Ion accumulation control for analyzer

Abstract

Method for operating an analytical instrument comprising a first ion storage and a second ion storage downstream of the first ion storage, wherein the method comprises: Determine whether a target accumulation time for the second ion storage is greater than a threshold accumulation time; If it is determined that the target accumulation time is less than the threshold accumulation time: accumulation of ions within the second ion storage using an accumulation time based on the target accumulation time; and If it is determined that the target accumulation time is greater than the threshold accumulation time: Accumulate ions within the first ion storage using a first accumulation time based on a difference between the target accumulation time and the threshold accumulation time, transfer the ions accumulated in the first ion storage to the second ion storage, and accumulate further ions within the second ion storage using a second accumulation time based on the threshold accumulation time.

Description

Field of invention

[0001] The present invention relates to the field of mass spectrometry, in particular methods of mass spectrometry in which ions are accumulated in an ion trap, such as Fourier transform (FT) mass spectrometry using electrostatic traps, such as electrostatic orbital traps. General state of the art

[0002] Ion traps are used in many types of mass spectrometers. For example, Thermo Fisher Scientific's Orbitrap™ instruments use a curved linear ion trap (“C-trap”) together with an electrostatic orbital trap to provide high-resolution, accurate mass analysis. The C-trap and the electrostatic orbital trap are positioned downstream of an ion source, with ions passing to the C-trap (and from there to the electrostatic orbital trap) via various ion-optical devices located between the ion source and the C-trap.

[0003] It is often necessary to precisely control the total number of ions accumulated in an ion trap, for example, to optimize the number of ions so that it remains below, but as close as possible to, a limit for the ion trap, such as a space charge limit. The number of ions accumulated in an ion trap is, in turn, controlled by using a gate to manage the accumulation time (e.g., filling time) of ions in the trap. In commercial instruments, a relatively sophisticated and fast ion gate can be provided to enable sufficiently precise control of the accumulation time in the trap.

[0004] Modern mass spectrometers operate at increasingly faster repetition rates, enabling high performance over shorter trials and a greater number of samples to be processed. Typically, the main limitations on the repetition rate are the instrument's sensitivity, as a certain accumulation time is often required to collect enough sample ions for analysis; the time required to process these ions for analysis; the analysis time itself; and / or the time the electronics need to switch between analyte targets.

[0005] It is assumed that there is still room for improvement in facilities and procedures for mass analysis. Brief description

[0006] A first aspect provides a method for operating an analytical instrument comprising a first ion storage and a second ion storage, wherein the second ion storage is downstream of the first ion storage, the method comprising: Determine whether a target accumulation time for the second ion storage is greater than a threshold accumulation time; If it is determined that the target accumulation time is less than the threshold accumulation time: accumulation of ions within the second ion storage using an accumulation time based on the target accumulation time; and If it is determined that the target accumulation time is greater than the threshold accumulation time: Accumulate ions within the first ion storage using a first accumulation time based on a difference between the target accumulation time and the threshold accumulation time, transfer the ions accumulated in the first ion storage to the second ion storage, and accumulate further ions within the second ion storage using a second accumulation time based on the threshold accumulation time.

[0007] Embodiments provide a method for operating an analytical instrument such as a mass spectrometer. The instrument includes a (second) ion reservoir (e.g., an ion trap) which may be located downstream of an ion source. Ions can be passed from the ion source to the second ion reservoir via one or more ion-optical devices (and via a first ion reservoir) arranged between the ion source and the second ion reservoir, and can accumulate within the second ion reservoir, e.g., before being ejected from the second ion reservoir into a mass analyzer for analysis. The instrument may include a relatively fast (and relatively accurate) ion gate configured to control the accumulation time of ions in the second ion reservoir. The instrument may be operated cyclically, e.g.,such that successive batches of ions are each accumulated in the second ion storage and then directed to the mass analyzer and analyzed by it.

[0008] During each instrument cycle, ions can be accumulated in the second ion reservoir according to a target accumulation time for that cycle. The target accumulation time can be determined (e.g., estimated) for each cycle (or for each set of multiple cycles) such that accumulating ions for the duration of the target accumulation time provides (approximately) a desired number of ions to be accumulated within the second ion reservoir for that cycle. The desired number of ions might be, for example, below, but as close as possible to, a limit such as a space charge limit for the second ion reservoir and / or for the mass analyzer.

[0009] During each instrument cycle, the instrument can be operated in a mode in which ions are accumulated in the second ion storage, as well as in a mode in which ions (do not reach the second ion storage and) are not accumulated in the second ion storage.

[0010] For example, the second ion storage unit can operate in both an accumulating and a non-accumulating (i.e., closed) operating mode during each instrument cycle. It may be necessary to operate the second ion storage unit in its non-accumulating mode for a certain minimum period of time during each cycle, e.g., to allow time for processing and / or transferring the accumulated ions to the mass analyzer for analysis, etc.

[0011] Additionally or alternatively, during each instrument cycle, one or more of the one or more ion-optical devices positioned between the ion source and the second ion storage (such as a mass filter) can be operated in both a mode in which ions are passed through to (and accumulate in) the second ion storage and a mode in which ions are not passed through to (and thus do not reach or accumulate in) the second ion storage. For example, during each cycle, a mass filter can be controlled such that its transmission window switches between several different m / z values ​​(a mean mass-to-charge ratio (m / z) of its transmission window).During periods when the transmission window is maintained at a specific m / z value, ions with mass-to-charge ratios corresponding to the m / z of the window are passed through the mass filter. During periods when the transmission window of the mass filter is changing, ions are not passed through the mass filter.

[0012] Thus, during each instrument cycle, the instrument can be operated in a mode in which ions are accumulated in the second ion storage for a certain period of time, which is less than or equal to a maximum accumulation time, where the maximum accumulation time is based on the difference between the total cycle time and the (e.g. necessary) time in which the instrument is operated in a mode (or modes) in which ions do not reach the second ion storage and are not accumulated in the second ion storage.

[0013] In certain experiments, it can be advantageous to operate the instrument at a relatively high repetition rate. This may be the case, for example, when the instrument is coupled to a separation device such as a liquid chromatography unit, particularly when the separation device operates with a relatively short gradient (e.g., of a few minutes or a few tens of minutes). In this case, operating the instrument at a relatively high repetition rate can ensure that relatively rapidly eluting peaks are correctly sampled by the instrument.

[0014] The inventors have now realized, however, that increasing the instrument's repetition rate can significantly reduce its sensitivity. This is because increasing the repetition rate decreases the time available for each cycle. If the instrument must operate in a mode (or modes) where ions are not accumulated in the secondary ion reservoir for at least a certain (e.g., fixed) minimum time during each cycle (as described above), increasing the repetition rate necessitates a reduction in the maximum available accumulation time in each cycle. Reducing the maximum accumulation time can, in turn, decrease the number of ions accumulated in the ion reservoir and thus the instrument's sensitivity.

[0015] In the methods described herein, a threshold accumulation time is defined for the second ion storage. The threshold accumulation time can be based on the maximum accumulation time, i.e., the difference between the total cycle time and the necessary non-accumulation time (e.g., equal to or approximately equal to). Alternatively, the threshold accumulation time could be set to a value smaller than the maximum accumulation time.

[0016] If the target accumulation time for the second ion reservoir is less than (or equal to) the threshold accumulation time, ions are accumulated within the second ion reservoir using the target accumulation time, e.g., in the "normal" way. Thus, the relatively fast (and accurate) gate connected to the second ion reservoir operates in its accumulating (e.g., open) mode for a time based on (e.g., equal to) the target accumulation time (and otherwise operates in its non-accumulating mode (e.g., closed mode)), such that ions are accumulated directly within the second ion reservoir for a time based on (e.g., equal to) the target accumulation time.

[0017] However, if the target accumulation time for the second ion reservoir is greater than the threshold accumulation time, the accumulation of ions within the second ion reservoir utilizes a first ion reservoir located upstream of the second ion reservoir within the instrument (i.e., closer to the ion source). Specifically, ions are initially accumulated within the first ion reservoir using a first accumulation time based on the difference between the target accumulation time and the threshold accumulation time (e.g., equal to or approximately equal to). That is, ions are "pre-accumulated" within the first ion reservoir. The pre-accumulated ions are then transferred from the first ion reservoir to the second ion reservoir. Then, additional ions are accumulated directly within the second ion reservoir (i.e.,In addition to the previously accumulated ions, a second accumulation time is used, based on the threshold accumulation time (e.g., equal to or approximately equal to). In this way, ions are accumulated within the second ion reservoir for a time based on the target accumulation time (e.g., equal to or approximately equal to).

[0018] Advantageously, pre-accumulating ions in this way means that the maximum permissible target accumulation time can be increased to be greater than the threshold accumulation time (i.e., greater than the difference between the total cycle time and the necessary non-accumulation time), and can, for example, approach the total cycle time. This, in turn, means that the instrument's repetition rate can be increased without requiring a significant reduction in sensitivity.

[0019] Furthermore, the methods described herein can be implemented in a manner that requires no physical modifications to existing instruments. In embodiments, the first ion storage is provided in one or more ion-optical devices arranged between the ion source and the second ion storage, and utilizes a relatively slow (and relatively inaccurate) ion gate that is (already) present in the instrument. For example, the first ion storage can be formed within an ion guide (such as a transfer ion guide) of the one or more ion-optical devices, and the relatively slow (and relatively inaccurate) ion gate can be an exit lens for this ion.

[0020] Furthermore, the inventors recognized that under these circumstances, it is advantageous to use pre-accumulation of ions within the first ion storage only when the target accumulation time exceeds the threshold accumulation time. This means that when the target accumulation time is relatively short, only the relatively fast and accurate gate associated with the second ion storage is used to control the overall accumulation time. The relatively slow and inaccurate gate associated with the first ion storage is used only when the target accumulation time is relatively long, in which case the error resulting from the use of a less accurate gate is proportionally smaller. Thus, embodiments provide highly accurate control over the number of ions accumulated within the second ion storage.

[0021] It is therefore understood that embodiments provide improved facilities and procedures for mass analysis.

[0022] The analytical instrument can be a mass spectrometer, which may include, for example, an ion source. Ions can be generated from a sample in the ion source. The ions can be guided from the ion source to the second ion storage via one or more ion-optical devices positioned between the ion source and the second ion storage.

[0023] The one or more ion-optical devices may comprise any suitable arrangement of one or more ion guides, one or more lenses, one or more gates, and the like. The one or more ion-optical devices may include one or more transfer ion guides for passing ions, and / or one or more mass selectors or filters for mass-selecting ions, and / or one or more ion-cooling ion guides for cooling ions, and / or one or more collision or reaction cells for fragmenting or converting ions, and so on. One or more, or each, of the ion guides may comprise a multipole ion guide, such as a quadrupole ion guide, hexapole ion guide, etc., a segmented multipole ion guide, a stacked ring ion guide, and the like.

[0024] The analytical instrument can include one or more mass analyzers, which may be located downstream of the second ion storage unit. Ions accumulated in the second ion storage unit can be directed to the mass analyzer and then analyzed by the mass analyzer, e.g., to determine a mass spectrum of the ions.

[0025] The mass analyzer(s) may include any suitable type(s) of mass analyzer, such as, in particular, an ion trap mass analyzer and / or a time-of-flight mass analyzer.

[0026] Where present, the ion trap mass analyzer can be an electrostatic orbital trap mass analyzer. The mass analyzer may have an inner electrode arranged along an axis and two outer detection electrodes spaced apart along the axis and surrounding the inner electrode. Ions trapped within the mass analyzer can oscillate at a frequency that may depend on their mass-to-charge ratio and can be detected using mirror current detection. The ions can essentially perform harmonic oscillations along the axis in an electrostatic field as they orbit the inner electrode. The mass analyzer may be an Orbitrap™ mass analyzer from Thermo Fisher Scientific. Further details of an Orbitrap™ mass analyzer can be found, for example, in U.S. Patent No. US 5,886,346 A.

[0027] Where applicable, the time-of-flight mass analyzer can be any suitable type of time-of-flight mass analyzer, such as, in particular, a multi-reflection time-of-flight mass analyzer. Ions within the mass analyzer can oscillate between a pair of ion mirrors until they reach a detector. Ions can travel through the mass analyzer with a time of flight determined by the mass-to-charge ratio of the ions. The multi-reflection time-of-flight mass analyzer can optionally be of the inclined-mirror type described in US Patent No. 9,136,101 B2.

[0028] In some embodiments, the instrument includes both an electrostatic ion trap mass analyzer and a time-of-flight mass analyzer, as described, for example, in US Patent No. US 10,699,888 B2.

[0029] The instrument includes a first ion storage and a second ion storage, with the second ion storage being located downstream of the first ion storage (i.e., further away from the ion source than the latter).

[0030] The first ion storage unit is located downstream of the ion source and can be configured to receive ions from the ion source. The first ion storage unit can form part of one or more ion-optical devices arranged between the ion source and the second ion storage unit. The first ion storage unit can be configured within an ion guide of one or more ion-optical devices, such as a transfer ion guide. In certain embodiments, the first ion storage unit is configured within a so-called "bent flat-pole" ion guide of one or more ion-optical devices, which may correspond to the design described in US Patent No. 9,536,722 B2.

[0031] The second ion storage unit is located downstream of the ion source and can be configured to receive ions from the ion source via one or more ion-optical devices (and via the first ion storage unit). The second ion storage unit can be an ion trap. The ion trap can comprise any suitable ion trap, such as a linear ion trap or a curved linear ion trap (C-trap). The ion trap can also be formed from a combination of several ion traps. The ion trap can be used to cool the accumulated ions before they are injected into a mass analyzer. The ion trap can be configured to pulse ions from the ion trap to the mass analyzer.

[0032] The ion trap may have an axis and may be operated to eject ions from the ion trap orthogonal to the axis leading to the mass analyzer. An example of a suitable ion trap for injection into an electrostatic orbital trap mass analyzer is a curved linear trap (C-trap), as described, for example, in WO 2008 / 081334 A2. Additionally or alternatively, the ion trap may be operated to eject ions from the ion trap in a direction parallel to the axis leading to the mass analyzer. In some embodiments, ions may be ejected either to a first (e.g., electrostatic ion trap) mass analyzer or to a second (e.g., time-of-flight) mass analyzer, as described, for example, in US Patent No. US 10,699,888 B2.

[0033] US 2006 / 0284072 A1 relates to a mass spectrometer comprising an ion source, a first linear ion trap for mass-selective ejection, a second linear ion trap for pulsed ejection, and a time-of-flight mass spectrometer (TOF), wherein a controller adjusts the ejection parameters of the second trap based on the mass range of the first trap to achieve high sensitivity and accuracy over a wide mass range.

[0034] US 2009 / 0294661 A1 relates to a mass spectrometer and a method for mass spectrometry with multiple linear ion trap sections in which the directions of resonant excitation of the ions in adjacent traps are different or substantially orthogonal to each other in order to optimize the trap capacity and mass accuracy simultaneously.

[0035] US 2009 / 0302215 A1 relates to a method for operating tandem ion traps to reduce space charge effects, in which ions are transferred between a first and a second ion trap in a time-shifted manner and in specific mass ranges and are mass-selectively ejected.

[0036] US 2011 / 0204221 A1 relates to a mass spectrometer and a method for mass spectrometry which minimizes ion losses and increases throughput in structural analysis by using a pre-ion trap unit between a mass filter and an ion dissociation chamber.

[0037] The first ion storage unit can operate in transmission mode and in accumulation mode. In transmission mode, ions can pass through the first ion storage unit without accumulating within it. In accumulation mode, ions can accumulate within the first ion storage unit without passing through it. The second ion storage unit can operate in accumulation mode and in a closed (non-accumulating) mode. In accumulation mode, ions can accumulate within the second ion storage unit. In closed mode, ions can be prevented from entering the second ion storage unit; that is, they are not accumulated within it.

[0038] The first ion storage device can have at least one first gate configured to control the accumulation time of ions in the first ion storage device. This first gate can be used to control the accumulation time by operating it in an accumulation mode for a desired period of time, while otherwise operating in a transmission mode.

[0039] The at least one first gate can comprise a single gate, but it is possible for the at least one gate to comprise multiple gates (e.g., two). Where multiple gates are present, there can be an inlet gate and an outlet gate. If the at least one first gate comprises a single gate, operating the first ion storage in transmission mode can involve operating the single gate in an open mode, and operating the first ion storage in accumulation mode can involve operating the single gate in a closed mode.

[0040] In certain embodiments, the at least one first gate is an exit lens of a transfer ion guide in which the first ion storage is formed. The first ion storage can be operated in its transmission / accumulation mode by applying suitable different voltages to the exit lens, e.g., whereby in accumulation mode the voltage applied to the exit lens causes ions to be trapped within the ion guide, and in transmission mode the voltage applied to the exit lens does not cause ions to be trapped in the ion guide.

[0041] The second ion storage device can have at least one second gate configured to control the accumulation time of ions in the second ion storage device. This second gate can be used to control the accumulation time by operating it in an accumulation mode for a desired period of time, while otherwise operating in an open mode.

[0042] The at least one second gate can comprise a single gate or multiple gates (e.g., two). Where multiple gates are present, there can be an inlet gate and an outlet gate. If the at least one second gate comprises a single gate, operating the second ion storage in accumulation mode can involve operating the single gate in an open mode, and operating the second ion storage in closed mode can involve operating the single gate in a closed mode. If the at least one second gate comprises multiple gates (e.g., two), operating the second ion storage in accumulation mode can involve operating the inlet gate in an open mode and operating the outlet gate in a closed mode; and operating the second ion storage in closed mode can involve operating the inlet gate in a closed mode.

[0043] In certain embodiments, the at least one second gate is a dedicated ion gate configured to precisely control the accumulation time of ions in the second ion reservoir (while, as described above, the at least one first gate is an ion guide exit lens). Thus, the response time (i.e., the time the ion gate takes to transition from the fully closed to the fully open state (and vice versa)) of the at least one second gate can be faster than the response time of the at least one first gate. For example, the response time of the at least one second gate can be on the order of a few microseconds or a few tens of microseconds, while the response time of the at least one first gate can be on the order of a few hundred microseconds.Thus, the accuracy of at least one second gate can be greater than the accuracy of at least one first gate.

[0044] The instrument can be operated cyclically, for example, by accumulating successive batches of ions in the second ion reservoir and then analyzing them with the mass analyzer. Suitable repetition rates for the instrument can be on the order of a few tens or hundreds of Hz.

[0045] As described above, a threshold accumulation time is defined for the second ion storage, where the threshold accumulation time may be based on the difference between the total cycle time for the instrument and a time per cycle (e.g., equal to, approximately equal to, or less than) in which the instrument is operated in a mode (or modes) in which ions are not accumulated in the second ion storage (elsewhere).

[0046] Modes in which ions are not accumulated (otherwise) in the second ion reservoir can include: (i) the closed (non-accumulating) mode of the second ion reservoir, i.e., when accumulated ions are processed and / or directed to the mass analyzer for analysis, and / or (ii) a non-transmission mode of a mass filter upstream of the second ion reservoir, i.e., when the m / z window of the mass filter is changed. Thus, the time per cycle that the instrument operates in a mode in which ions are not accumulated in the second ion reservoir can include: (i) a (e.g.,(fixed) time per cycle in which the second ion storage unit is operated in a non-accumulating (closed) operating mode while ions accumulated in the second ion storage unit are processed and / or directed to a mass analyzer for analysis, and / or (ii) a time per cycle in which a mass filter is operated in a non-transmission operating mode.

[0047] If it is determined that the target accumulation time is less than the threshold accumulation time, ions are accumulated within the second ion reservoir using an accumulation time based on the target accumulation time. The accumulation time may be equal to the target accumulation time or approximately equal to the target accumulation time (e.g., to account for other instrument delays, switching times, etc.).

[0048] If the target accumulation time is determined to be greater than the threshold accumulation time, ions are accumulated within the first ion reservoir using an initial accumulation time based on the difference between the target accumulation time and the threshold accumulation time. The initial accumulation time may be equal to the difference between the target accumulation time and the threshold accumulation time, or it may be approximately equal to the difference between the target accumulation time and the threshold accumulation time (e.g., to account for other instrument delays, switching times, etc.).

[0049] These accumulated ions are directed to the second ion reservoir, and further ions are then accumulated within the second reservoir using a second accumulation time based on the threshold accumulation time. The second accumulation time can be equal to the threshold accumulation time or approximately equal to it (e.g., to account for other instrument delays, switching times, etc.).

[0050] The second accumulation period can follow the first immediately, or there can be a (short) delay between the first and second accumulation periods, for example, to allow time for switching the electronics and / or directing ions to the second ion storage. The sum of the second and first accumulation periods can be equal to or approximately equal to the target filling time.

[0051] In embodiments, the accumulation of ions within the second ion storage device using an accumulation time based on the target accumulation time comprises operating the first ion storage device in its transmission operating mode during the accumulation time, such that ions pass through the first ion storage device during the accumulation time without being accumulated within the first ion storage device. The accumulation of ions within the second ion storage device using an accumulation time based on the target accumulation time may also comprise operating the second ion storage device in its accumulation mode during the accumulation time, such that ions are accumulated within the second ion storage device during the accumulation time.

[0052] Accumulating ions within the first ion storage using the first accumulation time may involve operating the first ion storage in its accumulation mode during the first accumulation time, such that ions are accumulated within the first ion storage during the first accumulation time.

[0053] Transferring ions accumulated in the first ion storage device to the second ion storage device can involve operating the first ion storage device in its transmission mode, such that ions accumulated in the first ion storage device are transferred to the second ion storage device. Alternatively, transferring ions accumulated in the first ion storage device to the second ion storage device can involve operating the second ion storage device in its accumulation mode, such that ions transferred from the first ion storage device to the second ion storage device are accumulated in the second ion storage device.

[0054] Accumulating further ions within the second ion storage using the second accumulation time can involve operating the first ion storage in its transmission mode during the second accumulation time, such that ions pass through the first ion storage during the second accumulation time without being accumulated within the first ion storage. Accumulating further ions within the second ion storage using the second accumulation time can also involve operating the second ion storage in its accumulation mode during the second accumulation time, such that ions are accumulated within the second ion storage during the second accumulation time.

[0055] The one or more ion-optical devices may include a first mass filter, such as a first quadrupole mass filter. The first ion storage device may be located upstream of the first mass filter. If, as described above, the mass filter is controlled such that its transmission window (with a mean mass-to-charge ratio (m / z)) switches between several different m / z values ​​during each instrument cycle, this allows the first ion storage device to be used for pre-accumulation at times when the mass filter's transmission window is changing, i.e., when ions are not passing through the mass filter.

[0056] The one or more ion-optical devices can also include a second mass filter, such as a second quadrupole mass filter. For example, the second mass filter can be a relatively low-resolution "pre-filter," while the first mass filter can be a relatively high-resolution analytical mass filter. The first ion reservoir can be positioned between the first and second mass filters. This allows the second mass filter to perform mass selection of ions entering the first ion reservoir, for example, to prevent overfilling of the first ion reservoir.

[0057] The method may include the first mass filter filtering ions according to their mass-to-charge ratio, wherein the first mass filter filters ions using an isolation window having a width of > about 2 Da, > about 3 Da, > about 5 Da, or > about 10 Da. The isolation window may have a width of no more than about 50 Da. The method may include fragmenting or converting the filtered ions such that the ions accumulated in the second ion reservoir are fragmented ions. The method may include directing the fragmented ions accumulated in the second ion reservoir to the mass analyzer and mass-analyzing the fragmented ions using the mass analyzer. Thus, in embodiments, the mass analysis is an MS2 mass analysis.The mass analyzer can be an electrostatic ion trap mass analyzer, and the mass analysis can be performed using an analyzer transient of < 100 ms, such as 64 ms, or < 50 ms, such as 32 ms, 16 ms, or 8 ms. The instrument can be operated at a repetition rate of > approximately 10 Hz, > approximately 20 Hz, > approximately 40 Hz, > approximately 60 Hz, or > approximately 80 Hz.

[0058] The procedure may include performing a data-independent acquisition (DIA) procedure, in which a set of MS2 fragmentation spectra is acquired, e.g., in a sweep over a mass / z range of interest. Thus, the procedure may include segmenting a mass range of interest into a plurality of precursor mass segments (e.g., each precursor mass segment having a mass range of no more than 5 Da), and for each precursor mass segment: (i) fragmenting the precursor ions within that precursor mass segment, and (ii) performing an MS2 mass analysis of the fragmented ions by: accumulating the fragmented ions in the second ion storage, directing the accumulated fragmented ions to the mass analyzer, and mass-analyzing the fragmented ions.

[0059] Any mass analysis can generate a time-varying transient signal. A mass spectrum, such as an MS2 mass spectrum (or an MS1 ​​mass spectrum), can be generated from any time-varying transient signal by deconvolution. In certain embodiments, the deconvolution technique is a high-resolution deconvolution technique such as the "phase-constrained spectrum deconvolution method" (also known as ΦSDM), i.e., as described in Grinfeld et al., "Phase-constrained spectrum deconvolution for Fourier transform mass spectrometry," Anal. Chem., 89 (2): 1202-1211 (2017), and also European patent application no. EP 3 086 354 A1, the entire contents of which are incorporated herein by reference.

[0060] Thus, in embodiments, the ΦSDM deconvolution technique is applied to relatively short MS2 transient signals, with the instrument operating at a relatively high repetition rate. Under these circumstances, the preaccumulation method allows the duty cycle to be maintained at short transient lengths, thereby mitigating sensitivity losses (as described above), while the ΦSDM deconvolution technique restores the resolution loss resulting from short transient lengths.

[0061] As described in European Patent Application No. EP 3 086 354 A1, in these embodiments a Fourier transform of the transient signal is performed to generate a first set of complex amplitudes, each of which corresponds to a specific frequency of a first set of frequencies. The first set of frequencies may be uniformly spaced. A second set of complex amplitudes is generated, each of which corresponds to a specific frequency of a second set of frequencies. The second set of frequencies may be uniformly spaced. The second set of frequencies may have a spacing (or minimum spacing) smaller than that of the first set of frequencies. The second set of frequencies may have a spacing (or minimum spacing) smaller than the inverse of the duration of the transient signal.The second set of complex amplitudes can cover the same frequency range as the first set of complex amplitudes (or exceed it or match it), and thus the second set can contain more complex amplitudes than the first set. Therefore, the second set of complex amplitudes can provide higher resolution.

[0062] The second set of complex amplitudes can be optimized to generate an improved second set of complex amplitudes. At least some of the complex amplitudes from the improved second set can be used to generate the mass spectrum. The improved second set of complex amplitudes can yield a mass spectrum of higher quality.

[0063] Optimizing the second set of complex amplitudes can involve varying at least one of the complex amplitudes from the second set based on (or depending on) an objective function. For example, the at least one complex amplitude can be varied with the goal of obtaining an essentially extreme value of the objective function. Optionally, all complex amplitudes from the second set can be varied as part of the optimization step, or a subset can be optimized as part of the optimization step.

[0064] The optimization can be performed under a constraint. That is, for at least some of the complex amplitudes of the second set, the phase of each of these at least some complex amplitudes can be constrained relative to one or more expected phases. The expected phases can be frequency-dependent. The objective function can depend on one or more complex amplitudes of the first set of complex amplitudes and one or more complex amplitudes of the second set of complex amplitudes. For each frequency of the first set of frequencies, the objective function can relate one or more complex amplitudes of the second set to the respective complex amplitude from the first set (e.g., by making the objective function a function of the one or more complex amplitudes of the second set and the respective complex amplitude from the first set).The restriction can be applied to all complex amplitudes of the second set that are varied as part of the optimization step, or to a subset of these complex amplitudes.

[0065] By generating and optimizing a second set of complex amplitudes, the transient can be thought of as decomposed into a finer frequency grid. Since the second set of complex amplitudes is not bound to the first set as a linear combination of these amplitudes, the resolution increases as the grid spacing of the second set of frequencies decreases. This leads to a significantly improved accuracy of the resulting mass spectrum. In other words, the ΦSDM method can be thought of as operating with two sets of frequencies. The first set of frequencies can include frequencies with a minimum spacing of 1 / T, where T is the duration of the transient signal. The second set of frequencies can include frequencies with a minimum spacing of less than 1 / T. The second set of frequencies can contain the first set as a subset.Since the minimum spacing of the second set of frequencies is smaller than that of the first set, the second set of complex amplitudes can provide a higher resolution.

[0066] It is understood that "complex" refers to a number that can be expressed with a real and an imaginary part. The imaginary part can be zero; that is, "complex," as used here, covers real numbers.

[0067] One advantage of the ΦSDM method is the integrability of the generated mass spectrum. In other words, the intensity of all peaks, both resolved and unresolved, is preserved. This avoids the suppression effects of the conventional Fourier transform approach, which are caused by the interference of neighboring peaks. Therefore, the ΦSDM method is particularly advantageous when highly accurate intensity information is required. Furthermore, calculations can be performed with shorter transients, thereby increasing the speed and throughput of the instrument.

[0068] In some embodiments, the step of performing a Fourier transform includes windowing the Fourier-transformed transient signal in the frequency domain, where the first set of complex amplitudes corresponds to the windowed Fourier-transformed transient signal. This windowing may involve applying a window function to the first set of complex amplitudes. Typically, applying a window function involves scaling each complex amplitude of the first set of complex amplitudes by the value of the window function at the respective frequency. Additionally or alternatively, the windowing may involve discarding the complex amplitudes whose respective frequencies lie outside one or more predefined ranges. For example, complex amplitudes of the first set of complex amplitudes whose respective frequencies are above the Nyquist frequency of the transient signal may be discarded and / or set to zero.

[0069] Advantageously, this can increase processing speed and reduce computational effort, as subsequent processing can be limited to regions of interest. For a sufficiently sparse spectrum or segments of interest, calculations can be performed only within spectral windows that encapsulate these regions.

[0070] Another aspect is a non-transitory, computer-readable storage medium that stores computer software code which, when executed on a processor, performs the procedure(s) described above.

[0071] Another aspect is providing a control system for an analytical instrument such as a mass spectrometer, wherein the control system is configured to cause the analytical instrument to perform the procedure(s) described above.

[0072] Another aspect is the provision of an analytical instrument, such as a mass spectrometer, which includes the control system described above.

[0073] Another aspect is provided by an analytical instrument, such as a mass spectrometer, which offers comprehensive support: a first ion storage; a second ion storage device, wherein the second ion storage device is located downstream of the first ion storage device; and a control system configured to: to determine whether a target accumulation time for the second ion storage is greater than a threshold accumulation time; If it is determined that the target accumulation time is less than the threshold accumulation time: to cause ions to accumulate within the second ion reservoir using an accumulation time based on the target accumulation time; and If it is determined that the target accumulation time is greater than the threshold accumulation time: to cause ions to accumulate within the first ion storage using a first accumulation time based on a difference between the target accumulation time and the threshold accumulation time; to cause the ions accumulated in the first ion storage to be directed to the second ion storage; and to cause further ions to accumulate within the second ion storage using a second accumulation time based on the threshold accumulation time.

[0074] These aspects and embodiments may include one or more or each of the optional features described herein, and include these in embodiments. Description of the drawings

[0075] With reference to the attached figures, various embodiments will now be described in more detail, wherein: Fig. 1 schematically shows a mass spectrometer which can be operated according to embodiments; Fig. 2 schematically shows a mass spectrometer which can be operated according to embodiments; Fig. 3 schematically shows a mass spectrometer which can be operated according to embodiments; Fig. 4 a known method for operating the mass spectrometer of Fig. 2 or Fig. 3 illustrated; Fig. 5A the procedure of Fig. 4 illustrates, Fig. 5B a method for operating the mass spectrometer of Fig. 2 or Fig. 3 according to embodiments illustrated, and Fig. 5C a method for operating the mass spectrometer of Fig. 2 or Fig. 3 illustrated according to embodiments; Fig. 6 illustrates a method for operating a mass spectrometer according to embodiments; Fig. 7 illustrates a method for operating a mass spectrometer according to embodiments; Fig. 8 shows the stresses applied to the half-lens, the curved flatapol exit lens, and the C-trap exit lens of the mass spectrometer of Fig. 2 or Fig. 3 are created when they are operated according to embodiments; Fig. 9 shows an estimate of the ion current for fluoranthene when using the mass spectrometer of Fig. 2 is analyzed, which is operated with and without the pre-accumulation mode activated; Fig. Figure 10A shows a fragmentation spectrum of an isolated m / z 524 MRFA peptide, which was determined using the mass spectrometer of Fig. 2 was obtained, which was operated with pre-accumulation mode disabled, and Fig. Figure 10B shows a fragmentation spectrum of the isolated m / z 524 MRFA peptide, which was measured using the mass spectrometer of Fig. 2 was obtained, which was operated with pre-accumulation mode activated; Fig. Figure 11 shows a graph of ion current losses versus repetition rate, obtained using the mass spectrometer of Fig. 2 was obtained, which was operated with and without the pre-accumulation mode activated; Fig. 12 head-to-head comparisons of the number of peptides and protein groups identified for a one-hour chromatographic separation of 200 ng of HeLa digest, using the mass spectrometer of Fig. 2 was obtained, which was operated with and without the pre-accumulation mode activated; and Fig. Figure 13A shows a fragmentation spectrum of the isolated m / z 524 MRFA peptide, which was determined using the mass spectrometer of Fig. 3 was obtained, which was operated at 200 Hz with pre-accumulation mode disabled, and Fig. Figure 13B shows a fragmentation spectrum of the isolated m / z 524 MRFA peptide, which was determined using the mass spectrometer of Fig. 3 was obtained, which was operated at 200 Hz with pre-accumulation mode activated. Detailed description

[0076] Fig. Figure 1 schematically illustrates a mass spectrometer that can be operated according to various embodiments. As in Fig. As shown in Figure 1, the mass spectrometer includes an ion source 10, one or more ion transfer stages 20, an ion trap 30 and a mass analyzer 40.

[0077] The Ion Source 10 is configured to generate ions from a sample. The Ion Source 10 can be any suitable continuous or pulsed ion source, such as an electrospray ionization (ESI) ion source, a MALDI (matrix-assisted laser desorption / ionization) ion source, an atmospheric pressure ionization (API) ion source, a plasma ion source, an electron ionization ion source, a chemical ionization ion source, and so on. More than one ion source can be provided and used. The ions can be any suitable type of ions to be analyzed, e.g., small and large organic molecules, biomolecules, DNA, RNA, proteins, peptides, fragments thereof, and the like.

[0078] The ion source 10 can be coupled to a separation device such as a liquid chromatography separation device or a capillary electrophoresis separation device (not shown), such that the sample that is ionized in the ion source 10 comes from the separation device.

[0079] The ion transfer stage(s) 20 is / are downstream of the ion source 10 and may include an atmospheric pressure interface and one or more ion guides, lenses, and / or other ion-optical devices configured such that some or all of the ions generated by the ion source 10 can be transferred from the ion source 10 to the ion trap 30. The ion transfer stage(s) 20 may include any suitable number and configuration of ion-optical devices, which may optionally include, for example, one or more RF and / or multipole ion guides, one or more ion-cooling ion guides, one or more mass-selective ion guides, and so on.

[0080] The ion trap 30 is located downstream of the ion transfer stage(s) 20 and is configured to receive and accumulate ions from the ion source 10 (via the one or more ion transfer stages 20). The ion trap 30 can comprise any suitable type of ion trap, such as a multipole (e.g., quadrupole) ion trap.

[0081] In some embodiments, the ion trap 30 is extended in an axial direction (thus defining a trap axis) in which the ions enter the trap. Ions can be captured radially in the trap 30 by applying RF voltage(s) to capture (e.g., rod) electrodes of the trap. The ion trap 30 can be or include a curved linear ion trap (C-trap), i.e., where the capture rod electrodes are curved. However, the ion trap 30 can be or include any other suitable type of ion trap, such as a linear ion trap.

[0082] The ion trap 30 comprises an inlet lens or gate 32 and an outlet lens or gate 34. The inlet lens 32 can be operated in an open mode, in which ions (from the ion source 10) can pass through the inlet lens and enter the ion trap 30, or in a closed mode, in which ions (from the ion source 10) cannot pass through the inlet lens 32 and cannot enter the ion trap 30. When the inlet lens 32 is operated in its closed mode, ions already inside the ion trap 30 cannot leave the ion trap via the inlet lens 32. Similarly, the exit lens 34 can be operated in an open mode, in which ions can pass through the exit lens and leave the ion trap 30, or in a closed mode, in which ions cannot pass through the entrance lens and do not leave the ion trap.The inlet lens 32 (the exit lens 34) can be closed or opened by applying a suitable voltage to the inlet lens 32 (to the exit lens 34).

[0083] Ions from the ion source 10 can be accumulated in the ion trap 30 by operating the exit lens 34 in its closed mode while the inlet lens 32 is operated in its open mode. After a desired ion filling time in the ion trap 30, the inlet lens 32 can be closed (by changing the voltage applied to the inlet lens 32) such that ions cannot exit the trap 30 and ions from the ion source 10 can no longer enter the ion trap 30. Thus, the mass spectrometer is configured to accumulate ions in the ion trap 30 with an adjustable accumulation time (filling time). By controlling the filling time of ions into the trap, where the ion flow into the trap 30 is known or can be approximated, the total number of ions accumulated in the ion trap 30 can be controlled.

[0084] Once ions have accumulated in the ion trap 30, they can be ejected from within the trap into the mass analyzer 40. Ions can be ejected from the ion trap 30 in an axial direction, or they can be ejected from the trap 30 in a direction orthogonal to the axis of the trap (orthogonal ejection), for example by applying one or more suitable DC voltages to the ion trap 30.

[0085] The mass analyzer 40 is downstream of the ion trap 30 and configured to receive ions from the ion trap 30. The mass analyzer is configured to analyze the ions to determine their mass-to-charge ratio and / or their mass, i.e., to generate a mass spectrum of the ions. The mass analyzer 40 can be an ion trap mass analyzer, such as an electrostatic orbital trap, and in particular an Orbitrap™-FT mass analyzer, as manufactured by Thermo Fisher Scientific. Alternatively, the mass analyzer 40 can be a time-of-flight (ToF) mass analyzer, such as a multireflection time-of-flight (MR-ToF) mass analyzer.

[0086] It should be noted that Fig. Figure 1 is merely schematic and the mass spectrometer can contain, and in some embodiments does contain, any number of one or more additional components. For example, in certain embodiments the mass spectrometer includes a collision or reaction cell. The instrument can contain a single mass analyzer or more than one (e.g., two) mass analyzer(s).

[0087] As also in Fig. As shown in Figure 1, the mass spectrometer is controlled by a control unit 50, such as a suitably programmed computer, which controls the operation of various components of the spectrometer and, for example, sets the voltages to be applied to the various components. The control unit 50 can also receive and process data from various components, including the detector(s), e.g., perform a Fourier transform on detected signals. The control unit 50 is configured, among other things, to determine the settings (e.g., the filling time of the ion trap 30, etc.) for the injection of ions into the mass analyzer 40 for analytical scans.

[0088] The mass spectrometer can be operated such that successive batches of ions from the ion source 10 are each analyzed by the mass analyzer 40. Each batch of ions is first accumulated in the ion trap 30, and then the accumulated ions (or, for example, fragment ions derived from the accumulated ions) are injected into the mass analyzer 40.

[0089] It may be desirable for each batch of ions analyzed by the mass analyzer 40 to contain as many ions as possible, for example, to improve the statistics of the mass spectrum. However, at relatively high ion concentrations, undesirable space charge effects can occur and limit the mass resolution and mass accuracy. Therefore, the total number of ions accumulated in the ion trap 30 is controlled to optimize the number of ions injected into the mass analyzer 40 so that it is below, but as close as possible to, a limit for the mass analyzer 40, such as a space charge limit. The total number of ions accumulated in the ion trap 30 can also be controlled, or alternatively, so that it is below a limit for the ion trap 30, such as the space charge limit. Typically, between 5 × 10 3 and 1 × 10 6Elementary charges are stored, for example between 1 × 10 4 and 1 × 10 6 or between 1 × 10 5 and 5 × 10 5 .

[0090] However, the ion flux from the ion source 10 may be highly variable. This is particularly true when the ion source 10 is coupled to a separation device such as a liquid chromatography or capillary electrophoresis apparatus, where the ion flux from the ion source 10 can vary by several orders of magnitude over time.

[0091] Therefore, embodiments employ so-called automatic gain control (AGC) techniques to accurately control the total number of ions accumulated in the ion trap 30, despite a variable ion flux into the trap 30. These techniques typically rely on an accurate and reliable real-time estimation of the current ion current or ion flux received by the ion trap 30. Then, by controlling the filling time T of the ion trap 30, the total number of ions or the total charge accumulated in the trap 30 (and injected into the mass analyzer 40) can be appropriately controlled.

[0092] Thus, for each batch of ions, a target accumulation time T can be determined based on an estimate of the current ion current or ion flow received by the ion trap 30, and ions can be accumulated in the ion trap 30 for a period of time equal to the target accumulation time T.

[0093] Fig. 2 and Fig. Figure 3 shows in more detail two exemplary mass spectrometers that can be operated according to the embodiments. It is understood that the Fig. 2 and Fig. The three instruments shown are not limiting examples and numerous variations are possible.

[0094] In the Fig. In the embodiment shown in Figure 2, the ion source 10 of the instrument is an electrospray ionization (ESI) ion source. The instrument includes a vacuum interface comprising a transfer tube 21, an ion funnel 22, a quadrupole pre-filter ion guide 23, and a curved flat-pole ion guide 24. The curved flat-pole ion guide 24 may have the design described in U.S. Patent No. US 9,536,722 B2.

[0095] The instrument also includes a mass filter in the form of a quadrupole mass filter 26, an ion trap 30a in the form of a curved linear ion trap (“C-trap”), and a collision cell 30b in the form of an ion-guided multipole collision cell (“IRM”). Ions from the ion source 10 can be accumulated in the C-trap 30a and / or collision cell 30b by opening and closing a gating electrode located in a charge detector assembly 27, which is positioned between the C-trap 30a and the mass filter 26.

[0096] The instrument also includes a mass analyzer 40a in the form of an orbital ion trap mass analyzer. As in Fig. As shown in Figure 2, the orbital trap 40a comprises an inner electrode 41 extending along the orbital trap axis and a split pair of outer electrodes 42, 43 surrounding the inner electrode 41 and defining a capture volume between them. Ions are trapped in this volume and oscillate by orbiting the inner electrode 41, to which a capture voltage is applied as it oscillates back and forth along the trap axis. The pair of outer electrodes 42, 43 act as detection electrodes to detect an image current induced by the oscillation of the ions in the capture volume, thereby providing a detected signal.

[0097] The outer electrodes 42, 43 typically function as a differential pair of detection electrodes and are connected to respective inputs of a differential amplifier (in Fig. 2 (not shown) is coupled, which in turn is part of a digital data acquisition system for receiving the detected signal. The detected signal can be processed using a Fourier transform to obtain a mass spectrum of ions inside the trap.

[0098] After accumulating in the ion trap 30a and / or collision cell 30b, ions can be ejected into the mass analyzer 40a. This can be achieved by ejecting the ions from the trap 30a in a direction orthogonal to the trap's axis (orthogonal ejection), for example, by applying one or more suitable DC voltages to the ion trap 30a. Alternatively, the ions can be injected into the mass analyzer 40a via one or more lenses and a deflector electrode. The mass analyzer 40a is located downstream of the ion trap 30a and is configured to receive ions from the ion trap 30a (via the one or more lenses and the deflector electrode).

[0099] The collision or reaction cell 30b is located downstream of the ion trap 30a. Ions collected in the ion trap 30a can either be ejected orthogonally to the mass analyzer 40a without entering the collision or reaction cell 30b, or the ions can be passed axially to the collision or reaction cell 30b for processing before being returned to the ion trap 30a for subsequent orthogonal ejection to the mass analyzer 40a. Processing can include, for example, fragmenting the ions by collisions with a collision gas and / or a reagent in the collision cell 30b, or further cooling the ions by collisions with a gas at lower energies that cause fragmentation.

[0100] With reference to Fig. 3 is the one in Fig. The 3 mass spectrometers shown are essentially the mass spectrometer of Fig. 2 similar. The one in Fig. However, the mass spectrometer shown in Figure 3 incorporates an additional time-of-flight (ToF) mass analyzer in the form of a multi-reflection time-of-flight (ToF) mass analyzer 40b, which has been added to the rear of the instrument. This hybridized instrument is described in more detail in U.S. Patent No. 10,699,888. In the Fig. The instrument shown in Figure 3 is the inclined mirror type analyzer described in US Patent No. US 9 136 101 B2, but it is understood that any type of ToF analyzer could be used.

[0101] As in Fig. As shown in Figure 3, the instrument includes a multipole ion guide 31 to allow ions to be guided from the collision cell 30b to the time-of-flight mass analyzer 40b. The time-of-flight mass analyzer 40b includes an extraction trap 44, through which ions are delivered from the collision cell 30b to the extraction trap 44 via the multipole ion guide 31. The ions are accumulated and cooled in the extraction trap 44.

[0102] The extraction trap 44 can comprise two capture regions: one at a relatively higher pressure for rapid ion cooling and a second, lower-pressure region for ion extraction. Ions are cooled in the high-pressure region and then transferred to the low-pressure region, where they are pulsedly ejected into the ToF analyzer via a pair of deflectors 45. Ions oscillate between a pair of mirrors 46 that are inclined relative to each other, so that the ion path is slowly deflected and reflected back to a detector 47. Correction strip electrodes 48 counteract the loss of ion focus that would otherwise be caused by the non-parallelism of the mirrors.

[0103] Modern mass spectrometers operate at increasingly faster repetition rates, enabling high performance over shorter trials and larger sample volumes. The main limitations on the repetition rate are instrument sensitivity (a certain accumulation time is required to collect enough sample ions for analysis), the time needed to process these ions for analysis, the analysis time itself, and the time the electronics require to switch between analyte targets.

[0104] In the Fig. In the instrument shown in Figure 2, ions generated by the electrospray ionization (ESI) ion source 10 must pass through the vacuum interface, i.e., the transfer tube 21, the ion funnel 22, the quadrupole pre-filter ion guide 23, and the curved flat-pole ion guide 24, before being mass-selected by the quadrupole mass filter 26 and accumulated and / or fragmented in the ion trap 30a and / or the collision cell 30b. Ions can then be returned to the C-trap 30a and pulsedly ejected into the mass analyzer 40a for analysis. In conventional operation, at the maximum permissible repetition rate of 40 Hz or every 25 ms, the maximum accumulation time is only 10 ms, which corresponds to a duty cycle of only 40%.

[0105] The longer ions are measured in the 40a mass analyzer, the higher the resolution and the greater the analyzer's sensitivity. For MS2 (ion fragmentation) measurements, which typically dominate most applications, very high resolution is not required, but a high repetition rate and sensitivity are desirable. Therefore, relatively short 16 ms transients of the mass analyzer are often used for these measurements, yielding a resolution of about 7500 at m / z 200. Shorter transients remain feasible, but at this point, the instrument's operating overhead and the required ion accumulation time for well-resolved spectra limit the instrument's repetition rate to about 40 Hz.

[0106] The operation of the instrument can be parallelized to maximize efficiency. In particular, the measurement period of ions in the mass analyzer 40a itself is very time-consuming and is typically decoupled from the process of loading and processing ions in the C-trap 30a and the collision cell 30b. Another parallelized stage is the switching of the voltages of the remaining ion optics and the transfer of ions through them to the ion gate.

[0107] Fig. Figure 4 illustrates these main parallelized operations and their approximate timing. It should be noted that the ion accumulation time is fully coupled to the relatively slow operation of the ion processing.

[0108] As described above, an important feature of commercial instruments is the precise control of the number of ions injected into the C-trap 30a and the mass analyzer 40a, a process known as automatic gain control (AGC). This is achieved by fine-tuning the filling time using a very fast beam deflection ion gate within the charge detection assembly 27 upstream of the C-trap 30a. This gate is typically accurate to about 30 µs (or less), although a more advanced dual-gate design, such as that described in US Patent No. US 8,026,475 B2, is accurate to about 1–2 µs. As described above, such precise control is necessary due to the large variations in ion beam intensity and the limited dynamic range of both the C-trap 30a and the mass analyzer 40a.

[0109] The inventors have now recognized that a problem with existing instrument designs lies in their relatively poor (< 50%) duty cycle when the instrument is operated at relatively high repetition rates. This can reduce sensitivity for rapid trials and / or those with low sample loads and can prevent even higher repetition rates from being achieved. The main reason for this problem is in Fig. 4 can be seen. The ion accumulation time runs in series with the C-trap / IRM ion processing and reset, which are very time-consuming operations, and which block the C-trap 30a for ion accumulation.

[0110] Although this problem occurs with a time-of-flight instrument, such as the one in Fig. While the instrument shown in section 3 may be less serious, these instruments have their own timing problems. In particular, the inventors recognized that one problem lies in the potentially long time intervals required to switch the front-end electronics and the quadrupole 26 in order to capture target ions with different m / z and transfer them through the C-trap 30a. In the instrument shown in Fig. In the instrument shown, the transfer and processing of ions in the extraction trap 44 block the trap for a relatively long period, about 3 ms. Combined with about 1 ms for preparing the quadrupole 26, this leaves very little time for ion accumulation at the desired repetition rate of 200 Hz / 5 ms.

[0111] Embodiments address the problem of sensitivity loss at high repetition rates due to ion accumulation time limitations imposed by non-parallelizable instrument operations. In particular, embodiments address the problems associated with the temporal overhead caused by the C-trap / IRM ion processing and reset sequence and the m / z target switching and ion transfer time for the front part of the instrument (i.e., ion funnel / pre-filter / bent flat pole / quadrupole).

[0112] According to various embodiments, a parallel ion accumulation stage is added within one or more ion transfer stages 20. The pre-accumulation stage can be provided within any suitable stage of the one or more ion transfer stages 20. With reference to Fig. 2 and Fig. For example, the ion funnel, the pre-filter 23, the curved flat-pole ion guide 24, or the mass filter 26 can be operated as a pre-accumulation ion trap. In other instrument designs, an equivalent or similar ion transfer stage (e.g., the second part of a two-stage ion funnel) can be used in this way. The ion pre-accumulation process can run in parallel with the slow ion processing operations of the C-trap / IRM arrangement. This allows for a significant additional effective filling time.

[0113] With reference to Fig. 2 and Fig. In certain embodiments, the parallel ion accumulation stage is provided within the curved flat-pole ion guide 24. The curved flat-pole ion guide 24 is particularly suitable for use as a high-capacity capture device, as it incorporates a quadrupole RF capture field and a superimposed DC current gradient for guiding ions. The curved flat-pole 24 also has an end lens 25, the voltage of which can be switched to function as a coarse ion gate.

[0114] Since this end lens 25 (or its equivalent in other instrument designs) is a relatively slow device compared to the dedicated ion gate in the charge detection arrangement 27, it can only roughly control the ion timing sequences and is therefore unsuitable for performing accurate AGC with short fill times. To maintain AGC accuracy, preaccumulation using the curved flatapol end lens 25 can thus be deactivated if the desired fill time falls below a threshold fill time, which corresponds, for example, to the maximum fill time that can support the desired repetition rate via the prior art accumulation method.

[0115] This can be achieved by defining the filling time through the open ion gate 27 as the primary filling time and the additional filling time within the curved flat-pole ion guide 24 as the auxiliary filling time. The entire filling time is then assigned to the primary filling time until a maximum (e.g., approximately 10 ms) is reached, and then the remaining time is assigned to the auxiliary filling time. This maintains linearity, and the absolute AGC accuracy is only lost for very long filling times, where this represents a small proportional loss.

[0116] Thus, in certain embodiments, ion accumulation is controlled at two independent locations. For the in Fig. 2 and Fig. In the mass spectrometer shown, ion accumulation is already carried out in the C-trap / IRM 30 and controlled by a gating electrode located within the charge detector assembly 27. An additional accumulation stage is implemented for ion accumulation within the bent flat pole 24 and is controlled by a voltage applied to the exit lens 25 of the bent flat pole. This additional capture sequence can run in parallel with a preceding ion packet being processed within the C-trap / IRM 30, when ions would otherwise be discarded and disposed of.

[0117] Fig. Figure 5 shows a comparison between the operating sequence of the state of the art ( Fig. 5A) and the present embodiment ( Fig. 5B). Each arrow describes the approximate movement of ions through the instrument in a series of processes, and each parallelized series of processes has a separate arrow. It is understood that Fig. 5 is a simplified description, as the instrument is in reality extremely complex, but it adequately shows the most relevant stages. In Fig. In stage 5B, only the curved Flatapol 24 had an additional filling time added. However, since it was added to the short first stage, there is plenty of free time before this stage catches up with the other two stages and begins to dominate.

[0118] It should be noted that a restriction of the display of Fig. 5 consists in the fact that it shows the primary and auxiliary filling times in parallel, although they actually draw ions from the same source 10 and must be shorter overall than the total repetition rate.

[0119] Fig. Section 5C demonstrates alternative time sequences that can be achieved using the techniques described herein. Fig. 5C allows the primary filling time to be reduced to 2 ms and the repetition rate to be increased to approximately 75 Hz. The unacceptable loss of duty cycle that would occur when using the prior art accumulation method is avoided by the relatively long auxiliary filling period.

[0120] Advantageously, the preaccumulation scheme can be seamlessly deactivated between scans. As described above, sufficiently accurate control of the ion population requires control of the fill times down to about 30 µs or less for intense ion streams. However, gating via a lens 25 according to the embodiments is much slower than this (and thus acts as a less accurate separation device), typically requiring about 100 µs for opening / closing. Therefore, for intense ion beams with relatively short target fill times, it is desirable to eliminate the preaccumulation process entirely. In embodiments where preaccumulation is controlled by a secondary fill time prior to the primary fill time, a fill time shorter than the maximum for the primary fill time results in an auxiliary fill time of zero, after which the exit lens 25 of the bent flatapole 24 never closes.For filling times that exceed this maximum, an auxiliary filling time can then be introduced.

[0121] Fig. Figure 6 shows this ranking for dividing the total ion accumulation time between the primary and auxiliary fillings. Essentially, the primary filling should be at or near its maximum before the auxiliary filling is used. In this way, the inaccuracy of the auxiliary filling only affects the ion current measurement for long accumulations, where the time error is relatively small and thus makes only a relatively small contribution to the overall error.

[0122] Additionally, this method should maximize the linear response of the ion load with changing fill time. However, it should be noted that corrections for small errors around the switching point can be provided and applied. For example, to account for the switching time of the curved flatapol exit lens 25, a small (e.g., about 100 µs) additional opening time may need to be added to its shortest fill times.

[0123] Fig. Figure 7 illustrates a method according to embodiments. As in Fig. As shown in Figure 7, a desired accumulation time T is first compared with a threshold accumulation time Tt for the ion trap (step 101), and it is determined whether the desired accumulation time T is greater than the threshold accumulation time Tt or not (step 102). As described above, the threshold accumulation time Tt can be set to be equal to, approximately equal to, or less than the difference between the total cycle time for the instrument and a time per cycle during which the instrument is operated in a mode (or modes) in which ions are not accumulated in the ion trap 30 (or elsewhere).

[0124] If the desired accumulation time T is less than or equal to the threshold accumulation time Tt, then ions are accumulated in the primary ion trap using the desired accumulation time T (step 103), i.e. in a “normal” way.

[0125] However, if the desired accumulation time T is greater than the threshold accumulation time Tt, ions are pre-accumulated in the auxiliary ion trap using an auxiliary accumulation time approximately equal to the difference between the desired accumulation time T and the threshold accumulation time Tt (i.e., T minus Tt) (step 104). These ions accumulated in the auxiliary ion trap are then directed to the primary ion trap (step 105). Finally, additional ions are accumulated in the primary ion trap (to replenish the ions accumulated in the auxiliary trap and directed to the primary trap) using an accumulation time approximately equal to the threshold accumulation time Tt (step 106). Thus, the total accumulation time for ions is approximately equal to the desired accumulation time, i.e., (T - Tt) + Tt = T.

[0126] Back to Fig. 5. Another advantage of providing preaccumulation at an early stage of the instrument before the mass filter (e.g., bent flat pole 24) can be seen in how this resolves the relatively long “electronics switching + ion transfer” stage. Naturally, ions require less time to travel from source 10 to bent flat pole 24 than from source 10 to IRM 30b. The switching time of the electronics can also be improved if, for example, the quadrupole electronics are the slowest part, as is the case with existing commercial instruments. This can reduce a stage that typically takes > 4 ms to parallelized stages that require only 1 or 2 ms in total.

[0127] Furthermore, in embodiments, the pre-accumulation stage is connected downstream of a pre-filter 23. Advantageously, the pre-filter 23 enables a coarse mass selection of ions entering the curved flat pole 24, thereby reducing the space charge load presented by unwanted ions, e.g., by about 90%, thus preventing overfilling of the device when it is operating in accumulation mode, which could otherwise also impede the effect of the main mass filter 26.

[0128] A simplified version of the preaccumulation procedure was programmed and applied to an Orbitrap™ instrument by the [company / organization / person] in [location / development]. Fig. The type shown in point 2 was applied. In this procedure, the primary filling time was set to a fixed 10 ms for all fragmentation spectra, and the auxiliary filling time was set to utilize the remainder of the available time defined by the instrument's repetition rate.

[0129] Fig. Figure 8 illustrates the timing sequences for opening and closing the half-lens 27 (“beam control”) and the exit lens 25 of the curved flatapole, which control the primary and secondary accumulation times, together with the timing sequence of the operation of the C-trap exit lens. As in Fig. As shown in Figure 8, at the moment the primary injection process (“beam control”) ends, the exit lens 25 of the curved flatapole is adjusted from a transmission voltage of -10 V to a capture voltage of +10 V. The voltages applied to the exit lens of the C-trap move from a small negative voltage when ions are loaded into the IRM 30b to a slightly positive ramp voltage when ions are returned from the IRM 30b to the C-trap 30a. The voltage is then pulsed to +250 V at the beginning of the analyzer 40 injection cycle and then set to reset.

[0130] Fig. Figure 9 shows an estimate of the ion current for fluoranthene, with the preaccumulation mode switched from disabled to enabled. Fig. 9. The ion current is estimated from mass analysis measurements using the orbital ion trap mass analyzer 40. It is evident that the ion current approximately doubles thanks to the doubling of the duty cycle.

[0131] Fig. Figure 10 shows fragmentation spectra of an isolated m / z 524 MRFA peptide with the pre-accumulation set to deactivated ( Fig. 10A) and activated ( Fig. 10B). It is evident that both fragment spectra are very similar with respect to the fragment ions present and relative ion abundances. However, the “normalized highest peak values”, a measurement of the ion current for the largest peak, are more than doubled when preaccumulation is activated due to the increase in the duty cycle.

[0132] Fig. Figure 11 shows a comparison of the ion current losses (normalized to 48 Hz) with the repetition rate when the pre-accumulation procedure is enabled or disabled. The instrument's repetition rate was increased by reducing the primary filling time, as described above in relation to Fig. 5C was described, which reduced the cycle time from 21 to 13 ms. The background ion current was measured via the estimated total filling time and the peak signal-to-noise ratio of the ions, allowing an estimation of the signal change at higher repetition rates.

[0133] It is evident that the sensitivity of the instrument without preaccumulation rapidly degrades at high repetition rates, but with preaccumulation activated, the sensitivity loses only about 10% of the signal above 70 Hz. This is thought to be related to the efficiency of flushing trapped ions from the bent flat pole 24 and passing them through the quadrupole 26 when only short primary filling times (< 3 ms) are used.

[0134] Fig. Figure 12 illustrates the pre-accumulation procedure used in a proteomics application, in this case a one-hour chromatographic separation of a complex sample, 200 ng HeLa digest, under several different procedure parameters. Fig. Figure 12 shows head-to-head comparisons of the number of identified peptides and protein groups for the pre-accumulation and standard procedures. The data on the left are for the instrument operating at a resolution of 120 K MS1 and 15 K MS2 and a fill time of 23 ms. The data in the middle are for the instrument operating at a resolution of 60 K MS1 and 7.5 K MS2 and a fill time of 10 ms. The data on the right are for the instrument operating at a resolution of 120 K MS1 and 7.5 K MS2 and a fill time of 10 ms.

[0135] Although these long, highly concentrated separations are relatively unattractive for this technique (since signal and time are not such extremely limiting factors), the number of identified peptides and protein groups is significantly and consistently increased. In one example, where the MS1 resolution was 120 K and the MS2 resolution was 7.5 K, the improvement in peptide identification reached a remarkable 29%.

[0136] The simple pre-accumulation method was adapted to a time-of-flight mass spectrometer of the in Fig. The instrument was ported to the type shown in the image. It was operated at 200 Hz with a timing overhead of 3 ms, which is approximately the lower limit for fast precursor switching and ion transfer with a bent flat-pole capture stage (the lower limit without is even higher, 3.5–4 ms). The remaining 2 ms were set as the ion injection time.

[0137] Fig. Figure 13 shows a comparison of the signal for fragmented MRFA with and without preaccumulation. Fig. Figure 13 shows that the ion signal for these time sequences is more than doubled when preaccumulation is used. For data-independent accumulation, where the precursor selection moves in only small steps, these time sequence overheads can be reduced to ~1.5 ms, and the duty cycle benefits will be more modest, but still substantial.

[0138] It is understood that embodiments refer to the use of two consecutive fill times, an optionally inaccurate one for pre-accumulation and an accurate one for main ion trap (C-trap / IRM) accumulation. This enables seamless switchable operation, where parallelized scans are mixed with non-parallelized scans for short fill times or for AGC pre-scans to maintain accurate ion population control and linearity.

[0139] Advantageously, this can double the instrument sensitivity for fast (e.g., 40 Hz) experiments and eliminates a major bottleneck for even faster acquisition, enabling sensitive measurements up to 75 Hz. With optimization, even higher rates of 80–100 Hz can be achieved. The technique can be applied to existing instruments without hardware modifications. The method is particularly suitable for fast experiments with low sample loading.

[0140] Although various special embodiments have been described above, several alternative embodiments are possible.

[0141] Although, for example, various embodiments have been described above with regard to Obitrap™ instruments, embodiments are applicable to other instrument designs. As above with reference to Fig. As described in Figure 1, a generalized instrument layout comprises an ion source, an interface, a gate, a trap, and an analyzer. Preaccumulation can occur in a portion of the interface or at least upstream of the gate that controls the accumulation. In these embodiments, instead of the bent flat pole 24 used for preaccumulation, ion-optical interface devices can be employed, which may include a wide range of multipole ion guides / traps, along with stacking ring, ion funnel, or ion carpet-type devices.

[0142] In these embodiments, the instrument may include a mass filter, for example, between the interface and the gate. The mass filter may be placed before or after the gate, but for accurate gating to function, at least one ion gate must be present, separate from the pre-accumulation device. This is present in some q-ToF instruments, which can exclude ions before they accumulate in their collision cell, which then acts as a trap.

[0143] The gate can also be integrated into the trap, for example, if gating is controlled by the exit lens of a collision cell prior to ToF extraction.

[0144] As described above, the "preaccumulation" method of various embodiments can significantly improve the sensitivity of the analytical instrument. However, a related problem arises in the context of Orbitrap™ analyzers, as the sensitivity benefits are most pronounced at relatively high repetition rates, i.e., at relatively short (e.g., 8 ms or 16 ms) transients of the Orbitrap™ analyzer (although at 32 ms the method still provides a 1 / 3 increase in the ion signal). At these transient lengths, the resolution of the Orbitrap™ analyzer is relatively low, particularly 3750 and 7500 at m / z 200, and drops off rapidly at higher m / z.

[0145] With the data-dependent acquisition (DDA) methods described above, the gains in sensitivity outweigh the losses resulting from the decreasing resolution. However, data-independent acquisition (DIA) methods are becoming increasingly important, especially at high throughput, where they demonstrate excellent results. DIA methods are almost never run with 16 ms transients on Orbitrap™ instruments, typically using 32 ms or 64 ms instead, even for short LC gradients. While sensitivity to low-level species is certainly an important consideration with these methods, resolution is a key factor, and 7500 dpi may be too low for some applications.Higher resolution may be required to distinguish interfering peaks in complex spectra, and fragment mass accuracy can also be an important factor in these methods at low signal-to-noise ratios.

[0146] Therefore, a problem arises due to the relative performance weakness of Orbitrap™ analyzers in high-throughput DIA experiments, caused by high resolution and sensitivity requirements that limit the repetition rate.

[0147] A major advancement in the signal processing of the Orbitrap™ analyzer was the development of the so-called "Phase-Constrained Spectrum Deconvolution" or "ΦSDM" method, as described, for example, in Grinfeld et al., Phase-constrained spectrum deconvolution for Fourier transform mass spectrometry, Anal. Chem., 2017, 89, 1202-1211, and also European Patent Application No. EP 3 086 354 A1, the entire contents of which are incorporated herein by reference. While computationally more expensive than the standard "eFT" method, it has the property of multiplying the resolution for a given transient length, enabling a more reliable assignment of peak frequency and position and reducing the influence of interfering peaks.

[0148] ΦSDM spectral processing with "super resolution" has a significant positive impact on isolated DIA proteomics experiments. However, a drawback of ΦSDM alone is that it does not improve the instrument's sensitivity. Thus, while it provides high resolution for shorter transients of the Orbitrap™ analyzer, sensitivity still decreases with conventional methods. It is therefore insufficiently compatible with conventional 16 ms and 8 ms transients, where the Orbitrap™ analyzer's duty cycle decreases in addition to the normal reduction in ion accumulation time and signal / noise, as required by the faster repetition rate (as described above). This limitation can be more significant than the requirement for a high level of processing power.

[0149] In some embodiments, the preaccumulation method described above and the ΦSDM process are combined, particularly for high-throughput DIA methods. This combination can also be applied to DIA-like DDA methods, for example, when the isolation window is wide and the challenges are similar. The preaccumulation method allows the duty cycle to be maintained at short transient lengths and reduces sensitivity loss, while the ΦSDM technique compensates for the resolution loss. Together, the two methods reduce the transient base from 32 ms to 16 ms or even 8 ms, enabling high repetition rates up to >70 Hz, which are advantageous for high throughput and / or short LC gradient analyses.

[0150] In these embodiments, an Orbitrap™ mass spectrometer such as the one described in Fig. 2 or Fig. The instrument shown in Figure 3 is used, coupled with a liquid chromatography device to provide a separated sample, typically tryptic digests of biological protein samples. As described above, these instruments typically have a configuration in which ion accumulation is blocked by the operation of the extraction trap (C-trap) 30a.

[0151] As also described above, the Fig. 5B and Fig.Section 5C describes the modified instrument operating sequence and timings for preaccumulation with 16 ms and 8 ms transients. By first injecting ions into the curved flat pole 24, a second parallel ion accumulation stage is created, which can operate while the C-trap / IRM 30 is active. At 40 Hz operation, the duty cycle more than doubles, and at 75 Hz, it increases fivefold. Ion capture and release are controlled by switching the voltage at the exit lens 25 of the curved flat pole, e.g., from +10 to -10 V.

[0152] The transient data collected by the Orbitrap™ analyzer, either MS and / or MS / MS spectra, can then be analyzed using the ΦSDM technique.

[0153] The instrument can operate an otherwise conventional DIA procedure, acquiring a series of preaccumulation MS / MS fragmentation spectra in a pre-programmed pass across the mass range, with an optional full MS scan for precursor quantification. The full MS scan does not have to use the preaccumulation technique, but it can.

[0154] The ΦSDM technique can be applied to MS and / or MS / MS spectra. ΦSDM with an external processor can slow down the entire system when long transients are used, as can be the case with full MS scans. Therefore, in some implementations, the ΦSDM technique is used only for MS / MS scans (and not for MS scans) to maintain speed.

[0155] Experiments that can particularly benefit from the combined technique include those using short LC gradients, e.g., less than 30 minutes, such as 3–15 minutes, where high repetition rates are required, and those using substantial sample loads (e.g., ~50–2000 ng). Very low sample loads (e.g., <10 ng or <1 ng) lead to sensitivity problems, which can be exacerbated by short transients, and are therefore often studied with wide isolation windows and long transients. Similarly, the isolation window should preferably not be very narrow, although down to 2 DIA can be used. The transient length of the Orbitrap™ analyzer can be approximately 8–32 ms for MS / MS to achieve particular improvements through the combination of preaccumulation and ΦSDM. The transient of the full-MS Orbitrap™ analyzer can be longer, e.g., B. 32-128 ms.

[0156] In some implementations, the ΦSDM technique can be applied only to specific regions of a spectrum, such as particularly congested regions (e.g., the precursor region) or areas with low signal-to-noise peaks that would benefit from greater mass accuracy. In this case, a regular profile spectrum can first be generated and then queried for regions to which ΦSDM should be applied. Such a filter can help reduce the computational load of the method.

[0157] It is understood that the combination of the preaccumulation method and the ΦSDM technique makes Orbitrap™ instruments compatible with short transients and thus fast DIA experiments. This advantage is a synergistic effect, as the two must be used together to function. Conventional Orbitrap™ instruments are generally unsuitable for high sampling rates, especially for DIA experiments, because the duty cycle, the signal, and the corresponding resolution loss lead to interference between closely spaced peaks. Applying the preaccumulation method solves the duty cycle problem, but the resolution is still reduced. However, applying the ΦSDM technique restores this to normal, suitable levels.

[0158] It is understood that due to the shorter transient there may still be a loss of ion accumulation time, however this is less of a problem for short LC gradients, as the ion current is usually considerably higher.

[0159] Although these embodiments are particularly suitable for DIA methods, they can also be advantageous for high-throughput DDA where the isolation window is sufficiently wide that spectra are complex and resolution becomes important.

[0160] Other similar high-resolution deconvolution techniques can be used in the same way, such as the least-squares fit method, i.e., as described in the article by Kozhinov et al. (2022), “Super-resolution mass spectrometry enables rapid, accurate, and highly-multiplexed proteomics at the MS2-level”, bioRxiv. This technique shows some similarities to ΦSDM in terms of properties and performance.

Claims

[1] Method for operating an analytical instrument comprising a first ion storage and a second ion storage downstream of the first ion storage, the method comprising: Determine whether a target accumulation time for the second ion storage is greater than a threshold accumulation time; If it is determined that the target accumulation time is less than the threshold accumulation time: accumulation of ions within the second ion storage using an accumulation time based on the target accumulation time; and If it is determined that the target accumulation time is greater than the threshold accumulation time: Accumulate ions within the first ion storage using a first accumulation time based on a difference between the target accumulation time and the threshold accumulation time, transfer the ions accumulated in the first ion storage to the second ion storage, and accumulate further ions within the second ion storage using a second accumulation time based on the threshold accumulation time. [2] Method according to claim 1, wherein the analysis instrument comprises at least one first gate configured to control an accumulation time of ions in the first ion storage, and at least one second gate configured to control an accumulation time of ions in the second ion storage, wherein a response time of the at least one second gate is faster than a response time of the at least one first gate. [3] Method according to claim 1 or 2, wherein the analytical instrument comprises an ion source and one or more ion-optical devices arranged between the ion source and the second ion storage, wherein the one or more ion-optical devices are configured to pass ions from the ion source to the second ion storage, and wherein the first ion storage is arranged within the one or more ion-optical devices. [4] Method according to claim 3, wherein the first ion storage is formed in a transfer ion guide of one or more ion-optical devices. [5] Method according to any of the preceding claims, wherein the second ion storage is an ion trap such as a linear ion trap. [6] Method according to one of the preceding claims, wherein the analysis instrument includes a first mass filter upstream of the second ion storage, and wherein the first ion storage is upstream of the first mass filter. [7] Method according to claim 6, wherein the analysis instrument includes a second mass filter upstream of the first mass filter, wherein the resolution of the second mass filter is smaller than the resolution of the first mass filter and wherein the first ion storage is arranged between the first mass filter and the second mass filter. [8] Method according to claim 6 or 7, further comprising that the first mass filter filters ions according to their mass-to-charge ratio, wherein the first mass filter filters ions using an isolation window with a width of > about 2 Da. [9] Method according to claim 8, further comprising fragmenting the filtered ions, wherein the ions accumulated in the second ion storage are fragmented ions. [10] Method according to one of the preceding claims, wherein the analytical instrument comprises a mass analyzer located downstream of the second ion storage, and wherein the method comprises directing ions accumulated in the second ion storage to the mass analyzer and analyzing the ions using the mass analyzer. [11] Method according to claim 10, wherein the mass analyzer is an electrostatic ion trap mass analyzer or a time-of-flight mass analyzer. [12] Method according to claim 10 or 11, wherein the mass analyzer analyzing the ions generates a time-varying transient signal, and wherein the method further comprises generating a mass spectrum from the time-varying transient signal using a phase-limited spectrum deconvolution (ΦSDM) method. [13] Method according to claim 12, wherein the time-varying transient signal has a duration of < 50 ms. [14] Method according to any of the preceding claims, wherein the analyzer is operated cyclically and wherein the threshold accumulation time is based on a difference between a total cycle time for the analyzer and a time per cycle in which the analyzer is operated in a mode in which ions are treated in a manner other than being accumulated in the second ion storage. [15] Method according to claim 14, wherein the time per cycle in which the analytical instrument is operated in the mode in which ions are treated other than being accumulated in the second ion storage comprises a time per cycle in which the second ion storage is operated in a non-accumulating mode while ions accumulated in the second ion storage are processed and / or directed to a mass analyzer for analysis. [16] Method according to claim 14 or 15, further comprising operating the analysis instrument with a repetition rate > 20 Hz, > 40 Hz, > 60 Hz or > 80 Hz. [17] Method according to any of the preceding claims, wherein the accumulation of ions within the second ion storage using an accumulation time based on the target accumulation time comprises: Operating the first ion storage device in a transmission operating mode during the accumulation period, such that ions pass through the first ion storage device during the accumulation period without being accumulated within the first ion storage device; and Operating the second ion storage in an accumulation mode during the accumulation time, so that ions are accumulated within the second ion storage during the accumulation time. [18] Method according to any of the preceding claims, wherein the process comprises directing the ions accumulated in the first ion storage to the second ion storage: Operating the first ion storage unit in transmission mode, such that ions accumulated in the first ion storage unit are directed to the second ion storage unit; and Operating the second ion storage in an accumulation mode such that ions passed from the first ion storage to the second ion storage are accumulated within the second ion storage. [19] Method according to any of the preceding claims, comprising accumulating further ions within the second ion storage using the second accumulation time: Operating the first ion storage unit in a transmission mode during the second accumulation period, such that ions pass through the first ion storage unit during the second accumulation period without being accumulated within the first ion storage unit; and Operating the second ion storage in an accumulation mode during the second accumulation time, so that ions are accumulated within the second ion storage during the second accumulation time. [20] Method according to any of the preceding claims, wherein, if it is determined that the target accumulation time is equal to the threshold accumulation time, the method comprises accumulating ions within the second ion storage using an accumulation time based on the target accumulation time. [21] Control system for an analytical instrument, such as a mass spectrometer, wherein the control system is configured to cause the analytical instrument to perform the method according to any one of claims 1-20. [22] Analytical instrument, such as a mass spectrometer, comprising the control system according to claim 21. [23] Analytical instrument, such as a mass spectrometer, comprising: a first ion storage; a second ion storage device, wherein the second ion storage device is located downstream of the first ion storage device; and a control system configured to: to determine whether a target accumulation time for the second ion storage is greater than a threshold accumulation time; If it is determined that the target accumulation time is less than the threshold accumulation time: to cause ions to accumulate within the second ion reservoir using an accumulation time based on the target accumulation time; and If it is determined that the target accumulation time is greater than the threshold accumulation time: to cause ions within the first ion reservoir to accumulate using an initial accumulation time based on a difference between the target accumulation time and the threshold accumulation time; to cause the ions accumulated in the first ion storage to be directed to the second ion storage, and to cause further ions to accumulate within the second ion storage, using a second accumulation time based on the threshold accumulation time. [24] Non-volatile, computer-readable storage medium that stores computer software code which, when executed on a processor, performs the method according to any one of claims 1 to 20 on an analysis instrument according to claim 22 or 23.

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