Acquisition of ion mobility-mass-to-charge ratio data

Abstract

A method of operating an analytical instrument is provided. The analytical instrument comprises: an ion mobility separator; a mass filter disposed downstream of the ion mobility separator; and a mass analyzer disposed downstream of the mass filter. The method includes an ion mobility separator performing a plurality of ion mobility separation scans, where in each ion mobility separation scan, the ion mobility separator receives ions and separates the ions according to their ion mobility. The method includes the mass filter filtering the separated ions using an isolation window, and during each ion mobility separation scan: (i) scanning the center mass-to-charge ratio (m / z) of the isolation window, and (ii) controlling the width [Delta] mz of the isolation window such that during the ion mobility separation scan, the center mass-to-charge ratio (m / z) of the isolation window is greater than the center mass-to-charge ratio (m / z) of the isolation window. Ions emerging from the ion mobility separator over an ion mobility arrival time range [Delta] T are transported by the mass filter. The method includes the mass analyzer performing one or more mass analysis scans during each ion mobility separation scan, where in each mass analysis scan, the mass analyzer receives and analyzes ions transmitted by the mass filter or ions sourced from the ions transmitted by the mass filter, wherein each mass analysis scan has a duration T, and wherein [Delta] Tlt; t.

Description

Technical Field

[0001] The present invention relates to the field of mass spectrometry and in particular to mass spectrometry combined with ion mobility separation. Background Art

[0002] In modern mass spectrometry, Fourier transform (FT) and multi-reflection time-of-flight (mrTOF) mass spectrometers (MS) offer particularly high performance. FT-MS utilizes transient acquisition on time scales up to hundreds of milliseconds, while mrTOF-MS utilizes transient acquisition on time scales up to 1 to 2 milliseconds. Meanwhile, high-resolution ion mobility separator (hrIMS) devices produce peptide ion arrival time distributions between <1 and a few milliseconds wide.

[0003] Due to this time mismatch, when FT-MS or mrTOF is used as a detector for high-resolution ion mobility separation, ion packets of different ions separated in ion mobility space can be detected in the same FT-MS or mrTOF spectrum, thereby reducing the separation achieved using ion mobility. This problem is compounded when the combination of FT-MS or mrTOF with hrIMS is applied to proteomics or metabolomics workflows, where fast liquid chromatography (LC) is used to reduce sample complexity.

[0004] Therefore, there is a need to interface a "slow" mass analyzer to a hrIMS device without sacrificing the resolution of the hrIMS device, nor sacrificing the analytical throughput and analytical depth in a high performance MS. In general, a "slow" mass analyzer can be defined as a mass analyzer in which the duration of a single mass analysis scan is high enough to reduce the temporal resolution of the ion mobility separator (IMS) or hrIMS.

[0005] hrIMS devices are described, for example, in US 6,791,078, US 7,838,826, US 8,507,852, US 8,552,366, US 8,835,839, US 9,552,969, US 10,741,375 and GB 2,494,562.

[0006] There are different ways of operating such hrIMS devices with a "slow" mass analyser, as described, for example, in GB 2,494,562. Figure 1 , where the horizontal axis represents the mass-to-charge ratio (m / z) of the ions and the vertical axis represents the time over which the ions elute from the hrIMS device. The shaded band illustrates a typical distribution of peptide ions with different charge states (from +1 to +4). Typically, the duration T IMS maxThe hrIMS scan may comprise N MS scans (where N may range, for example, from 1 to >100, depending on the hrIMS device and analyzer). Figure 1 As shown, for simplicity, each MS scan may have the same duration T.

[0007] The most straightforward way to acquire a two-dimensional ion mobility-m / z map would be to select a narrow time slice ΔT of ions during each MS scan and then detect their full spectrum using a mass analyzer. As is known in the art, the detection itself can be delayed relative to this time, e.g. Figure 1 Ions selected during "MS Scan 1" can be detected during "MS Scan 2" or even "MS Scan 3". By gating the start scan time relative to the ion mobility (IM) separation (e.g. Figure 1 The data for the entire 2D map can be collected in steps of approximately T / ΔT (shown as the double shaded line in the figure). In practice, for an FT-MS operating with a period of T = 64 ms and a desired time resolution of ΔT = 1 ms, this would require a step size of L = T / ΔT = 64. Even if T ≥ T IMS max , which also results in a total cycle of > 4 seconds, or even slower, sometimes making it incompatible with LC.

[0008] Another approach is to apply multiple gating windows within each MS scan with variable periods, followed by deconvolution of the 2D map using, for example, a Hadamard transform (Poltash et al., Anal. Chem., 2018, 90, 10472-10478).

[0009] Different approaches are described in EP 1,271,138 and GB 2,494,562. Here, a quadrupole mass filter after the hrIMS device is scanned synchronously with the elution of ions from the hrIMS device so that only ions of a specific mobility-m / z ratio enter the downstream analyzer for detection. A fast orthogonal acceleration ToF mass analyzer is used for detection, which provides sufficient time resolution to be compatible with ion mobility separation. This so-called "linked scanning" method effectively allows the removal of a selected range of ion mobilities.

[0010] It is believed that there is still room for improvement in mass spectrometry methods and equipment. Summary of the invention

[0011] A first aspect provides a method of operating an analytical instrument, the analytical instrument comprising: an ion mobility separator; a mass filter, the mass filter being arranged downstream of the ion mobility separator; and a mass analyzer, the mass analyzer being arranged downstream of the mass filter, the method comprising:

[0012] The ion mobility separator performs a plurality of ion mobility separation scans, wherein in each ion mobility separation scan, the ion mobility separator receives ions and separates the ions according to their ion mobility;

[0013] The mass filter filters the separated ions using the isolation window, and during each ion mobility separation scan: (i) scans the central mass-to-charge ratio (m / z) of the isolation window, and (ii) controls the width Δmz of the isolation window so that during the ion mobility separation scan, ions emerging from the ion mobility separator within the ion mobility arrival time range ΔT are transmitted by the mass filter; and

[0014] The mass analyzer performs one or more mass analysis scans during each ion mobility separation scan, wherein in each mass analysis scan, the mass analyzer receives and analyzes ions transmitted by the mass filter or ions derived from the ions transmitted by the mass filter, wherein each mass analysis scan has a duration T, and wherein ΔT <T。

[0015] Embodiments relate to methods of operating analytical instruments such as mass spectrometers. The instrument may include: an ion source configured to generate ions from a sample; an ion mobility separator arranged downstream of the ion source and configured to separate the received ions according to the ion mobility of the received ions; a mass filter arranged downstream of the ion mobility separator and configured to filter the received ions according to the mass-to-charge ratio (m / z) of the received ions (i.e., using an isolation window with a central mass-to-charge ratio (m / z) and a width Δmz); and a mass analyzer arranged downstream of the mass filter and configured to perform mass analysis on the received ions. The instrument may optionally include a fragmentation device arranged downstream of the mass filter and configured to selectively fragment the received ions. Including ion mobility (IM) separation is beneficial because it improves the duty cycle and sensitivity of the instrument.

[0016] In the method, an ion mobility separator performs multiple ion mobility separation scans, and during each ion mobility separation scan, a mass filter scans the central mass-to-charge ratio of its isolation window, optionally allowing ions with a specific charge state to be transmitted by the mass filter. In addition, the width Δmz of the isolation window is controlled so that during each ion mobility separation scan, ions emerging from the ion mobility separator within an ion mobility arrival time range ΔT are transmitted by the mass filter, wherein ΔT is less than the duration T of the mass analysis scan.

[0017] The method is particularly suitable for instruments in which the mass analyzer is a relatively "slow" mass analyzer, i.e., in which the mass analyzer does not provide sufficient time resolution to be compatible with ion mobility separation. In this method, time resolution is provided by controlling the quadrupole mass filter. In effect, in each ion mobility separation scan, ions corresponding to "bands" of two-dimensional (2D) ion mobility arrival time-m / z space are analyzed, where each band has a range ΔT in the ion mobility arrival time dimension equal to the desired time resolution.

[0018] In some embodiments, by performing multiple such ion mobility separation scans in which ions from adjacent bands in the 2D ion mobility arrival time-m / z space are analyzed, ions within a region of interest in the 2D ion mobility arrival time-m / z space can be analyzed in a particularly direct and efficient manner.

[0019] It will therefore be appreciated that the embodiments provide improved methods of operating analytical instruments.

[0020] The analytical instrument may be a mass spectrometer, for example including an ion source. Ions may be generated from a sample in the ion source. The ion source may optionally be coupled to a chromatographic separation device, such as a liquid chromatography (LC) separation device, so that the sample ionized in the ion source comes from the separation device. Therefore, the analytical instrument may include: an ion source, which is arranged upstream of the ion mobility separator; and a chromatographic separation device, which is arranged upstream of the ion source, and the method may include: the chromatographic separation device performs chromatographic separation on the sample; and the ion source ionizes the chromatographically separated sample to produce ions.

[0021] The analytical instrument includes an ion mobility separator, which can be arranged downstream of an ion source, and is configured to perform an ion mobility separation scan to separate the ions received from the ion source according to the ion mobility of the ions received from the ion source. The ion mobility separator can be operated in a cyclic manner, i.e., to repeatedly perform an ion mobility separation scan. In each ion mobility separation scan, the ion mobility separator can receive ions from the ion source, and, for example, accumulate ion packets in an accumulation region. Alternatively, the ion packets can be accumulated in an ion trap upstream of the ion mobility separator. Then, the ion mobility separator can separate the ion packets according to the ion mobility of the ions, for example, by passing the ion packets through the ion mobility separation region. Ions with higher ion mobility arrive at the end of the ion mobility separation region (and leave the separator) before ions with lower ion mobility.

[0022] Each ion mobility separation scan may have a duration T IMS In other words, the ion mobility separator may have a cycle time T IMS Duration TIMS The time required to accumulate ion packets and the time required to separate ions may be included. Alternatively, the duration T IMS The duration T may correspond only to the time required to separate the ions, where the accumulation of ion packets is performed in parallel with the separation of previously accumulated ion packets. IMS It can be about hundreds of milliseconds or thousands of milliseconds. The duration T IMS It may be constant in any given experiment, but can be varied between experiments by appropriate control of the instrument.

[0023] The analytical instrument includes a mass filter, which is arranged downstream of the ion mobility separator and is configured to receive separated ions from the ion mobility separator. The mass filter can be any suitable mass filter that can be operated to filter the ion according to the m / z of the ion, such as a quadrupole mass filter. Other types of filters can also be used, such as Wien filters or time-of-flight filters (for example, as described in US 7,999,223). The mass filter can be configured so that the received ions with the m / z within the m / z isolation window of the mass filter are isolated and forwardly transmitted by the mass filter, and the received ions with the m / z outside the m / z isolation window are attenuated by the mass filter, for example, not forwardly transmitted by the mass filter. The width and / or center m / z of the isolation window are controllable (variable) by, for example, appropriately controlling the RF voltage and DC voltage applied to the mass filter. Thus, for example, the mass filter may be capable of operating in a transmission mode of operation, whereby most or all ions within a relatively wide m / z isolation window are transmitted forward by the mass filter, and may be capable of operating in a filtering mode of operation, whereby only ions within a relatively narrow m / z isolation window (centered about the desired m / z) are isolated by the mass filter and transmitted forward.

[0024] The analytical instrument may optionally include a fragmentation device, which is arranged downstream of the mass filter and is configured to receive ions transmitted by the mass filter. The fragmentation device may be configured to selectively fragment some or all of the received ions, i.e., to produce fragment ions. The fragmentation device may be capable of operating in a fragmentation operating mode, whereby most or all of the received ions are fragmented to produce fragment ions (which can then be transmitted onward from the fragmentation device), and may be capable of operating in a non-fragmentation operating mode, whereby most or all of the received ions are transmitted onward without being (intentionally) fragmented. It is also possible to achieve a non-fragmentation operating mode by causing ions to bypass the fragmentation device.

[0025] The analytical instrument includes a mass analyzer, which is arranged downstream of the mass filter (and, where present, downstream of the fragmentation device) and is configured to perform a mass analysis scan to determine the mass-to-charge ratio (m / z) of the received ions. The mass analyzer may be capable of operating in a cyclic manner, i.e., to repeatedly perform mass analysis scans. In each mass analysis scan, the mass analyzer receives ions and performs mass analysis on the ions. In an embodiment, the mass analyzer is an ion trap mass analyzer, such as an electrostatic orbital trap, and more specifically an Orbitrap TM FT mass analyser. Alternatively, the mass analyser may be a time of flight (ToF) mass analyser, such as a multi-reflection time of flight (mrTOF) mass analyser.

[0026] It is possible to configure the instrument so that ions can be delivered to the mass analyzer in the form of an ion beam, for example, without accumulating before being delivered to the mass analyzer. Therefore, in an embodiment, ions are directly accumulated in the mass analyzer. In these embodiments, the number of ions accumulated in the mass analyzer can be controlled by controlling the accumulation time (for example, filling time) of ions entering the mass analyzer. This can then be controlled by operating the door or lens of the mass analyzer and / or the door or lens in the instrument upstream (between the ion source and the mass analyzer) of the mass analyzer in the open (transmission) operating mode for the desired amount of time (and otherwise operating the door or lens in the closed (non-transmission) operating mode). The example of such a door can be found in, for example, GB 2,585,472. Such one or more doors can be used to attenuate the intensity of the incoming ion peak using pulse width modulation, wherein the pulse width is significantly shorter than ΔT. This allows to increase the dynamic range of the spectrum collected by the mass analyzer.

[0027] In a specific embodiment, ions are transferred to the mass analyzer from an ion trap arranged upstream of the mass analyzer. The ions may initially accumulate in the ion trap, and then, for example, be transferred to the mass analyzer in the form of ion packets. The ion trap may be referred to as an injection device for injecting ions into the mass analyzer. The ion trap may include any suitable ion trap, such as a linear ion trap or a curved linear ion trap (C-trap), for example, as described in WO 2008 / 081334. The ion trap may be used for cooling the accumulated ions before they are injected into the mass analyzer. The ion trap may also or alternatively (under the MS2 operating mode) be used as a fragmentation device to fragment ions before the fragment ions are injected into the mass analyzer.

[0028] In these embodiments, the number of ions accumulated in the mass analyzer can be controlled by controlling the accumulation time (e.g., fill time) of ions entering the ion trap. This in turn can be controlled by operating the door or lens of the ion trap and / or a door or lens in the instrument upstream of the ion trap (between the ion source and the ion trap) in an open (transmitting) operating mode for a desired amount of time (and otherwise operating the door or lens in a closed (non-transmitting) operating mode).

[0029] Each mass analysis scan has a duration T. In other words, the mass analyzer has a cycle time T. T may include all overhead associated with the operation of the mass analyzer. The duration T may include the time required to accumulate ion packets (and optionally cool and / or fragment those ions, and optionally inject those ions into the mass analyzer) and the time required to mass analyze those ions. Alternatively, the duration T may correspond only to the time required to mass analyze the ion packets, or only to the time required to accumulate ion packets (and optionally cool and / or fragment those ions), where the accumulation of ion packets is performed in parallel with the mass analysis of previously accumulated ion packets.

[0030] In certain embodiments, the mass analysis scan duration T is less than the ion mobility separation scan duration T IMS , that is, T <T IMS In an embodiment, the mass analyzer is of a type in which the mass analyzer scan time T is relatively long, for example, such that two or more, tens or hundreds of mass analysis scans may be performed during each ion mobility separation scan. The duration T may be on the order of a few milliseconds, tens of milliseconds, or hundreds of milliseconds. For example, the duration T may be: (i) ≥ 1 ms; (ii) ≥ 2 ms; (iii) ≥ 5 ms; (iv) ≥ 10 ms; (v) ≥ 50 ms; or (vi) ≥ 100 ms. The duration T may be constant in any given experiment, but may vary between experiments by appropriate control of the instrument.

[0031] The analytical instrument may be capable of operating in at least an MS1 ​​mode of operation in which the instrument performs one or more MS1 mass analysis scans and in an MS2 mode of operation in which the instrument performs one or more MS2 mass analysis scans.

[0032] In each MS1 mass analysis scan, the mass filter is operated in its transmission mode or its filtering mode with a relatively wide isolation window width (e.g., on the order of hundreds or thousands of Th), and the ions are not (intentionally) fragmented, so that ions in a relatively wide m / z range generated by the ion source are received by the mass analyzer and mass analyzed.

[0033] In each MS2 mass analysis scan, the mass filter is operated in its filtering mode with a relatively narrow (e.g., about one or tens of Th) isolation window width to isolate ions, and the isolated ions are fragmented in a fragmentation device, so that ions in a relatively narrow m / z range generated by the ion source are isolated and fragmented, and the resulting fragment ions are received by the mass analyzer and mass analyzed.

[0034] Each MS1 mass analysis scan may have a duration T MS1 , and each MS2 mass analysis scan may have a duration T MS2 Usually, T MS1 >T MS2 , because high resolution data is relatively more important for MS1 scans, while high speed is relatively more important for MS2 scans (e.g., so that many more MS2 scans can be collected per unit time). In the case where the instrument is operated in a cyclic manner, typically T MS2 Can be set to T MS1 A fraction of MS2 / T MS1 =1 / 2, 1 / 4, 1 / 8, 1 / 16, etc.

[0035] In this method, an ion mobility separator performs a plurality of ion mobility separation scans, and during each ion mobility separation scan, the center m / z of the isolation window of the mass filter is scanned (changed).

[0036] In some embodiments, this is done so that ions of a particular charge state are transmitted by the mass filter. That is, in each ion mobility separation scan, ions of a particular charge state may be preferentially transmitted by the mass filter compared to ions of any other charge state. For example, only ions of a particular charge state may be transmitted and / or most or all ions of other charge states may be attenuated by the mass filter.

[0037] To this end, the center m / z of the isolation window of the mass filter can be scanned along a curve or a set of curve segments in the ion mobility arrival time-m / z space so that ions with a particular charge state are transmitted by the mass filter. The curve or the set of curve segments can be defined relative to the known relationship between the ion mobility arrival time and the m / z of ions of a particular charge state.

[0038] In this regard, it has long been recognized that for ions of various different charge states (e.g., singly, doubly, triply, etc.), there is a relationship between ion mobility arrival time and m / z. These relationships can take the form of different bands for each different charge state. Each band follows a characteristic curve in ion mobility arrival time-m / z space and has a characteristic (e.g., average) spread in ion mobility arrival time, Tb Therefore, the relationship between the ion mobility arrival time and the m / z of an ion with a particular charge can be described by a curve or a set of curve segments in the ion mobility arrival time-m / z space and a characteristic extension of the arrival time T b Thus, one or more curves or one or more groups of curve segments (and one or more characteristic arrival time extensions T b ), where each curve or group of curve segments (and each characteristic arrival time spread T b ) corresponds to a specific ionic charge state.

[0039] As described in more detail in co-pending application US 63 / 468,170, the entire contents of which are incorporated herein by reference, the one or more curves or one or more groups of curve segments (and the one or more characteristic arrival time spreads T b ) can be determined by performing a calibration on the instrument.

[0040] During each ion mobility separation scan, the width Δmz of the isolation window of the mass filter is controlled so that during each ion mobility separation scan, ions emerging from the ion mobility separator within the ion mobility arrival time range ΔT are transmitted by the mass filter.

[0041] In some embodiments, the width Δmz of the isolation window of the mass filter, and thereby the ion mobility arrival time range ΔT, is controlled so that during each ion mobility separation scan, (only) ions with a particular charge state are transmitted by the mass filter. To this end, the width Δmz can be configured such that the ion mobility arrival time range ΔT is less than the characteristic (e.g., average) spread T of the bands corresponding to ions with a particular charge state. b , that is, ΔT <T b .

[0042] Accordingly, for a given mobility separation time T and a given sample, Δmz may be significantly smaller than the spread of m / z of the analytes of the sample at that time T, ie, such that only a fraction of the singly charged states are selected per scan.

[0043] Thus, in each ion mobility separation scan, the mass filter is controlled to transmit ions in "bands" of two-dimensional (2D) ion mobility arrival time-m / z space, where each band optionally tracks a particular ion charge state band and has an average ion mobility arrival time spread T in the ion mobility arrival time dimension that is less than that band's average ion mobility arrival time spread T b The range of ΔT.

[0044] In addition, the width Δmz of the isolation window of the control filter is such that ΔT is less than the duration T of the mass analysis scan, i.e., ΔT < T. In this regard, the method is particularly suitable for instruments in which the mass analyzer is a relatively "slow" mass analyzer, i.e., in which the mass analyzer does not provide sufficient time resolution to be compatible with ion mobility separation. In this method, time resolution is provided by controlling the quadrupole filter. In fact, in each ion mobility separation scan, ions corresponding to a "strip" in the two-dimensional (2D) ion mobility arrival time - m / z space are analyzed, where each strip has a range ΔT in the ion mobility arrival time dimension equal to the required time resolution.

[0045] In some embodiments, by performing multiple such ion mobility separation scans in which ions from adjacent strips in the 2D ion mobility arrival time - m / z space are analyzed, ions within each band of interest in the 2D ion mobility arrival time - m / z space can be analyzed in a particularly direct and efficient manner.

[0046] Thus, in some embodiments, the method includes performing multiple ion mobility separation scans with respect to a particular ion charge state band, where in each ion mobility separation scan, different strips from that band are analyzed.

[0047] Thus, the multiple ion mobility separation scans can include at least a first ion mobility separation scan and a second ion mobility separation scan, and the method can include:

[0048] During the first ion mobility separation scan: scanning the center mass-to-charge ratio of the isolation window along a first curve or a first set of curve segments in the ion mobility arrival time - m / z space such that ions having a first charge state are transmitted by the filter; and

[0049] During the second ion mobility separation scan: scanning the center mass-to-charge ratio of the isolation window along a second different curve or a second set of curve segments in the ion mobility arrival time - m / z space such that ions having the same first charge state are transmitted by the filter.

[0050] More generally, the multiple ion mobility separation scans can include a first set of K ion mobility separation scans, where K is an integer ≥ 2, and the method can include:

[0051] During each of the K ion mobility separation scans: scanning the center mass-to-charge ratio of the isolation window along a corresponding curve or a corresponding set of curve segments in the ion mobility arrival time - m / z space such that ions having a first charge state are transmitted by the filter.

[0052] Multiple strips for a particular charge state may cover most of all bands of interest in the 2D ion mobility arrival time-m / z space. Multiple strips may be adjacent to each other and may cover the bands of interest without leaving any gaps. Multiple strips may overlap and / or not overlap.

[0053] Thus, most or all of the second curve or second set of curve segments may be separated from the first curve or first set of curve segments by an offset in ion mobility arrival times, wherein the offset in ion mobility arrival times may correspond to (i.e., be equal to or approximately equal to) the ion mobility arrival time range ΔT. Similarly, for each of the K corresponding curves or K sets of curve segments, most or all of the curve or set of curve segments may be separated from an adjacent curve or an adjacent set of curve segments by an offset in ion mobility arrival times, wherein the offset in ion mobility arrival times may correspond to (i.e., be equal to or approximately equal to) the ion mobility arrival time range ΔT.

[0054] The second curve or second set of curve segments may have substantially the same shape in the ion mobility arrival time-m / z space as the first curve or first set of curve segments, and / or all K curves or all sets of curve segments may have substantially the same shape in the ion mobility arrival time-m / z space. One or more shapes of the curves or curve segments may be configured to correspond to the shape of the charge state band of interest.

[0055] In certain embodiments, the product (K×ΔT), which may be approximately equal to the characteristic (eg, average) spread T of the band of interest, b ) is less than the duration T of each mass analysis scan. As described further below, this means that the information of interest can be acquired in a particularly efficient manner.

[0056] In an embodiment, during each ion mobility separation scan, the center m / z of the isolation window is scanned within the m / z range of interest. For different ion mobility separation scans, the m / z range of interest may be the same, or may be different for some or each ion mobility separation scan. The m / z range of interest or each m / z range of interest may be continuous. Alternatively, the m / z range of interest or each m / z range of interest may include a set of different and separated m / z sub-ranges. Therefore, each strip may be continuous or discontinuous.

[0057] In some embodiments, during one or more or each ion mobility separation scan, the center m / z of the isolation window is scanned substantially continuously and / or smoothly. For example, the center m / z of the isolation window can be scanned continuously and / or smoothly over (at least) the m / z range of interest. However, in the case of a mass filter with digital control, the scan can be approximately continuous and / or approximately smooth, since the voltage on the mass filter will actually be changed in (small) steps.

[0058] For each stripe, during the corresponding mobility separation scan, the center m / z of the isolation window of the mass filter can be scanned along a curve or a set of curve segments in the ion mobility arrival time-m / z space, so that the ions in the "strip" of the two-dimensional (2D) ion mobility arrival time-m / z space are transmitted and analyzed. The curve or the group of curve segments can be configured to provide a one-to-one (bijective) relationship between the ion mobility arrival time and m / z. Therefore, in some embodiments, during one or more or each ion mobility separation scan, the center m / z of the isolation window is bijectively scanned within the m / z range of interest (i.e., using a one-to-one relationship). Similarly, in some embodiments, the curve may have or the group of curve segments may each have a positive (or negative) derivative at all points along the curve or the group of curve segments.

[0059] As discussed above, in each ion mobility separation scan, the width Δmz of the isolation window of the mass filter is controlled so that ions emerging from the ion mobility separator within the ion mobility arrival time range ΔT are transmitted by the mass filter. The width Δmz of the isolation window of the mass filter can be controlled so that at some, most, or all of the time during the ion mobility separation scan, ions emerging from the ion mobility separator within the ion mobility arrival time range ΔT are transmitted by the mass filter. For example, the width Δmz of the isolation window of the mass filter can be controlled so that at least when the center m / z of the isolation window is scanned within the m / z range of interest (e.g., at all times), ions emerging from the ion mobility separator within the ion mobility arrival time range ΔT are transmitted by the mass filter.

[0060] In some embodiments, during each ion mobility separation scan, e.g., at least during a portion of the ion mobility separation scan, ΔT remains substantially constant as the center m / z of the isolation window is scanned within the m / z range of interest. Thus, the method may include, during one or more or each ion mobility separation scan: controlling a width Δmz of the isolation window such that the ion mobility separation arrival time range ΔT remains substantially constant as the center m / z of the isolation window is scanned within the m / z range of interest.

[0061] Alternatively, ΔT may vary during the ion mobility separation scan, for example in a predetermined manner. Thus, the method may comprise during one or more or each ion mobility separation scan: (ii) controlling the width Δmz of the isolation window such that the ion mobility separation arrival time range ΔT varies when the center m / z of the isolation window is scanned over the m / z range of interest.

[0062] In some embodiments, ΔT is configured to be the same between different ion mobility separation scans.Thus, the method may include controlling the width Δmz of the isolation window such that the ion mobility separation arrival time range ΔT is the same during two or more or all ion mobility separation scans.

[0063] Alternatively, ΔT may vary between different ion mobility separation scans, for example in a predetermined manner.Thus, the method may comprise controlling the width Δmz of the isolation window such that the ion mobility separation arrival time range ΔT varies between two or more ion mobility separation scans.

[0064] In certain embodiments, each ion mobility separation scan has a duration T IMS , and the method includes controlling the width Δmz of the isolation window during each ion mobility separation scan according to the following equation:

[0065]

[0066] The ion mobility separation arrival time range ΔT may have a duration of: (i) ≤ 0.5 ms; (ii) ≤ 1 ms; (iii) ≤ 2 ms; (iv) ≤ 5 ms; (v) ≤ 10 ms; or (vi) ≤ 50 ms.

[0067] In some embodiments, only a single charge state band needs to be analyzed. However, it is also possible to analyze multiple different charge state bands in the same manner as described above for a single charge state.

[0068] Thus, the multiple ion mobility separation scans may include a second set of K 2 Secondary ion mobility separation scan, where K 2 is an integer ≥ 2, and the method may include:

[0069] In K 2 During each ion mobility separation scan in the secondary ion mobility separation scan: the center m / z of the isolation window is scanned along a corresponding curve or a corresponding set of curve segments in the ion mobility arrival time-m / z space so that ions with a second different charge state are transmitted by the mass filter.

[0070] The second group K 2The K secondary ion mobility separation scans can be configured in a similar manner to the first set of K secondary ion mobility separation scans, except that the second set of K 2 The secondary ion mobility separation scan can target different charge state bands.

[0071] So, for example, for K 2 The corresponding curves or K 2 Each of the group of curve segments, most or all of the curve or the group of curve segments may be separated from an adjacent curve or an adjacent group of curve segments by an offset in ion mobility arrival time, wherein the offset in ion mobility arrival time may correspond to (i.e., be equal to or approximately equal to) the ion mobility arrival time range ΔT. K 2 curve or K 2 Most or all of the curve segments in the group may have substantially the same shape in the ion mobility arrival time-m / z space, and the product (K 2 ×ΔT) may be less than the duration T of each mass analysis scan.

[0072] In these embodiments, the first charge state can be one of the following: (i) singly charged ions; (ii) doubly charged ions; (iii) triply charged ions; (iv) quadruple charged ions; and (iv) more than quadruple charged ions. The second charge state can be one of the following: (i) singly charged ions; (ii) doubly charged ions; (iii) triply charged ions; (iv) quadruple charged ions; and (iv) more than quadruple charged ions, wherein the second charge state is a different charge state from the first charge state.

[0073] Similarly, a third charge state band or additional charge state bands may be analyzed in a similar manner.

[0074] In some embodiments, the intensity of one or more or each band may be adjusted, for example, to prevent detector saturation and / or space charge effects for a particular band and / or to improve the dynamic range of the overall analysis. For example, where it is known that a particular band will contain ions with a relatively high abundance, the intensity of the band may be reduced (and / or where it is known that a particular band will contain ions with a relatively low abundance, the intensity of the band may not be reduced or may be reduced by a smaller amount). Thus, the method may include, during one or more or each ion mobility separation scan: adjusting (e.g., reducing) the intensity of ions transmitted by the mass filter based on the expected intensity of these ions.

[0075] The intensity of the ions may be adjusted in any suitable manner, for example using the techniques described in GB 2,585,472, which is incorporated herein by reference. Thus, for example, the attenuation device may be arranged downstream (or upstream) of the mass filter and may be configured to attenuate the ions, for example by rapid pulse width modulation at a rate much less than ΔT. A constant attenuation factor may be used for the entirety of each strip, and / or the attenuation factor may be varied across the strip in a predetermined manner.

[0076] Although, as described above, in certain embodiments, each curve or group of curve segments is shaped based on the shape of the charge state band of interest, it is also possible to determine the shape of part or all of a curve or group of curve segments based on one or more other factors.

[0077] For example, in some embodiments, a targeted analysis method can be used. In these embodiments, the curve or the group of curve segments about the strip can be configured to include (i.e., pass through) ions of one or more isotope-labeled internal standards in the sample. For example, the curve or the group of curve segments can be configured to include (pass through) ions of multiple isotope-labeled internal standards of interest in the sample. Then, during the ion mobility separation scan, when the internal standard of interest is detected, a targeted scan for the corresponding target analyte can be activated.

[0078] Thus, the method may include:

[0079] adding one or more internal standards of one or more target analytes to the sample;

[0080] ionizing the sample and internal standard to produce ions;

[0081] During a first ion mobility separation scan: (i) scanning the central mass-to-charge ratio (m / z) of the isolation window such that ions of the internal standard are transmitted by the mass filter; and

[0082] determining whether ions of the internal standard are detected in the mass analysis scan;

[0083] Wherein when it is determined that ions of the internal standard are detected in the mass analysis scan, the method further comprises performing one or more target scans, each target scan having a target isolation window including an m / z representative of the target analyte.

[0084] For example, a target scan for a target analyte may be performed during a second ion mobility separation scan, which may be an ion mobility separation scan immediately following a first ion mobility separation scan. In some embodiments, the curve or set of curve segments for the second ion mobility separation scan may be locally deformed to include (pass through) the target analyte.

[0085] In some embodiments, the curve or set of curve segments for a strip may be configured to include (i.e., pass through) different charge states of the same molecule. Thus, the method may include: during an ion mobility separation scan: (i) scanning the central mass-to-charge ratio (m / z) of the isolation window such that ions of the same analyte having a plurality of different charge states are transmitted by the mass filter. Multiple such ion mobility separation scans may be performed, wherein each ion mobility separation scan is for a different analyte.

[0086] Another aspect provides a non-transitory computer readable storage medium storing computer software code which, when executed on a processor, performs the method described above.

[0087] Another aspect provides a control system for an analytical instrument such as a mass spectrometer, the control system being configured to cause the analytical instrument to perform the method described above.

[0088] Another aspect provides an analytical instrument, such as a mass spectrometer, comprising a control system as described above.

[0089] Another aspect provides an analytical instrument such as a mass spectrometer, the analytical instrument comprising:

[0090] an ion mobility separator, wherein the ion mobility separator is configured to perform a plurality of ion mobility separation scans, wherein in each ion mobility separation scan, the ion mobility separator receives ions and separates the ions according to their ion mobility;

[0091] a mass filter disposed downstream of the ion mobility separator; and

[0092] a mass analyzer disposed downstream of the mass filter, wherein the mass analyzer is configured to perform a mass analysis scan, wherein in each mass analysis scan, the mass analyzer receives ions and performs mass analysis on the ions; and

[0093] A control system, the control system being configured to:

[0094] causing the instrument to perform multiple ion mobility separation scans;

[0095] During each ion mobility separation scan, causing the mass filter to: (i) scan the central mass-to-charge ratio (m / z) of the isolation window, and (ii) controlling the width Δmz of the isolation window so that during the ion mobility separation scan, ions emerging from the ion mobility separator within the ion mobility arrival time range ΔT are transmitted by the mass filter; and

[0096] causing the mass analyzer to perform one or more mass analysis scans during each ion mobility separation scan, wherein each mass analysis scan has a duration T, and wherein ΔT <T。

[0097] These aspects and embodiments may be combined with, and in embodiments with, any one or more or each of the aspects, embodiments and / or optional features described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] Various embodiments will now be described in more detail with reference to the accompanying drawings, in which:

[0099] Figure 1 Exemplifying known methods;

[0100] Figure 2 schematically illustrates a mass spectrometer operable in accordance with an embodiment;

[0101] Figure 3 A method according to an embodiment is illustrated; and

[0102] Figure 4 The method according to the embodiment is illustrated. DETAILED DESCRIPTION

[0103] Embodiments utilize a "slow" mass analyzer to improve the resolution of IMS determinations and reduce the analysis time required to create a 2D mobility-m / z map.

[0104] This is achieved by realizing that due to the strong correlation between ion mobility and m / z, only a small fraction of the 2D mobility-m / z space is actually occupied by the ions of interest (typically <10% to 20%). In addition, for many analytes of interest, especially in metabolomics, only one charge state needs to be analyzed, typically a singly or doubly charged ion. This means that ions typically occupy a relatively narrow band with an ion mobility arrival time width Tb(m / z), where in some cases Tb <T。

[0105] Embodiments provide an optimization method similar to the linked scanning method, where the discrete changes in the central m / z of the quadrupole are minimized by matching the mass selection to the desired mobility time resolution ΔT.

[0106] Figure 2 Schematically illustrates analytical instruments such as a mass spectrometer (MS) that can be operated according to the embodiments. Figure 2 As shown, the instrument includes an ion source 10 , an ion mobility (IM) separator 20 (such as a high resolution ion mobility separator (hrIMS)), a mass filter 30 , a fragmentation device 40 and a mass analyzer 50 .

[0107] 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 ion source, and an atmospheric pressure ionization (API) ion source, a plasma ion source, an electron ionization ion source, a chemical ionization ion source, etc. More than one ion source can be provided and used. The ions can be any suitable type of ions to be analyzed, such as small and large organic molecules, biomolecules, DNA, RNA, proteins, peptides, fragments thereof, etc.

[0108] The ion source 10 may be coupled to a separation device, such as a liquid chromatography separation device or a capillary electrophoresis separation device (not shown), so that the sample ionized in the ion source 10 is from the separation device.

[0109] Ion mobility separator 20 is arranged at the downstream of ion source 10, and is configured to receive ions from ion source 10. Ion mobility separator 20 is configured to separate the received ions according to the ion mobility of the received ions. Ion mobility separator 20 may include any suitable type of ion mobility separator. For example, electric fields such as DC voltage gradient and / or traveling DC voltage waves may be arranged to promote ions along the length of separator and make ions pass through gas so that ions are separated according to their ion mobility. Optionally facing the countercurrent of gas or perpendicular to the countercurrent of gas promotes ions. Alternatively, airflow may be arranged to promote ions along the length of separator, and electric fields such as DC voltage gradient and / or traveling DC voltage waves may be arranged to be opposite to airflow so that ions are separated according to their ion mobility. Ion mobility separator 20 may be a linear separator or circulation (closed loop) separator with straight or folded path.

[0110] The mass filter 30 is arranged downstream of the ion mobility separator 20 and is configured to receive ions from the ion source 10 (via the ion mobility separator 20). The mass filter 30 is configured to filter the received ions according to the mass-to-charge ratio (m / z) of the received ions. The mass filter 30 can be configured so that the received ions with m / z within the m / z isolation window (or "transmission window") of the mass filter are forwarded by the mass filter, while the received ions with m / z outside the m / z isolation window are attenuated by the mass filter, for example, not forwarded by the mass filter. The width and / or center m / z of the isolation window are controllable (variable), for example, by appropriately controlling the RF voltage and DC voltage applied to the mass filter 30. Thus, for example, the mass filter 30 may be capable of operating in a transmission mode of operation, whereby most or all ions within a relatively wide m / z window are transmitted forwardly by the mass filter 30, and may be capable of operating in a filtering mode of operation, whereby only ions within a relatively narrow m / z window (centered about a desired m / z) are transmitted forwardly by the mass filter 30. The mass filter 30 may be any suitable type of mass filter, such as a quadrupole mass filter.

[0111] The fragmentation device 40 is arranged downstream of the mass filter 30 and is configured to receive most or all of the ions transmitted by the mass filter 30. The fragmentation device 40 can be configured to selectively fragment some or all of the received ions, i.e., to produce fragment ions. The fragmentation device 40 can be operated in a fragmentation operating mode, whereby most or all of the received ions are fragmented to produce fragment ions (which can then be transmitted forward from the fragmentation device 40), and can be operated in a non-fragmentation operating mode, whereby most or all of the received ions are transmitted forward without being (intentionally) fragmented. It is also possible to achieve a non-fragmentation operating mode by causing the ions to bypass the fragmentation device 40. The fragmentation device 40 can also be operated in one or more intermediate operating modes, for example, whereby the degree of fragmentation is controllable (variable).

[0112] The fragmentation device 40 may be any suitable type of fragmentation device, such as, for example, a collision induced dissociation (CID) fragmentation device, an electron induced dissociation (EID) fragmentation device, a photodissociation fragmentation device, etc. Many other types of fragmentation are possible.

[0113] The mass analyzer 50 is arranged downstream of the ion mobility separator 20 and is configured to receive ions from the ion source 10 (via the ion mobility separator 20 and the mass filter 30, and optionally via the fragmentation device 40). The mass analyzer 50 is configured to analyze the received ions to determine their mass-to-charge ratio and / or mass, i.e., to generate a mass spectrum of the ions. The mass analyzer 50 can be an ion trap mass analyzer, such as an electrostatic orbital trap mass analyzer, and more specifically an Orbitrap TM FT mass analyzer.

[0114] Thus, the mass analyser 50 may comprise an inner electrode extending along the axis of the orbital trap and a pair of separated outer electrodes, the pair of outer electrodes surrounding the inner electrode and defining a capture volume therebetween, in which ions are captured and oscillated by orbiting around the inner electrode, to which a capture voltage is applied while oscillating back and forth along the axis of the trap. The pair of outer electrodes serve as detection electrodes to detect image currents caused by the oscillation of ions in the capture volume and thereby provide a detected signal. The outer electrodes are typically used as a differential detection electrode pair and are coupled to respective inputs of a differential amplifier, which in turn forms part of a digital data acquisition system to receive the detected signal. The detected signal may be processed using a Fourier transform to obtain a mass spectrum of the ions within the trap.

[0115] Alternatively, the mass analyser 50 may be a time of flight (ToF) mass analyser, such as a multi-reflection time of flight (mrToF) mass analyser.

[0116] It should be noted that Figure 2 These are merely illustrative, and the apparatus may, and in embodiments does, include any number of one or more additional components.

[0117] For example, the instrument may include one or more ion transfer stages or ion capture stages, for example, arranged between the various shown devices. The one or more ion transfer stages may include, for example, an atmospheric pressure interface and / or one or more ion guides, lenses and / or other ion optical devices, which are configured so that ions can be transferred between the various shown devices. The ion transfer stage may include any suitable number and configuration of ion optical devices, for example, optionally including one or more RF ion guides and / or multipole ion guides, one or more ion guides for cooling ions, one or more mass selective ion guides, etc.

[0118] like Figure 2 As also shown, the instrument is under the control of a control unit 60 (such as a suitably programmed computer) which controls the operation of the various components of the instrument. The control unit 60 may also receive and process data from the various components, including the analyzer 50. The control unit 60 is particularly configured to determine the settings for the ion mobility separator 20, the mass filter 30, the fragmentation device 40 and the mass analyzer 50 for the analytical scan.

[0119] For example, the control system 60 may cause the instrument to perform one or more MS1 scans, wherein in each MS1 scan the mass filter 30 is operated in its transmission mode or in its filtering mode and the ions are not (intentionally) fragmented, such that ions over a wide m / z range produced by the ion source 10 are mass analyzed by the mass analyzer 50.

[0120] The control system 60 can also cause the instrument to perform one or more MS2 scans, in which, in each MS2 scan, the mass filter 30 operates in its filtering mode and ions are fragmented in the fragmentation device 40, so that ions in a narrow m / z range generated by the ion source 10 are selected and fragmented, and the resulting fragment ions are mass analyzed by the mass analyzer 50.

[0121] Thus, in an embodiment, ions are formed by the ion source 10 and supplied to the hrIMS device 20, which may have a capture and release region to provide a 100% duty cycle for collecting ions from the source 10. There may be an ion gate and / or additional mass filters before the hrIMS device 20 to control the total number of ions therein. When ions are eluted from the hrIMS device 20, they are filtered by the quadrupole mass filter 30 according to their m / z, and after optional fragmentation in the collision cell 40, they are detected by the mass analyzer 50. The mass analyzer 50 can be an Orbitrap TM mass analyzer, an FT ICR mass analyzer, or any type of ToF or mrToF mass analyzer. The mass analyzer can be integrated with an ion storage device to collect and buffer all ions leaving the quadrupole mass filter 30.

[0122] In operation, for each scan of the hrIMS device 20, the quadrupole 30 follows a specific smooth line or curve T IMS (m / z) on the 2D mobility-m / z plot, such that at each point on the curve, the width Δmz of the isolation window of the quadrupole mass filter is defined by the desired mobility time resolution ΔT as:

[0123]

[0124] Using the hard sphere approximation, the relationship between the ion mobility μ and the mass m and charge z of the ion is: μ ∼ z / m 2 / 3 and T IMS ∼ 1 / μ, so the equation can be rewritten as:

[0125]

[0126] For the mobility band with an average spread of the mobility time Tb, K = Tb / ΔT steps will be taken to create a 2D mobility-m / z plot of the compound classes of interest (e.g., lipids, glycans, nucleotides, or peptides, etc. with a specific charge state). Since Tb can be established during method development or calibration, it can be selected to be substantially less than the analysis duration T, so K < L and less time is required to cover the region of interest with high mobility resolution.

[0127] ΔT and therefore Δmz may also or alternatively depend on m / z.

[0128] As long as the curve T IMS The derivative of (m / z) remains completely positive (or completely negative), the curve can have a change in shape (hence the IMS resolution is encoded in m / z), and its smoothness ensures that quadrupole filtering can accurately follow the curve without tailing, ion loss or mobility time gaps. The quadrupole mass filter 30 may be capable of scanning at speeds >1000 Th / sec or >10,000 Th / sec.

[0129] As described above, a set of adjacent curves / fringe can be collected in this manner. When the entire set of adjacent curves / fringe is collected to cover the region of interest, all known interpolation and data extraction methods can be used. For example, if the isotopic distribution of a particular analyte is spread over 2 to 3 adjacent fringes, this can be used to determine the ion mobility with a higher accuracy than ΔT, thereby potentially achieving a resolving power of mobility>500.

[0130] It should be understood that the embodiments provide for the use of coupled hrIMS quadrupole scanning with a narrow and smoothly varying mass selection window to improve the performance of the system. The embodiments increase the acquisition speed of 2D mobility-m / z spectra when a slow mass analyzer is used for detection, and especially when only a specific class of compounds is analyzed.

[0131] While various specific embodiments have been described above, various alternative embodiments are possible.

[0132] For example, a non-standard mass filter (eg, a mass filter having several narrow stopband filters within its transmission mass window) can be used to acquire not one band, but two or three or more bands in parallel, further increasing acquisition speed.

[0133] The method can be used for both data dependent acquisition and data independent acquisition (DDA and DIA, respectively).

[0134] This approach is particularly suitable for targeted analysis, where the curve is shaped during method development in such a way that it passes through all isotopically labeled internal standards in the mixture. The analysis is then performed along this curve. Once one or more internal standards are detected, the targeted analysis of the corresponding analyte can be activated. This can also be done by locally distorting the curve to pass over the target compound ( Figure 3 ).

[0135] This method can also be used with a pulsed ion source such as MALDI to focus on specific low-abundance components. This is useful when T>T IMS maxThis is particularly true for the case of fast IMS, but also works well for lossless ion manipulation (SLIM), trapped ion IMS, and cyclic IMS configurations, where T IMS max It can reach hundreds of milliseconds or even seconds.

[0136] Will Figure 1 Methods and Figure 3 A combination of methods may be beneficial. For example, a prior art method may be run at a lower resolution (larger ΔT) to give a coarse but panoramic 2D map, and then Figure 3 The method can be used to obtain higher resolution mobility information for specific regions of interest depending on the specific analytical problem (e.g., for separation of isomers, etc.).

[0137] The method may involve scanning the center m / z of the isolation window over a continuous m / z range (e.g. Figure 3 ), but may alternatively involve scanning the center m / z of the isolation window within a set of different and separate m / z sub-ranges. This may allow, for example, high dynamic range scanning, target multiplexing analysis, selection of different charge states of the same multi-charged analyte, etc.

[0138] In some embodiments, the intensity through the transmission window may be adjusted based on its (a priori measured) intensity.The intensity of the ions may be adjusted in any suitable manner, for example using the technique described in GB 2,585,472, ie rapid pulse width modulation at a rate much smaller than ΔT.

[0139] Although, as described above, in various embodiments each curve is shaped based on the shape of the charge state band of interest, it is also possible to determine the shape of some or all of the curves based on one or more other factors.

[0140] For example, in some embodiments, the method may include separating multiple charges together. IMS This is particularly effective when T is equal to or less than T. In this case, different charge states z of the same molecule (e.g., protein) can be separated and analyzed together because they appear at different m / z and different mobility arrival times.

[0141] Figure 4 An example of selecting multiple charge states of the same analyte by coupled quadrupole / IMS scanning according to this method is illustrated. Figure 4 In FIG. 1 , the double dashed line and the solid line show two different analytes. The analyte of interest can be gated for selection or collection with other analytes.

[0142] In these embodiments, for each analyte, (m / z)*z=m is constant, but the ion mobility arrival time depends on the collision cross section of the charge state. This can be known a priori or by calibration. Thus, Figure 4 As shown, during each ion mobility separation scan, the center m / z of the isolation window can be scanned within a plurality of different charge state bands, so that ions of the same analyte (e.g., the same protein) having a plurality of different charge states are transmitted by the mass filter. Multiple such ion mobility separation scans can be performed, wherein each ion mobility separation scan is for a different analyte (e.g., a different protein).

[0143] Although the invention has been described with reference to various embodiments, it should be understood that various changes may be made without departing from the scope of the invention as set forth in the following claims.

Claims

1. A method of operating an analytical instrument, the analytical instrument comprising: Ion mobility separators; a mass filter disposed downstream of the ion mobility separator; and a mass analyzer disposed downstream of the mass filter, the method comprising: The ion mobility separator performs a plurality of ion mobility separation scans, wherein in each ion mobility separation scan, the ion mobility separator receives ions and separates the ions according to their ion mobility; The mass filter uses an isolation window to filter the separated ions, and during each ion mobility separation scan: (i) scans the central mass-to-charge ratio (m / z) of the isolation window, and (ii) controls the width Δmz of the isolation window so that during the ion mobility separation scan, ions emerging from the ion mobility separator within an ion mobility arrival time range ΔT are transmitted by the mass filter; and The mass analyzer performs one or more mass analysis scans during each ion mobility separation scan, wherein in each mass analysis scan, the mass analyzer receives and analyzes ions transmitted by the mass filter or ions derived from the ions transmitted by the mass filter, wherein each mass analysis scan has a duration T, and wherein ΔT <T。 2. The method of claim 1, wherein the plurality of ion mobility separation scans comprises at least a first ion mobility separation scan and a second ion mobility separation scan, and wherein the method comprises: During the first ion mobility separation scan: scanning the central mass-to-charge ratio of the isolation window along a first curve or a first set of curve segments in ion mobility arrival time-m / z space such that ions having a first charge state are transmitted by the mass filter; and During the second ion mobility separation scan: the central mass-to-charge ratio of the isolation window is scanned along a second different curve or a second set of curve segments in the ion mobility arrival time-m / z space so that ions having the same first charge state are transmitted by the mass filter.

3. The method of claim 2, wherein most or all of the second curve or second set of curve segments are separated from the first curve or first set of curve segments by a shift in ion mobility arrival times. The method of claim 3 , wherein the shift in ion mobility arrival times corresponds to the ion mobility arrival time range ΔT.

5. The method of claim 2, 3 or 4, wherein the second curve or second set of curve segments has substantially the same shape in the ion mobility arrival time-m / z space as the first curve or first set of curve segments.

6. The method of any one of the preceding claims, wherein the plurality of ion mobility separation scans comprises a first set of K ion mobility separation scans, and wherein the method comprises: During each of the K ion mobility separation scans: scanning the central mass-to-charge ratio of the isolation window along a corresponding curve or a corresponding set of curve segments in the ion mobility arrival time-m / z space so that ions having a first charge state are transmitted by the mass filter; Wherein for each of the K respective curves or groups of curve segments, most or all of the curve or group of curve segments are separated from an adjacent curve or group of curve segments by a shift in ion mobility arrival times.

7. The method according to claim 6, wherein: The shift in ion mobility arrival time corresponds to the ion mobility arrival time range ΔT; and / or Most or all of the K curves or K groups of curve segments have substantially the same shape in the ion mobility arrival time-m / z space.

8. The method of claim 6 or 7, wherein the product (K x ΔT) is less than the duration T of each mass analysis scan.

9. The method of any one of claims 6 to 8, wherein the plurality of ion mobility separation scans comprises a second set of K2 ion mobility separation scans, and wherein the method comprises: During each ion mobility separation scan in the K2 ion mobility separation scan: scanning the central mass-to-charge ratio of the isolation window along a corresponding curve or a corresponding set of curve segments in the ion mobility arrival time-m / z space so that ions with a second different charge state are transmitted by the mass filter; Wherein for each of K2 respective curves or K2 groups of curve segments, most or all of the curve or the group of curve segments are separated from an adjacent curve or an adjacent group of curve segments by a shift in ion mobility arrival times.

10. The method according to any one of claims 2 to 9, wherein: The first charge state is one of: (i) a singly charged ion; (ii) a doubly charged ion; (iii) a triply charged ion; (iv) a quadruple charged ion; and (iv) more than quadruple charged ions; and / or The second charge state is one of: (i) a singly charged ion; (ii) a doubly charged ion; (iii) a triply charged ion; (iv) a quadruple charged ion; and (iv) more than quadruple charged ions, and the second charge state is a different charge state from the first charge state.

11. The method of any one of the preceding claims, wherein the plurality of ion mobility separation scans comprises at least a first ion mobility separation scan, and wherein the method further comprises: adding one or more internal standards of one or more target analytes to the sample; ionizing the sample and the internal standard to generate ions; as well as During the first ion mobility separation scan: (i) scanning the central mass-to-charge ratio (m / z) of the isolation window so that ions of the internal standard are transmitted by the mass filter, and (ii) determining whether ions of the internal standard are detected in the mass analysis scan; Wherein when it is determined that ions of the internal standard are detected in the mass analysis scan, the method further comprises performing one or more target scans, each target scan having a target isolation window including an m / z representing the target analyte.

12. The method according to any one of the preceding claims, further comprising: During an ion mobility separation scan in the plurality of ion mobility separation scans: (i) scanning the central mass-to-charge ratio (m / z) of the isolation window so that ions of the same analyte having multiple different charge states are transmitted by the mass filter.

13. A method according to any one of the preceding claims, comprising: During one or more or each ion mobility separation scan: (i) scanning the center m / z of the isolation window over an m / z range of interest; wherein the m / z range of interest is continuous; or Wherein the m / z range of interest comprises a set of distinct and separate m / z sub-ranges.

14. The method according to claim 13, comprising: During one or more or each ion mobility separation scan: (i) the central m / z of the isolation window is scanned substantially smoothly and / or substantially continuously and / or bijectively over the m / z range of interest.

15. The method according to claim 13 or 14, comprising: During one or more or each ion mobility separation scan: (ii) controlling the width Δmz of the isolation window so that when the center m / z of the isolation window is scanned within the m / z range of interest, ions emerging from the ion mobility separator within the ion mobility arrival time range ΔT are transmitted by the mass filter.

16. The method according to claim 13, 14 or 15, comprising: During one or more or each ion mobility separation scan: (ii) controlling the width Δmz of the isolation window so that the ion mobility separation arrival time range ΔT remains substantially constant when the center m / z of the isolation window is scanned over the m / z range of interest; and / or During one or more or each ion mobility separation scan: (ii) controlling the width Δmz of the isolation window such that the ion mobility separation arrival time range ΔT varies as the center m / z of the isolation window is scanned over the m / z range of interest.

17. The method according to any one of claims 13 to 16, comprising: controlling the width Δmz of the isolation window so that the ion mobility separation arrival time range ΔT is the same during two or more or all of the plurality of ion mobility separation scans; and / or The width Δmz of the isolation window is controlled such that the ion mobility separation arrival time range ΔT varies between two or more ion mobility separation scans of the plurality of ion mobility separation scans.

18. A method according to any one of the preceding claims, wherein each ion mobility separation scan has a duration T IMS , and wherein the method comprises controlling the width Δmz of the isolation window during each ion mobility separation scan according to the following equation:

19. The method of any of the preceding claims, further comprising, during an ion mobility separation scan in the plurality of ion mobility separation scans: adjusting the intensity of ions transmitted by the mass filter based on an expected intensity of the ions.

20. The method according to any one of the preceding claims, wherein the duration T is: (i)≥1ms; (ii)≥2ms; (iii)≥5ms; (iv)≥10ms; (v)≥50ms; Or (vi) ≥ 100ms.

21. The method according to any one of the preceding claims, wherein the ion mobility arrival time range ΔT is: (i) ≤ 0.5 ms; (ii) ≤ 1 ms; (iii) ≤ 2 ms; (iv) ≤ 5 ms; (v) ≤ 10 ms; or (vi) ≤ 50 ms.

22. The method of any one of the preceding claims, wherein the mass analyser is a Fourier transform (FT) mass analyser or a multi-reflection time-of-flight (mrTOF) mass analyser.

23. A non-transitory computer readable storage medium storing computer software code which, when executed on a processor, performs the method according to any one of the preceding claims.

24. A control system for an analytical instrument, such as a mass spectrometer, the control system being configured to cause the analytical instrument to perform a method according to any one of claims 1 to 22.

25. An analytical instrument, such as a mass spectrometer, comprising a control system according to claim 24.

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