Mass spectrometry method, method for manipulating ions using ion storage device, ion storage device, mass spectrometer and computer software

Abstract

The invention provides a mass spectrometry method. The method comprises, for each sub-range of a plurality of sub-ranges selected from a total m / z range, the steps of: injecting a sample of precursor ions into a first ion storage device via an access aperture region, the precursor ions having an m / z value within the sub-range; holding a first portion of the sample of precursor ions within the first ion storage device; and ejecting a second portion of the sample of precursor ions from the first ion storage device via an exit region.

Description

Field of the Invention

[0001] The field of the present invention is liquid chromatography mass spectrometry (LC-MS). Specifically, the present invention relates to tandem mass spectrometry in which precursor ions and fragment ions are analyzed. More specifically, the present invention relates to improving the dynamic range of MS1 scans in tandem mass spectrometry. The present invention relates specifically but not exclusively to advanced hybrid mass spectrometers having multiple analyzers. Background Art

[0002] Standard tandem liquid chromatography mass spectrometry (LC-MS) methods involve performing a "MS1" scan, in which ions having a wide m / z range are analyzed by a mass analyzer to produce a MS1 spectrum (containing information about precursor ions). Methods of operating an LC-MS also involve isolating and fragmenting ions from eluted analyte species to perform a "MS2" scan, in which ions within a narrow m / z range are isolated (e.g., using a quadrupole mass filter), those ions are fragmented, and the fragment ions are mass analyzed to produce a MS2 spectrum, which contains structural and quantitative information about the fragment ions.

[0003] In an LC-MS method, multiple MS2 scans are typically performed during chromatographic separation, where the m / z isolation range is different for each MS2 scan. The MS2 (or "MS / MS") spectrum is supported by a MS1 (or "MS" or "full MS") survey scan, which provides high quality peak information such as accurate mass data and precursor intensities of a wide range of unfragmented precursor ions.

[0004] For data independent acquisition (DIA) methods, the MS1 scan is optional and can be skipped to allow time to generate additional MS2 spectra. The list of m / z targets for each of the multiple MS2 scans can be a stepwise increasing / decreasing m / z list across the m / z range of interest. One such example is described in EP 3,410,463, which is incorporated herein by reference. The resulting precursor information can be used for quantification, while the fragment information can be used for identification.

[0005] In data dependent acquisition (DDA), an MS1 step is required to generate a list of precursor targets for MS2 analysis. The list of m / z targets for each of the multiple MS2 scans corresponds to the list of precursor ions identified in the MS1 scan.

[0006] To perform mass analysis of ions, the ions (fragment ions or precursor ions) are typically first accumulated in an ion trap and then the accumulated ions are ejected as a packet into a mass analyzer for mass analysis. Accumulation improves the sensitivity of the instrument, but care is required during the fill time of the ion trap for the ions (so-called "auto gain control" AGC) to avoid harmful space charge effects.

[0007] One problem with existing methods is that the dynamic range of the MS1 spectrum is limited. Depending on the sample, the concentration of digested peptides varies by more than 10 orders of magnitude, but in a single-shot spectrum in, for example, an electrostatic Orbitrap mass analyzer (such as an Orbitrap TM manufactured by Thermo Fisher Scientific TM FT mass analyzer), the dynamic range may be limited to approximately 4 orders of magnitude. In addition, the number of ions that can be injected into the Orbitrap mass analyzer is limited to approximately 10 5 by the capacity of the accumulation C-trap. Thus, low-intensity peptide precursor signals may be suppressed. For DDA experiments, these problems are significant and may lead to precursor targets being missed. For DIA experiments, the lack of good precursor data hinders identification and quantification.

[0008] One way to improve the dynamic range of the MS1 spectrum is the "Boxcar" method (as described in Meier et al., Nature Methods, 2018, Vol. 15, pp. 440-448) and the high dynamic range (HDR) method described in UK Patent Application No. 2211790.7, which is incorporated herein by reference. In these methods, the broad mass range being analyzed is subdivided into multiple narrower isolation windows. For each isolation window, a separate injection is made into the C-trap, with different fill times depending on the ion current. By this method, densely populated m / z regions are attenuated while sparsely populated m / z regions are amplified. Thus, the detection sensitivity for relatively weak peaks is improved, and the effective dynamic range of the scan is increased. However, the use of these methods significantly increases the time required to accumulate ions, especially when a large number of isolation windows are needed. Due to the long fill times for low-level windows and the time required to switch the quadrupole and ion source voltages, HDR scans may require a large amount of additional time and affect the rate at which MS2 scans can be performed. Thus, these methods can reduce the time available for MS2 scans in rapid LC-MS methods, which is limited by the time it takes for the sample to elute from the column.

[0009] The multi-window HDR method also has the disadvantage of discarding a large number of ions due to quadrupole isolation. The loss of ions can be addressed via a pre-accumulation process. For example, a trapped ion mobility device can be used to pre-accumulate ions before the quadrupole and release the ions in a mass / mobility-dependent manner synchronized with the mass filter, thus greatly reducing ion loss (as described in Meier et al., Molecular Cellular Proteomics, 2018, Vol. 17, pp. 2524-2545).

[0010] Differential mobility filtering can be used to improve proteomics performance by removing analytically less useful singly charged ions from the accumulated population (as described in Hebert et al., Anal. Chem., 2018, Vol. 90, pp. 9529-9537). This can be advantageous in low sample or single cell experiments where the analyte signal is small compared to the singly charged solvent background signal.

[0011] In DIA experiments, the MS2 spectra can retain a proportion of unfragmented precursor ions (in an amount not sufficient to be analytically useful). Some instruments offer an optional feature called "stepped collision energy" described in US 9,536,717. This feature provides multiple separate injections into the collision cell (also known as the "fragmentation chamber") at a range of collision energies, and then the summed ion population is transferred to a C-trap / Fourier transform mass analyzer and analyzed together. This variation in energy increases the probability that one of the energies used is optimal for fragmenting the precursor ions. However, this method also uses many collision energies that are not optimal for fragmenting the precursor ions, along with the associated cost of ion beam time. The actual act of changing the collision energy and making a second or third injection in the collision cell may not be overly time-intensive, and in some cases, may have an additional 1-3 ms overhead on a scan cycle of >40 ms.

[0012] US 8,686,350 describes a method where different types of ions can be accumulated in an ion trap before being ejected into a mass analyzer. In one example, a combination of two types of ions with the same narrow mass range is injected into the ion trap, one ion being fragmented and one ion remaining as an intact precursor. This allows for greater confidence that the precursor ions can be detected and mass measured accurately. However, quantification may be affected by the proportion of unfragmented precursors left by the MS2 injection. SUMMARY OF THE INVENTION

[0013] The present invention provides a mass spectrometry method. The method includes the following steps for each of a plurality of sub-ranges selected from a total m / z range:

[0014] Injecting a sample of precursor ions having an m / z value within the sub-range into a first ion storage device via an entrance orifice region;

[0015] Retaining a first portion of the precursor ion sample within the first ion storage device, where the first portion includes ions having an m / z value within the sub-range; and

[0016] Ejecting a second portion of the precursor ion sample from the first ion storage device via an exit region,

[0017] The second part includes ions having m / z values within a sub-range.

[0018] In option a), the method further includes analyzing a sample of fragmented precursor ions in a first mass analyzer, wherein the sample of fragmented precursor ions is formed by fragmentation of a second part of the precursor ions.

[0019] In option b), the method further includes accumulating in a second ion storage device a sample of fragmented precursor ions to be analyzed in the second ion storage device for analysis in a first mass analyzer, wherein the sample of fragmented precursor ions is formed by fragmentation of a second part of the precursor ions.

[0020] The proposed method facilitates slicing an ion beam proportionally based on the energy distribution of the ion beam. A barrier in the first ion storage device can be used to slice the ion beam. The sliced ion beam can be used to create a parallel accumulation region for simultaneously accumulating precursor ions for HDR MS1 scans (SIM injection) alongside a series of MS2 scans.

[0021] The sample of precursor ions can be sliced based on the energy of each ion. Thus, each of the first and second parts can include ions from the same m / z sub-range. This is in contrast to some prior art methods that slice an ion sample into several parts based on the m / z value of the ions such that each of these parts includes ions from different m / z sub-ranges.

[0022] The methods described herein can be used to establish high-quality HDR scans (or equivalent precursor data from multiple MS1 scans, where each scan spans multiple sub-ranges).

[0023] The proposed method is suitable for quantification over a wide dynamic range of analyte ions. Normally performing separate MS1 and MS2 scans would involve extended delays to switch the ion source and quadrupole to scan through the mass range independently of the DIA cycle used to obtain fragment data. The number of ions to be processed for HDR scans is high and thus always consumes a significant proportion of the ion beam time.

[0024] The fragmented precursor ions in the sample of fragmented precursor ions can be formed by fragmentation of precursor ions having m / z values within a sub-range. More specifically, the sample of fragmented precursor ions can consist of fragmented precursor ions formed by fragmentation of precursor ions having m / z values within that sub-range.

[0025] The second part of the precursor ion sample can be the remainder of the ion grouping after the first part of the ion grouping has been sliced. In other words, the sample of precursor ions can consist of a first part and a second part.

[0026] The sample of precursor ions can consist of precursor ions having m / z values within a sub-range.

[0027] The inlet region can include an inlet orifice.

[0028] The outlet region can include an outlet orifice.

[0029] Each of the plurality of sub-ranges can have the same width.

[0030] The width of each sub-range can be 20 Thomson or less.

[0031] Preferably, the injected ions are mass-filtered quite narrowly. In some examples, the width of each sub-range can be 5 Thomson or less.

[0032] The plurality of sub-ranges can be contiguous.

[0033] A DC barrier can be provided at the outlet region.

[0034] In some examples, a second DC barrier can be provided at the inlet region.

[0035] The method can further include, for each sub-range, determining the charge state of the precursor ion sample and adjusting the DC barrier based on the charge state of the precursor ion sample.

[0036] The method can further include, for each sub-range, adjusting the DC barrier based on the m / z sub-range. In other words, the level of the DC barrier can be customized based on the m / z value of the injected precursor ions.

[0037] The method can further include, for each sub-range, adjusting the DC barrier to compensate for space charge conditions.

[0038] The first ion storage device can include one or more intermediate barriers. A first portion of the precursor ion sample can be held below the one or more intermediate barriers. The one or more intermediate barriers can be relatively small compared to the DC barrier at the outlet region.

[0039] By providing intermediate barriers for holding ions, the held ions can be more evenly distributed throughout the first ion storage device.

[0040] The one or more intermediate barriers can be provided by one or more auxiliary trapping electrodes.

[0041] The method can further include, for each sub-range, adjusting the ion energy of the precursor ion sample to compensate for space charge conditions.

[0042] As the amount of ions accumulated / held increases, space charge conditions occur in the ion storage device. This can increase the local potential, redistribute the incident ion energy, and / or cause the ions already trapped to be axially and / or radially ejected. Accordingly, operating parameters (such as the level of the DC barrier and / or the ion injection energy) can be adjusted to compensate for these effects and control the proportion of ions held in the first ion storage device.

[0043] The level of the intermediate barrier can be adjusted to redistribute the held ions within the storage device and affect the space charge conditions within the ion storage device.

[0044] The method can also include, for each sub-range, adjusting the ion energy of the precursor ion sample based on the m / z sub-range. In other words, the ion injection energy can be customized based on the m / z value of the precursor ions being injected.

[0045] Holding the first portion of the precursor ion sample within the first ion storage device can include holding the first portion of the precursor ion sample away from the main axis of the first ion storage device such that the precursor ion sample of the subsequent sub-range is not blocked.

[0046] The first ion storage device can include a weak potential saddle such that the first portion of the precursor ion sample is stored away from the main axis of the first ion storage device.

[0047] The first ion storage device can have a sufficient length such that most of the first portion of the precursor ion sample is held within the first ion storage device away from the exit region.

[0048] Samples of precursor ions of each sub-range among the plurality of sub-ranges can be combined together within the first ion storage device such that the first ion storage device contains precursor ions having m / z values from the total m / z range.

[0049] Samples of precursor ions of each sub-range among the plurality of sub-ranges can be combined together within the first ion storage device such that the first ion storage device contains precursor ions having m / z values from the total m / z range. In other words, ions from each sub-range may be present within the first ion storage device.

[0050] The method can also include analyzing the combined sample of precursor ions having m / z values from the total m / z range in the first mass analyzer or the second mass analyzer. In other words, the held portions of multiple SIM injections can be combined together within the first ion storage device and analyzed together in the mass analyzer. Although the precursor accumulation step is performed for each sub-range, the step of analyzing the combined sample of precursor ions can be performed once for the total m / z range.

[0051] In a first example, the method further includes ejecting precursor ions from a first ion storage device into a second mass analyzer. In other words, there can be two mass analyzers in series.

[0052] The precursor ions ejected from the first ion storage device into the second mass analyzer can include a combined first portion of precursor ions from multiple sub-ranges. In some examples, the precursor ions ejected from the first ion storage device into the second mass analyzer can include a combined first portion of the retained precursor ions for each of the multiple sub-ranges.

[0053] The method can further include analyzing the precursor ions in the second mass analyzer.

[0054] The first mass analyzer (for analyzing fragment ions) can be a time-of-flight mass analyzer.

[0055] The second mass analyzer (for analyzing precursor ions) can be a Fourier transform mass analyzer.

[0056] In a second example, the method can further include transferring the precursor ions held in the first ion storage device to a first mass analyzer (in option a) or a second ion storage device (in option b). In the second example, the combined precursor ions (the combined first portion of the retained precursor ions) held in the first ion storage device are sent to the first mass analyzer. Thus, a second mass analyzer may not be required.

[0057] The first mass analyzer (for analyzing fragment ions and precursor ions) can be a time-of-flight mass analyzer.

[0058] In the case where the sample of fragmented precursor ions accumulates in the second ion storage device and the precursor ions held in the first ion storage device are transferred to the second ion storage device (option b), the method can further include ejecting the precursor ions from the second ion storage device into the first mass analyzer.

[0059] The precursor ions transferred from the first ion storage device (transferred to the first mass analyzer or the second ion storage device) can include a first portion of the precursor ion samples for each of the multiple sub-ranges.

[0060] The transferred precursor ions can include precursor ions having m / z values from the total m / z range. In other words, the transferred precursor ions can include a combined sample from each sub-range.

[0061] In the case where the sample of fragmented precursor ions accumulates in the second ion storage device (option b), the method can further include (for each sub-range) ejecting the sample of fragmented precursor ions from the second ion storage device into the first mass analyzer.

[0062] After each accumulation (before accumulating fragment ions formed by fragmentation of precursor ions from the next sub-range), a sample of the fragmentation precursor ions can be ejected from the second ion storage device.

[0063] A sample of the fragmentation precursor ions can be ejected from the second ion storage device before the precursor ions are transferred to the second ion storage device.

[0064] After samples of the fragmentation precursor ions for each of the plurality of sub-ranges have been ejected from the second ion storage device into the first mass analyzer, the precursor ions can be transferred from the first ion storage device to the second ion storage device.

[0065] The method may also include, for each of the plurality of sub-ranges, analyzing a first portion of the precursor ion sample in the first mass analyzer or the second mass analyzer. In this alternative, the precursor ions for each sub-range are analyzed separately in a SIM scan, rather than accumulating all SIM injections together in one scan and analyzing the precursor ions from the total m / z range. The SIM scan can be interleaved with the fragment scan, which can advantageously mean that no reconfiguration of the ion filter is required between the SIM scan and the fragment scan for each sub-range. The method may also include obtaining scan data related to the precursor ions for each sub-range. The method may also include combining the scan data related to the precursor ions for each sub-range to form a high-definition scan for the total m / z range. In other words, the SIM scan data is stitched together to provide a high-resolution MS scan. In this case, a pre-scan may not be required (since the sub-ranges are contiguous and cover the total m / z range).

[0066] In another example, an HDR MS scan can be sliced into a plurality of scans, each of the plurality of scans being related to precursor ions from a plurality of contiguous sub-ranges. In this alternative, each scan includes simultaneously analyzing precursor ions skimmed from multiple SIM injections. The method may also include combining the scan data from each of the plurality of scans to form a high-definition scan of the total m / z range. In this case, a pre-scan may not be required because a) the sub-ranges are contiguous and cover the total m / z range and b) each sub-range within the sub-ranges is analyzed in a corresponding one of the plurality of scans.

[0067] In some examples, the first ion storage device can be configured to operate under pure molecular flow conditions. Pure molecular flow conditions (also known as free molecular flow or Knudsen diffusion) are observed when the Knudsen number Kn is greater than 20 or more preferably Kn > 10.

[0068] The method may also include fragmenting a second portion of the precursor ion sample to produce a sample of fragmented precursor ions.

[0069] In the case where the sample of fragmented precursor ions accumulates in the second ion storage device (option b), the ions can be fragmented in the second ion storage device.

[0070] In other words, the precursor ions can be directed to the second ion storage device, and then once the ions have accumulated in the second ion storage device, they can be fragmented.

[0071] Alternatively, a multipole collision cell (e.g., an IRM collision cell) can be used to fragment the ions.

[0072] The multipole collision cell can be downstream of the first ion storage device such that a second portion of the sample of precursor ions is directed to the multipole collision cell, which then transfers the fragmented ions to the first mass analyzer (in option a) or the second ion storage device (in option b). In other words, in option a), the multipole collision cell can be between the first ion storage device and the first mass analyzer, or in option b), the multipole collision cell can be between the first ion storage device and the second ion storage device.

[0073] In option b), the fragmented ions can accumulate in the second ion storage device and then be ejected from the second ion storage device into the first mass analyzer.

[0074] The method can also include configuring an ion filter for each of a plurality of subranges to transmit precursor ions having m / z values within that subrange. A sample of precursor ions can be received from the configured ion filter. The sample of fragmented precursor ions can be formed by fragmentation of the precursor ions (the second portion of the precursor ion sample) received from the configured ion filter.

[0075] In other words, the ion filter may not be reconfigured between filling the first ion storage device and the second ion storage device.

[0076] The sample of fragmented precursor ions can be formed by fragmentation of the second portion of the precursor ion sample ejected from the first ion storage device.

[0077] Configuring the ion filter can include setting a transmission window of the ion filter. The transmission window can be adjusted between each of the plurality of subranges. For each subrange, the transmission window for the step of injecting the sample of precursor ions can be the same as the transmission window for the step of analyzing the sample of fragmented precursor ions (in option a) or the step of accumulating the sample of fragmented precursor ions in the second ion storage device (in option b).

[0078] A front-end accumulation device, such as an ion mobility separator, such as a trapped ion mobility separator (e.g., incorporated into the Bruker TIMS-ToF series mass spectrometers) (and would be advantageous for the methods described herein), can be used. Such devices can release ions within an m / z range synchronized with the quadrupole isolation window. Thus, ion transport is greatly facilitated. Also thereby, the required injection time can be reduced (due to a brighter isolated ion beam). This reduction in injection time can be used to offset the additional time overhead of SIM injection.

[0079] The method can further include configuring the ion mobility separator to transfer precursor ions having m / z values within that sub-range to an ion filter.

[0080] The method can further include, for each of a plurality of sub-ranges within the total m / z range, controlling the ion mobility separator such that the precursor ions transferred to the ion filter correspond to the transmission window of the ion filter.

[0081] The sample injecting the precursor ions can include controlling the fill time of the precursor ions based on the relative abundances of the precursor ion species within the corresponding sub-range.

[0082] In the case where the sample of fragmented precursor ions is accumulated in a second ion storage device (option b), the method can further include ejecting the sample of fragmented precursor ions from the second ion storage device into a first mass analyzer and analyzing the sample of fragmented precursor ions in the first mass analyzer for each of the plurality of sub-ranges.

[0083] The first mass analyzer can be a time-of-flight mass analyzer.

[0084] The first mass analyzer can be a multi-reflection time-of-flight MR-ToF mass analyzer.

[0085] The first ion storage device can be a curved linear trap (e.g., c-trap).

[0086] In the case where the sample of fragmented precursor ions is accumulated in a second ion storage device (option b), the second ion storage device can be a linear trap (e.g., DP-R trap).

[0087] The method can further include ionizing the sample to generate precursor ions.

[0088] The sample of fragmented precursor ions can be formed by fragmentation of precursor ions having m / z values within that sub-range at a plurality of different collision energies.

[0089] The method can further include:

[0090] Configuring the ion filter to transmit precursor ions having m / z values from the total m / z range;

[0091] Transfer an initial sample of precursor ions having m / z values from a total m / z range to a first mass analyzer or a second mass analyzer;

[0092] Analyze the initial sample of precursor ions; and

[0093] Obtain scan data for the total m / z range from the analysis of the initial sample of precursor ions.

[0094] In other words, the method can include a pre-scan (e.g., AGC pre-scan) of the total m / z range before analyzing ions from multiple sub-ranges. Optionally, the method can include accumulating (in a single fill and in a first ion storage device or a second ion storage device) an initial sample of precursor ions having m / z values from the total m / z range. Alternatively, the initial sample can be directed to the first mass analyzer or the second mass analyzer. Analyzing an initial sample of precursor ions having m / z values from the total m / z range can include obtaining scan data for the total m / z range (which can be low-resolution, full MS scan data rather than SIM scan data).

[0095] The method can further include, for each sub-range, adjusting the injection time of the precursor ion sample based on scan data obtained from the analysis of the initial sample of precursor ions (having m / z values from the total m / z range).

[0096] More specifically, the injection time for each sub-range can be based on the relative abundance of precursor ion species in each sub-range to improve the dynamic range of MS1 resulting from analyzing the first portion of the hold of the combination of precursor ions from SIM injections.

[0097] The method can further include determining the plurality of sub-ranges from the total m / z range based on scan data obtained from the analysis of the initial sample of precursor ions (having m / z values from the total m / z range). This can be referred to as a data-dependent acquisition (DDA) method.

[0098] The method can be performed within a time period based on the chromatographic peak width when the sample elutes from the chromatographic system.

[0099] There is also provided a method of manipulating ions using an ion storage device. The ion storage device includes: an inlet region; an outlet region; a trapping volume between the inlet region and the outlet region. The method includes:

[0100] Injecting a packet of ions via the inlet region, wherein the packet of ions contains ions having m / z values within a sub-range selected from a total m / z range;

[0101] Applying a DC barrier at the inlet region and the outlet region;

[0102] Keep a first portion of the ion packet within a trapping volume, where the first portion includes ions having m / z values within a sub-range;

[0103] Eject a second portion of the ion packet via an exit region, where the second portion includes ions having m / z values within a sub-range.

[0104] The proposed method can be used to split an ion beam within a known ion storage device, such as a C-trap. The operation of the proposed method can be achieved via the transmission of an ion beam (or ion packet) across a portion of an exit lens. The proposed method provides a beam splitter based on collision cooling, which can also be extended to other ion guiding structures.

[0105] Many other uses of the split ion beam are also possible. For example, the split ions are used for any other purpose, such as alternative fragmentation.

[0106] Many other uses of the beam splitting method are also possible. For example, the method can be used to split ions for any other purpose, such as alternative fragmentation. Thus, the proposed method has applications other than the provided examples (e.g., preparing ions for HDR scanning). For example, the retained ions can be sent to another detector for quantification (e.g., a secondary electron multiplier with single ion detection, as taught by US9812307).

[0107] The DC potential applied at the exit region can create a potential barrier that causes the first portion of the ion packet to be retained in the ion storage device.

[0108] The trapping volume can be between an entrance region and an exit region.

[0109] The entrance region can include an entrance hole.

[0110] The exit region can include an exit hole.

[0111] The DC barrier applied at the exit region can be greater than the DC potential within the trapping volume.

[0112] The DC barrier applied at the entrance region can be greater than the DC potential within the trapping volume.

[0113] The m / z range of the ion packet can be 20 Thomson (Th) or less. Preferably, the injected ions are mass-filtered quite narrowly. In some examples, the m / z range of the ion packet can be 5 Thomson or less.

[0114] The method can further include determining the charge state of the ion packet and adjusting the DC barrier at the exit region based on the determined charge state of the ion packet.

[0115] The method may further include adjusting the ion energy of an ion packet based on the m / z range of the ion packet. In other words, the level of the DC barrier can be customized based on the m / z value of the injected ions.

[0116] The method may further include adjusting the DC barrier at the exit aperture based on the m / z range of the ion packet.

[0117] The method may further include adjusting the ion energy of the ion packet and / or adjusting the DC barrier at the exit aperture to compensate for space charge conditions.

[0118] Holding a first portion of the ion packet within the ion storage device may include holding the first portion of the ion packet away from the main axis of the ion storage device.

[0119] The ion storage device may include a weak potential saddle such that the first portion of the ion packet is stored away from the main axis of the ion storage device.

[0120] The ion storage device may have a sufficient length such that a majority of the first portion of the ion packet is held within the ion storage device away from the exit region.

[0121] The ion storage device may include one or more intermediate barriers (between the inlet region and the exit region). The first portion of the ion packet may be held below the one or more intermediate barriers. The one or more intermediate barriers may be relatively small compared to the DC barrier at the exit region (and / or relatively small compared to the DC barrier at the inlet region).

[0122] The one or more intermediate barriers may be provided by one or more auxiliary trapping electrodes.

[0123] A mass spectrometer configured to perform the above method is also provided.

[0124] An ion storage device configured to perform the above method is also provided.

[0125] A computer software including instructions is also provided, which when executed by a processor of a computer causes the computer to perform the above method. BRIEF DESCRIPTION OF THE DRAWINGS

[0126] The present invention may be implemented in various ways and specific embodiments will now be described by way of example only and with reference to the following drawings.

[0127] Figure 1 A schematic diagram of a mass spectrometer suitable for performing the method according to an embodiment of the present invention is shown.

[0128] Figure 2 A beam limiting method within an ion storage device based on discrimination by ion energy is shown.

[0129] Figure 3A Shows the relationship of signal intensity versus time in a Fourier transform mass analyzer where ions held across multiple sub-ranges perform a full MS scan when the barrier at the exit hole of the C-trap (“exit lens potential”) increases over time.

[0130] Figure 3B Shows the relationship of signal intensity versus time in a time-of-flight mass analyzer where MS2 fragmentation scans are performed for each of multiple sub-ranges when the barrier at the exit hole of the C-trap (“exit lens potential”) increases over time.

[0131] Figure 4 Shows the MS1 spectra generated by a Fourier transform mass analyzer during a DIA run for three different values of the barrier at the exit region of an ion storage device.

[0132] Figure 5 Shows a DIA method for parallel accumulation of SIM injections in a curved linear ion storage device, constructed as a single HDR scan using a Fourier transform mass analyzer, interleaved between a series of MS2 scans in a time-of-flight mass analyzer. Detailed Description

[0133] Figure 1 Shows a schematic arrangement of a mass spectrometer 1 suitable for performing the methods according to embodiments of the present invention. The mass spectrometer 1 can be a hybrid Fourier transform / multi-reflection time-of-flight mass spectrometer (MR-ToF) as described in US10,699,888, which is incorporated by reference. Details of the mass analyzer are described in US 9,136,101, which is incorporated by reference herein.

[0134] In Figure 1 a sample to be analyzed (e.g., from an autosampler) is supplied to a chromatographic device such as a liquid chromatography (LC) column ( Figure 1 not shown in). One such example of an LC column is the ProSwift monolithic column from Thermo Fisher Scientific, Inc, which provides high performance liquid chromatography (HPLC) by forcing a sample transported in a mobile phase through a stationary phase of irregular or spherical shaped particles that make up the stationary phase under high pressure. In an HPLC column, sample molecules elute at different rates depending on their degree of interaction with the stationary phase.

[0135] Chromatography can be generated by measuring the number of sample molecules eluted from an HPLC column over time using a detector (e.g., a mass spectrometer). The sample molecules eluted from the HPLC column will be detected as peaks above the baseline measurement on the chromatogram. When different sample molecules have different elution rates, multiple peaks on the chromatogram can be detected. Preferably, individual sample peaks are separated in time from other peaks in the chromatogram such that different sample molecules do not interfere with each other.

[0136] In chromatography, the presence of a chromatographic peak corresponds to the period of time during which the sample molecules are present at the detector. Thus, the width of the chromatographic peak is equal to the period of time during which the sample molecules are present at the detector. Preferably, the chromatographic peak has a Gaussian shape distribution, or can be assumed to have a Gaussian shape distribution. Accordingly, the width of the chromatographic peak can be determined based on multiple standard deviations calculated from the peak. For example, the peak width can be calculated based on 4 standard deviations of the chromatographic peak. Alternatively, the peak width can be calculated based on the width at half of the maximum height of the peak. Other methods known in the art for determining peak width may also be suitable.

[0137] Then, the sample molecules separated by liquid chromatography are ionized using an electrospray ionization source (ESI source) 2 at atmospheric pressure.

[0138] The sample ions then enter the vacuum chamber of the mass spectrometer 1 and are guided by a capillary 25 into a solely RF S-lens 3 (also known as an ion funnel). The ions are focused by the S-lens 3 into an injection flat electrode 4 (also known as a quadrupole pre-filter) which injects the ions into a curved flat electrode 5 with an axial field. The curved flat electrode 5 guides (charged) ions along a curved path passing through it, while unwanted neutral molecules (such as entrained solvent molecules) are not guided along the curved path and are lost. For example, the curved path can be a 90-degree bend or an S-shaped wobble.

[0139] A TK lens 6 is located at the distal end of the curved flat electrode 5. The ions enter a downstream mass selector in the form of a quadrupole mass filter 7 from the curved flat electrode 5. The TK lens serves as an edge field corrector for the quadrupole mass filter 7. The quadrupole mass filter 7 is typically but not necessarily segmented and serves as a bandpass filter, thereby allowing selected mass numbers or a limited mass range to pass while excluding ions of other mass-to-charge ratios (m / z). The filter can also operate in a solely RF mode in which the filter has no mass selectivity, i.e., it transmits substantially all m / z ions. For example, the quadrupole mass filter 7 can be controlled by a controller to select precursor ions within a certain range of mass-to-charge ratios that are allowed to pass while filtering out (attenuating) other ions in the precursor ion stream. Alternatively, the S-lens 3 can operate as an ion gate and the ion gate (TK lens) 6 can be an electrostatic lens.

[0140] Although Figure 1A quadrupole mass filter is shown, but those skilled in the art will understand that other types of mass selection devices may also be suitable for selecting precursor ions within the mass range of interest. For example, the ion separator depicted in US-A-2015287585, the ion trap depicted in WO-A-2013076307, the ion mobility separator described in US-A-2012256083, the ion gate mass selection device described in WO-A-2012175517, or the charged particle trap described in US799223, which is incorporated herein by reference. Those skilled in the art should understand that other methods of selecting precursor ions based on ion mobility, differential mobility, and / or transverse modulation are also suitable.

[0141] The isolation of multiple ions of different masses or mass ranges in an ion trap can also be performed using a method called synchronous precursor scan (SPS). Additionally, in some embodiments, more than one ion selection device or mass selection device may be provided. For example, another mass selection device may be provided downstream of the fragmentation chamber 12. In this way, MS 3 or MS n scans can be performed (usually using a ToF mass analyzer for mass analysis) when necessary.

[0142] The ions then pass through a quadrupole exit lens / segmented lens arrangement 8, which acts as an ion gate to control the passage of ions into the first transfer multipole 9 optionally via a charge detector (not shown). The first transfer multipole 9 guides the mass-filtered ions from the quadrupole mass filter 7 into a curved linear ion trap (C-trap) 10. The C-trap (first ion storage device) 10 has a longitudinally extending curved electrode supplied with an RF voltage and end caps supplied with a DC voltage. As a result, a potential well extends along the curved longitudinal axis of the C-trap 10. In a first operating mode, a DC end cap voltage is set for the C-trap such that ions arriving from the first transfer multipole 9 are trapped in the potential well of the C-trap 10, where the ions are cooled. The injection time (IT) of the ions into the C-trap determines the number of ions (ion packet) subsequently ejected from the C-trap into the mass analyzer.

[0143] The cooled ions accumulate into a cloud towards the bottom of the potential well and are then orthogonally ejected from the C-trap towards the second mass analyzer 11. As Figure 1 shown, the second mass analyzer is a Fourier transform mass analyzer, such as an Orbitrap mass analyzer 11, for example, the Orbitrap sold by Thermo Fisher Scientific. TMMass analyzer. The Fourier transform mass analyzer 11 has an off-center injection hole, and ions are injected into the orbitrap mass analyzer 11 as coherent groups through the off-center injection hole. Then, the ions are trapped in the orbitrap mass analyzer by a hyperlogarithmic electric field, and the ions perform back-and-forth motion in the longitudinal direction while orbiting around the inner electrode.

[0144] The axial (z) component of the movement of the ion packet in the orbitrap mass analyzer is (more or less) defined as a simple harmonic motion, where the angular frequency in the z direction is related to the square root of the mass-to-charge ratio of a given ion species. Thus, over time, the ions are separated according to their mass-to-charge ratio.

[0145] Ions in the orbitrap mass analyzer are detected by using an image current detector (not shown), which generates a "transient" containing information about all ion species in the time domain when the ion species pass through the image current detector. Then, this transient undergoes a fast Fourier transform (FFT), thereby generating a series of peaks in the frequency domain. Based on these peaks, a mass spectrum representing the abundance / ion intensity versus m / z can be generated.

[0146] In the above configuration, without fragmentation, sample ions (more specifically, a mass range segment of the sample ions within the mass range of interest selected by the quadrupole mass filter 7) are analyzed by the orbitrap mass analyzer 11. The resulting mass spectrum is denoted as MS1.

[0147] Although Figure 1 the orbitrap mass analyzer 11 is shown, alternatively, other mass analyzers can be used, including other Fourier transform mass analyzers. For example, a Fourier transform ion cyclotron resonance (FTICR) mass analyzer can be used as the mass analyzer for MS1 scans. Mass analyzers such as orbitrap mass analyzers and ion cyclotron resonance mass analyzers can also be used in the present invention even when other types of signal processing different from Fourier transform are used to obtain mass spectrometry information from the transient signal (see, for example, WO 2013 / 171313, Thermo Fisher Scientific).

[0148] In the second operating mode of the C-trap 10, the ions that pass through the quadrupole exit lens / segmented lens arrangement 8 and the first transfer multipole 9 and enter the C-trap 10 can also continue their path through the C-trap and enter the fragmentation chamber 12, which can be an "ion routing multipole" (IRM) collision cell. Thus, the C-trap effectively acts as an ion guide in the second operating mode. Alternatively, the cooled ions in the C-trap 10 can be ejected axially from the C-trap into the fragmentation chamber 12. In Figure 1In the mass spectrometer 1, the fragmentation chamber 12 is a high-energy collision dissociation (HCD) device to which collision gas is supplied. The precursor ions reaching the fragmentation chamber 12 collide with the collision gas molecules, causing the precursor ions to fragment into fragment ions.

[0149] Although the HCD fragmentation chamber 12 is shown in Figure 1 , other fragmentation devices using methods such as collision-induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), photodissociation, etc. can alternatively be used. In addition, ion fragmentation can be performed in the high-voltage region of the extraction trap 14.

[0150] The fragmented ions can be ejected from the fragmentation chamber 12 to the C-trap 10 at the relative axial ends. The ejected fragmented ions enter the second transfer multipole 13. The second transfer multipole 13 guides the fragmented ions from the fragmentation chamber 12 into the extraction trap (second ion trap) 14. The extraction trap 14 is a radio-frequency voltage-controlled trap containing buffer gas. For example, a suitable buffer gas is argon with a pressure range from 5×10 -4 mbar to 1×10 -2 mbar. The extraction trap can quickly cut off the applied RF voltage and apply a DC voltage to extract the trapped ions. A suitable flat extraction trap, also known as a rectangular ion trap, is further described in US9,548,195, which is incorporated herein by reference. Alternatively, the C-trap is also suitable for use as the second ion trap.

[0151] The extraction trap 14 is provided to form ion packets of the fragmented ions before they are injected into the time-of-flight mass analyzer 15. The extraction trap 14 accumulates the fragmented ions before they are injected into the time-of-flight mass analyzer 15.

[0152] Although the extraction trap (ion trap) is shown in the Figure 1 embodiment, those skilled in the art will understand that other methods of forming ion packets of the fragmented ions will be equally suitable for the present invention. For example, the relative slow transfer of ions through a multipole can be utilized to affect the aggregation of ions, and the ions can then be ejected as a single group into the ToF mass analyzer. Alternatively, the orthogonal displacement of ions can be utilized to form groups. Other details of these alternatives can be found in US 2003 / 0001088, which describes the traveling-wave ion accumulation method and is incorporated herein by reference.

[0153] In Figure 1In the figure, the time-of-flight mass analyzer 15 shown is a multi-reflection time-of-flight mass analyzer (MR-ToF) 15. The MR-ToF 15 is constructed around two opposing ion mirrors 16, 162 that are elongated in the drift direction. The mirrors are opposed in a direction orthogonal to the drift direction. The extraction trap 14 injects ions into the first mirror 16, and then the ions oscillate between the two mirrors 16, 162. The ejection angles of the ions from the extraction trap 14 and additional deflectors 17, 172 allow control of the ion energy in the drift direction such that the ions are guided along the length of the mirrors 16, 162 during oscillation, thereby generating a Z-shaped trajectory. The mirrors 16, 162 are tilted relative to each other, thereby generating a potential gradient that delays the drift velocity of the ions and causes the ions to reflect back and converge on the detector 18 in the drift dimension. The tilting of the opposing mirrors generally has the negative side effect of changing the period of ion oscillation as the ions travel along the drift dimension. This is corrected using a strip electrode 19 (as a compensation electrode) that changes the flight potential of the portion of the space between the mirrors that varies along the length of the opposing mirrors 16, 162. The combined variation in the width of the strip electrode 19 and the change in the distance between the mirrors 16, 162 allows ion reflection and spatial convergence on the detector 18 and maintains good time focusing. A suitable MR-ToF 15 for use in the present invention is further described in US2015028197(A1), which is incorporated herein by reference.

[0154] In one example, an MS1 scan can be performed by the second mass analyzer (orbitrap mass analyzer 11). In a second example, precursor ions can be fragmented and an MS2 scan can be performed by the second mass analyzer (orbitrap mass analyzer 11) or the first mass analyzer (time-of-flight mass analyzer), depending on whether the fragmentation chamber is controlled to eject the ions back towards the C-trap 10 or forward into the second transfer multipole 13. In additional operating modes, the second mass analyzer (time-of-flight mass analyzer 15) can perform an MS1 scan of the ions. In this operating mode, the ions are axially guided through the C-trap 10 to the fragmentation chamber, but no fragmentation gas is introduced and the ions are guided to the second transfer multipole 13 without fragmentation. The ions can then be grouped and accumulated in the extraction trap 14 as described above.

[0155] Once a predetermined number of ions have accumulated in the extraction trap, the ions accumulated in the extraction trap are injected into the MR-ToF analyzer 15 as ion packets. By ensuring that each packet of ions injected into the MR-ToF 15 has at least a predetermined (minimum) number of ions, the resulting ion packets reaching the detector will represent the entire mass range of interest of the MS1 or MS2 spectrum. A single group of precursor ions or fragmented ions is sufficient to obtain the MS1 or MS2 spectrum of the corresponding ions. For MS2, this represents an increased sensitivity compared to the conventional acquisition of a time-of-flight spectrum, in which multiple spectra are typically acquired and summed for each given mass range segment. Preferably, the minimum total ion current (TIC) in each mass window is accumulated in the extraction trap before injection into the time-of-flight mass analyzer. In some examples, the time-of-flight mass analyzer acquires at least N spectra (scans) per second in the MS2 domain, where N = 50, or more preferably 100 or 200 or more.

[0156] Preferably, at least X% of the MS2 scans contain more than Y ion counts (where X = 30 or 50 or 70 or most preferably 90 or more, and Y = 200 or 500 or 1000 or 2000 or 3000 or 5000 or more). Most preferably, at least 90% of the MS2 scans contain more than 500 ion counts, or more preferably more than 1000 ion counts, and ideally more than 5000 ion counts. This provides an increased dynamic range of the MS2 spectrum. The desired ion count for each MS2 scan can be provided by adjusting the number of ions included in each group of fragmented ions. For example, in Figure 1 an embodiment, the accumulation time of the extraction trap can be adjusted to ensure that a sufficient number of ions have been accumulated. Thus, the controller can be configured to determine that a suitable packet of fragmented ions has been formed when a predetermined number of ions are present in the extraction trap or when a predetermined time period has elapsed. The predetermined time period can be specified to ensure that the time-of-flight mass analyzer operates at the desired frequency when the ion current to the extraction trap is relatively low.

[0157] The mass spectrometer 1 is under the control of a controller that is configured, for example, to control the ejection timing of the trapping assembly; set appropriate potentials on the electrodes of the quadrupole, etc. to focus and filter ions; capture mass spectrometry data from the orbital trapping device 11; obtain mass spectrometry data from the MR-ToF 15; control the sequence of MS1 scans and MS2 scans, etc. It should be understood that the controller can include a computer that can operate according to a computer program that contains instructions to cause the mass spectrometer to perform the method steps according to the present invention.

[0158] It should be understood that Figure 1The specific arrangement of the components shown is not necessary for the methods described subsequently. In fact, other arrangements for the methods implementing the embodiments of the present invention are also suitable. In some examples, all scans (MS1, MS2, and / or SIM) are performed by an MR-ToF analyzer, which is faster than an orbitrap analyzer.

[0159] A front-end accumulation device, such as an ion mobility separator (e.g., a trapped ion mobility separator TIMS), can be configured to release ions in the m / z range corresponding to the quadrupole isolation window. As a result, the ion transmission of the quadrupole filter is improved. Due to the ion mobility separator, the injection time required can also be reduced (since the isolated ion beam is brighter). This reduction in injection time can be used to at least partially offset the additional time overhead of SIM injection.

[0160] The ion mobility separator can include a stacked-ring ion guide that applies a DC gradient in one direction to push ions and passes them in the opposite direction through a gas flow.

[0161] In one example of the ion mobility separator, an electric field barrier in the gas flow is used to block ions according to their ion mobility. As the ion mobility increases, the field barrier decreases, releasing the ions.

[0162] TIMS is described in detail in U.S. Patent 7,838,826, U.S. Patent 9,891,194, and Meier et al. (2018, Molecular Cellular Proteomics, Vol. 17, pp. 2534 - 2545), which are incorporated herein by reference.

[0163] The extended ion funnel consists of a plurality of segmented electrodes assembled around a common axis. The extended ion guide can be considered in three sections:

[0164] An entrance focusing section,

[0165] A mobility analysis section, and

[0166] An exit focusing section.

[0167] In the focusing section, the distance between adjacent electrodes is approximately equal to the thickness of the electrodes. The diameter of the orifices in the electrodes is a function of the position of the electrodes in the ion funnel assembly. For example, the segmented electrode with the largest orifice is located at the entrance end of the ion funnel, and the segmented electrode with the smallest orifice is located at the exit end of the ion funnel.

[0168] In some examples, the orifice diameter can be a linear function of the position of the segmented electrodes. In other examples, the function can be non-linear. The angle formed between the common axis and the inner boundary of the ion funnel (i.e., formed by the inner edges of the segmented electrodes) can be approximately 19°. However, any angle between 0° and 90° can be used.

[0169] In the mobility analysis section of the ion funnel, the segmented electrodes can all have the same inner diameter. The space between adjacent electrodes can be filled with a dielectric or resistive gasket. The thickness of the segmented electrodes should be less than their inner diameter, and the spacing between the electrodes should be less than the thickness of the segmented electrodes to maintain a uniform RF field such that the axial DC field is uniform near the axis.

[0170] The gasket or O-ring between the electrodes forms a substantially airtight seal such that the orifices in the electrodes form airtight channels through which gas can flow. Gas enters the channels in the focusing section, forms a laminar flow that uniformly flows through the mobility analysis section, contracts by leaving the focusing section, and then exits through the orifices in the final electrode. The orifices are substantially cylindrically symmetric to maintain a cylindrically symmetric flow profile. In operation, the symmetric laminar flow of the gas means that all ions of a given type at a given position along the axis will experience a given force due to the gas flow, substantially independent of their lateral position relative to the axis.

[0171] The quadrupole ion filter consists of four rods equally spaced at a predetermined radius around a central axis. A radio frequency (RF) (e.g., 1 MHz sine wave) electric potential is applied between these rods. The electric potentials on adjacent rods are 180° out of phase. The rods on opposite sides of the axis of the quadrupole are electrically connected such that the quadrupole is formed as two pairs of rods. Ions travel along the axis of the quadrupole and leave the quadrupole through an orifice. The RF electric potential applied between these rods tends to radially confine the ions. When only RF is applied between these rods, substantially all ions are transmitted through the quadrupole. Applying DC as well as RF electric potential between the pairs of rods allows only ions within a limited mass range to be transmitted through the quadrupole. Ions outside this mass range are filtered out and do not reach the exit end.

[0172] The DC electric field strength varies as a function of the position along the axis. However, at some positions in the analysis section, the field strength reaches a maximum, thereby forming a potential barrier that ions must overcome in order to reach the exit end of the funnel. Near the position of this maximum field strength, the uniformity of the DC field is important because this is the point at which ions are selected based on their mobility. Therefore, the DC field should be cylindrically symmetric.

[0173] An example method of operation includes the following steps:

[0174] Form a DC barrier in the analysis section;

[0175] Apply an RF field for focusing ions towards the axis;

[0176] Generate ions in an ion source;

[0177] Introduce ions in a carrier gas into an extended ion funnel;

[0178] Introduce ions into a focusing section by applying an electric potential to the electrodes of the focusing section and / or deflection electrodes;

[0179] Transfer ions into an analysis section by applying a DC potential to the electrodes of the focusing section;

[0180] Optionally, prevent additional ions from entering the analysis section by applying a DC potential to the deflection electrodes and / or the electrodes of the focusing section;

[0181] Use a pump located downstream of the exit end of the funnel to induce a carrier gas flow through the channel;

[0182] Gradually reduce the DC barrier in the analysis section to allow the carrier gas flow to push ions from an ion packet over the DC barrier in order of ion mobility; and

[0183] Focus ions through an orifice in an exit electrode.

[0184] In another example, a DC gradient is used to push ions out, against the gas wind. As the DC potential increases, ions are released in order of mobility.

[0185] In one example method, a full mass scan is performed using a Fourier transform mass analyzer 11 with a long acquisition transient to generate a high-resolution MS1 spectrum. Meanwhile, the MR-ToF 15 analyzer performs a series of MS2 acquisitions at a very fast scan rate and high sensitivity.

[0186] Example methods for combining ion injection and analysis are described in US 8,686,350, which is incorporated herein by reference.

[0187] The exemplary methods described herein accumulate different types of ions for separate analysis: first, precursor ions that have not been fragmented; and second, fragment ions formed by fragmentation of the precursor ions. The ions can be from the same ion source and have the same quadrupole isolation window but different fragmentation energies (the collision energy of the precursor ions is zero). The fragment ions are analyzed in a mass analyzer to provide a fragment spectrum. The precursor ions are accumulated in an ion storage device and combined with precursor ions from other quadrupole isolation windows. The combined precursor ions are then analyzed in the mass analyzer to provide analysis scan data. Such scan data provides precursor information in addition to the fragment spectrum. The accumulation of precursor ions can be performed quickly together with normal MS2 fragment analysis because there is no additional quadrupole switching time, and the additional injection time for the precursor ion (SIM) component should be lower than the time for fragmentation injection.

[0188] When using a hybrid instrument (such as Figure 1 the instrument shown), it is preferred to measure the SIM component (the unfragmented precursor ions of each sub-range) in a high-resolution MS1 scan. To do this, the unfragmented SIM component needs to be separated from the fragmented MS2 component and then collected for separate analysis.

[0189] In some examples, the ions can be switched between two separate ion destinations using a beam switching device to create a branched ion path. A switchable path ion guide is described in UK Patent Application No. 2209555.8, which is incorporated herein by reference. Other suitable devices are described in Patent Publications US7829850B2, US20190103261A1, US8581181B2, and US9984861B2. As described above, in this DIA method, for each injection performed with fragmentation, there is an additional injection with the same quadrupole isolation window and no fragmentation energy. As is normal for DIA, the target mass is scanned over a predetermined range and isolation step.

[0190] To further improve efficiency, it is preferred to eliminate the overhead due to the need to actively switch the beam path. One way to do this is to separate a portion of the ion injection. This can be achieved by differentiating the properties of the ions (such as position or energy). Then, a single (longer) injection can be separated and used to supply two ion destinations. Even more preferably, the conditions are set such that a certain proportion of the ion beam is separated, either as a function of collision cooling or spatial distribution, and the split ion beam is delivered to separate ion destinations. This then further saves the time overhead between injections. Thus, a method of beam splitting is provided by skimming cooled ions at a barrier.

[0191] Figure 2Shows an ion beam limiting method according to the present disclosure. Based on the discrimination of ion energy, ion beam limiting is performed within an ion storage device (such as a C-trap). Ions are injected through an entrance hole (or lens). At the exit of the hole (or lens), most of the injected ions are ejected. However, low-energy ions will not be able to pass through the potential barrier and are trapped within the ion storage device.

[0192] An example method for limiting an ion beam utilizes the energy distribution of the ion beam. When the ion beam passes through a relatively high DC potential barrier, such as at a fragment of a deceleration lens or an ion guide, a certain proportion of ions with low kinetic energy may be reflected by the potential barrier. Figure 2 Displays a method whereby an ion beam passes from an ion guide through a C-trap to an IRM for fragmentation. The fragmented ions are then sent to an MR-ToF analyzer for MS2 analysis. Both the entrance lens and the exit lens are set to a higher electric potential than the C-trap body, and there is a normal DC drop between the ion optics to keep the ions moving. The exit lens is set to a sufficient electric potential (about +5 V) such that a certain proportion of ions (preferably about 5%-20%) are prevented from leaving and are trapped in the C-trap. This is aided by the fact that ions passing through the C-trap may lose energy via collisions with the buffer gas in the C-trap. In some examples, the buffer gas in the C-trap can be nitrogen at a pressure of about 2×10 -3 mbar. This can correspond to a preferred "collision thickness" of 0.05 mbar*mm of nitrogen, which is significantly lower than the typical requirements for collision cooling (usually, collision cooling is performed at a collision thickness of about 0.2 mbar*mm, as described in US4963736). Preferably, for small molecules and peptides, the "collision thickness" is in the range of 0.02 mbar*mm - 0.10 mbar*mm of nitrogen, and for other types of molecules (e.g., proteins and protein complexes), the "collision thickness" is appropriately scaled with the collision cross-section.

[0193] In the DIA method, during a series of MS2 scans, most ions will be sent for analysis while a certain proportion will be held and accumulated in the ion guide. During the course of multiple MS2 scans, a large ion accumulation will exist in the C-trap and can be ejected into a Fourier transform mass analyzer in a manner similar to the "Boxcar" method described in the background art to provide an HDR scan. Advantageously, this method can be performed on Figure 1 instruments without significant modification.

[0194] Can be used Figure 1 within the C-trap 100 in a Figure 2 hybrid mass spectrometer to perform the method described relative to Figure 1DIA method of a hybrid mass spectrometer. The C-trap lens is set such that a certain proportion of the ion beam passing through the C-trap is limited by the exit lens and retained within the C-trap. Ions having m / z values within a series of sub-ranges are sequentially injected into the C-trap. The injection time for each sub-range is proportional to the local ion intensity within that sub-range (in terms of the total number of target ions hitting that sub-range). In this way, highly prevalent species will be attenuated, thus preventing them from dominating the entire population in a manner similar to the Boxcar / HDR method. At the end of each DIA cycle, the accumulated population of precursor ions held by the C-trap can be pulsed out to a second mass analyzer (e.g., a Fourier transform mass analyzer) for high-resolution full MS analysis.

[0195] One challenge faced by this method is caused by the mass / charge-dependent characteristics and space charge response of the method. The energy distribution of the ion beam at the exit lens can be mass- and mobility-dependent, as ions are collisionally cooled to different extents based on these characteristics. Taking these effects into account for each sub-range, it is preferred that the injected ions are very narrowly mass-filtered. Additionally, the charge state of the injected ions can be determined and compensated for. Calibration of the device parameters between sub-ranges can be performed to address these effects. In one example, the ion energy (via the DC path) can be adjusted based on the m / z value of the ions within the sub-range. In another example, the exit lens DC barrier can be adjusted between each sub-range. For example, for injected ions with higher m / z values, it may be necessary to increase the exit lens DC barrier.

[0196] After a large number of ions have accumulated within the C-trap, the strong space charge conditions within the trap can raise the local electric potential, redistribute the incident ion energy, and axially and radially eject the trapped ions. These effects can be compensated for by tuning the device parameters (e.g., DC barrier level, internal trap electric potential, ion cooling, ion injection energy, etc.).

[0197] Another method of compensating for the space charge effect is to store the retained ions in another type of storage device where there is a weak electric potential saddle along the main axis of the device. This can allow slow ions (with lower energy) to "roll away" from fast ions along the main axis along which they travel.

[0198] In another example, the ions can be stored along the main axis but away from the barrier at the exit region. In this case, a long system can be provided.

[0199] In another example, ions can be stored under a small intermediate potential barrier, such as that generated by an auxiliary trapping electrode, while a second stronger potential barrier is generated at the exit region. Then, the space charge of the retained ions will accumulate on the periphery of the storage device, thereby reducing the impact on the flight of other ions. Assuming that the pressure control is relatively slow, ion trapping can be controlled by setting the levels of the intermediate potential barrier and the potential barrier at the exit aperture.

[0200] In one example experiment, a Pierce Flexmix calibration solution was infused into a Fourier transform / MR-ToF hybrid mass spectrometer of the Figure 1 described class. The DIA method was run, where the instrument cycled through a series of MR-ToF MS2 mass analysis operations for sub-ranges within the total m / z range of 350 Th - 980 Th. Additionally, for each DIA cycle, a single full MS scan of the m / z range 150 - 2000 was performed using the Fourier transform mass analyzer. During this DIA method, the C-trap entrance lens was covered to +5 V to allow the transmission of the -6 eV ion beam but retain the trapped ions. The DC potential at the barrier at the exit region (exit lens) of the C-trap was increased from 0 V to 2 V to 4 V to 6 V to 8 V, and the DIA cycle was repeated for each potential.

[0201] Figure 3 shows the relative intensities of the mass spectra in the Fourier transform mass analyzer and the MR-ToF mass analyzer when the DC potential at the barrier at the exit region is increased.

[0202] Figure 3A Shows the relationship of the signal intensity in the Fourier transform mass analyzer, which performs a full MS scan of the ions retained across multiple sub-ranges, relative to time as the potential barrier ("exit lens potential") at the exit aperture of the C-trap increases over time.

[0203] Figure 3B Shows the relationship of the signal intensity in the time-of-flight mass analyzer, which performs an MS2 fragmentation scan for each of multiple sub-ranges, relative to time as the potential barrier ("exit lens potential") at the exit aperture of the C-trap increases over time.

[0204] It can be seen that the MR-ToF signal is not greatly affected until stepped up to 6 V, which is related to the 6 eV energy injected into the C-trap. However, even at 8 V some ions can still pass through, although only a small proportion. This is to be expected since the true potential barrier is determined not only by the DC potential applied to the exit barrier but also by the lower potential of the surrounding ion optics. The Fourier transform mass analyzer signal increases significantly at the 6 V point, meaning that the trapped ions from the DIA cycle are being injected into the Fourier transform mass analyzer for the full MS scan, as expected.

[0205] Figure 4 Shows the full MS mass spectra obtained on a Fourier transform mass analyzer during the time when the C-trap exit lens was held at 2 V, 4 V, and 6 V. At 2 V, the 150 - 2000 full MS scans were similar to the Flexmix distribution. There was an anomalous attenuation of the doubly charged MRFA peptide at m / z 262, although this was most likely the result of defective timing in the prototype software build and could be ignored.

[0206] At 4 eV, the ions in the DIA cycle range of 350 - 980 became more prominent, while the higher m / z super-tagged envelopes decreased relatively.

[0207] With the 6 V spectrum, the super-tagging was greatly proportionally reduced, and the spectrum was completely dominated by the ions captured from the DIA cycle. A higher relative proportion of the smaller peaks within the DIA m / z range was also noted, which implies an improvement in the dynamic range, even within this basic experiment.

[0208] Figure 5 Shows a graphical illustration of the DIA method that can be implemented on a Figure 1 mass spectrometer. In the DIA method according to some specific examples, for each injection performed in the case of fragmentation into a second ion storage device, the precursor ions are held within a first ion storage device having the same quadrupole isolation window (since these are precursor ions, the collision energy is reduced or there is no collision energy). As normal for DIA, the target mass is scanned through a predetermined range and isolation step. Figure 5 Shows such a scan sequence from m / z 300 Th - 900 Th, with a 5 Th isolation window, as can be used for "bottom-up" measurements of protein samples for digestion. An optional AGC (automatic gain control) pre-scan can be performed (preferably in a time-of-flight mass analyzer) to help determine the appropriate ion injection time in subsequent scans. Alternatively, a Fourier transform mass analyzer (e.g., Orbitrap TM mass analyzer) can be used for an optional MS1 full scan. The MS2 injection is transferred to a linear trap and extracted to a time-of-flight (e.g., MR-ToF) mass analyzer for a series of scans (T1 to T61). SIM injections are accumulated together in an ion storage device, such as a curved linear ion storage device (C-trap) or other ion trapping devices. An HDR MS1 scan can be generated by analyzing the combined precursor ions (e.g., in a Fourier transform mass analyzer).

[0209] SIM and MS2 injections are performed simultaneously to minimize quadrupole and source switching time overheads, which could otherwise significantly reduce the time available for ion accumulation. At the end of the cycle, the series of accumulated SIM injections are then extracted into a Fourier transform mass analyzer for long transient analysis, e.g., at 240K resolution.

[0210] Advantageously, AGC is used to control the number of ions in the curved linear ion storage device and the Fourier transform mass analyzer such that the number of precursor ions held in the first ion storage device for each SIM injection does not add up to a level that overwhelms the ion storage device once all the injections have been performed. For example, if there are 60 SIM injections per cycle, the number of ions held from each SIM injection in the first ion storage device can be limited to about 1500 ions.

[0211] For longer cycles, additional scans using the Fourier transform mass analyzer can be performed in the middle of the cycle to increase the dynamic range of the precursor scan data at the cost of reducing the maximum transient time by half. In other words, the precursor ions held in the first ion storage device from the first half of the SIM injections from multiple sub-ranges can be analyzed together in the first scan, and the precursor ions held in the first ion storage device from the second half of the SIM injections from multiple sub-ranges can be analyzed together in the second scan.

[0212] More generally, rather than performing one scan of the precursor ions from the total m / z range, the method can include multiple scans of the precursor ions, where each scan includes precursor ions from multiple sub-ranges.

[0213] Figure 5 The method can be performed on Figure 1 the combined Fourier transform mass analyzer and MR-ToF instrument shown. Alternatively, the method can be performed on a single mass analyzer instrument, such as a ToF-only instrument or a Fourier transform mass analyzer only (and potentially taking longer).

[0214] The timing of the different operations can be configured such that certain operations are performed in parallel to reduce the total time taken. Figure 1 The Fourier transform / MR-ToF instrument shown in may be able to operate at approximately 200Hz for single injections, with an injection time (also known as "fill time") of 3ms and an overhead of 2ms. Additional overheads will eat into the duty cycle and reduce the instrument sensitivity.

[0215] Ions are transported from the ion source to the analyzer. Multiple separate ion packets can be processed simultaneously in different components.

[0216] Fragmentation can be performed in the high-pressure region of the IRM 120 or the second ion storage device (also referred to as the "extraction trap" 140). During operation according to some example methods, the collision energy of the fragmentation chamber can be adjusted.

[0217] Because the first ion storage device holds precursor ions for a first ion destination (e.g., a Fourier transform mass analyzer) and transports the precursor ions to a second ion destination (e.g., a second ion storage device or a ToF mass analyzer), fragmentation is performed downstream of the first ion storage device (e.g., via the high-pressure region of the extraction trap 140 or the IRM collision cell 120).

[0218] In another variant of the DIA process shown in FIG. 3, a stepped fragmentation energy can be applied to each MS2 scan. In other words, MS2 ions are added to the second ion storage device in multiple injections, each injection having a different fragmentation energy.

[0219] In some variants of the method, in the case where a pre-scan is performed, it is preferably not to perform SIM injection on the m / z sub-ranges of the total precursor mass range that have been densely filled. This can be determined based on a full MS pre-scan, and the full MS scan should collect sufficient data for such regions. Omitting SIM injection for these sub-ranges can further reduce the time required for the entire method and improve the resolution of the scans for combined SIM injection. SIM injection can be omitted by operating the ion beam separator in an operating mode such that substantially all ion packets are directed to the second exit region. The HDR MS1 scan can be obtained by combining the full MS pre-scan with the scans for combined SIM injection.

[0220] Using a front-end accumulation device such as a trapped ion mobility separator (as incorporated in the Bruker TIMS-ToF series mass spectrometers) or a structure for non-destructive ion manipulation (SLIM) can also be beneficial for the above examples. Such devices can release ions in an m / z range synchronized with the quadrupole isolation window. Thus, ion transmission can be improved. Also thus, the required injection time can be reduced (due to a brighter isolated ion beam). This reduction in injection time can be used to offset the additional time overhead of SIM injection.

[0221] The above examples are described in the context of a C-trap and an instrument including a C-trap, which is particularly suitable for performing the disclosed methods. However, these methods can be performed using any instrument having a suitable collision thickness and arranged to produce a controlled potential barrier. Many different other types of ion storage devices are suitable. In addition, multipole devices (e.g., IRM) can also be suitable for performing the proposed methods. The typical operating conditions for the multi-device are 1×10 at a length of 120 mm -2mbar. To promote the proportional cooling of the ion beam used to implement the proposed method, the usual multipole conditions can be scaled down to about one-half to one-third.

[0222] In this document, the term mass may be used to refer to the mass-to-charge ratio m / z. Unless otherwise stated, the resolution of a mass analyzer is understood to refer to the mass analyzer resolution determined at a mass-to-charge ratio of 200.

[0223] In the description of the present invention herein, it should be understood that unless otherwise implicitly or explicitly understood or stated, words in the singular form encompass their plural counterparts, and words in the plural form encompass their singular counterparts. Further, it should be understood that unless otherwise implicitly or explicitly understood or stated, for any given component or embodiment described herein, any possible candidates or alternatives listed for that component can generally be used individually or in combination with each other. Further, it should be understood that the drawings shown herein are not necessarily drawn to scale, where only some elements may be drawn for the clarity of the present invention. Also, reference numerals may be repeated in the respective drawings to indicate corresponding or similar elements. Additionally, it should be understood that any such list of candidates or alternatives is illustrative only and not restrictive unless otherwise implicitly or explicitly understood or stated.

[0224] Unless otherwise defined, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification (including definitions) shall prevail. It should be understood that there is an implied "about" before quantitative terms mentioned in the description of the present invention, such that minor and non-substantial deviations are within the scope of the present invention. In this application, unless otherwise specifically stated, the use of the singular includes the plural. Further, the use of "comprising", "including" and "containing" is not intended to be limiting and does not exclude the possibility of also including other elements. In the case of using the word "consisting", this is intended to mean excluding other elements. As used herein, "a" may also refer to "at least one" or "one or more". Further, the use of "or" is inclusive, such that the phrase "A or B" is true when A is true, B is true, or both A and B are true.

[0225] As used in this document, when used as a noun, the term "scan" means a mass spectrum, regardless of the type of mass analyzer used to generate and acquire the mass spectrum. When used as a verb in this document, the term "scan" refers to generating and acquiring a mass spectrum by a mass analysis method, regardless of the type of mass analyzer or mass analysis used to generate and acquire the mass spectrum. As used herein, the term "full scan" refers to a mass spectrum that covers a range of mass-to-charge (m / z) values including multiple mass spectral peaks.

[0226] As used in this document, each of the terms "liquid chromatography" and "liquid chromatography method" (both abbreviated as "LC") and the term "liquid chromatography mass spectrometry" (abbreviated as "LC-MS") is intended to apply to any type of liquid separation system capable of separating a liquid sample carrying multiple analytes into various "fractions" or "isolates", where the chemical composition of each such "fraction" or "isolate" is different from that of each other such fraction or isolate, and where the term "chemical composition" refers to the quantity, concentration, and / or type of the various analytes in the fraction or isolate. Thus, the terms "liquid chromatography", "liquid chromatography method", "liquid chromatography mass spectrometry", "LC", and "LC-MS" are intended to include and refer to, but not be limited to, liquid chromatography, high performance liquid chromatography, ultra-high performance liquid chromatography, size exclusion chromatography, and capillary electrophoresis devices.

[0227] In addition to LC devices, any other separation device, including ion mobility devices, HPLC, GC, or ion chromatography, can be connected to a mass spectrometer. Any known fragmentation method (including collision-activated dissociation, photon-induced dissociation, electron capture, or electron transfer dissociation) also produces data suitable for the present invention.

[0228] Although embodiments in accordance with the present disclosure have been described with reference to specific types of devices and applications (notably mass spectrometers), and in such cases the embodiments have specific advantages as discussed herein, the methods in accordance with the present disclosure can be applied to other types of devices and / or applications. Specific manufacturing details of the ion guide and associated uses are not only potentially advantageous (notably in view of known manufacturing constraints and capabilities), but can vary significantly to obtain devices with similar or identical operation. Unless otherwise specified, each feature disclosed in this specification can be replaced with an alternative feature for the same, equivalent, or similar purpose. Thus, unless otherwise specified, each feature disclosed is only one example of a series of equivalent or similar attribute features.

[0229] Unless otherwise stated, the use of any and all examples or exemplary language ("e.g.", "such as", and similar language) herein is intended merely to better illustrate the invention and does not limit the scope of the invention. No language in the specification should be construed as indicating any non-claimed element as essential for practicing the invention.

[0230] Unless otherwise specified or the context otherwise requires, any steps described in this specification can be performed in any order or simultaneously.

[0231] All aspects and / or features disclosed in this specification can be combined in any combination, except for combinations where at least some of such features and / or steps are mutually exclusive. As described herein, there can be specific combinations of aspects that have additional benefits, such as aspects for an ion guide for a mass spectrometer and / or an ion mobility spectrometer. Specifically, the preferred features of the present invention apply to all aspects of the present invention and can be used in any combination. Similarly, features described in non-essential combinations can be used individually (not in combination).

Claims

1. A mass spectrometry method, comprising the following steps: For each of a plurality of sub-ranges selected from a total m / z range: Injecting a sample of precursor ions having an m / z value within the sub-range into a first ion storage device via an inlet region; Holding a first portion of the sample of precursor ions within the first ion storage device, wherein the first portion comprises ions having an m / z value within the sub-range; Ejecting a second portion of the sample of precursor ions from the first ion storage device via an outlet region, wherein the second portion comprises ions having an m / z value within the sub-range; Any one of the following: a) Analyzing the sample of fragmented precursor ions in a first mass analyzer, or b) Accumulating the sample of fragmented precursor ions in a second ion storage device for analysis in a first mass analyzer, wherein the sample of fragmented precursor ions is formed by fragmentation of the second portion of the precursor ions.

2. The method according to claim 1, wherein each of the plurality of sub-ranges has the same width, and the width of each sub-range is 20 Thomson or less.

3. The method according to claim 1 or claim 2, wherein a DC barrier is provided at the outlet region.

4. The method according to claim 3, further comprising for each sub-range: Determine the charge state of the sample of the precursor ion and adjust the DC barrier based on the charge state of the sample of the precursor ion; and / or Adjusting the DC barrier based on the m / z sub-range; and / or Adjusting the DC barrier to compensate for space charge conditions.

5. The method according to claim 3 or claim 4, wherein the first ion storage device comprises one or more intermediate barriers, and the first portion of the sample of precursor ions is held below the one or more intermediate barriers, and the one or more intermediate barriers are relatively small compared to the DC barrier at the outlet region.

6. The method according to any one of the preceding claims, further comprising for each sub-range: Adjusting the ion energy of the sample of precursor ions and / or adjusting the DC barrier at the outlet aperture to compensate for space charge conditions; and / or Adjusting the ion energy of the sample of precursor ions based on the m / z sub-range.

7. The method according to any one of the preceding claims, wherein: Holding the first portion of the sample of precursor ions within the first ion storage device comprises holding the first portion of the sample of precursor ions away from the main axis of the first ion storage device such that the sample of precursor ions of a subsequent sub-range is not blocked; and / or The first ion storage device comprises a weak potential saddle such that the first portion of the sample of precursor ions is stored away from the main axis of the first ion storage device; and / or The first ion storage device has a certain length, and the length of the first ion storage device is sufficient such that most of the first portion of the sample of precursor ions is held within the first ion storage device away from the outlet region.

8. The method according to any one of the preceding claims, wherein the method further comprises transferring the precursor ions held in the first ion storage device to a) the first mass analyzer or b) the second ion storage device, wherein the precursor ions transferred from the first ion storage device comprise the first portion of the sample of the precursor ions of each sub-range of the plurality of sub-ranges.

9. The method according to any one of the preceding claims, wherein the first ion storage device is configured to operate under pure molecular flow conditions.

10. The method according to any one of the preceding claims, further comprising, for each of the plurality of sub-ranges: Configuring an ion filter to transmit precursor ions having m / z values within the sub-range; Wherein the sample of the precursor ions is received from the configured ion filter, Wherein the sample of the fragmented precursor ions is formed by fragmentation of the precursor ions received from the configured ion filter.

11. The method according to claim 10, wherein configuring the ion filter comprises setting a transmission window of the ion filter, wherein the transmission window is adjusted between each of the plurality of sub-ranges, and wherein for each sub-range, the transmission window for the step of injecting the sample of the precursor ions is the same as the transmission window for the step of a) analyzing the sample of the fragmented precursor ions or b) accumulating the sample of the fragmented precursor ions.

12. The method according to claim 10 or claim 11, further comprising configuring an ion mobility separator to transfer precursor ions having m / z values within the sub-range to the ion filter.

13. The method according to claim 12, further comprising, for each of the plurality of sub-ranges within the total m / z range, controlling the ion mobility separator such that the precursor ions transferred to the ion filter correspond to the transmission window of the ion filter.

14. The method according to any one of the preceding claims, wherein injecting the sample of the precursor ions comprises controlling the fill time of the precursor ions based on the relative abundances of the precursor ion species in the corresponding sub-range.

15. The method according to any one of the preceding claims, further comprising: Configuring an ion filter to transmit precursor ions having m / z values from the total m / z range; Transferring an initial sample of precursor ions having m / z values from the total m / z range to the first mass analyzer or the second mass analyzer; Analyzing the initial sample of the precursor ions; And Obtaining scan data of the total m / z range from the analysis of the initial sample of the precursor ions.

16. The method according to claim 15, further comprising: For each sub-range, adjusting the injection time of the sample of the precursor ions based on the scan data obtained from the analysis of the initial sample of the precursor ions.

17. A method of manipulating ions using an ion storage device, the ion storage device comprising: An inlet region; An outlet region; A trapping volume between the inlet region and the outlet region, The method comprising: Inject an ion packet via the inlet region, where the ion packet comprises ions having m / z values within a sub-range selected from a total m / z range; Apply a DC barrier at the inlet region and at the outlet region; Retain a first portion of the ion packet within the trapping volume, where the first portion comprises ions having m / z values within the sub-range; and Eject a second portion of the ion packet via the outlet region, where the second portion comprises ions having m / z values within the sub-range.

18. The method according to claim 17, wherein: The DC barrier applied at the outlet region is greater than the DC electric potential in the trapping volume; and / or The DC barrier applied at the inlet aperture is greater than the DC electric potential in the trapping volume.

19. The method according to claim 17 or claim 18, wherein the width of the sub-range is 20 Thomson or less.

20. The method according to any one of claims 17 to 19, further comprising one or more of the following: Determine the charge state of the ion packet and adjust the DC barrier at the outlet region based on the determined charge state of the ion packet; Adjust the ion energy of the ion packet based on the m / z range of the ion packet; Adjust the DC barrier at the outlet aperture based on the m / z range of the ion packet; And Adjust the ion energy of the ion packet and / or adjust the DC barrier at the outlet aperture to compensate for space charge conditions.

21. The method according to any one of claims 17 to 20, wherein: Retaining the first portion of the ion packet within the ion storage device comprises retaining the first portion of the ion packet away from the main axis of the ion storage device; and / or The ion storage device comprises a weak potential saddle such that the first portion of the ion packet is stored away from the main axis of the ion storage device; and / or The ion storage device has a sufficient length such that a majority of the first portion of the ion packet is retained within the ion storage device away from the outlet region.

22. The method according to any one of claims 17 to 21, wherein the ion storage device comprises one or more intermediate barriers, where the first portion of the ion packet is retained below the one or more intermediate barriers, and where the one or more intermediate barriers are relatively small compared to the DC barrier at the outlet region.

23. A mass spectrometer configured to perform the method according to any one of claims 1 to 16.

24. An ion storage device configured to perform the method according to any one of claims 17 to 22.

25. A computer software comprising instructions that, when executed by a processor of a computer, cause the computer to perform the method according to any one of claims 1 to 22.

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