans and MS2 scans can be performed based on any of the three possible activation-dissociation regions available in the new design architecture, thus enabling the development of advanced workflows optimized for the analysis of different classes of complex analytes.

[0106] Similarly, a unique workflow incorporating MS3 scanning can be achieved by combining any two of the three available activation-dissociation regions (i.e., at least one of the ion activation-dissociation regions adjacent to the trapping device, the piecewise linear quadrupole ion trap, and the collision cell).

[0107] In an example operating mode (referred to herein as the second MS3 mode), MS2 is performed using one of the activation-dissociation regions coupled to the trapping device, while at least a portion of the mass-selected product ions using the first quadrupole mass filter undergo MS3 activation-dissociation in the piecewise linear quadrupole ion trap using electrons, photons, or other dissociation means.

[0108] In an example operating mode (referred to herein as the third MS3 mode), MS2 is performed on at least a portion of the analyte ions selected using a first quadrupole mass filter in a piecewise linear quadrupole ion trap, while the MS3 mass selection and MS3 CID steps are performed using a second quadrupole mass filter and a second collision cell, respectively. Alternative combinations are possible, for example, by performing MS2 using one of the activation-dissociation regions adjacent to the trapping device, followed by mass selection and MS3 collision activation using a collision cell. Thus, complex analytical methods or DDAMSn experiments can be designed that combine different ion activation-dissociation modes in successive and / or subsequent scans.

[0109] Scan families including the DDA method (i.e., scan cycles or loops) may include one or more consecutive MS-level full scans to drive a series of MS2 scans, followed by a series of MS3 scans. In any operating mode, analyte ions, intermediate product ions, and final product ions are all mass-analyzed by a mass analyzer (preferably a time-of-flight mass analyzer).

[0110] MS4

[0111] In an example operating mode (referred to herein as the first MS4 mode), MS4 experiments can be performed with high repetition rates. The first-stage full scan requires both the piecewise linear quadrupole ion trap and the collision cell to operate without activating or fragmenting the analyte ions, while both the first and second quadrupole mass filters operate in transport or pass-through mode to provide the necessary list of analyte ions to drive subsequent MS2, MS3, and / or MS4 experiments. This MS-first-stage full scan information is first generated, followed by a series of MSn scans, preferably using a time-of-flight mass analyzer positioned downstream of the collision cell. To determine the mass-to-charge ratio of the fragment ions undergoing MS3 analysis, one or more MS2 scans are performed after the MS-first-stage full scan, thereby measuring the m / z ratio of the MS2 fragments using a time-of-flight mass analyzer. Preferably, the MS2 scans are performed using the activation-dissociation region of the trapping device, and this information at the MS2 level is further utilized to define the isolation window of the first quadrupole mass filter to select ions for the MS3 fragmentation phase, which is typically performed in a piecewise linear quadrupole ion trap. In one example, the MS3 fragmentation stage is performed by reacting mass-selected MS2 fragment ions with externally injected low-energy electrons, for example, in a piecewise linear quadrupole ion trap. In another example, larger proteins or protein fragments undergo charge reduction upon electron capture to form radical species without producing an informative MS3 fragmentation mass spectrum. Enhanced information can be achieved in MS4 experiments by selecting at least one charge state from the charge-reduced ion population using a second quadrupole mass filter, which can be further activated and / or dissociated in a collision cell. In this example where electrons are used to perform the MS3 fragmentation step, the mass window selected to drive the second quadrupole mass filter can be calculated by the mass of MS2 fragments that have undergone electron reaction and charge reduction. The same applies to electron-induced dissociation (EID), where the charge state of the analyte or fragment increases via electron desorption (electron ionization). The new instrument architecture can support different MSn workflows, in which one or more sequential MS scans, MS2 scans, MS3 scans and MS4 scans forming a cycle are combined and applied sequentially to the analysis of analyte ions, specifically enhancing the analytical depth to characterize structurally complex molecules.

[0112] Ion mobility separation

[0113] In one example, the capture device includes an ion mobility separation device comprising an ion mobility separation region for separating ions based on their ion mobility, for example, at pressure levels above 0.1 mbar (0.01 kPa). In another example, the capture device includes a captured ion mobility spectrometer and / or a captured ion mobility spectrometer comprising: an ion storage region for storing and / or accumulating an ion beam; and an ion mobility analyzer region for separating and, optionally selectively, transferring the separated ions. In a preferred example, the capture device is a captured ion mobility spectrometer configured with a first capture region and a second capture region for accumulating ions, the second capture region being used to receive accumulated ions, separate the received ions, and sequentially release the separated ions based on their low-field (i.e., ion) mobility. In one example, the ion mobility separation device includes an ion mobility gate to enable mobility selection of the separated ions to be transferred downstream to a first quadrupole mass filter. In this way, a portion of the ions separated by ion mobility can be selectively transferred downstream to the first quadrupole mass filter.

[0114] scanning

[0115] In one example, the ion analysis apparatus is configured to perform a first scan, including performing one or more first processing steps in a piecewise linear quadrupole ion trap and / or one or more second processing steps in a collision cell, performing one or more mass selection steps in a first quadrupole mass filter and / or a second quadrupole mass filter, and optionally performing one or more ion mobility selection steps in a trapping device, wherein the trapping device includes a trapping ion mobility spectrometer and / or a trapping ion mobility spectrometer.

[0116] In one example, the ion analysis device is configured to perform a second scan, including performing one or more first processing steps in a piecewise linear quadrupole ion trap and / or one or more second processing steps in a collision cell, performing one or more mass selection steps in a first quadrupole mass filter and / or a second quadrupole mass filter, and optionally performing one or more ion mobility selection steps in a capture device, wherein one or more of the first processing steps, second processing steps, mass selection steps and / or ion mobility selection steps of the first scan and the second scan are different from each other.

[0117] Quality Analyzer

[0118] In one example, the quality analyzer includes an orthogonal time-of-flight quality analyzer and / or an orthogonal time-of-flight quality analyzer that includes at least one reflector unit.

[0119] MSn Workflow

[0120] The preferred MSn workflow (including the ion mobility separation step) includes the following steps:

[0121] (a) Collision activation, used to disperse, dedissolve or dissociate ions via collision or photon in one of the ion activation-dissociation regions adjacent to the trapped ion mobility spectrometer and upstream of the first quadrupole mass filter;

[0122] (b) Ions separated by using ion mobility gate-gated mobility and / or activated product ions or MS2 fragments by using a first quadrupole mass filter to select product ions or fragments by the m / z ratio;

[0123] (c) Using electronic, photonic, or other ion activation-dissociation methods known in the art to activate and dissociate at least a portion of the MS3 activation-dissociation product ions or MS2 fragments in a piecewise linear quadrupole ion trap;

[0124] (d) Using a second quadrupole mass filter, MS3 selection is performed on the active ions or MS3 fragments based on the m / z ratio of the active ions (charge-reduced or ionized species) or MS3 fragments;

[0125] (e) MS4 collision-activated dissociation-selected species; and

[0126] (f) Use a time-of-flight mass analyzer to perform mass analysis on the final product ions.

[0127] One or more of the steps above can be omitted to reduce the MS4 DDA workflow to an MS3 or MS2 experiment.

[0128] Most preferably, the experimental cycle comprises a single full MS scan, followed by one or more consecutive MS2 scans, and then one or more sequentially applied MS3 and MS4 scans. Once such a cycle consisting of MS, MS2, MS3, and MS4 scans is completed, the MSn DDA method cycle is restarted by selecting new precursor or analyte ions, or by selecting new fragment ions generated at the MS2 level, or by selecting new fragment ions generated at the MS3 level while maintaining the same MS and MS2 steps. In such a DDA workflow, a mobility / mass-charge selection process is performed using an ion mobility gate and / or a first quadrupole mass filter and / or a second quadrupole mass filter.

[0129] The sequential steps of mass selection and ion activation / dissociation allow for more in-depth characterization of target regions, interaction interfaces, or domains of macromolecular ions in terms of sequence, and more detailed characterization of these regions, interfaces, or domains in terms of structure, which would otherwise be impossible using the standard MS2 workflow. Furthermore, the new instrument architecture allows for the execution of MSn (e.g., n=1, 2, 3, and / or 4; e.g., n=1, 2, 3, 4, and / or 5) experiments at potentially maximum scan rates exceeding 10 Hz, which improves the average signal-to-noise ratio while also accommodating front-end in-line separation methods.

[0130] Preferably, the MSn workflow disclosed herein was developed within the context of a Data Dependency Acquisition (DDA) analysis workflow. The DDA method includes processing loops, each loop combining a first-level full scan followed by multiple consecutive MS2 scans. Advanced DDA methods can be developed using novel design architectures, where a first-level full scan is used to generate information driving the MS2 scans, and the information generated from the MS2 scans can be further processed in real-time to drive MS3 and MS4 scans.

[0131] The novel design architecture enables the execution of an MS2 CID step within the collision cell as part of a series of MS2 scans, and then allows the reproduction of the same set of CID fragment ions by applying the CID step to the same analyte population in different regions of the geometry (e.g., in a collision-activated region adjacent to the trapping device) as part of a series of MS3 scans. This approach allows for the use of all ion optics downstream of the trapping device to support more involved MS3 and MS4 scans while maintaining the highest possible MS2 CID scan rate performed within the collision cell.

[0132] In one example, the second quadrupole mass filter is replaced by an RF-only ion guide, wherein the RF ion guide is one of a quadrupole RF ion guide, a hexapole RF ion guide, an octapole RF ion guide, or a higher-order RF ion guide, wherein the mass selection step at the MS3 level or higher is achieved by a piecewise linear quadrupole ion trap.

[0133] controller

[0134] It should be understood that ion analysis devices include controllers configured to, for example, synchronously and / or simultaneously (i.e., in parallel, concurrently) control an ionization source, an ion trap, a first quadrupole mass filter, a piecewise linear ion trap, a second quadrupole mass filter, and / or a collision cell, for example, to control the processing of corresponding ions therein. In one example, the controller includes a processor and memory, and optionally, control electronics. It should be understood that ion analysis devices include one or more power supplies electrically coupled to an ionization source, an ion trap, a first quadrupole mass filter, a piecewise linear ion trap, a second quadrupole mass filter, and / or a collision cell, for example, wherein the controller is configured to control one or more power supplies to control the processing of corresponding ions.

[0135] Transfer ions

[0136] In one example, a first quadrupole mass filter, a segmented linear quadrupole ion trap, a second quadrupole mass filter, and / or a collision cell are configured to transfer ions, for example, for a first-level full scan.

[0137] In one example, the first quadrupole mass filter is configured to receive ions transferred from the capture device and transfer the received ions, for example, without mass filtration or substantially without mass filtration. In this way, the first quadrupole mass filter acts as an ion guide for the transfer of ions.

[0138] In one example, the piecewise linear quadrupole ion trap is configured to receive ions transferred from a first quadrupole mass filter and to transfer the received ions, for example, without performing a first processing step. In this way, the piecewise linear quadrupole ion trap acts as an ion guide for the transfer of ions.

[0139] In one example, the second quadrupole mass filter is configured to receive ions transferred from the trapping device and to transfer the received ions, for example, without mass filtration or substantially without mass filtration. In this way, the second quadrupole mass filter acts as an ion guide for the transfer of ions.

[0140] In one example, the collision cell is configured to receive ions transferred from a first quadrupole mass filter and to transfer the received ions, for example, by modulating the transfer of the received ions. For example, if the collision cell is coupled to a time-of-flight (TOF) mass analyzer, a signal can be applied to the collision cell to modulate or aggregate the ions into packets, and the arrival times of these packets can be synchronized with the TOF extraction pulses, for example, to increase the duty cycle of the TOF analyzer. In this way, the collision cell acts as an ion guide for the transfer of ions.

[0141] In one example, a first quadrupole mass filter, a piecewise linear quadrupole ion trap, a second quadrupole mass filter, and / or a collision cell are configured to transfer ions downstream. In another example, the first quadrupole mass filter, the piecewise linear quadrupole ion trap, the second quadrupole mass filter, and / or a collision cell are configured to transfer ions upstream.

[0142] Example scan mode

[0143] Tables 1 through 4 summarize example scanning modes of exemplary embodiments. Preferred scanning modes are shown in bold. Accumulation or IMS is preferred. Transfer-indicating ions are transferred by the ion optics without additional processing. For linear quadrupole ion traps, the activation of MSn+1 ions and / or the dissociation of MSn (e.g., n=1, n=2, or n=3) product ions, the generation of MSn+1 product ions from MSn product ions, the mass selection of MSn+1 product ions, and the activation and / or dissociation of MSn+2 ions of the mass-selected MSn+1 product ions require two ion activation-dissociation steps, and may require an accumulation step in linear quadrupole ion traps. Accumulation and IMS can be combined, or IMS can be used alone or accumulation can be used alone.

[0144] Storage—capturing and / or accumulating ions in collision pools and / or piecewise linear ion traps.

[0145] As described in more detail below, the collision cell can be used not only for collision activation, thermalization, and / or modulation of ion transfer, but also additionally and / or alternatively for storing, capturing, and / or accumulating ions, similar to the storage, capture, and / or accumulation capabilities of a piecewise linear ion trap. In this way, the relatively large space charge capacity of the collision cell can be used for storing, capturing, and / or accumulating ions. Additionally and / or alternatively, the collision cell can be used for storing, capturing, and / or accumulating mass-filtered ions, for example, by a first quadrupole mass filter and / or a second quadrupole mass filter, preferably mass-filtered by the first quadrupole mass filter.

[0146] In one example, the ion analysis apparatus is configured to perform a first processing step on ions in a piecewise linear quadrupole ion trap, store-capture and / or accumulate the processed ions (optionally filtered by a second quadrupole mass filter) in a collision cell, transfer the stored-captured and / or accumulated ions (optionally filtered by a second quadrupole mass filter) upstream back to the piecewise linear quadrupole ion trap, perform another first step on the ions in the piecewise linear quadrupole ion trap, and transfer these processed ions (optionally filtered by a second quadrupole mass filter) downstream back to the collision cell for another second processing step to repeat the first processing step on the ions (preferably those accumulated in the piecewise linear quadrupole ion trap and / or m / z selected ions) and repeat the second processing step on the ions in the collision cell. The first processing step and the storage-capture and / or accumulation can be repeated within an experimental cycle. In this way, different first processing steps can be performed on the ions, and / or a sufficient number of ions can be stored, captured and / or accumulated for a specific first processing step, such as an additional first processing step or a final first processing step, before the processed ions are transferred to the mass analyzer via a second quadrupole mass filter and a collision cell.

[0147] The collision cell is configured to: receive at least a second subset of ions transferred from a second quadrupole mass filter; perform a second processing step on the received second subset of ions; and transfer the processed ions, for example, downstream or upstream. In one example, the second subset of ions is, or substantially, a first subset of ions. For example, the collision cell may therefore store-capture and / or accumulate a first subset of ions that has been filtered by a first quadrupole mass filter and subsequently transferred to the collision cell via a piecewise linear quadrupole ion trap and a second quadrupole mass filter.

[0148] In one example, the second processing step includes collision activation, thermalization, storage-capture, accumulation, and / or regulation of the transfer of at least a second subset of the received ions. That is, the collision cell can be used not only for collision activation, thermalization, and / or regulation of ion transfer, but also additionally and / or alternatively for storage-capture and / or accumulation of ions.

[0149] In one example, the collision cell is configured to store, capture, and / or accumulate at least a second subset of ions transferred from the second quadrupole mass filter. For example, processed ions transferred from a piecewise linear quadrupole ion trap may be filtered by the second quadrupole mass filter and subsequently stored, captured, and / or accumulated in the collision cell.

[0150] In one example, the collision cell is configured to store, capture, and / or accumulate at least a second subset of ions transferred from the second quadrupole mass filter. For example, processed ions transferred from a piecewise linear quadrupole ion trap can be filtered by the second quadrupole mass filter and subsequently stored, captured, and / or accumulated in the collision cell repeatedly during experimental cycles (i.e., within experimental cycles). In this way, a relatively large number of ions can be stored, captured, and / or accumulated in the collision cell during experimental cycles.

[0151] In one example, the collision cell is configured to transfer the processed ions downstream, for example, to a mass analyzer.

[0152] In one example, the collision cell is configured to transfer the processed ions upstream, for example, via a second quadrupole mass filter to a piecewise linear quadrupole ion trap.

[0153] In one example, the collision cell is configured to transfer the processed ions upstream during experimental cycles (i.e., within experimental cycles), for example, via a second quadrupole mass filter to a piecewise linear quadrupole ion trap, and then transfer the further processed ions downstream, for example, to a mass analyzer.

[0154] In one example, the collision cell is configured to: receive at least a second subset of ions transferred from the second quadrupole mass filter, perform a second processing step on the received at least a second subset of ions, and transfer the processed ions upstream.

[0155] In one example, the second quadrupole mass filter is configured to receive processed ions transferred from the collision cell and to transfer at least a third subset of the processed ions upstream.

[0156] In one example, the piecewise linear quadrupole ion trap is configured to: receive at least a third subset of ions transferred from a second quadrupole mass filter, perform a first processing step on the received at least a third subset of ions, and transfer the processed ions downstream.

[0157] In one example, a piecewise linear quadrupole ion trap and / or collision cell are configured to repeatedly perform a first processing step and a second processing step on ions transferred therebetween.

[0158] In one example, the ion analysis device includes:

[0159] An ionization source, configured to generate an ion beam;

[0160] The capturing device is configured to receive an ion beam and transfer the ions axially upward and downstream.

[0161] A first quadrupole mass filter is configured to receive ions transferred from a capture device and to transfer at least a first subset of the received ions.

[0162] A piecewise linear quadrupole ion trap is configured to: receive at least a first subset of ions transferred from a first quadrupole mass filter, perform a first processing step, such as a first first processing step, on the received at least a first subset of ions, and transfer the processed ions.

[0163] A second quadrupole mass filter is configured to receive processed ions transferred from a piecewise linear quadrupole ion trap and to transfer at least a second subset of the processed ions.

[0164] A collision cell configured to: receive at least a second subset of ions transferred from a second quadrupole mass filter; perform a second processing step on the received at least a second subset of ions; and transfer the processed ions; and

[0165] A mass analyzer is configured to receive processed ions from a collision cell and perform mass analysis on the received processed ions.

[0166] The second processing step includes collision activation, thermalization, storage-capture, accumulation and / or regulation of the transfer of at least a second subset of the received ions;

[0167] The collision cell is configured to: store at least a second subset of ions transferred from the second quadrupole mass filter and / or process ions, and transfer the stored-captured and / or accumulated ions upstream to the second quadrupole mass filter.

[0168] The second quadrupole mass filter is configured to: receive stored-captured and / or accumulated ions transferred from the collision cell, and upstream transfer at least a third subset of the stored-captured and / or accumulated ions to the piecewise linear quadrupole ion trap; and

[0169] The piecewise linear quadrupole ion trap is configured to: receive at least a third subset of ions transferred from a second quadrupole mass filter, perform a first processing step (e.g., a second first processing step) on the received at least a third subset of ions, and transfer the processed ions downstream to the second quadrupole mass filter.

[0170] Optionally, the piecewise linear quadrupole ion trap and / or collision cell are configured to repeatedly perform the first and second processing steps on the ions transferred therebetween, for example, including performing an additional or final first processing step in the piecewise linear quadrupole ion trap, and transferring these processed ions to the mass analyzer via a second quadrupole mass filter and the collision cell, thereby storing, capturing and / or accumulating a sufficiently large number of processed ions in the collision cell before performing additional or final first processing steps on these stored-captured and / or accumulated processed ions in the piecewise linear quadrupole ion trap.

[0171] In one example, the ion analysis device includes:

[0172] An ionization source, configured to generate an ion beam;

[0173] The capturing device is configured to receive an ion beam and transfer the ions axially upward and downstream.

[0174] A first quadrupole mass filter is configured to receive ions transferred from a capture device and to transfer at least a first subset of the received ions.

[0175] A piecewise linear quadrupole ion trap is configured to: receive at least a first subset of ions transferred from a first quadrupole mass filter, and transfer at least a first subset of ions;

[0176] The second quadrupole mass filter is configured to receive at least a first subset of ions transferred from the piecewise linear quadrupole ion trap, and to transfer the first subset of ions.

[0177] A collision cell is configured to: receive a first subset of ions transferred from a second quadrupole mass filter, perform a first and second processing step on the received first subset of ions, and transfer the processed ions; and wherein the collision cell is configured to: perform at least one additional second processing step on a further subset of ions, and transfer the processed ions.

[0178] A mass analyzer is configured to receive processed ions from a collision cell and perform mass analysis on the received processed ions.

[0179] The second processing step, such as the first and second processing steps, includes collision activation, thermalization, storage-capture and / or accumulation of at least a first subset of the received ions;

[0180] The collision cell is configured to: store at least a first subset of ions transferred from the second quadrupole mass filter and / or process ions, and transfer the stored, captured and / or accumulated and / or processed ions upstream to the second quadrupole mass filter.

[0181] The second quadrupole mass filter is configured to: receive stored-captured and / or accumulated and / or processed ions transferred from the collision cell, and upstream transfer at least a second subset of the stored-captured and / or accumulated and / or processed ions to the piecewise linear quadrupole ion trap; and

[0182] The piecewise linear quadrupole ion trap is configured to: receive at least a second subset of ions transferred from the second quadrupole mass filter, perform a first processing step, such as a first first processing step, on the received at least a second subset of ions, and transfer the processed ions downstream back to the second quadrupole mass filter.

[0183] The second quadrupole mass filter is configured to: receive a second subset of ions and transfer at least a third subset of ions to the collision cell; and

[0184] The collision cell is configured to: store, capture and / or accumulate at least a third subset of ions transferred from the second quadrupole mass filter, and perform a third processing step;

[0185] The third processing step includes collision activation, thermalization, storage-capture, accumulation and / or regulation of the transfer of at least a third subset of the received ions.

[0186] In one example, accumulation of mass-selected ions using a quadrupole mass filter is performed in a collision cell. In another example, mass-selected ions are stored in the collision cell, and the accumulation period is controlled by deflecting the incident ion beam at specified time intervals, calculated in real-time based on the space charge capacity of downstream ion optics (e.g., the collision cell) and related to the ionic strength or ion current of the mass-selected ions measured in an MS-first-level full scan. In one example, controlling the accumulation period allows for optimal loading of the collision cell to receive mass-selected analyte ions initially entrained in a continuous ion beam. In another example, the accumulated mass-selected ions are thermalized in the collision cell and transferred back to a piecewise linear ion trap for use in MS2 activation-dissociation or MSn experiments. In another example, upstream ion transfer is possible by switching the direction of the axial DC gradient established across the collision cell. In another example, MS2 product ions generated in the piecewise linear ion trap are then transferred back to the collision cell and ultimately ejected toward a TOF mass analyzer to measure the mass-to-charge ratio.

[0187] For example, when the space charge capacity of the collision cell is greater than that of the trapping device, accumulating mass-selected ions in the collision cell rather than the trapping device may be preferred. MS and MS2 mass spectrometry, derived from larger ion populations, offer higher signal-to-noise ratios, leading to enhanced sensitivity and increased identification of fragment ions. Accumulation is made possible by the collision gas present in the collision cell, which typically operates under hydrostatic pressure conditions of approximately 0.01 mbar.

[0188] The advantage of accumulating ions in a collision cell compared to accumulating ions in a trapping device is that, in the former case, the accumulated population will only include mass-selected ions that can individually occupy the charge capacity of the collision cell, while in the latter case, the capacity of the trapping device will accommodate a variety of ions generated in the ionization source.

[0189] For example, if the pressure in a piecewise linear ion trap is dynamically controlled using one or more gas pulse valves, such as by injecting gas pulses at a lower repetition rate, and when the accumulation period exceeds the residence time of the gas in the trap volume of the piecewise linear ion trap, it may be preferable to accumulate mass-selected ions in a collision cell rather than in the piecewise linear ion trap. Extended accumulation periods are more conveniently implemented in devices operating under hydrostatic conditions to improve the signal-to-noise ratio and detect low-abundance ions. Alternatively and additionally, ion accumulation in a piecewise linear quadrupole ion trap can be achieved by operating one or more gas pulse valves at a higher repetition rate to establish a quasi-steady-state pressure level throughout the accumulation period. The advantage of directly accumulating ions in a piecewise linear ion trap compared to accumulating them in a collision cell is that, in the former case, the accumulated population can be activated and / or fragmented in the piecewise linear ion trap by different methods (e.g., by the reaction of analyte ions with electrons and / or photons), while in the latter case, activation is limited to collision-induced dissociation in the collision cell.

[0190] Different types of MS activation-dissociation experiments can be performed in this exemplary workflow. For example, accumulated mass-selected ions can be transferred back to the piecewise linear ion trap via a second quadrupole mass filter (i.e., from the collision cell) for electron- or photon-based fragmentation. Alternatively, mass-selected ions can be ejected from the collision cell back to the piecewise linear ion trap with sufficient kinetic energy to undergo collision-induced dissociation. Furthermore, mass selection using a second quadrupole mass filter when ions are transferred back to the piecewise linear ion trap from the collision cell can be used in fast MS3 or higher MSn workflows.

[0191] method

[0192] The second aspect provides ion analysis methods, including:

[0193] Generate an ion beam;

[0194] The ion beam is received by a capture device and the ions are transferred axially upwards and downstreams.

[0195] At least a first subset of the transferred ions are mass filtered by a first quadrupole mass filter.

[0196] The first processing step is performed on at least a first subset of mass-filtered ions using a piecewise linear quadrupole ion trap.

[0197] At least a second subset of the treated ions are mass filtered by a second quadrupole mass filter;

[0198] A second processing step is performed on at least a second subset of the mass-filtered ions using a collision cell; and

[0199] Mass analysis is performed on at least a second subset of the processed ions using a mass analyzer.

[0200] The second aspect may include any steps as described with respect to the first aspect.

[0201] Additionally and / or alternatively, the second aspect provides an ion analysis method comprising:

[0202] Generate an ion beam;

[0203] It receives the ion beam and transfers the ions axially upwards and downstream;

[0204] Mass filtration is performed on at least a first subset of the transferred ions;

[0205] Perform a first processing step on at least a first subset of the mass-filtered ions;

[0206] Mass filtration is performed on at least a second subset of at least a first subset of the treated ions;

[0207] Perform a second processing step on at least a second subset of the mass-filtered ions; and

[0208] Mass analysis was performed on at least a second subset of the treated ions.

[0209] Additionally and / or alternatively, the second aspect provides an ion analysis method comprising:

[0210] Generate an ion beam;

[0211] It receives the ion beam and transfers the ions axially upwards and downstream;

[0212] The transferred ions are subjected to mass filtration;

[0213] Perform the first treatment step on the mass-filtered ions;

[0214] The treated ions are then subjected to mass filtration.

[0215] A second processing step is performed on the mass-filtered ions; and

[0216] Mass analysis was performed on the treated ions.

[0217] definition

[0218] Ion processing in a piecewise linear quadrupole ion trap may include radially confining ions (e.g., using a pair of out-of-phase RF waveforms applied to the poles of the quadrupole) and optionally axially manipulating ions (e.g., using a DC potential that forms an axial gradient to transfer ions between different segments and / or transferring ions by forming a barrier for capturing and / or ejecting ions with DC and / or RF potentials).

[0219] The treatment of ions in a piecewise linear quadrupole ion trap may additionally and / or alternatively include activating and / or dissociating ions by various means, including collisions with buffer gas molecules, reactions with photons, variable-energy electrons, reactive neutrals, and / or reagent ions of the same or opposite polarity.

[0220] Ion processing in a segmented linear quadrupole ion trap can further involve MSn multi-stage tandem ion activation-dissociation, wherein, in each stage of the MSn experiment, preferably in different segments of the linear ion trap, different ion activation-dissociation methods are applied. The combination of ion activation-dissociation methods in each stage of the MSn experiment is also enabled by a controller.

[0221] In one example, the first processing step includes: storing-capturing, accumulating, isolating, and / or activating-dissociating at least a first subset of the received ions; preferably storing-capturing, isolating, and activating-dissociating at least a first subset of the received ions; more preferably storing-capturing and activating-dissociating at least a first subset of the received ions. That is, the piecewise linear quadrupole ion trap can be configured to store-capturing, accumulating, isolating, and / or activating-dissociating at least a first subset of the received ions. For example, a first quadrupole mass filter can be used to mass-select the ions, the mass-selected ions can be accumulated in the piecewise linear quadrupole ion trap, and the accumulated mass-selected ions can be processed in the piecewise linear quadrupole ion trap according to the first processing step.

[0222] In one example, the first processing step performed in the piecewise linear ion trap includes accumulating ions, such as single or multiple charge states of proteins or oligonucleotides or any other class of molecules, selecting them using a first quadrupole mass filter, and further performing additional processing steps on these selected and accumulated ions to produce activated product species and / or fragments.

[0223] Processing of ions in the collision cell can include collision cooling or thermalizing, transferring thermally thermalized ions to downstream optics (i.e., upstream), and / or fragmentation via high-energy collisions with buffer gas molecules. Additionally and / or alternatively, processing of ions in the collision cell can include storing-capturing and / or accumulating ions (including collision cooling or thermalizing ions and / or fragmentation). Optionally, the stored-capturing and / or accumulated ions are transferred upstream, for example, via a second quadrupole filter to a piecewise linear quadrupole ion trap. If the collision cell is coupled to a time-of-flight (TOF) mass analyzer, the processing can also involve applying additional signals to modulate or aggregate the processed ions into packets, and synchronizing the arrival times of these packets with the TOF extraction pulses to improve the duty cycle of the TOF analyzer.

[0224] The treatment of ions in the collision cell may additionally and / or alternatively include applying at least one additional RF signal and further modulating the signal to form an axial barrier to selectively eject ions from the collision cell while all ions across a wide range of mass-to-charge ratios exhibit similar time of flight from the collision cell to the extraction region of the TOF analyzer.

[0225] In one example, the second processing step includes collision activation, thermalization, storage-capture, accumulation, and / or regulation of the transfer of at least a second subset of the received ions.

[0226] Ion activation can include: reacting ions with electrons to form radical ions in a lower charge state due to electron capture, or in a higher charge state due to electron separation (ionization), which may further lead to the dissociation of radical ions to form fragments with or without supplemental collisions or IR activation; colliding to activate ions to increase the internal energy of ions at levels below or above the dissociation threshold; using IR activation to increase the internal energy of ions at levels below or above the dissociation threshold; colliding to activate ions to induce unfolding, which may result in conformational annealing; and also reacting ions with UV photons to form product / fragment ions.

[0227] A single scan involves: receiving ions from an ionization source in a trapping device; using the trapping device in pass-through mode, accumulation mode, and / or ion mobility separation mode; transferring at least a portion of the ions through a first quadrupole mass filter; processing the ions in a piecewise linear quadrupole ion trap; transferring at least a portion of the ions through a second quadrupole mass filter; processing the ions in a collision cell; and acquiring a mass spectrum. Multiple mass spectra can be acquired during a single scan, particularly when using a high-speed time-of-flight (TOF) mass spectrometer for ion detection, because the duration of each scan can be several milliseconds, and TOF mass spectra are acquired on a microsecond timescale. Since ions can be processed simultaneously in different ion optics within a newly designed architecture, separate scan sequences are performed and can overlap temporally. Such parallel processing of ions is highly desirable to achieve the highest possible duty cycle. A single scan can include a single full scan to provide accurate ion intensity and mass-to-charge ratio information across a wide range of analytes present in the sample. A single scan may additionally and / or alternatively include an MSn ion processing workflow, where n = 2, 3, 4, and / or 5, to provide product ion information of a specific analyte that can be linked back to the MS-first-level full scan. A single cycle may include at least one first-level full scan followed by one or more MS2 scans. A single cycle may also include one or more MS3 scans following one or more MS2 scans based on MS-first-level full scan information. A single cycle may also include one or more MS4 scans subsequently linked back to one or more MS2 and one or more MS3 scans following MS-first-level full scans. The ion activation-dissociation region adjacent to the trapping device includes regions upstream or downstream of the trapping device, including additional RF ion guides and DC and / or RF electrodes capable of operating at higher pressures and located upstream and / or capable of operating at lower pressures and located downstream of the trapping device. In one example, the ion analysis device includes one or more RF ion guides between the trapping device and a first quadrupole mass filter and / or between the first quadrupole mass filter and a piecewise linear ion trap.

[0228] The instrument duty cycle measures the percentage of the ion beam effectively utilized by the mass spectrometer and is defined as the ratio of the effectively utilized portion of the ion signal detected by the mass spectrometer and used to generate m / z information. This enables the identification and characterization of molecular ions relative to the total ion signal generated in the ionization source. The duty cycle of the ion optics measures the percentage of the ion beam effectively utilized by the ion optics and is defined as the ratio of the effectively utilized portion of the ion signal processed by the ion optics. For example, the duty cycle can also be estimated based on the timing characteristics of a switching DC lens that deflects the incident ion beam entering and leaving the trapping device, and for example, for a 20 ms deflection and an 80 ms accumulation time, the duty cycle of the trapping device becomes 80%. Attached Figure Description

[0229] To better understand the invention and to illustrate how exemplary embodiments of the invention can be implemented, reference will be made to the accompanying drawings by way of example only, in which:

[0230] Figure 1 A mass spectrometer according to an exemplary embodiment is schematically depicted;

[0231] Figure 2 A mass spectrometer according to an exemplary embodiment is schematically depicted in use;

[0232] Figure 3 A method according to an exemplary embodiment is schematically depicted;

[0233] Figure 4 A method according to an exemplary embodiment is schematically depicted;

[0234] Figure 5 A method according to an exemplary embodiment is schematically depicted;

[0235] Figure 6 A mass spectrometer according to an exemplary embodiment is schematically depicted in use;

[0236] Figure 7 A mass spectrometer according to an exemplary embodiment is schematically depicted in use;

[0237] Figure 8 A mass spectrometer according to an exemplary embodiment is schematically depicted in use; and

[0238] Figure 9 A method according to an exemplary implementation is illustrated. Detailed Implementation

[0239] Figure 1 A mass spectrometer 100 according to an exemplary embodiment is shown.

[0240] Ion analysis device 100 includes:

[0241] Ionization source 101 is configured to generate an ion beam;

[0242] The capture device 102 is configured to receive an ion beam and transfer the ions axially upward and downstream.

[0243] The first quadrupole mass filter 103 is configured to receive ions transferred from the capture device 102 and to transfer at least a first subset of the received ions;

[0244] A piecewise linear quadrupole ion trap 104 is configured to receive at least a first subset of ions transferred from a first quadrupole mass filter 103, perform a first processing step on the received at least a first subset of ions, and transfer the processed ions.

[0245] The second quadrupole mass filter 105 is configured to receive processed ions transferred from the segmented linear quadrupole ion trap 104, and to transfer at least a second subset of the processed ions.

[0246] The collision cell 106 is configured to receive at least a second subset of the ions transferred from the second quadrupole mass filter 105, perform a second processing step on the received at least a second subset of the ions, and transfer the processed ions.

[0247] The mass analyzer 107 is configured to receive the processed ions from the collision cell and perform mass analysis on the received processed ions.

[0248] The ion beam 101 is introduced axially along the ion optical axis of the trapping device 102, or perpendicularly to the ion optical axis of the trapping device 102. Preferably, the trapping device 102 includes a set of annular electrodes supplied with a pair of anti-phase sinusoidal RF waveforms to form alternating dipole fields to radially confine the ions. Radial trapping can also be established using a quadrupole field formed by a quadrupole electrode structure to increase the radial compression of the ion beam. Higher-order RF multipole is another alternative, which increases the space charge capacity of the trapping device. Combining a higher-order RF field at the inlet end of the device and across the trapping region of accumulated ions with a lower-order RF field at the outlet end to enhance transmission through the differential aperture 109 is highly desirable. The number of charges 101 flowing into the trapping device 102 can be controlled by switching the DC potential applied to the electrode 108 between transmission and deflection levels. Charge groups spanning a wide range of mass-to-charge ratios can be accumulated and stored in the trapping device 102, radially confined by the RF field and axially confined by an appropriate DC potential applied to the electrode stack. This allows ions to flow into the trapping device, and the accumulated implantation period can be adjusted by controlling the duration of the DC potential applied to electrode 108 to match the capacity of trapping device 102, and avoids ion loss and uncontrolled fragmentation typically observed when limiting high space charge loads. Preferably, the operating pressure of trapping device 102 is >0.1 mbar. Most preferably, the trapping device operates in a pressure range of 1 mbar and 10 mbar controlled by a mechanical pump and valves to regulate the pumping rate. Alternatively, a turbomolecular pump can be used to achieve lower pressures, such as 0.01 mbar or lower. When the trapping device operates at >0.1 mbar, ions are focused through differential aperture 109 into ion funnel 110, which operates at lower pressure, and through a second differential aperture 111 into RF ion guide 112 designed with an axial DC gradient.

[0249] Once the accumulated population stored in the trapping device 102 is ejected to downstream optics for further processing, the ion beam from the ionization source can be redirected back into the trapping device 102 using a DC potential applied to electrode 108. The accumulation period can be matched with the downstream processing period, thus significantly increasing the duty cycle of the method. In another preferred operating mode, the trapping device 102 operates in a transport or pass-through mode to avoid the mass and / or mobility-related discrimination effects observed when capturing large amounts of charge, thus providing accurate information about ion abundance and accurate quantification of analytes across the entire mass-to-charge ratio range. Most preferably, the pass-through mode is applied to a single MS full scan, typically followed by MSn (n=2, 3, 4) scans, where ion capture in the trapping device is crucial for focusing the ion beam, increasing the duty cycle, and enhancing the overall sensitivity of the method.

[0250] Differential orifice 113 and additional pumping are provided to reduce the pressure at lower settings, typically in areas established by turbomolecular pumps, at depths of <10. -4 The pressure in the first quadrupole mass filter 103 operates at millibar pressure. The first quadrupole receives aggregated ion populations and can selectively deliver subsets of ions within a narrow mass range, or deliver a wide range of mass-to-charge ratios, depending on the applied method. Wide mass range delivery is desired in MS-first-level full scans, preferably when the capture device is operating in pass-through mode, while narrow m / z windows or selection of individual charge states of high-quality ions (e.g., proteins) are desirable for rapid MSn experiments when the capture device is operating in capture mode. The high-speed MSn workflow is achieved by the novel instrument architecture disclosed in this invention.

[0251] A set of DC lenses 114 and an additional RF ion guide 115 are disposed between the first quadrupole mass filter 103 and the piecewise linear quadrupole ion trap 104. Preferably, the piecewise linear quadrupole ion trap is differentially pumped, with the differential pumping volume defined by differential apertures 116 and 117. Ion processing in the piecewise linear quadrupole ion trap 104 includes radially confining ions by applying a set of rectangular RF waveforms to the quadrupoles. Individual DC signals are applied to each of the quadrupole segments to axially manipulate ions. Additional analytical DC signals and AC waveforms with single or multiple frequency components can be applied to different segments to isolate, collision-activated, dissociate ions, and selectively stop ions at different positions on the stability graph aq coordinates. Ion processing in the piecewise linear quadrupole ion trap 104 also includes activating and / or dissociating ions with electrons, photons, reactive neutrals, and / or reagent ions of the same or opposite polarity. These activation-dissociation tools can be applied sequentially or simultaneously during a single scan, wherein an ion isolation step is applied between activation-dissociation steps to improve the level of structural characterization and maximize sequencing information of complex biomolecular ions.

[0252] The second quadrupole mass filter 105 is positioned downstream of the segmented linear quadrupole ion trap 104 to enable rapid selection of fragment ions generated upstream. Although mass selection capability is available in the linear ion trap, the isolation step in capture mode is still slower than performing the isolation step using the second quadrupole mass filter 105, which can deliver the selected mass-to-charge ratio on a microsecond timescale, thus reducing scan time and increasing duty cycle.

[0253] The collision cell 106 can be used for collision activation, which induces dissociation products detected using the mass analyzer 107. The collision cell operates at approximately 0.01 mbar pressure, and the collision or buffer gas is confined by two lens electrodes, wherein differential apertures 118 are provided at the inlet and outlet of the device. When the outlet lens 118 is supplied with DC and / or RF modulated signals to control the ejection of ions toward the mass analyzer 107, an axial DC field is required to drive the ions forward. Furthermore, the collision cell thermalizes the analyte and fragment ions to form a collimated ion beam, thereby improving the mass resolution of the mass analyzer 107, preferably the mass resolution of a time-of-flight mass analyzer.

[0254] Figure 2 A mass spectrometer 200 according to an exemplary embodiment is shown. The mass spectrometer 200 is generally as described with respect to mass spectrometer 100, and a detailed description of that mass spectrometer 100 is omitted for brevity. An ion beam 201 is received by a trapping device 202 configured with an accumulation region 211 for storing large ion populations. The ion beam is modulated by a switching DC signal applied to electrode 210 to deflect ions along 209 into the trapping device 202 or along 208 away. The number of ions accumulated in the trapping device 211 is determined by the timing characteristics of the switching DC signal. Typical accumulation times range from <1 ms to >100 ms, depending on the analyte concentration and the intensity of the incident ion beam. The intensity of the incident ion beam can be periodically measured during analysis (preferably at the beginning of each cycle comprising multiple scans) to define the accumulation period. The intensity of the incident ion beam can be measured by performing an MS-level full scan and / or by collecting ion current on one of the electrodes of a geometry (preferably electrode 208 or any alternative electrode structure upstream of the trapping device). If multiple analyte ions are processed within a cycle, an equal number of different accumulation periods are preferred and adjusted according to the ion intensity of each analyte measured in the first full scan. Therefore, all subsequent MSn scans arranged for each analyte are performed with the optimal charge number. This is achieved by dynamically controlling the switching DC signal applied to electrode 210 in each scan to optimally fill the capture device 202.

[0255] A first ion activation-dissociation region 216 located upstream of the trapping device 202 and / or a second ion activation-dissociation region 217 located downstream can be used to generate MS2 fragments from at least a subset of ions generated from the ionization source. If the upstream ion activation-dissociation region is utilized, the accumulation region 211 will be primarily filled with MS2 fragments, depending on the degree of fragmentation induced, for example, by high-energy collisions with background gas molecules or by interaction with externally injected UV photons. Alternatively, analyte ions can accumulate in the ion activation-dissociation region 217 located downstream of the trapping device 202 and subsequently fragment. In this exemplary embodiment, MS2 fragment ions are always generated upstream of the first quadrupole mass filter 203.

[0256] The quadrupole mass filter 203 selectively delivers at least a subset of ions 212 based on their mass-to-charge ratio. In a preferred method, a first MS-first-stage full scan is performed with the quadrupole mass filter 203, configured to deliver ions across a wide range of mass-to-charge ratios 212. Multiple consecutive MS-first-stage full scans may be required to generate high signal-to-noise ratio mass spectra containing high-fidelity isotopic distributions to facilitate spectral annotation or assignment, particularly for high-quality analytes observed at higher charge states. The MS-first-stage full scan precedes the MS2 fragmentation step, which can be performed in regions 216 and 217 before or after the capture device, while the quadrupole mass filter 203 is configured to deliver all MS2 fragment ions 212 distributed across a wide range of mass-to-charge ratios. Similarly, multiple consecutive MS2 scans may be required to generate high signal-to-noise ratio fragmentation spectra containing high-fidelity isotopic distributions to facilitate spectral annotation.

[0257] In this preferred method or operating mode, subsequent MS3 scans can be configured in real time using MS2 scan information, for example by selecting specific MS2 fragment ions using a first quadrupole mass filter 203, which can then be further transferred and processed in a piecewise linear quadrupole ion trap 204. Figure 2A mass-selected population 213 of MS2 fragment ions, stored in one of the linear ion trap segments, is shown to facilitate reaction with electrons injected from an external source 218. As an example of the MS3 step in such a high-speed MSn ion activation-dissociation workflow, a multicharged protein can react with electrons at near-zero kinetic energy to produce a series of charge-reduced MS2 radical fragment ions that would normally undergo dissociation to produce an MS3 counterpart. An additional MS4 fragmentation stage is achieved at the highest possible speed in this novel instrument geometry by mass-selecting product ions 214 generated in the segmented linear quadrupole ion trap from a series of charge-reduced MS2 radical fragment ions and / or from their MS3 dissociation products using a second quadrupole mass filter 205. The DC offset of the segmented linear quadrupole ion trap 204 can be increased relative to the collision cell, thus increasing the kinetic energy of the mass-selected ions 214 above the dissociation threshold to generate and thermalize MS4 fragments 215 in the collision cell 206. The fragment ions 215 eventually accumulate at the exit of the collision cell and are released toward the time-of-flight mass analyzer 207.

[0258] With MS2 fragments or product ions undergoing charge reduction and ionization selected by the first quadrupole mass filter 203 and further activated by kinetic-controlled ion-electron reactions in the piecewise linear quadrupole ion trap 204, the mass-charge window of the second quadrupole mass filter 205 can be directly calculated from the mass of the MS2 fragment ions undergoing charge reduction or ionization. Therefore, no additional real-time processing of the MS3 mass spectrum is required to further improve the analysis speed. In contrast, the selection of MS3 fragment ions requires additional real-time processing of the MS3 ion population. To maximize the analysis speed, real-time calculations of MS and MSn mass spectral characteristics are performed on the MS timescale, preferably less than 50 ms, and most preferably less than 10 ms.

[0259] Different types of ion-activated dissociation can occur at the MS3 stage in a piecewise linear quadrupole ion trap 204, for example by injecting UV or IR photons through an ion trapping volume in a dedicated segment 219 configured with a window for optical access. Other types of fragmentation at the MS3 stage known in the prior art are conceivable.

[0260] Table 5 summarizes the example scanning modes of the ion analysis device 100, as shown in the reference table. Figure 3 , Figure 4 and Figure 5Described. Preferred scanning modes are in bold. Accumulation or IMS is preferred. Transfer-indicating ions are transferred by the ion optics without additional processing. For linear quadrupole ion traps, activation and / or dissociation of MSn+1 ions for MSn (e.g., n=1, n=2, or n=3) product ions, generation of MSn+1 product ions from MSn product ions, mass selection of MSn+1 product ions, and activation and / or dissociation of MSn+2 ions for mass-selected MSn+1 product ions require two ion activation-dissociation steps, and may require an accumulation step in linear quadrupole ion traps. Accumulation and IMS can be combined, IMS can be used alone, or accumulation can be used alone.

[0261] Figure 3 An example of a series of MS4 scans 300 according to an exemplary embodiment is shown, which are performed in parallel and visualized using 50 ms long snapshots, for example, acquired using a mass spectrometer 100 or 200. The duration of a single scan is approximately 105 ms. Each MS4 scan involves transferring a subset of ions generated in an ionization source to a mass analyzer and detector via ion optics with a novel architecture, and includes a 50 ms long accumulation period performed in a capture device 301 also configured with a fragmentation region for performing an MS2 step, an additional 50 ms ion-electron reaction period performed in a piecewise linear quadrupole ion trap 302, and a 5 ms period for the MS4 stage and extraction to the TOF performed in a collision cell 303. A first quadrupole mass filter 304 and a second quadrupole mass filter 305 are configured to perform the MS3 and MS4 mass selection steps, respectively. Parallelization of continuous scans enables operation at a repetition rate of 20 Hz by temporally overlapping the accumulation period of subsequent scans with the processing steps of the previous scan performed in the piecewise linear quadrupole ion trap and collision cell. The repetition rate of the MS4 experiment is determined by the slowest operation or function performed within a single scan, and in this example, this is determined by both the 50 ms accumulation period and the 50 ms ion-electron reaction time. For example, higher repetition rate MS4 experiments are possible by reducing the accumulation time and increasing the reaction rate between electrons and ions. Preferably, the individual scans within each cycle can be configured with different accumulation periods to optimize the ion population stored in the trapping device, which is determined by the intensity of the analyte measured in the previous MS-first full scan. Ideally, the previous MS-first full scan is performed with the trapping device 301 operating in pass mode to achieve accurate quantification of the analyte. Most preferably, the inflow of ions from the ionization source into the first trapping region is uninterrupted, wherein the trapping device duty cycle is close to 100%.

[0262] In the DDA workflow, Figure 3The MS4 scan shown is preceded by a series of MS-level full scans, and a series of MS2 and MS3 scans, which together form the experimental cycle.

[0263] Figure 4 A method 400 for performing MS4 analysis on complex samples, according to an exemplary embodiment, is illustrated. In a typical analytical workflow, the analyte is separated by LC / CE and subsequently ionized by electrospray ionization. An example of the mass spectrometer 400 includes three co-eluting charged state envelopes 401, 402, and 403, each envelope corresponding to a different multi-charged analyte.

[0264] A single cycle 408 includes at least one leading MS-level full scan 409, followed by a series of MSn sub-cycles 405, 406, and 407, each sub-cycle dedicated to at least one analyte or at least one precursor ion charge state from the corresponding charge state envelopes 401, 402, and 403 observed in the leading MS-level full scan 409. The first step in each cycle is to generate the MS-level full scan 409 to identify the different analyte species and their abundances present in the mass spectrometer. This calculation is performed in real time, and particularly in the case of high-quality, high-charge-state analytes, requiring a fast charge-state deconvolution algorithm to extract features from the mass spectrum and define instrument parameters in subsequent scans. Preferably, for samples with a high dynamic range in analyte concentration, precise quantification is achieved by operating the capture device in straight-through mode when performing the MS-level full scan.

[0265] Following the lead-in MS-1 full scan 409, a single or, most preferably, series of MS2 scans are then set in the DDA method, whereby individual charge states selected using a first quadrupole mass filter are collisionally activated, for example, in a piecewise linear quadrupole ion trap or collision cell. The resulting MS2 spectra are then processed in real-time or offline to identify fragment ions in the MS2 spectra, which can be further interrogated or processed through additional MS3 / MS4 fragmentation stages. Where real-time processing of the MS2 mass spectrometry features is feasible, the method is arranged to initiate a series of MS3 scans. This is achieved, for example, by collisionally activating ions coupled to either of the two fragmentation regions of the trapping device, and transferring selected MS2 fragments to a piecewise linear quadrupole ion trap using a first quadrupole mass filter, thereby achieving the MS3 fragmentation step, which is selected from a series of alternative dissociation methods.

[0266] Perform a single or, most preferably, a series of MS3 scans, in which mass-selected MS2 fragments are subjected to reactions with electrons, photons, or other fragmentation mechanisms known in the art. MS3 mass spectra are generated using a mass analyzer, preferably a time-of-flight analyzer, and the characteristics of the MS3 spectra can be processed in real time using a fast algorithm to drive the MS4 fragmentation stage. Alternatively, MS3 products can be predicted directly from the MS2 mass spectra, and MS4 experiments can be set up without real-time reading / processing of MS3 information.

[0267] The MS4 step may involve mass selection of MS3 product ions using a second quadrupole mass filter and collisional activation in a collision cell, generating a large number of MS4 fragments. Typically, a single subcycle (e.g., subcycle 405) includes multiple MS4 scans to improve the signal-to-noise ratio through averaging and to address low fragment ion intensities at this final stage of the analytical workflow. MS3 and MS4 experiments may also require a prior accumulation step to raise the ion population to a sufficiently high level to produce a detectable signal. Ions may be accumulated in one of the segments of a linear quadrupole ion trap prior to performing the MS3 / MS4 fragmentation stage.

[0268] In the case of offline processing of the MS2 dataset, a second target LC / CE run can be performed on the same sample, wherein the mass-to-charge ratio of MS2 fragment ions for MS3 and MS4 processing is defined in the DDA method prior to analysis.

[0269] Preferably, similar to Figure 3 The workflow disclosed in the description, in which each scan 404 is executed sequentially within each of sub-loops 405, 406 and 407, is configured to overlap, thereby enhancing the instrument duty cycle.

[0270] Simplified versions of the MS4 workflow are also disclosed, such as MS2 CID followed by MS3 EID, or MS2 CID followed by MS3 UVPD MS3 scan, where the MS3 step is always performed in a piecewise linear quadrupole ion trap, and a first quadrupole mass filter is used to select MS2 fragments for further processing. In yet another example, the MS3 fragmentation stage can be performed in a collision cell, while the MS2 fragmentation stage can be performed in a piecewise linear quadrupole ion trap, in which case a second quadrupole mass filter is used to select MS2 fragments for further processing. Various different ion activation workflows are realized through the novel instrument architecture disclosed in this invention, and these can be mixed and matched to design novel DDA analysis workflows to optimize the analysis of different classes of analytes.

[0271] More specifically, the analysis of complex glycan or glycopeptide samples requires an MS2 collision-induced dissociation (CID) fragmentation stage, followed by an MS3 electron-induced dissociation (EID) stage applied to selected MS2 CID fragments to fully characterize the molecular structure, glycan topology, and / or amino acid sequence. This can be achieved using a scheduled DDA method, where the MS2 scan is configured with a first quadrupole mass filter to perform mass selection of precursor or analyte ions, and a segmented linear ion trap or collision cell is configured to perform the subsequent MS2 CID step. The subsequent MS3 scan is configured with an activation-dissociation region coupled to a trapping device that generates the MS2 CID fragments, which are then mass-selected using the first quadrupole mass filter, and the MS3 scan is configured with a segmented linear ion trap to perform the MS3 EID fragmentation stage. The MS3 fragmentation stage is achieved by real-time reading of the MS2 spectrum characteristics and defining instrument parameters, such as the position of the first quadrupole mass filter and / or the reaction time of the EID step applied in the subsequent MS3 scan. Typically, a first-level full scan precedes the MS2 and MS3 scans and their corresponding sub-cycles, forming a DDA cycle. The DDA method involves sequentially applying such cycles to process and characterize all or most analytes present in complex samples.

[0272] Reference Figure 5 A first exemplary description of different scans comprising a single cycle in the DDA MS3 method disclosed above is provided. In the MS-first-level full scan 500, a continuous ion beam 507 is received in a capture region 501 operating in pass mode 508, eliminating space charge capacity limitations and associated mass discrimination effects. All precursor or analyte ions 509 are transferred through a first quadrupole mass filter 502 without mass selection. Similarly, all ions 510 are transferred toward a mass analyzer, preferably a time-of-flight analyzer 506, through a piecewise linear ion trap 503, a second quadrupole mass filter 504, and a collision cell 505, producing a panoramic view of the mass spectrum with representative ion abundances of different analytes present in the complex sample and the corresponding incident ion beam 507. Preferably, ions undergoing soft collisions in the collision cell 505 are gated into the TOF by switching the voltage applied to the exit lens electrode of the collision cell between storage and ejection levels. The switching of the lens electrode is synchronized with the application of TOF extraction pulses to the sample ions over a wide m / z range. Most preferably, the AC signal is further applied to the exit lens electrode of the collision cell, and the ions ejected from the collision cell arrive at the extraction region of the TOF mass analyzer simultaneously and independently of their m / z ratios, thereby maximizing the m / z range recorded in the mass spectrum. Single or multiple TOF spectra are generated during the MS-first full scan, which is typically faster than subsequent MSn scans.

[0273] The cumulative period in subsequent MS2 CID scans or continuous MS2 CID scans 520 is determined by the total ion intensity or total ion current measured in the MS-first full scan 500 to optimally fill the capture device 521, now operating in capture mode, receiving analyte ions 528 from the ionization source. According to this preferred DDA MS3 method, the first quadrupole mass filter 522 is configured to select a first analyte based on the relative ion intensity of all analytes identified in the MS-first full scan 500. In a non-targeted analysis workflow, the first analyte to be selected is typically the most abundant substance, but other selection criteria known in the art can be applied; for example, selection rules can be applied based on the analyte's charge state and / or a specified mass range of interest. The mass-selected analyte ions 530 are transferred through a piecewise linear quadrupole ion trap 523 and a second quadrupole mass filter 524, and undergo CID in the collision cell 525. MS2 CID fragment ions 531 are then gated or ejected toward the mass analyzer 526, producing an MS2 CID spectrum with fragment ion information corresponding to the specific analyte.

[0274] Real-time or offline data processing of MS2 CID data allows subsequent MS3 scans to be scheduled either within the same LC / CE run or in a second LC / CE run. Most preferably, rapid processing of MS2 CID mass spectrometry allows for the definition of an MS3 scan 540 performed in the same run, whereby the incident ion beam 547 is fragmented via collisions in one of the two activation-dissociation regions 548, 549 of the trapping device 541. Similar to MS2 CID scan 520, the amount of ions 550 stored in the trapping device 541 is determined by the total ion intensity or total ion current measured in the MS-first full scan 500. The first quadrupole mass filter 542 is now scheduled to select MS2 fragment ions 551 based on a different set of selection criteria compared to those applied in the MS2 CID scan. For example, when analyzing glycans or glycopeptides, quadrupole mass selection can be based on a predefined list of m / z values ​​corresponding to trisaccharides or pentasaccharides. Other selection rules can be applied to drive the first quadrupole mass filter 542 in MS3 scans known in the art. The mass-selected MS2 CID fragment ions 553 are then stored in a segmented linear quadrupole ion trap 543 and subjected to MS3 EID by injecting a 35 eV electron beam from an external electron source 553 into the linear ion trap. All MS3 EID fragment ions are then transferred 554 through a second quadrupole mass filter 544 to a collision cell 545 and aggregated 555 before being ejected toward a mass analyzer 546.

[0275] Reference Figure 5The description relates to a set of three separate scans that may comprise a single cycle in a DDA, MS2, CID, MS3, and EID workflow. Subcycles or each scan within a cycle can be performed multiple times to optimize the quality of the resulting mass spectra, while multiple subcycles or cycles are applied sequentially to analyze different analytes delivered by a front-end separation method and / or ionization source. Preferably, multiple subcycles can be performed, where each subcycle includes an MS2 step and an MS3 step to process multiple analytes identified in the leading MS-first full scan. Variations of this workflow are envisioned depending on the category of analytes, such as performing ECD or UVPD instead of EID during the MS3 fragmentation stage. In another variation of the method, operating the capture device in capture mode during the MS-first full scan may be desirable to enhance the measurement sensitivity for low-concentration samples. Most preferably, when switching between the MS-first full scan and the MSn scan, a continuous ion flow into the capture region remains uninterrupted, where the duty cycle of the capture device is close to 100%.

[0276] Also refer to Figure 5 A second exemplary description of different scans comprising a single cycle is provided in the DDA MS3 method disclosed above. In this variation of the workflow, the MS2 scan is configured to accumulate mass-selected ions 529 using a quadrupole mass filter 522 in a segmented linear quadrupole ion trap 523, while the trapping device 521 operates in a straight-through mode or ion transport mode, i.e., ion accumulation is not performed in this portion of the device. Ions accumulated in the segmented linear quadrupole ion trap 523 during the MS2 scan 520 can undergo electron-electron reactions and further generate ECD or EID fragments, which are subsequently transferred to the TOF mass analyzer 526 via a second quadrupole mass filter 524 and a collision cell 525. The accumulation of ions in the segmented linear quadrupole ion trap 523 can be performed in single or multiple consecutive segments and in different portions of the device under the influence of a collision cooling gas. The cooling gas can be continuously introduced or injected using a pulsed valve operating at a high repetition rate to establish a quasi-steady-state pressure level throughout the accumulation period. Figure 5The subsequent MS3 scan 540 presented in the preferred workflow can be omitted, thus limiting the method to only MS1 scan 500 and MS2 scan 520, or it can be applied in a modified format to extend the analytical information already provided by the previous MS2 scan discussed in this preferred workflow. Specifically, MS3 scan 540 may involve accumulating mass-selected ions 551 into a piecewise linear quadrupole ion trap 543 using a first quadrupole mass filter 542, similar to MS2 scan 520 in this preferred operating mode, wherein mass-selected ions 529, 551 are accumulated in piecewise linear quadrupole ion traps 523, 543. In MS3 scan 540, for example, accumulated ions 553 that have undergone reactions with electrons to generate charge-reduced analyte ions and / or ExD fragments can be further accelerated toward the collision cell 545 with sufficient kinetic energy to generate additional CID fragments 555. All product ions generated via electron reactions with the piecewise linear quadrupole ion trap 543 and the collision cell 545 are then gated into the TOF mass analyzer 546. Preferably, a second mass selection step in the MS3 scan 540 is applied using a second quadrupole mass filter 544 to mass select for charge-reduced analyte ions and / or fragments generated via electron reactions with the piecewise linear quadrupole ion trap 543.

[0277] In another MSn workflow supported by a new design architecture, the analysis of complex samples containing antibodies or Fab units requires multiple first-level full scans to improve statistical data, followed by multiple MS2 CID scans or MS2 ECD scans to generate complete light chains or N-terminal b-type ions from both the light and heavy chains, all containing hypervariable domains, also known as complementarity-determining regions (CDR1, CDR2, and CDR3) responsible for antigen binding. De novo sequencing of these regions is crucial for the development of new therapies. In the case of a DDA method configured with MS2 CID scans, the subsequent MS3 scans consist of MS2 CIDs coupled to either of the two collision-activated regions of the trapping device, followed by mass selection using a first quadrupole mass filter, and finally an MS3 fragmentation phase performed in a piecewise linear quadrupole ion trap, which may involve reactions with electrons, such as ECD, EID, or interactions with photons, such as UVPD, or other types of dissociation known in the art. The MS2 ECD fragmentation stage of the MS2 scan and the MS3 ECD / EID fragmentation stage of the MS3 scan may also include a collision activation step, which can be achieved by resonant excitation or beam-type CID performed respectively in a piecewise linear quadrupole ion trap or in a collision cell. Such an MS3 collision activation scan can be configured as an MS4 scan by adding a mass selection step, for example by using the isolation capability of a piecewise linear quadrupole ion trap or an optimal second quadrupole mass filter.

[0278] The new design architecture supports more advanced workflows, where light chains generated in the MS2 CID scan are further processed by MS3 ECD and MS4 CID, while b-type heavy chain fragments generated in the subsequent MS2 CID scan, starting with the same precursor / analyte ions, are further processed by MS3 UVPD. Additionally, ion accumulation can be performed in a piecewise linear quadrupole ion trap prior to the MS3 / MS4 scan to enhance the signal-to-noise ratio in the resulting mass spectra. In all these variations, the trapping device can operate in trap / accumulation and / or straight-through modes, as described above. Figure 5 The two exemplary descriptions disclosed in the discussion.

[0279] according to Figure 6 The preferred geometry and operation of the ion analysis apparatus 600 of the present invention are shown. An ion beam 601 is received by a trapping device 602 configured to operate as a trapped ion mobility spectrometer. The trapping device 602 has an accumulation region 610 for storing a large number of ions and a mobility separation region, in which accumulated ions are separated according to differences in their ion mobilities 611. In this way, accumulated ions can be transferred from the accumulation region 610 to the ion mobility region 611 and separated therein, while other incident ions can accumulate simultaneously in the accumulation region 610. The incident ion beam 601 can be modulated by a switching DC signal applied to an electrode 612, which deflects ions along 209 into the trapped ion mobility spectrometer 602 or along 208 away. The number of ions accumulated in the trapped ion mobility spectrometer is determined by the timing characteristics of the switching DC signal. The mass spectrometer 600 is generally as described with respect to mass spectrometers 100 and / or 200; detailed descriptions thereof are omitted for brevity.

[0280] Most preferably, the processing cycle in the MS-first full scan and MSn scan is equal to the cumulative cycle that results in a 100% duty cycle in the capture device. Under these conditions, the DC potential applied to electrode 612 is preferably static, maintaining a continuous ion flow from the ionization source into the capture device, which acts as an aggregator, converting the continuous ion flow into packets delivered to downstream ion optics for processing, mass analysis, and detection.

[0281] A first ion activation region 613 located upstream of the captured ion mobility spectrometer 602 and / or a second ion activation region 614 located downstream can be used to generate MS2 fragments from at least a subset of ions generated in the ionization source. Analyte and / or fragment ions separated in the captured ion mobility spectrometer 602 can be selected based on their ion mobilities by switching the DC signal applied to the electrode gate 615 between the transport and stop potentials. Similarly, an ion migration gate using a switching DC signal for mobility-based ion selection can be integrated into the captured ion mobility spectrometer 602. The mobility-selected ions can undergo mass selection 616 in a first quadrupole mass filter 603 and further processing in a piecewise linear quadrupole ion trap 604, for example, by injecting electrons from an external source 617 to generate fragments 617. Additional mass selection steps can be performed on fragment ions 619 using a second quadrupole mass filter 605, while collisional activation and aggregation of product ions 620 can be achieved in a collision cell 606, with mass analysis preferably performed using a time-of-flight mass analyzer.

[0282] When ion 618 is processed by ExD or UVPD in one of the segments of the segmented linear quadrupole ion trap 604, subsequent portions of ions 621 from the same or subsequent packets ejected from the captured ion mobility spectrometer can be stored in the upstream segment, thereby increasing the duty cycle of the analysis.

[0283] All publicly disclosed DDA methods and scan loop combinations to date, such as those mentioned above. Figure 3 , Figure 4 and Figure 5 The operating mode discussed is directly applicable to the design of a capture device with two capture regions or the capture ion mobility spectrometer 602, including the ability to select ions based on ion mobility, similar to utilizing... Figure 6 The newly disclosed instrument architecture provides a description of mass-selected ions.

[0284] Table 6 summarizes example scan modes for the ion analysis apparatus 600. Preferred scan modes are shown in bold. Accumulation or IMS is preferred. The transfer indicator ions are transferred by this ion optics without additional processing. For a linear quadrupole ion trap, the activation and / or dissociation of MSn+1 ions from MSn (e.g., n=1, n=2, or n=3) product ions, the generation of MSn+1 product ions from MSn product ions, the mass selection of MSn+1 product ions, and the activation and / or dissociation of MSn+2 ions from the mass-selected MSn+1 product ions require two steps of ion activation-dissociation, and may require an accumulation step in the linear quadrupole ion trap. Accumulation and IMS can be combined, IMS can be used alone, or accumulation can be used alone.

[0285] Reference Figure 7 An exemplary description is provided of different scans that include a single loop in yet another DDA MS2 method.

[0286] In MS-Level 1 Full Scan 700 (see also...) Figure 9 Example 1), receiving a continuous ion beam 707 in a capture device 701 operating in mode 708 eliminates space charge capacity limitations and associated mass discrimination effects. All precursor or analyte ions 709 are transferred through a first quadrupole mass filter 702 without mass selection. It is desirable to modulate the RF waveform amplitude to control the low-mass cutoff of the device and transfer only higher-quality analyte ions. Similarly, all ions or higher m / z fragments including the charge state envelope of analyte ion 710 are transferred toward a mass analyzer, preferably a time-of-flight analyzer 706, through a piecewise linear ion trap 703, a second quadrupole mass filter 704, and a collision cell 705, producing a panoramic view of the mass spectrum with representative ion abundances of different analytes present in complex samples and corresponding incident ion beams 707. Single or multiple TOF spectra are generated during the MS-first full scan, which is typically faster than the subsequent MSn scan, and the exit lens of the collision cell can be modulated between the capture potential and the ejection potential at a repetition rate equal to the repetition rate of the TOF extraction pulse to improve the duty cycle of the method.

[0287] In another MS2 workflow supported by the new design architecture, the accumulation of mass-selected ions 729, mass-selected using a quadrupole mass filter 722, is performed in a collision cell 725. The mass-selected ions are stored in a collision cell 731, while the accumulation period is controlled by deflecting the incident ion beam 727 at specified time intervals, calculated in real time based on the space charge capacity of downstream ion optics (e.g., collision cell 725) and related to the ion strength or ion current of the mass-selected ions 729 measured in an MS-1 full scan 700. In this exemplary embodiment, controlling the accumulation period allows for optimal filling of the collision cell 725, receiving the mass-selected analyte ions 730 initially entrained in the continuous ion beam 727. The accumulated mass-selected ions 731 are thermalized in the collision cell and can be transferred back to the piecewise linear ion trap 723 for MS2 activation-dissociation or for MSn experiments. Ions can be transferred upstream by switching the direction of the axial DC gradient established on the collision cell. MS2 product ions generated in the piecewise linear ion trap 723 are then transferred back to the collision cell 725 and are eventually sprayed toward the TOF mass analyzer 726 for measuring the mass-to-charge ratio.

[0288] For example, when the space charge capacity of the collision cell is greater than the capacity of the trapping device, it is preferable to accumulate mass-selected ions in the collision cell 725 rather than in the trapping device 721. MS and MS2 mass spectrometers derived from larger ion populations have higher signal-to-noise ratios, thereby improving sensitivity and increasing the number of fragment ion recognitions. The presence of collision gas in the collision cell enables accumulation; the collision cell typically operates under hydrostatic pressure conditions of approximately 0.01 mbar.

[0289] The significant advantage of accumulating ions in the collision cell 805, in contrast to accumulating ions in the trapping device 721, is that in the former case, the accumulated population will consist only of mass-selected ions that can individually occupy the charge capacity of the collision cell, while in the latter case, the capacity of the trapping device will accommodate a wide range of ions generated in the ionization source.

[0290] For example, if the pressure in the segmented linear ion trap 723 is dynamically controlled using one or more gas pulse valves, and when the accumulation period exceeds the residence time of the gas in the trap volume of the segmented linear ion trap, it is preferable to accumulate mass-selected ions in the collision cell 725 rather than in the segmented linear ion trap 723. Extended accumulation periods are more convenient to execute in devices operating under hydrostatic conditions to improve the signal-to-noise ratio and detect low-abundance ions.

[0291] Different types of MS activation-dissociation experiments can be performed in this exemplary workflow. For example, accumulated mass-selected ions 731 can be transferred back to the piecewise linear ion trap 723 732 via a second quadrupole mass filter 724 for electron- or photon-based fragmentation. Alternatively, the mass-selected ions 732 can be ejected from the collision cell 725 back to the piecewise linear ion trap 723 with sufficient kinetic energy to undergo collision-induced dissociation. Furthermore, the mass selection using the second quadrupole mass filter 724 when ions 732 are transferred from the collision cell 725 back to the piecewise linear ion trap 723 can be used in fast MS3 workflows or higher MSn workflows.

[0292] The new design architecture supports additional workflows, thereby enabling ion accumulation in the collision pool. (See reference...) Figure 7 An exemplary description of MS2 scans performed in collision cells 702 and 722 is discussed, while... Figure 8 An exemplary description of an MS3 or MS4 scan is presented, wherein a quadrupole quality filter 802 is used to selectively transmit 809 from upstream 813 of the capture device 801 (see also...). Figure 9 Example 27) or downstream 814 (see also) Figure 9Example 29) Generated MS2 fragments. A continuous mass-selected beam of MS2 CID fragment ions 810 is thus transported to downstream optics within a predetermined period, i.e., the MS2 ion accumulation period, to optimally fill the collision cell 805. The accumulated MS2 CID fragments 811 are then transferred upstream 812 through a second quadrupole mass filter 804 into a piecewise linear ion trap 803 for the MS3 activation-dissociation stage. In effect, the MS2 CID step initially performed in the collision cell 805 at the MS2 stage is reproduced at the MS3 stage upstream of the quadrupole mass filter 802, enabling rapid selection of MS2 product ions at high resolution. Finally, MS3 and / or MS4 product ions are ejected towards a TOF analyzer 806 to produce a product ion mass spectrum.

[0293] Advanced DDA methods will be combined with new design architectures. As an example of top-down or mid-bottom-up LC / MS experiments, the DDA algorithm includes the following steps:

[0294] (a) Identify the different charge state envelopes of co-eluted analyte ions present in MS-first full scan or in mass spectra generated by averaging multiple MS-first full scans;

[0295] (b) Define a first isolation window for the quadrupole mass filter to select at least one charge state from a single charge state envelope of multiple charge state envelopes present in the mass spectrometer and corresponding to different co-eluted analyte ions, preferably a charge state that does not overlap with any other charge state corresponding to different analyte ions;

[0296] (c) subjecting at least one selected charge state to an MS2 activation-dissociation step and performing multiple scans on the same charge state to improve the statistics and signal-to-noise ratio of the MS2 spectrum corresponding to the selected charge state, thereby improving the fidelity of fragment ion isotope distribution and facilitating data analysis and interpretation;

[0297] (d) Define a second isolation window for the quadrupole mass filter based on the information available in step (a) to select at least one additional charge state from multiple charge state envelopes present in the mass spectrometer and corresponding to different co-eluted analyte ions;

[0298] (e) subjecting the charge state selected in step (d) to an MS2 activation-dissociation step, and performing multiple scans on the same charge state to improve the statistics and signal-to-noise ratio of the MS2 spectrum; and

[0299] (f) Before completing the cycle and before initiating the subsequent MS-level full scan as described in step (a), repeat steps (b) and (c) or steps (d) and (e) for additional charge states belonging to different charge state envelopes and / or the same charge state envelope;

[0300] Steps (d) and (e) can be omitted. For example, step (f) can follow steps (b) and (c), where a charge state different from the envelope of the same charge state can be selected and subjected to MS2 ion activation-dissociation.

[0301] The new DDA method, combined with a novel design architecture, differs from the standard TOP N method, where analyte ions are selected based on their intensity, typically starting with the most abundant analyte ion and progressively selecting lower-intensity ions from a list of ion intensities generated based on MS-level full scan information, which is also combined with a precursor or analyte ion exclusion list. In this new operating mode, the process of selecting at least one charge state corresponding to an analyte is based on identifying all charge states belonging to the same charge state envelope and using a quadrupole mass filter to drive the mass-charge selection process in a way that avoids the co-selection of overlapping charge states belonging to different charge state envelopes and corresponding different analytes. For example, if the most abundant charge state from the first charge state envelope overlaps with another charge state from the second charge state envelope, the new DDA algorithm will search and drive a quadrupole mass filter to select the highest and most abundant charge state from the first charge state envelope that does not overlap and can be mass-selected without excluding different analyte ions within the quadrupole isolation window. This method allows for the generation of high-quality top-down data during LC / MS experiments of whole proteins, where all fragment ions belong to a single analyte ion, facilitating data analysis and interpretation.

[0302] The DDA method may also include the following parameterization options:

[0303] Before the charge-state deconvolution algorithm can be applied, the number of MS-level full scans is defined independently and these scans are summed to generate an average mass spectrum.

[0304] The Charge-State Deconvolution (CSD) algorithm is applied to compute the richest charge states corresponding to the richest or all charge states in each charge state envelope and / or all different charge state envelopes, including neutral mass and ionic strength information. The CSD algorithm can also return a predetermined number of charge state envelopes and / or provide information only about a limited number of non-overlapping charge states, which are then analyzed at the MS2 level to improve computational speed and maintain a high measurement duty cycle.

[0305] The number of MS2 scans is defined by: selecting (a) the total duration of the MS2 scans and / or the number of MS2 TOF extraction pulses, for example, adapting to fixed or variable cumulative times and fixed or variable ion activation-dissociation times; (b) the number of analyte or precursor charge states to be subjected to fragmentation, starting with the most abundant analyte or precursor ions that can be effectively separated and do not have overlapping material in the mass spectrometer; and (c) defining an exclusion period for handling less abundant precursor or analyte charge states from previous steps.

[0306] Preferably, MS2 scans of the same precursor or analyte charge state are performed sequentially, rather than alternating between the first and second precursors.

[0307] The new DDA method may also include defining the ExD reaction time, where x = C, I, D (C = capture, I = induction, D = separation), for example based on a calibration table, where the charge state and / or quantity of ions are related to the ion-electron reaction cycle. For example, under ECD conditions, ions in higher charge states require shorter electron reaction times to undergo charge reduction and generate ECD fragments. The ExD reaction time can also be adjusted based on the quantity of ions accumulated in the piecewise linear ion trap. Preferably, lower density ion populations require shorter reaction times. The new DDA method may also include defining the accumulation time based on ion current measurements using MS-level full scan information or an electrometer connected to the electrode along the ion path. Most preferably, the ExD reaction time can be dynamically adjusted based on both the precursor ion charge state and the quantity of charge accumulated in the piecewise linear ion trap.

[0308] The new DDA method may also include the option to define the number of MS-primary full scans and MS2 or MSn scans independently and depending on the analyte ion intensity information. Higher abundance analyte ions may require fewer MS2 scans compared to low abundance substances, where statistical analysis and averaging of multiple scans are crucial for producing meaningful fragmentation spectra. The new DDA method may also include the option to co-separate charge states from envelopes of different charge states that may correspond to the same proteome. For example, two distinct charge state envelopes may be equidistant on the m / z scale, exhibiting a constant difference in neutral mass due to amino acid sequence modifications or adduct ion formation. The DDA method may also include a rapid pre-scan option performed with a short accumulation time to optimally define the accumulation time for subsequent MS-primary full scans.

[0309] Figure 9 A method according to an exemplary implementation is illustrated.

[0310] in short, Figure 9Fifty-nine methods according to exemplary embodiments (including Examples 2 to 60) are schematically depicted, which include accumulating ions in a collision cell (typically, storing-capturing and / or accumulating ions). Example 1 is also a method according to an exemplary embodiment, particularly a first-level full scan. Typically, methods according to the exemplary embodiments in Tables 1 to 5 may also include accumulating ions in a collision cell (typically, storing-capturing and / or accumulating ions). Figure 9 Implementations 1 to Example 60 are shown as flow charts (swimlane diagrams) instead of tables (see Tables 1 to 5) to more clearly represent the upstream and downstream transfer of ions within the experimental cycle. Figure 9 Examples 1 through 60 may include ion accumulation in a linear quadrupole ion trap and / or in a collision cell (typically, storage-capture and / or ion accumulation).

[0311] The novel DDA method disclosed in this paper has been developed as a driving reference. Figure 2 , Figure 3 , Figure 5 , Figure 6 , Figure 7 and Figure 8 Describes different workflows. Similar to a reference. Figure 4 The disclosed method, the novel DDA method described herein, can also be applied to workflows with multiple fragmentation stages. Furthermore, the DDA workflow based on the novel instrument architecture can be configured using standard top N DDA methods known in the art. Additionally, the method DDA can be used to configure the novel instrument architecture to achieve combinations of different MS scans, MS2 scans, MS3 scans, and MS4 scans, which involve accumulating ions using different trapping devices and / or combining these with pass-through mode scans in each stage of an MSn workflow. For example, m / z and / or mobility-selective ions, using a first quadrupole mass filter and / or a trapping ion mobility device, can be accumulated in a piecewise linear ion trap and further activated / dissociated using different fragmentation methods. These m / z and / or mobility-selective and accumulated ions can undergo MS2 CID using one or more consecutive scans, followed by MS2 ECD using one or more consecutive scans, to produce complementary information about the same one or more analytes. Typically, these two different MS2 scans are part of a scan cycle that includes an MS-first full scan. Additional scans may also be included to perform the MS3 activation / dissociation steps.

[0312] Although preferred embodiments have been shown and described, those skilled in the art will understand that various changes and modifications may be made without departing from the scope of the invention as defined by the appended claims and as described above.

[0313] At least some of the exemplary embodiments described herein can be constructed, in part or in whole, using dedicated, specialized hardware. Terms used herein, such as “component,” “module,” or “unit,” may include, but are not limited to, hardware devices such as circuit systems in the form of discrete or integrated components, field-programmable gate arrays (FPGAs), or application-specific integrated circuits (ASICs) that perform certain tasks or provide associated functionality. In some embodiments, the described elements may be configured to reside on tangible, persistent, addressable storage media and may be configured to execute on one or more processors. In some embodiments, these functional elements may include, for example, components such as software components, object-oriented software components, class components and task components, processes, functions, attributes, procedures, subroutines, program code segments, drivers, firmware, microcode, circuit systems, data, databases, data structures, tables, arrays, and variables.

[0314] Although example embodiments have been described with reference to the components, modules, and units discussed herein, such functional elements may be combined into fewer elements or separated into additional elements. Various combinations of optional features have been described herein, and it will be understood that the described features can be combined in any suitable combination. In particular, features of any example embodiment may be suitably combined with features of any other embodiment, unless such combinations are mutually exclusive. Throughout this specification, the terms "comprising" or "including" mean including the specified components, but do not exclude the presence of other components.

[0315] Please note all papers and documents submitted concurrently with or prior to this specification in connection with this application, and these papers and documents are publicly available together with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0316] All features disclosed in this specification (including any appended claims, abstract, and drawings) and / or all steps of any method or process so disclosed may be combined in any combination except where at least some of such features and / or steps are mutually exclusive.

[0317] Unless otherwise expressly stated, each feature disclosed in this specification (including any appended claims, abstract, and drawings) may be replaced by an alternative feature for the same, equivalent, or similar purpose. Therefore, unless otherwise expressly stated, each disclosed feature is merely one example of a general set of equivalent or similar features.

[0318] This invention is not limited to the details of the foregoing embodiments. The invention extends to any novel feature or any novel combination thereof disclosed in this specification (including any appended claims, abstract, and drawings), or to any novel step or any novel combination thereof in any method or process so disclosed.

[0319] scanning ion source Capture device First four-stage quality filter Linear quadrupole ion trap Second-stage quality filter Collision pool Quality Analyzer Level 1 MS Precursor ions Transfer, accumulation and / or IMS transfer transfer transfer transfer Mass spectrometry of all precursor ions MS2 Precursor ions Transfer, accumulation and / or IMS transfer Optional accumulation; MS2 ion activation and / or dissociation of precursor ions to generate MS2 product ions from precursor ions. transfer transfer Mass spectra of all MS2 product ions MS2 Precursor ions Transfer, accumulation and / or IMS transfer Optional accumulation; MS2 ion activation and / or dissociation of precursor ions to generate MS2 product ions from precursor ions. Mass filtration of MS2 product ions transfer Mass spectrometry of MS2 product ions after mass filtration MS2 Precursor ions Transfer, accumulation and / or IMS transfer transfer transfer CID of precursor ions, and the formation of MS2 product ions from precursor ions. Mass spectra of all MS2 product ions MS2 Precursor ions Transfer, accumulation and / or IMS Mass filtering of precursor ions (e.g., identified from a first-stage full scan) transfer transfer CID of mass-filtered precursor ions, and MS2 product ions generated from mass-filtered precursor ions. Mass spectrometry of MS2 product ions obtained from mass-filtered precursor ions. MS2 Precursor ions Transfer, accumulation and / or IMS Mass filtering of precursor ions (e.g., identified from a first-stage full scan) Optional accumulation; MS2 ion activation and / or dissociation of mass-filtered precursor ions to generate MS2 product ions from mass-filtered precursor ions. transfer transfer Mass spectrometry of MS2 product ions obtained from mass-filtered precursor ions. MS2 Precursor ions Transfer, accumulation and / or IMS Mass filtering of precursor ions (e.g., identified from a first-stage full scan) Optional accumulation; MS2 ion activation and / or dissociation of mass-filtered precursor ions to generate MS2 product ions from mass-filtered precursor ions. Mass filtration of MS2 product ions transfer Mass spectrometry of MS2 product ions after mass filtration MS3 Precursor ions Transfer, accumulation and / or IMS transfer Optional accumulation; activation and / or dissociation of precursor ions MS2 ions to generate MS2 product ions from precursor ions. Mass filtration of MS2 product ions CID of mass-filtered MS2 product ions, and the formation of MS3 product ions from mass-filtered MS2 product ions. Mass spectrometry of MS3 product ions MS3 Precursor ions Transfer, accumulation and / or IMS Mass filtering of precursor ions (e.g., identified from a first-stage full scan) Optional accumulation; MS2 ion activation and / or dissociation of mass-filtered precursor ions to generate MS2 product ions from mass-filtered precursor ions. Mass filtration of MS2 product ions CID of mass-filtered MS2 product ions, and the formation of MS3 product ions from mass-filtered MS2 product ions. Mass spectrometry of MS3 product ions

[0320] Table 1: Example scanning modes of exemplary embodiments. Preferred scanning modes are shown in bold. Accumulation or IMS is preferred. Transfer-indicated ions are transferred by this ion optics without additional processing.

[0321] scanning ion source Capture device First four-stage quality filter Linear quadrupole ion trap Second-stage quality filter Collision pool Quality Analyzer Level 1 MS Precursor ions IMS Transfer IMS precursor ions Transfer IMS precursor ions Transfer IMS precursor ions Transfer IMS precursor ions Mass spectrometry of all IMS precursor ions MS2 Precursor ions IMS Transfer IMS precursor ions Optional accumulation; MS2 ion activation and / or dissociation of precursor ions to generate MS2 product ions from IMS precursor ions. Transfer MS2 product ions Transfer MS2 product ions Mass spectra of all MS2 product ions MS2 Precursor ions IMS Transfer IMS precursor ions Optional accumulation; MS2 ion activation and / or dissociation of precursor ions to generate MS2 product ions from precursor ions. Mass filtration of MS2 product ions Transfer of mass-filtered MS2 product ions Mass spectrometry of MS2 product ions after mass filtration MS2 Precursor ions IMS Transfer IMS precursor ions Optional accumulation; transfer of IMS precursor ions Transfer IMS precursor ions CID of precursor ions, and the formation of MS2 product ions from precursor ions. Mass spectra of all MS2 product ions MS2 Precursor ions IMS Mass filtering of IMS precursor ions (e.g., identified from a first-level full scan) Optional accumulation; transfer of mass-filtered IMS precursor ions Transfer of filtered IMS precursor ions CID of mass-filtered IMS precursor ions, and MS2 product ions generated from mass-filtered precursor ions. Mass spectrometry of MS2 product ions obtained from mass-filtered precursor ions. MS2 Precursor ions IMS Mass filtering of IMS precursor ions (e.g., identified from a first-level full scan) Optional accumulation; MS2 ion activation and / or dissociation of mass-filtered IMS precursor ions to generate MS2 product ions from mass-filtered IMS precursor ions. Transfer MS2 product ions Transfer MS2 product ions Mass spectrometry of MS2 product ions obtained from mass-filtered precursor ions. MS2 Precursor ions IMS Mass filtering of IMS precursor ions (e.g., identified from a first-level full scan) Optional accumulation; MS2 ion activation and / or dissociation of mass-filtered IMS precursor ions to generate MS2 product ions from mass-filtered IMS precursor ions. Mass filtration of MS2 product ions Transfer of mass-filtered MS2 product ions Mass spectrometry of MS2 product ions after mass filtration MS3 Precursor ions IMS transfer Optional accumulation; MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from the precursor ions. Mass filtration of MS2 product ions CID of mass-filtered MS2 product ions, and the formation of MS3 product ions from mass-filtered MS2 product ions. Mass spectrometry of MS3 product ions MS3 Precursor ions IMS Mass filtering of IMS precursor ions (e.g., identified from a first-level full scan) Optional accumulation; MS2 ion activation and / or dissociation of mass-filtered IMS precursor ions to generate MS2 product ions from mass-filtered IMS precursor ions. Mass filtration of MS2 product ions CID of mass-filtered MS2 product ions, and the formation of MS3 product ions from mass-filtered MS2 product ions. Mass spectrometry of MS3 product ions

[0322] Table 2: Example scanning modes of exemplary embodiments, detailing ion mobility separation. IMS precursor ions are precursor ions that have undergone ion mobility separation. Ion mobility separation is maintained in each ion optics downstream of the IMS. Preferred scanning modes are shown in bold. Transfer indicates that ions are transferred by this ion optics without additional processing. Accumulation and IMS can be combined, or IMS can be used alone, or accumulation can be used alone.

[0323] scanning ion source Capture device Activation-dissociation zone (before or after the capture device) First four-stage quality filter Linear quadrupole ion trap Second-stage quality filter Collision pool Quality Analyzer Level 1 Precursor ions Transfer, accumulation and / or IMS transfer transfer transfer transfer transfer Mass spectrometry of all precursor ions MS2 Precursor ions Transfer, accumulation and / or IMS transfer Mass filtering of precursor ions (e.g., identified from a first-stage full scan) Optional accumulation; MS2 ion activation and / or dissociation of mass-filtered precursor ions to generate MS2 product ions from mass-filtered precursor ions. transfer transfer Mass spectrometry of MS2 product ions obtained from mass-filtered precursor ions. MS2 Precursor ions Transfer, accumulation and / or IMS transfer Mass filtering of precursor ions (e.g., identified from a first-stage full scan) transfer transfer CID of mass-filtered precursor ions, and MS2 product ions generated from mass-filtered precursor ions. Mass spectrometry of MS2 product ions obtained from mass-filtered precursor ions. MS2 Precursor ions Transfer, accumulation and / or IMS transfer Mass filtering of precursor ions (e.g., identified from a first-stage full scan) Optional accumulation; MS2 ion activation and / or dissociation of mass-filtered precursor ions to generate MS2 product ions from mass-filtered precursor ions. transfer transfer Mass spectrometry of MS2 product ions obtained from mass-filtered precursor ions. MS2 Precursor ions Transfer, accumulation and / or IMS transfer transfer Optional accumulation; MS2 ion activation and / or dissociation of precursor ions to generate MS2 product ions from precursor ions. transfer transfer Mass spectra of all MS2 product ions MS2 Precursor ions Transfer, accumulation and / or IMS transfer transfer Optional accumulation; MS2 ion activation and / or dissociation of precursor ions to generate MS2 product ions from precursor ions. Mass filtration of MS2 product ions transfer Mass spectrometry of MS2 product ions after mass filtration MS2 Precursor ions Transfer, accumulation and / or IMS transfer transfer transfer transfer CID of precursor ions, and the formation of MS2 product ions from precursor ions. Mass spectra of all MS2 product ions MS2 Precursor ions Transfer, accumulation and / or IMS MS2 ion activation and / or dissociation of precursor ions, generating MS2 product ions from precursor ions. transfer transfer transfer transfer Mass spectra of all MS2 product ions MS2 Precursor ions Transfer, accumulation and / or IMS MS2 ion activation and / or dissociation of precursor ions, generating MS2 product ions from precursor ions. Mass filtration of MS2 product ions transfer transfer transfer Mass spectrometry of MS2 product ions after mass filtration MS2 Precursor ions Transfer, accumulation and / or IMS MS2 ion activation and / or dissociation of precursor ions, generating MS2 product ions from precursor ions. transfer transfer Mass filtration of MS2 product ions transfer Mass spectrometry of MS2 product ions after mass filtration MS3 Precursor ions Transfer, accumulation and / or IMS MS2 ion activation and / or dissociation of precursor ions, generating MS2 product ions from precursor ions. Mass filtration of MS2 product ions Optional accumulation; MS3 ion activation and / or dissociation of mass-filtered MS2 product ions to generate MS3 product ions from MS2 product ions. transfer transfer Mass spectra of all MS3 product ions MS3 Precursor ions Transfer, accumulation and / or IMS MS2 ion activation and / or dissociation of precursor ions, generating MS2 product ions from precursor ions. Mass filtration of MS2 product ions Optional accumulation; MS3 ion activation and / or dissociation of mass-filtered MS2 product ions to generate MS3 product ions from MS2 product ions. Mass filtration of MS3 product ions transfer Mass spectrometry of MS3 product ions after mass filtration MS3 Precursor ions Transfer, accumulation and / or IMS MS2 ion activation and / or dissociation of precursor ions, generating MS2 product ions from precursor ions. Mass filtration of MS2 product ions transfer transfer CID of MS2 product ions after mass filtration, and the generation of MS3 product ions from MS2 product ions. Mass spectrometry of MS3 product ions MS4 Precursor ions Transfer, accumulation and / or IMS MS2 ion activation and / or dissociation of precursor ions, generating MS2 product ions from precursor ions. Mass filtration of MS2 product ions Optional accumulation; MS3 ion activation and / or dissociation of mass-filtered MS2 product ions to generate MS3 product ions from MS2 product ions. transfer CID of MS3 product ions, and the formation of MS4 product ions from MS3 product ions. MS4 product ion mass spectrometry MS4 Precursor ions Transfer, accumulation and / or IMS MS2 ion activation and / or dissociation of precursor ions, generating MS2 product ions from precursor ions. Mass filtration of MS2 product ions Optional accumulation; MS3 ion activation and / or dissociation of MS2 product ions to generate MS3 product ions from MS2 product ions. Mass filtration of MS3 product ions CID of MS3 product ions after mass filtration, and the generation of MS4 product ions from MS3 product ions. MS4 product ion mass spectrometry MS4 Precursor ions Transfer, accumulation and / or IMS MS2 ion activation and / or dissociation of precursor ions, generating MS2 product ions from precursor ions. Mass filtration of MS2 product ions Optional accumulation; MS3 ion activation and / or dissociation of MS2 product ions to generate MS3 product ions from MS2 product ions; mass selection of MS3 product ions and MS4 ion activation and / or dissociation of the mass-selected MS3 product ions to generate MS4 product ions from MS3 product ions. transfer transfer MS4 product ion mass spectrometry MS4 Precursor ions Transfer, accumulation and / or IMS MS2 ion activation and / or dissociation of precursor ions, generating MS2 product ions from precursor ions. Mass filtration of MS2 product ions Optional accumulation; MS3 ion activation and / or dissociation of MS2 product ions to generate MS3 product ions from MS2 product ions; mass selection of MS3 product ions and MS4 ion activation and / or dissociation of the mass-selected MS3 product ions to generate MS4 product ions from MS3 product ions. Mass filtration of MS4 product ions transfer Mass spectrometry of MS4 product ions after mass filtration MS5 Precursor ions Transfer, accumulation and / or IMS MS2 ion activation and / or dissociation of precursor ions, generating MS2 product ions from precursor ions. Mass filtration of MS2 product ions Optional accumulation; MS3 ion activation and / or dissociation of MS2 product ions to generate MS3 product ions from MS2 product ions; mass selection of MS3 product ions and MS4 ion activation and / or dissociation of the mass-selected MS3 product ions to generate MS4 product ions from MS3 product ions. transfer CID of MS4 product ions, and the generation of MS5 product ions from MS5 product ions. MS5 product ion mass spectrometry MS5 Precursor ions Transfer, accumulation and / or IMS MS2 ion activation and / or dissociation of precursor ions, generating MS2 product ions from precursor ions. Mass filtration of MS2 product ions Optional accumulation; MS3 ion activation and / or dissociation of MS2 product ions to generate MS3 product ions from MS2 product ions; mass selection of MS3 product ions and MS4 ion activation and / or dissociation of the mass-selected MS3 product ions to generate MS4 product ions from MS3 product ions. Mass filtration of MS4 product ions CID of MS4 product ions, and the generation of MS5 product ions from MS5 product ions. Mass spectrometry of MS5 product ions after mass filtration

[0324] Table 3: Example scanning modes of exemplary embodiments. Preferred scanning modes are shown in bold. Accumulation or IMS is preferred. Transfer indicates that ions are transferred by the ion optics without additional processing. For linear quadrupole ion traps, the activation and / or dissociation of MSn+1 ions of MSn (e.g., n=1, n=2, or n=3) product ions, the generation of MSn+1 product ions from MSn product ions and the mass selection of MSn+1 product ions, and the activation and / or dissociation of MSn+2 ions of the mass-selected MSn+1 product ions require two steps of ion activation-dissociation, and may require an accumulation step in a linear quadrupole ion trap.

[0325] scanning ion source Capture device Activation-dissociation zone (before or after the capture device) First four-stage quality filter Linear quadrupole ion trap Second-stage quality filter Collision pool Quality Analyzer Level 1 Precursor ions IMS Transfer IMS precursor ions Transfer IMS precursor ions Transfer IMS precursor ions Transfer IMS precursor ions Transfer IMS precursor ions Mass spectrometry of all IMS precursor ions MS2 Precursor ions IMS Transfer IMS precursor ions Mass filtering of IMS precursor ions (e.g., identified from a first-level full scan) Optional accumulation; MS2 ion activation and / or dissociation of mass-filtered precursor ions to generate MS2 product ions from mass-filtered IMS precursor ions. Transfer product ions Transfer product ions Mass spectrometry of MS2 product ions obtained from mass-filtered precursor ions. MS2 Precursor ions IMS Transfer IMS precursor ions Mass filtering of IMS precursor ions (e.g., identified from a first-level full scan) Transfer of mass-filtered IMS precursor ions Transfer of mass-filtered IMS precursor ions CID of mass-filtered IMS precursor ions, and the formation of MS2 product ions from mass-filtered IMS precursor ions. Mass spectrometry of MS2 product ions obtained from mass-filtered precursor ions. MS2 Precursor ions IMS Transfer IMS precursor ions Mass filtering of IMS precursor ions (e.g., identified from a first-level full scan) Optional accumulation; MS2 ion activation and / or dissociation of mass-filtered precursor ions to generate MS2 product ions from mass-filtered precursor ions. Transfer MS2 product ions Transfer MS2 product ions Mass spectrometry of MS2 product ions obtained from mass-filtered precursor ions. MS2 Precursor ions IMS Transfer IMS precursor ions Transfer IMS precursor ions Optional accumulation; MS2 ion activation and / or dissociation of precursor ions to generate MS2 product ions from IMS precursor ions. Transfer MS2 product ions Transfer MS2 product ions Mass spectra of all MS2 product ions MS2 Precursor ions IMS Transfer IMS precursor ions Transfer IMS precursor ions Optional accumulation; MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Mass filtration of MS2 product ions Transfer of mass-filtered MS3 product ions Mass spectrometry of MS2 product ions after mass filtration MS2 Precursor ions IMS Transfer IMS precursor ions Transfer IMS precursor ions Transfer IMS precursor ions Transfer IMS precursor ions CID of IMS precursor ions, and the formation of MS2 product ions from precursor ions. Mass spectra of all MS2 product ions MS2 Precursor ions IMS MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Transfer MS2 product ions Transfer MS2 product ions Transfer MS2 product ions Transfer MS2 product ions Mass spectra of all MS2 product ions MS2 Precursor ions Transfer, accumulation or IMS MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Mass filtration of MS2 product ions Transfer of mass-filtered MS2 product ions Transfer of mass-filtered MS2 product ions Transfer of mass-filtered MS2 product ions Mass spectrometry of MS2 product ions after mass filtration MS2 Precursor ions IMS MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Transfer MS2 product ions Transfer MS2 product ions Mass filtration of MS2 product ions Transfer of mass-filtered MS2 product ions Mass spectrometry of MS2 product ions after mass filtration MS3 Precursor ions IMS MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Mass filtration of MS2 product ions Optional accumulation; MS3 ion activation and / or dissociation of mass-filtered MS2 product ions to generate MS3 product ions from MS2 product ions. Transfer MS3 product ions Transfer MS3 product ions Mass spectra of all MS3 product ions MS3 Precursor ions IMS MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Mass filtration of MS2 product ions Optional accumulation; MS3 ion activation and / or dissociation of mass-filtered MS2 product ions to generate MS3 product ions from MS2 product ions. Mass filtration of MS3 product ions Transfer of mass-filtered MS3 product ions Mass spectrometry of MS3 product ions after mass filtration MS3 Precursor ions IMS MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Mass filtration of MS2 product ions Transfer of mass-filtered MS2 product ions Transfer of mass-filtered MS2 product ions CID of MS2 product ions after mass filtration, and the generation of MS3 product ions from MS2 product ions. Mass spectrometry of MS3 product ions MS4 Precursor ions IMS MS2 ion activation and / or dissociation of the precursor ion, generating MS2 product ions from the IMS precursor ion. Mass filtration of MS2 product ions Optional accumulation; MS3 ion activation and / or dissociation of mass-filtered MS2 product ions to generate MS3 product ions from MS2 product ions. Transfer of mass-filtered MS3 product ions CID of MS3 product ions, and the formation of MS4 product ions from MS3 product ions. MS4 product ion mass spectrometry MS4 Precursor ions IMS MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Mass filtration of MS2 product ions Optional accumulation; MS3 ion activation and / or dissociation of MS2 product ions to generate MS3 product ions from MS2 product ions. Mass filtration of MS3 product ions CID of MS3 product ions after mass filtration, and MS4 product ions generated from MS2 product ions. MS4 product ion mass spectrometry MS4 Precursor ions IMS MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Mass filtration of MS2 product ions Optional accumulation; MS3 ion activation and / or dissociation of MS2 product ions to generate MS3 product ions from MS2 product ions; mass selection of MS3 product ions and MS4 ion activation and / or dissociation of the mass-selected MS3 product ions to generate MS4 product ions from the mass-selected MS3 product ions. Transfer MS4 product ions Transfer MS4 product ions MS4 product ion mass spectrometry MS4 Precursor ions IMS MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Mass filtration of MS2 product ions Optional accumulation; MS3 ion activation and / or dissociation of MS2 product ions to generate MS3 product ions from MS2 product ions; mass selection of MS3 product ions and MS4 ion activation and / or dissociation of the mass-selected MS3 product ions to generate MS4 product ions from the mass-selected MS3 product ions. Mass filtration of MS4 product ions Transfer of mass-filtered MS4 product ions Mass spectrometry of MS4 product ions after mass filtration MS5 Precursor ions IMS MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Mass filtration of MS2 product ions Optional accumulation; MS3 ion activation and / or dissociation of MS2 product ions to generate MS3 product ions from MS2 product ions; mass selection of MS3 product ions and MS4 ion activation and / or dissociation of the mass-selected MS3 product ions to generate MS4 product ions from the mass-selected MS3 product ions. Transfer MS4 product ions CID of MS4 product ions, and the generation of MS5 product ions from MS5 product ions. MS5 product ion mass spectrometry MS5 Precursor ions IMS MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Mass filtration of MS2 product ions Optional accumulation; MS3 ion activation and / or dissociation of MS2 product ions to generate MS3 product ions from MS2 product ions; mass selection of MS3 product ions and MS4 ion activation and / or dissociation of the mass-selected MS3 product ions to generate MS4 product ions from the mass-selected MS3 product ions. Mass filtration of MS4 product ions CID of MS4 product ions, and the generation of MS5 product ions from MS5 product ions. Mass spectrometry of MS5 product ions after mass filtration

[0326] Table 4: Example scanning modes of exemplary embodiments, detailing ion mobility separation. Preferred scanning modes are shown in bold. IMS precursor ions are precursor ions separated by ion mobility. Ion mobility separation is maintained in each ion optics downstream of IMS. Transfer-indicating ions are transferred from this ion optics without additional processing. For linear quadrupole ion traps, the activation and / or dissociation of MSn+1 ions from MSn (e.g., n=1, n=2, or n=3) product ions, the generation of MSn+1 product ions from MSn product ions, the mass selection of MSn+1 product ions, and the activation and / or dissociation of MSn+2 ions from the mass-selected MSn+1 product ions require two steps of ion activation-dissociation, and may require a cumulative step in the linear quadrupole ion trap. Accumulation and IMS can be combined, or IMS can be used alone or accumulation can be used alone.

[0327] scanning Ion source 101 Capture device 102 Ion funnel 110 RF Ion Director 112 First four-stage quality filter 103 DC Lens 114 RF Ion Director 115 Linear quadrupole ion trap 104 Second-stage quality filter 105 Collision Pool 106 Quality Analyzer 107 Level 1, for example, 409 Precursor ions Transfer, accumulation and / or IMS transfer transfer transfer transfer transfer transfer transfer transfer Mass spectrometry of all precursor ions MS2, for example, 404 Precursor ions Transfer, accumulation and / or IMS transfer transfer Mass filtering of precursor ions (e.g., precursor ions identified from a first-stage full scan) transfer transfer Optional accumulation; MS2 ion activation and / or dissociation of mass-filtered precursor ions to generate MS2 product ions from mass-filtered precursor ions. transfer transfer Mass spectrometry of MS2 product ions obtained from mass-filtered precursor ions. MS2, e.g., 404 Precursor ions Transfer, accumulation and / or IMS transfer transfer Mass filtering of precursor ions (e.g., precursor ions identified from a first-stage full scan) transfer transfer transfer transfer CID of mass-filtered precursor ions, and MS2 product ions generated from mass-filtered precursor ions. Mass spectrometry of MS2 product ions obtained from mass-filtered precursor ions. MS2 Precursor ions (activation and / or dissociation of in-source and / or post-source ions, or treatment of precursor ions as an alternative to ion funnels to generate MS2 product ions from precursor ions – applicable to all) Transfer, accumulation and / or IMS MS2 ion activation and / or dissociation of precursor ions, generating MS2 product ions from precursor ions. transfer transfer transfer transfer transfer transfer transfer Mass spectra of all MS2 product ions MS2 Precursor ions Transfer, accumulation and / or IMS MS2 ion activation and / or dissociation of precursor ions, generating MS2 product ions from precursor ions. transfer Mass filtration of MS2 product ions transfer transfer transfer transfer transfer Mass spectrometry of MS2 product ions after mass filtration MS2 Precursor ions Transfer, accumulation and / or IMS MS2 ion activation and / or dissociation of precursor ions, generating MS2 product ions from precursor ions. transfer transfer transfer transfer transfer Mass filtration of MS2 product ions transfer Mass spectrometry of MS2 product ions after mass filtration MS2 Precursor ions Transfer, accumulation and / or IMS transfer transfer transfer transfer transfer Optional accumulation; MS2 ion activation and / or dissociation of precursor ions to generate MS2 product ions from precursor ions. transfer transfer Mass spectra of all MS2 product ions MS2 Precursor ions Transfer, accumulation and / or IMS transfer transfer transfer transfer transfer Optional accumulation; MS2 ion activation and / or dissociation of precursor ions to generate MS2 product ions from precursor ions. Mass filtration of MS2 product ions transfer Mass spectrometry of MS2 product ions after mass filtration MS2 Precursor ions Transfer, accumulation and / or IMS transfer transfer transfer transfer transfer transfer transfer CID of precursor ions, and the formation of MS2 product ions from precursor ions. Mass spectra of all MS2 product ions MS3, for example, 404 Precursor ions Transfer, accumulation and / or IMS MS2 ion activation and / or dissociation of precursor ions, generating MS2 product ions from precursor ions. transfer Mass filtration of MS2 product ions transfer transfer Optional accumulation; MS3 ion activation and / or dissociation of mass-filtered MS2 product ions to generate MS3 product ions from MS2 product ions. transfer transfer Mass spectra of all MS3 product ions MS3 Precursor ions Transfer, accumulation and / or IMS MS2 ion activation and / or dissociation of precursor ions, generating MS2 product ions from precursor ions. transfer Mass filtration of MS2 product ions transfer transfer Optional accumulation; MS3 ion activation and / or dissociation of mass-filtered MS2 product ions to generate MS3 product ions from MS2 product ions. Mass filtration of MS3 product ions transfer Mass spectrometry of MS3 product ions after mass filtration MS3 Precursor ions Transfer, accumulation and / or IMS MS2 ion activation and / or dissociation of precursor ions, generating MS2 product ions from precursor ions. transfer Mass filtration of MS2 product ions transfer transfer transfer transfer CID of MS2 product ions after mass filtration, and the generation of MS3 product ions from MS2 product ions. Mass spectrometry of MS3 product ions MS3 Precursor ions Transfer, accumulation and / or IMS MS2 ion activation and / or dissociation of precursor ions, generating MS2 product ions from precursor ions. transfer Mass filtration of MS2 product ions transfer transfer Optional accumulation; MS3 ion activation and / or dissociation of mass-filtered MS2 product ions to generate MS3 product ions from MS2 product ions. transfer CID of MS3 product ions, and the formation of MS4 product ions from MS3 product ions. MS4 product ion mass spectrometry MS4, for example, 404 Precursor ions Transfer, accumulation and / or IMS MS2 ion activation and / or dissociation of precursor ions, generating MS2 product ions from precursor ions. transfer Mass filtration of MS2 product ions transfer transfer Optional accumulation; MS3 ion activation and / or dissociation of MS2 product ions to generate MS3 product ions from MS2 product ions. Mass filtration of MS3 product ions CID of MS3 product ions after mass filtration, and the generation of MS4 product ions from MS3 product ions. MS4 product ion mass spectrometry MS4 Precursor ions Transfer, accumulation and / or IMS MS2 ion activation and / or dissociation of precursor ions, generating MS2 product ions from precursor ions. transfer Mass filtration of MS2 product ions transfer transfer Optional accumulation; MS3 ion activation and / or dissociation of MS2 product ions to generate MS3 product ions from MS2 product ions; mass selection of MS3 product ions, and MS4 ion activation and / or dissociation of the mass-selected MS3 product ions to generate MS4 product ions from the mass-selected MS3 product ions. transfer transfer MS4 product ion mass spectrometry MS4 Precursor ions Transfer, accumulation and / or IMS MS2 ion activation and / or dissociation of precursor ions, generating MS2 product ions from precursor ions. transfer Mass filtration of MS2 product ions transfer transfer Optional accumulation; MS3 ion activation and / or dissociation of MS2 product ions to generate MS3 product ions from MS2 product ions; mass selection of MS3 product ions, and MS4 ion activation and / or dissociation of the mass-selected MS3 product ions to generate MS4 product ions from the mass-selected MS3 product ions. Mass filtration of MS4 product ions transfer Mass spectrometry of MS4 product ions after mass filtration MS5 Precursor ions Transfer, accumulation and / or IMS MS2 ion activation and / or dissociation of precursor ions, generating MS2 product ions from precursor ions. transfer Mass filtration of MS2 product ions transfer transfer Optional accumulation; MS3 ion activation and / or dissociation of MS2 product ions to generate MS3 product ions from MS2 product ions; mass selection of MS3 product ions, and MS4 ion activation and / or dissociation of the mass-selected MS3 product ions to generate MS4 product ions from the mass-selected MS3 product ions. transfer CID of MS4 product ions, and the generation of MS5 product ions from MS5 product ions. MS5 product ion mass spectrometry MS5 Precursor ions Transfer, accumulation and / or IMS MS2 ion activation and / or dissociation of precursor ions, generating MS2 product ions from precursor ions. transfer Mass filtration of MS2 product ions transfer transfer Optional accumulation; MS3 ion activation and / or dissociation of MS2 product ions to generate MS3 product ions from MS2 product ions; mass selection of MS3 product ions, and MS4 ion activation and / or dissociation of the mass-selected MS3 product ions to generate MS4 product ions from the mass-selected MS3 product ions. Mass filtration of MS4 product ions CID of mass-filtered MS4 product ions, and the generation of MS5 product ions from mass-filtered MS4 product ions. MS5 product ion mass spectrometry

[0328] Table 5: Example scanning modes of exemplary embodiments. Preferred scanning modes are shown in bold. Accumulation or IMS is preferred. Transfer-indicating ions are transferred by this ion optics without additional processing. For linear quadrupole ion traps, the activation and / or dissociation of MSn+1 ions of MSn (e.g., n=1, n=2, or n=3) product ions, the generation of MSn+1 product ions from MSn product ions and the mass selection of MSn+1 product ions, and the activation and / or dissociation of MSn+2 ions of the mass-selected MSn+1 product ions require two steps of ion activation-dissociation, and may require an accumulation step in a linear quadrupole ion trap. See also Table 3.

[0329] scanning Ion source 101 Capture device 102 Ion funnel 110 RF Ion Director 112 First four-stage quality filter 103 DC Lens 114 RF Ion Director 115 Linear quadrupole ion trap 104 Second-stage quality filter 105 Collision Pool 106 Quality Analyzer 107 Level 1, for example, 409 Precursor ions IMS Transfer IMS precursor ions Transfer IMS precursor ions Transfer IMS precursor ions Transfer IMS precursor ions Transfer IMS precursor ions Transfer IMS precursor ions Transfer IMS precursor ions Transfer IMS precursor ions Mass spectrometry of all IMS precursor ions MS2, e.g., 404 Precursor ions IMS Transfer IMS precursor ions Transfer IMS precursor ions Mass filtering of IMS precursor ions (e.g., IMS precursor ions identified from a first-level full scan) Transfer of mass-filtered IMS precursor ions Transfer of mass-filtered IMS precursor ions Optional accumulation; MS2 ion activation and / or dissociation of mass-filtered IMS precursor ions to generate MS2 product ions from mass-filtered IMS precursor ions. Transfer MS2 product ions Transfer MS2 product ions Mass spectrometry of MS2 product ions obtained from mass-filtered precursor ions. MS2, e.g., 404 Precursor ions IMS Transfer IMS precursor ions Transfer IMS precursor ions Mass filtering of IMS precursor ions (e.g., IMS precursor ions identified from a first-level full scan) Transfer of mass-filtered IMS precursor ions Transfer of mass-filtered IMS precursor ions Transfer of mass-filtered IMS precursor ions Transfer of mass-filtered IMS precursor ions CID of mass-filtered IMS precursor ions, and the formation of MS2 product ions from mass-filtered IMS precursor ions. Mass spectrometry of MS2 product ions obtained from mass-filtered precursor ions. MS2 Precursor ions (activation and / or dissociation of in-source and / or post-source ions, or treatment of precursor ions as an alternative to ion funnels to generate MS2 product ions from precursor ions – applicable to all) IMS MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Transfer MS2 product ions Transfer MS2 product ions Transfer MS2 product ions Transfer MS2 product ions Transfer MS2 product ions Transfer MS2 product ions Transfer MS2 product ions Mass spectra of all MS2 product ions MS2 Precursor ions IMS MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Transfer MS2 product ions Mass filtration of MS2 product ions Transfer MS2 product ions Transfer MS2 product ions Transfer MS2 product ions Transfer MS2 product ions Transfer MS2 product ions Mass spectrometry of MS2 product ions after mass filtration MS2 Precursor ions IMS MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Transfer MS2 product ions Transfer MS2 product ions Transfer MS2 product ions Transfer MS2 product ions Transfer MS2 product ions Mass filtration of MS2 product ions Transfer of mass-filtered MS2 product ions Mass spectrometry of MS2 product ions after mass filtration MS2 Precursor ions IMS Transfer IMS precursor ions Transfer IMS precursor ions Transfer IMS precursor ions Transfer IMS precursor ions Transfer IMS precursor ions Optional accumulation; MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Transfer MS2 product ions Transfer MS2 product ions Mass spectra of all MS2 product ions MS2 Precursor ions IMS Transfer IMS precursor ions Transfer IMS precursor ions Transfer IMS precursor ions Transfer IMS precursor ions Transfer IMS precursor ions Optional accumulation; MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Mass filtration of MS2 product ions Transfer of mass-filtered MS2 product ions Mass spectrometry of MS2 product ions after mass filtration MS2 Precursor ions IMS Transfer IMS precursor ions Transfer IMS precursor ions Transfer IMS precursor ions Transfer IMS precursor ions Transfer IMS precursor ions Transfer IMS precursor ions Transfer IMS precursor ions CID of IMS precursor ions, and the formation of MS2 product ions from IMS precursor ions. Mass spectra of all MS2 product ions MS3, for example, 404 Precursor ions IMS MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Transfer MS2 product ions Mass filtration of MS2 product ions Transfer of mass-filtered MS2 product ions Transfer of mass-filtered MS2 product ions Optional accumulation; MS3 ion activation and / or dissociation of mass-filtered MS2 product ions to generate MS3 product ions from MS2 product ions. Transfer of mass-filtered MS3 product ions Transfer of mass-filtered MS3 product ions Mass spectra of all MS3 product ions MS3 Precursor ions IMS MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Transfer MS2 product ions Mass filtration of MS2 product ions Transfer of mass-filtered MS2 product ions Transfer of mass-filtered MS2 product ions MS3 product ions are activated and / or dissociated from the MS2 product ions after mass filtration, generating MS3 product ions from the MS2 product ions. Mass filtration of MS3 product ions Transfer of mass-filtered MS3 product ions Mass spectrometry of MS3 product ions after mass filtration MS3 Precursor ions IMS MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Transfer MS2 product ions Mass filtration of MS2 product ions Transfer of mass-filtered MS2 product ions Transfer of mass-filtered MS2 product ions Transfer of mass-filtered MS2 product ions Transfer of mass-filtered MS2 product ions CID of MS2 product ions after mass filtration, and the generation of MS3 product ions from MS2 product ions. Mass spectrometry of MS3 product ions MS3 Precursor ions IMS MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Transfer MS2 product ions Mass filtration of MS2 product ions Transfer of mass-filtered MS2 product ions Transfer of mass-filtered MS2 product ions Optional accumulation; MS3 ion activation and / or dissociation of mass-filtered MS2 product ions to generate MS3 product ions from MS2 product ions. Transfer of mass-filtered MS3 product ions CID of MS3 product ions, and the formation of MS4 product ions from MS3 product ions. MS4 product ion mass spectrometry MS4, for example, 404 Precursor ions IMS MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Transfer MS2 product ions Mass filtration of MS2 product ions Transfer of mass-filtered MS2 product ions Transfer of mass-filtered MS2 product ions Optional accumulation; MS3 ion activation and / or dissociation of MS2 product ions to generate MS3 product ions from MS2 product ions. Mass filtration of MS3 product ions CID of MS3 product ions after mass filtration, and the generation of MS4 product ions from MS3 product ions. MS4 product ion mass spectrometry MS4 Precursor ions IMS MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Transfer MS2 product ions Mass filtration of MS2 product ions Transfer of mass-filtered MS2 product ions Transfer of mass-filtered MS2 product ions Optional accumulation; MS3 ion activation and / or dissociation of MS2 product ions to generate MS3 product ions from MS2 product ions; mass selection of MS3 product ions, and MS4 ion activation and / or dissociation of the mass-selected MS3 product ions to generate MS4 product ions from the mass-selected MS3 product ions. Transfer MS4 product ions Transfer MS4 product ions MS4 product ion mass spectrometry MS4 Precursor ions IMS MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Transfer MS2 product ions Mass filtration of MS2 product ions Transfer of mass-filtered MS2 product ions Transfer of mass-filtered MS2 product ions Optional accumulation; MS3 ion activation and / or dissociation of MS2 product ions to generate MS3 product ions from MS2 product ions; mass selection of MS3 product ions, and MS4 ion activation and / or dissociation of the mass-selected MS3 product ions to generate MS4 product ions from the mass-selected MS3 product ions. Mass filtration of MS4 product ions Transfer of mass-filtered MS4 product ions Mass spectrometry of MS4 product ions after mass filtration MS5 Precursor ions IMS MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Transfer MS2 product ions Mass filtration of MS2 product ions Transfer of mass-filtered MS2 product ions Transfer of mass-filtered MS2 product ions Optional accumulation; MS3 ion activation and / or dissociation of MS2 product ions to generate MS3 product ions from MS2 product ions; mass selection of MS3 product ions, and MS4 ion activation and / or dissociation of the mass-selected MS3 product ions to generate MS4 product ions from the mass-selected MS3 product ions. Transfer MS4 product ions CID of MS4 product ions, and the generation of MS5 product ions from MS4 product ions. MS5 product ion mass spectrometry MS5 Precursor ions IMS MS2 ion activation and / or dissociation of IMS precursor ions to generate MS2 product ions from IMS precursor ions. Transfer MS2 product ions Mass filtration of MS2 product ions Transfer of mass-filtered MS2 product ions Transfer of mass-filtered MS2 product ions Optional accumulation; MS3 ion activation and / or dissociation of MS2 product ions to generate MS3 product ions from MS2 product ions; mass selection of MS3 product ions, and MS4 ion activation and / or dissociation of the mass-selected MS3 product ions to generate MS4 product ions from the mass-selected MS3 product ions. Mass filtration of MS4 product ions CID of mass-filtered MS4 product ions, and the generation of MS5 product ions from mass-filtered MS4 product ions. MS5 product ion mass spectrometry

[0330] Table 6: Example scanning modes of exemplary embodiments, detailing ion mobility separation. Preferred scanning modes are shown in bold. IMS precursor ions are precursor ions separated by ion mobility. Ion mobility separation is maintained in each ion optics downstream of the IMS. Transfer-indicating ions are transferred from this ion optics without additional processing. For linear quadrupole ion traps, the activation and / or dissociation of MSn+1 ions from MSn (e.g., n=1, n=2, or n=3) product ions, the generation of MSn+1 product ions from MSn product ions and the mass selection of MSn+1 product ions, and the activation and / or dissociation of MSn+2 ions from the mass-selected MSn+1 product ions require two steps of ion activation-dissociation and may require a cumulative step in the linear quadrupole ion trap. Accumulation and IMS can be combined, or IMS can be used alone or accumulation can be used alone. See also Table 4.

Claims

1. An ion analysis device, comprising: The ionization source is configured to generate an ion beam; A capture device is configured to receive the ion beam and transfer the ions axially upstream and downstream; A first quadrupole mass filter is configured to receive ions transferred from the capture device and to transfer at least a first subset of the received ions; A piecewise linear quadrupole ion trap is configured to receive at least a first subset of the ions transferred from the first quadrupole mass filter, perform a first processing step on the received at least a first subset of the ions, and transfer the processed ions. A second quadrupole mass filter is configured to receive the processed ions transferred from the segmented linear quadrupole ion trap, and to transfer at least a second subset of the processed ions; The collision cell is configured to receive at least a second subset of the ions transferred from the second quadrupole mass filter, perform a second processing step on the received at least a second subset of the ions, and transfer the processed ions. A mass analyzer is configured to receive the processed ions from the collision cell and perform mass analysis on the received processed ions; as well as The controller is configured to synchronously and / or simultaneously control the processing of the corresponding ions in the capture device, the first quadrupole mass filter, the segmented linear ion trap, the second quadrupole mass filter, and / or the collision cell.

2. The ion analysis apparatus according to claim 1, wherein, The ion analysis device can be configured with a set of operating modes, including MS operating mode, MS2 operating mode, MS3 operating mode and / or MS4 operating mode; wherein the ion analysis device is configured to switch between the set of operating modes of the ion analysis device between scans.

3. The ion analysis apparatus according to claim 2, wherein, The corresponding set of operating modes of the ion analysis device is implemented by the corresponding set of configurations of the ion analysis device; optionally, the ion analysis device is configured to change between the corresponding set of operating modes of the ion analysis device and the corresponding set of configurations of the ion analysis device between scans.

4. The ion analysis apparatus according to any of the preceding claims, wherein, The capture device can be configured to operate in ion transport mode and / or ion accumulation mode.

5. The ion analysis apparatus according to any of the preceding claims, comprising an ion activation-dissociation region adjacent to the capture device and upstream of the first quadrupole mass filter.

6. The ion analysis apparatus according to any of the preceding claims, wherein, The capture device includes a captured ion mobility spectrometer and / or a captured ion mobility spectrometer, the captured ion mobility spectrometer including: an ion storage region for storing and / or accumulating the ion beam; and an ion mobility analyzer region for separating and transferring ions from the ion mobility analyzer region, optionally selectively transferring the separated ions.

7. The ion analysis apparatus according to any of the preceding claims, wherein, The first processing step includes: storing-capturing, accumulating, isolating and / or activating-dissociating at least a first subset of the received ions; preferably storing-capturing, isolating and activating-dissociating at least a first subset of the received ions; more preferably storing-capturing and activating-dissociating at least a first subset of the received ions.

8. The ion analysis apparatus according to any of the preceding claims, wherein, The second processing step includes collision activation, thermalization, storage-capture, accumulation and / or regulation of the transfer of at least a second subset of the received ions.

9. The ion analysis apparatus according to any preceding claim, configured to perform a first scan, the first scan comprising performing one or more first processing steps in the segmented linear quadrupole ion trap and / or performing one or more second processing steps in the collision cell, performing one or more mass selection steps in the first quadrupole mass filter and / or the second quadrupole mass filter, and optionally performing one or more ion mobility selection steps in the trapping device, wherein, The capture device includes a captured ion mobility spectrometer and / or a captured ion mobility spectrometer.

10. The ion analysis apparatus of claim 9, configured to perform a second scan, the second scan comprising performing one or more first processing steps in the segmented linear quadrupole ion trap and / or performing one or more second processing steps in the collision cell, performing one or more mass selection steps in the first quadrupole mass filter and / or the second quadrupole mass filter, and optionally performing one or more ion mobility selection steps in the trapping device, wherein, One or more of the first processing step, the second processing step, the mass selection step, and / or the ion mobility selection step in the first scan and the second scan are different from each other.

11. The ion analysis apparatus according to any of the preceding claims, wherein, The collision cell is configured to transfer the processed ions upstream to the segmented linear quadrupole ion trap via the second quadrupole mass filter during experimental cycles, such as within a scan, and subsequently transfer additional processed ions downstream to the mass analyzer.

12. The ion analysis apparatus according to claim 11, wherein, The segmented linear quadrupole ion trap and / or the collision cell are configured to repeatedly perform the first processing step and the second processing step on the ions transferred between the segmented linear quadrupole ion trap and the collision cell, respectively.

13. The ion analysis apparatus according to any of the preceding claims, wherein, The second quadrupole mass filter is configured to perform mass selection on processed ions received from the segmented linear quadrupole ion trap; and wherein the collision cell is configured to accumulate at least a second subset of ions transferred from the second quadrupole mass filter.

14. The ion analysis apparatus according to any of the preceding claims, wherein, The trapping device can be configured to operate in ion accumulation mode and transfer the accumulated ions as a package of ions axially upward. Wherein, the segmented linear quadrupole ion trap can be configured to perform the first processing step on a first packet of ions transferred from the trapping device, and / or wherein, the collision cell can be configured to perform the second processing step on the first packet of ions transferred from the trapping device; and The capturing device can be configured to accumulate a second packet of ions in parallel with the first processing step and / or the second processing step.

15. An ion analysis method, comprising: Generate an ion beam; The ion beam is received by a capture device and the ions are transferred axially upward and downstream. At least a first subset of the transferred ions are mass filtered by a first quadrupole mass filter. A first processing step is performed on at least a first subset of the mass-filtered ions using a piecewise linear quadrupole ion trap. At least a second subset of the processed ions are mass filtered by a second quadrupole mass filter; A second processing step is performed on at least a second subset of the mass-filtered ions through a collision cell; At least a second subset of the processed ions are subjected to mass analysis using a mass analyzer; as well as The processing of corresponding ions in the capture device, the first quadrupole mass filter, the segmented linear ion trap, the second quadrupole mass filter, and / or the collision cell is controlled synchronously and / or simultaneously.

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