Data independent acquisition (DIA) using ion separation

Abstract

The present disclosure relates to data independent acquisition (DIA) using ion separation. A method of operating an analytical instrument includes ionizing a sample to produce sample ions; (i) performing a first ion separation scan by separating the sample ions according to a first physicochemical property, and analyzing the separated sample ions by performing one or more MS1 mass analysis scans; and (ii) performing a second ion separation scan by separating the sample ions according to the first physicochemical property, and analyzing the separated sample ions by performing a plurality of MS2 mass analysis scans. Each MS2 scan in the plurality of MS2 mass analysis scans uses one MS2 isolation window in the plurality of MS2 isolation windows. The method further includes analyzing MS1 data acquired from the one or more MS1 mass analysis scans, and configuring the plurality of MS2 isolation windows based on the analysis of the MS1 data.

Description

Technical Field

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

[0002] Mass spectrometry applications in proteomics and similar fields are typically performed using two different data acquisition approaches: data-dependent acquisition (DDA) and data-independent acquisition (DIA).

[0003] Figure 1 A typical DIA workflow is schematically shown, where samples are separated by liquid chromatography (LC) and ionized into a mass spectrometer (MS). The mass spectrometer is configured to perform a single MS1 scan covering the entire m / z region of interest ( Figure 1 ), and this scan is followed by a series of MS2 scans using various small mass filter m / z isolation windows ( Figure 1 (Dark shaded box in the figure), this small mass filter m / z isolation window, when integrated across the entire series of MS2 scans, covers the m / z region analyzed by the initial MS1 scan. This combination of an MS1 ​​scan and a subsequent MS2 scan is called a cycle, and this cycle is repeated multiple times throughout the LC gradient. Because a single LC elution profile is on the order of multiple seconds, and cycle times are typically 1-2 seconds, multiple cycles can occur along the elution profile, facilitating identification and quantification of sample ions.

[0004] It is believed that there is still room for improvement in mass spectrometry methods and apparatus. Summary of the Invention

[0005] A first aspect provides a method of operating an analytical instrument, the method comprising:

[0006] ionizing the sample to produce sample ions;

[0007] (i) performing a first ion separation scan by separating sample ions according to a first physicochemical property, and analyzing the separated sample ions by performing one or more MS1 mass analysis scans; and

[0008] (ii) performing a second ion separation scan by separating the sample ions according to the first physicochemical property, and analyzing the separated sample ions by performing a plurality of MS2 mass analysis scans, wherein each MS2 scan in the plurality of MS2 mass analysis scans uses one MS2 isolation window from the plurality of MS2 isolation windows.

[0009] The method may further include analyzing MS1 data acquired from the one or more MS1 mass analysis scans, and configuring a plurality of MS2 isolation windows based on the analysis of the MS1 data.

[0010] Embodiments relate to methods for operating analytical instruments such as mass spectrometers. The instrument may include an ion source configured to generate ions from a sample; an ion separator arranged downstream of the ion source and configured to separate the received ions according to a first physicochemical property; a mass filter arranged downstream of the ion separator and configured to filter the received ions according to the mass-to-charge ratio (m / z) of the received ions (i.e., using an isolation window with a central mass-to-charge ratio (m / z) and a width); a fragmentation device arranged downstream of the mass filter and configured to selectively fragment the received ions; and a mass analyzer arranged downstream of the mass filter and / or the fragmentation device and configured to perform mass analysis on the received ions. It is beneficial to include an ion separation device (such as an ion mobility (IM) separator, a differential ion mobility separator, or a device configured to separate ions according to the mass-to-charge ratio (m / z) of the ions) because this improves the duty cycle and sensitivity of the instrument.

[0011] The present inventors have recognized that for analytical instruments that include ion separation, such as ion mobility (IM) separation, conventional DIA workflows will no longer work. As described in more detail below, this is because the previous correlation between MS1 ​​scans and MS2 scans no longer exists, so that the sample ions of interest will generally not be present in the MS2 scan. The embodiments described herein provide a new data acquisition strategy that facilitates DIA methods using instruments with ion separation devices, such as IM-MS instruments.

[0012] In this method, the analytical instrument can perform a data-independent acquisition (DIA) method by performing one or more MS1 mass analysis scans during a first ion isolation scan, followed by performing multiple MS2 mass analysis scans during a second (e.g., immediately following) ion isolation scan. The instrument can perform one or more additional multiple MS2 mass analysis scans during one or more (immediately following) subsequent ion isolation scans. These steps can be repeated, for example, such that the analytical instrument repeatedly switches between performing MS1 mass analysis scans and MS2 mass analysis scans.

[0013] In this method, each of the multiple MS2 mass analysis scans uses a different MS2 isolation window from the multiple MS2 isolation windows. That is, the isolation window of the mass filter is controlled to be different in each of the multiple MS2 mass analysis scans performed during the second (or third, or fourth, etc.) ion isolation scan.

[0014] Furthermore, each MS2 isolation window is configured at least in part based on an analysis of MS1 data collected from one or more MS1 mass analysis scans. As further described below, by configuring multiple MS2 isolation windows based on an analysis of MS1 data, the sensitivity and / or duty cycle of the instrument can be significantly improved. Thus, this method provides an improved DIA workflow for instruments that include ion separators.

[0015] The analytical instrument can be a mass spectrometer, for example, including an ion source. Ions can be generated from a sample in the ion source. The ion source can be coupled to a chromatographic separation device, such as a liquid chromatography (LC) separation device, a gas chromatography (GC) separation device, etc., so that the sample ionized in the ion source comes from the chromatographic separation device.

[0016] Analytical instrument may include an ion separator, which is arranged in the downstream of an ion source, and is configured to perform ion separation scanning to separate the ion received from the ion source according to the first physicochemical property. The ion separator may be an ion mobility separator, in which case each ion separation scanning is an ion mobility separation scanning, and the first physicochemical property is ion mobility. Alternatively, the ion separator may be a differential ion mobility separator, in which case each ion separation scanning is a differential ion mobility separation scanning, and the first physicochemical property is differential ion mobility. Alternatively, the ion separator may be a device configured to separate the ion according to the mass-to-charge ratio (m / z) of the ion, in which case each ion separation scanning is a mass-to-charge ratio (m / z) separation scanning, and the first physicochemical property is a mass-to-charge ratio (m / z). The further details of the ion separator of various possible types are provided below.

[0017] The ion separator can be operated in a cyclic manner, i.e., repeatedly performing ion separation scans. In each ion separation scan, the ion separator can receive ions from an ion source and, for example, accumulate ion packets in an accumulation region. Alternatively, the ion packets can be accumulated in an ion trap upstream of the ion separator. The ion separator can then separate the ion packets according to a first physicochemical property of the ions, for example, by passing the ion packets through the ion separation region. Ions with higher first physicochemical property values ​​arrive at the end of the ion separation region (and leave the separator) before ions with lower first physicochemical property values ​​(or vice versa).

[0018] Each ion isolation scan may have a duration T IMS In other words, the ion separator may have a cycle time T IMS Duration T IMS The time required to accumulate the ion packets and the time required to separate the ions may be included. Alternatively, the duration T IMSThe duration T may correspond only to the time required to separate the ions, wherein the accumulation of ion packets is performed in parallel with the separation of previously accumulated ion packets. IMS It can be about hundreds of milliseconds or thousands of milliseconds. IMS It may be constant in any given experiment, but can be varied between experiments by appropriate control of the instrument.

[0019] The analytical instrument may include a mass filter, which is arranged in the downstream of the ion separator and is configured to receive the separated ions from the ion separator. The mass filter can be any suitable mass filter that can operate to filter the ions according to the m / z of the ions, such as a quadrupole mass filter. The mass filter can be configured to isolate and forward transmit the received ions with the m / z within the m / z isolation window, while the received ions with the m / z outside the m / z isolation window are attenuated by the mass filter, for example, not forward transmit the ions with the m / z outside the m / z isolation window. The width and / or center m / z of the isolation window are controllable (variable) by suitably controlling the RF voltage and DC voltage applied to the mass filter. Therefore, for example, the mass filter can be operated in a transmission operation mode, whereby most or all ions in a relatively wide m / z isolation window are forward transmit the ions by the mass filter, and can be operated in a filtering operation mode, whereby only ions in a relatively narrow m / z isolation window (centered on the desired m / z) are isolated and forward transmit the ions by the mass filter.

[0020] The analytical instrument may comprise a fragmentation device arranged downstream of the mass filter and configured to receive ions transmitted by the mass filter. The fragmentation device may be configured to selectively fragment some or all of the received ions, i.e. to produce fragment ions. The fragmentation device is capable of operating in a fragmentation mode of operation, whereby most or all of the received ions are fragmented to produce fragment ions (which can then be transmitted onward from the fragmentation device), and is capable of operating in a non-fragmentation mode of operation, whereby most or all of the received ions are transmitted onward without being (intentionally) fragmented. It is also possible to achieve the non-fragmentation mode of operation by causing the ions to bypass the fragmentation device.

[0021] The analytical instrument may include a mass analyzer that is arranged downstream of the fragmentation device and is configured to perform a mass analysis scan to determine the mass-to-charge ratio (m / z) of the received ions. The mass analyzer can be operated in a cyclic manner, i.e., to repeatedly perform mass analysis scans. In each mass analysis scan, the mass analyzer receives ions and performs mass analysis on the ions. The mass analyzer can be an ion trap mass analyzer, such as an electrostatic orbital trap, and more specifically an Orbitrap TMFT mass analyser. Other types of mass analysers would be possible. For example, the mass analyser could be a time of flight (ToF) mass analyser, such as a multi-reflection time of flight (MR-ToF) mass analyser.

[0022] In some embodiments, the ion beam is passed to the mass analyzer in the form of an ion beam, for example, without being accumulated before being passed to the mass analyzer. Therefore, in embodiments, ions are directly accumulated in the mass analyzer. In these embodiments, the number of ions accumulated in the mass analyzer can be controlled by controlling the accumulation time (for example, fill time) of the ions entering the mass analyzer. This can then be controlled by operating the door or lens of the mass analyzer and / or the upstream (between ion source and mass analyzer) of the mass analyzer in the open (transmission) operating mode for a desired amount of time (and otherwise operating the door or lens in the closed (non-transmission) operating mode).

[0023] However, in a specific embodiment, ions are transferred to the mass analyzer from an ion trap arranged upstream of the mass analyzer. The ions may initially accumulate in the ion trap and then be transferred to the mass analyzer, for example, in the form of ion packets. The ion trap may be referred to as an injection device for injecting ions into the mass analyzer. The ion trap may comprise any suitable ion trap, such as a linear ion trap or a curved linear ion trap (C-trap), for example, as described in WO 2008 / 081334. The ion trap may be used to cool the accumulated ions before they are injected into the mass analyzer. The ion trap may also or alternatively (in the MS 2 In the ion trap mode of operation, the ion trap is used as a fragmentation device to fragment the ions before injecting them into the mass analyzer. Multiple ion traps can also be used.

[0024] In these embodiments, the number of ions accumulated within the mass analyzer can be controlled by controlling the accumulation time (e.g., fill time) of ions entering the ion trap. This, in turn, can be controlled by operating the door or lens of the ion trap and / or a door or lens within the instrument upstream of the ion trap (between the ion source and the ion trap) in an open (transmitting) mode of operation for a desired amount of time (and otherwise operating the door or lens in a closed (non-transmitting) mode of operation).

[0025] Each mass analysis scan has a duration T MA In other words, the mass analyzer has a cycle time T MA . T MA All overhead associated with the operation of the mass analyzer may be included. Duration T MAThe time required to accumulate ion packets (and optionally cool and / or fragment those ions, and optionally inject those ions into a mass analyzer) and the time required to mass analyze those ions may be included. Alternatively, the duration T MA It may correspond only to the time required to mass analyze an ion packet, or only to the time required to accumulate ion packets (and optionally cool and / or fragment those ions), wherein accumulation of ion packets is performed in parallel with mass analysis of previously accumulated ion packets.

[0026] In certain embodiments, the mass analysis scan duration T MA Less than the ion separation scan duration T IMS , that is, T MA <T IMS In an embodiment, the mass analyzer is wherein the mass analyzer scan time T MA Relatively long, for example, of a type such that two or more, tens or hundreds of mass analysis scans may be performed during each ion isolation scan. Duration T MA It can be about tens of milliseconds or hundreds of milliseconds. MA It may be constant in any given experiment, but can be varied between experiments by appropriate control of the instrument.

[0027] The analytical instrument is capable of operating in at least an MS1 ​​mode of operation in which the instrument performs one or more MS1 mass analysis scans and in an MS2 mode of operation in which the instrument performs one or more MS2 mass analysis scans.

[0028] In each MS1 mass analysis scan, the mass filter is operated in its transmission mode or its filtering mode with a relatively wide isolation window width (e.g., on the order of hundreds or thousands of Th), and the ions are not (intentionally) fragmented, so that a relatively wide m / z range of ions generated by the ion source is mass analyzed by the mass analyzer.

[0029] In each MS2 mass analysis scan, the mass filter is operated in its filtering mode with a relatively narrow (e.g., about one or tens of Th) isolation window width to isolate ions, and the isolated ions are fragmented in a fragmentation device, so that ions in a relatively narrow m / z range generated by the ion source are isolated and fragmented, and the resulting fragment ions are mass analyzed by the mass analyzer.

[0030] Each MS1 mass analysis scan may have a duration T MS1 , and each MS2 mass analysis scan may have a duration T MS2 . Usually, T MS1 >T MS2, because high resolution data is relatively more important for MS1 scans, while high speed is relatively more important for MS2 scans (e.g., so that many more MS2 scans can be collected per unit time). In the case where the instrument is operated in a cyclic manner, typically T MS2 Can be set to T MS1 A fraction of, for example, T MS2 / T MS1 = 1 / 2、 1 / 4、 1 / 8, 1 / 16, etc.

[0031] In the methods of various embodiments, an ion source ionizes a sample (e.g., received from a (LC) separation device) to produce sample ions, and an ion separator performs a plurality of repeated ion separation scans, wherein in each ion separation scan, the ion separator receives the sample ions and separates them according to a first physicochemical property. Simultaneously, a mass analyzer performs a plurality of repeated mass analysis scans, wherein in each mass analysis scan, the mass analyzer receives the separated sample ions or fragment ions obtained from the separated sample ions and performs mass analysis on the received ions.

[0032] In particular, the analytical instrument may (i) perform one or more MS1 mass analysis scans during a first ion isolation scan, and (ii) perform multiple MS2 mass analysis scans during a second ion isolation scan. In the multiple repeated ion isolation scans, the second ion isolation scan may be immediately followed by the first ion isolation scan, but this is not required. Steps (i) and (ii) may be performed repeatedly, for example, such that the analytical instrument repeatedly switches between performing an MS1 ​​mass analysis scan in one ion isolation scan and performing an MS2 mass analysis scan in a subsequent ion isolation scan (and then performing an MS1 ​​mass analysis scan in the subsequent ion isolation scan, and so on).

[0033] In another embodiment, each cycle includes more than two ion separation scans. Therefore, for example, the method may include that the analytical instrument performs a plurality of repeated cycles, wherein each cycle includes any number of (such as three, four, five or more times) ion separation scans in the multiple repeated ion separation scans. Each ion separation scan in the cycle may be for MS1 analysis or MS2 analysis. In each cycle, there may be one or more (e.g., twice, three times, four times, etc.) MS1-guided ion separation scans, and in each cycle, there may be one or more (e.g., twice, three times, four times, etc.) MS2-guided ion separation scans. The timing of the mass analysis scan and the isolation window for each mass analysis scan may be configured so that each ion separation scan in the cycle is directed to different regions of ion arrival time-m / z space (i.e., first physicochemical property-m / z space). However, it is also possible that the region of ion arrival time-m / z space is analyzed (isolated again) more than once during the cycle.

[0034] In some embodiments, each cycle includes two or more ion isolation scans, and an MS1 ​​mass analysis scan is performed during an earlier ion isolation scan of each cycle, and an MS2 mass analysis scan is performed during a later ion isolation scan of each cycle. For example, each cycle can include the analytical instrument (i) performing one or more MS1 mass analysis scans during a first ion isolation scan, (ii) performing a first plurality of MS2 mass analysis scans during a second (e.g., immediately subsequent) ion isolation scan, (iii) performing a second plurality of MS2 mass analysis scans during a third (e.g., immediately subsequent) ion isolation scan, (iv) optionally performing a third plurality of MS2 mass analysis scans during a fourth (e.g., immediately subsequent) ion isolation scan, and (v) optionally performing one or more additional plurality of MS2 mass analysis scans during each of one or more additional (e.g., immediately subsequent) ion isolation scans.

[0035] Other orderings of MS1 scans and MS2 scans in each cycle are possible. For example, one or more additional MS1-guided ion isolation scans may be performed in each cycle (e.g., after the first ion isolation scan and before the MS2-guided ion isolation scan). Thus, while in some embodiments the "second" ion isolation scan follows the "first" ion isolation scan immediately, in other embodiments there may be one or more ion isolation scans between the "first" and "second" ion isolation scans in each cycle (and similarly for the "third" and "fourth" ion isolation scans).

[0036] Analytical instruments may perform multiple repetitive cycles during a complete chromatographic separation run on a (LC) chromatographic separation device.

[0037] During the first ion isolation scan of each cycle (and / or during any other MS1-directed ion isolation scan), the instrument may perform only one MS1 mass analysis scan, or may perform multiple MS1 mass analysis scans. The one or more MS1 mass analysis scans performed during the first (and / or other MS1) ion isolation scans of the cycle may together span approximately the duration T of the first ion isolation scan. IMS Thus, in the case where only a single MS1 mass analysis scan is performed during the first (and / or further MS1) ion isolation scan, T MS1 ≈T IMS Where multiple MS1 mass analysis scans are performed during the first (and / or further MS1) ion isolation scan, approximately N≈T may be performed during the first (and / or further MS1) ion isolation scan. IMS / T MS1 MS1 mass analysis scan.

[0038] The one or more MS1 mass analysis scans performed during the first (and / or additional MS1) ion isolation scan of a cycle can together span the m / z range of interest. The m / z range of interest can be any suitable m / z range, such as, for example, between about 100 and 2000, or the like. Thus, where only a single MS1 mass analysis scan is performed during the first (and / or additional MS1) ion isolation scan, the mass filter can be operated in its transmission mode or its filtering mode with a wide isolation window width that covers the m / z range of interest, such that the MS1 mass analysis scan can determine the mass-to-charge ratio (m / z) of ions within the entire m / z range of interest.

[0039] Where multiple MS1 mass analysis scans are performed during the first (and / or additional MS1) ion isolation scans, each mass analysis scan can be configured to determine the mass-to-charge ratio (m / z) of ions within a sub-range of the m / z range of interest. Thus, the m / z range of interest can be divided into a plurality (N) of overlapping or non-overlapping m / z sub-ranges, wherein the plurality of sub-ranges spans the entire m / z range of interest, and wherein each mass analysis in the plurality of MS1 mass analysis scans is configured to determine the mass-to-charge ratio (m / z) of ions within a respective different one of the sub-ranges.

[0040] To this end, each of the multiple MS1 mass analysis scans can use an MS1 ​​isolation window from a plurality of different MS1 isolation windows. That is, the isolation window of the mass filter can be controlled to be different in each of the multiple MS1 mass analysis scans performed during the first (and / or other MS1) ion isolation scans.

[0041] The multiple MS1 isolation windows can differ from each other in their center m / z. That is, each MS1 scan in the multiple MS1 mass analysis scans can use an MS1 ​​isolation window center m / z from a plurality of different MS1 isolation window center m / z (and the isolation window center m / z of the mass filter can be controlled to be different in each MS1 scan in the multiple MS1 mass analysis scans).

[0042] The multiple MS1 isolation windows may also differ from one another in their width, although this is not required and, instead, each MS1 scan in the multiple MS1 mass analysis scans may use the same MS1 isolation window width.

[0043] The MS1 isolation window center m / z and width can be selected so that multiple MS1 scans together span the entire m / z range of interest. Additionally, each MS1 isolation window can be selected based on one or more trend lines.

[0044] In some embodiments, each trend line corresponds to an ion charge state, and each trend line provides a relationship between ion arrival time and m / z for ions having that charge state. For example, each trend line can provide a relationship between ion mobility arrival time and m / z for ions having that charge state.

[0045] It has been recognized that for ions of various different charge states (e.g., singly, doubly, triply charged, etc.), there is a relationship between the ion mobility arrival time and m / z. These relationships can take the form of a "trend line" for each different charge state. The relationship between the ion mobility arrival time and m / z can be roughly linear for ions with a specific charge state, and therefore can be described by a linear trend line, such as in the form of a slope and an intercept. However, depending on the properties of the sample ions, nonlinear trend lines are also possible. In practice, for ions with a specific charge and a specific m / z, there will be some spread in the arrival time, but typically the spread is small enough to make it possible to distinguish ions of different charge states in most ion mobility arrival time-m / z spaces.

[0046] In some embodiments, in addition to or instead of charge state, each trend line corresponds to a particular chemical class, wherein each trend line provides a relationship between ion arrival time and m / z for ions of that chemical class and, optionally, that charge state. That is, different chemical classes can have different (charge-dependent) trend lines. Examples of chemical classes include: peptides derived from tryptic digests of proteins; a proteome or mixture; a lipid or lipidome; a metabolite or metaboliteome; a nucleotide, etc.

[0047] Thus, each trend line can correspond to (i) a particular charge state (e.g., singly, doubly, triply, etc.), (ii) a particular chemical class, or (iii) a particular combination of charge state and chemical class. Each trend line can provide an approximate relationship between ion arrival times and m / z for ions having that charge state and / or that chemical class. Each trend line can be used to determine an approximate expected ion arrival time for a sample ion that (i) depends on the m / z and charge state of the sample ion, (ii) depends on the m / z and chemical class of the sample ion, or (iii) depends on the m / z, charge state, and chemical class of the sample ion.

[0048] Trend lines can also be defined and used based on some other properties of the sample ions.

[0049] As described further below, and as described in more detail in co-pending application US 63 / 468,170 (the entire contents of which are incorporated herein by reference), one or more trend lines may be determined by performing a calibration on the instrument.

[0050] In an embodiment, each of the plurality of MS1 isolation windows is selected based on one or more of these trend lines. By selecting the MS1 isolation window based on the one or more trend lines during the first (and / or further MS1) ion isolation scan (e.g., by controlling the isolation window of the mass filter to track one or more of the trend lines), it can be ensured that (only) sample ions of interest (e.g., having one or more specific charge states and / or having a specific chemical class of interest) are present in the MS1 scan.

[0051] Thus, each MS1 isolation window center m / z can be selected based on one or more of the trend lines. That is, the center m / z of the isolation window of the mass filter can be configured to track one or more of the trend lines. Thus, for example, where each MS1 isolation window center m / z is selected based on a single trend line, the isolation window center m / z of each MS1 scan can be (approximately) equal to the m / z value given by the single trend line at the center (average) arrival time of the MS1 scan. Where each MS1 isolation window center m / z is selected based on two or more trend lines, the isolation window center m / z of each MS1 scan can be (approximately) equal to the average m / z value given by those two or more trend lines at the center (average) arrival time of the MS1 scan.

[0052] Likewise, each MS1 isolation window width can be selected based on one or more trend lines. Thus, for example, where each MS1 isolation window width is selected based on a single trend line, each MS1 isolation window width can be configured such that most or all ions having a charge state and / or chemical class associated with the single trend line are transmitted by the mass filter, and such that most or all ions having a charge state and / or chemical class other than the charge state and / or chemical class associated with the single trend line are attenuated (not transmitted) by the mass filter. Where each MS1 isolation window width is selected based on two or more trend lines, each MS1 isolation window width can be configured such that most or all ions having one of the charge states and / or chemical classes associated with the two or more trend lines are transmitted by the mass filter, and such that most or all ions having a charge state and / or chemical class other than the charge state and / or chemical class associated with the two or more trend lines are attenuated (not transmitted) by the mass filter.

[0053] Thus, in general, multiple MS1 isolation windows can be configured such that separated sample ions having charge states and / or chemical classes corresponding to charge states and / or chemical classes associated with one or more trend lines are isolated, while separated sample ions having charge states and / or chemical classes other than charge states and / or chemical classes associated with one or more trend lines are attenuated.

[0054] The MS1 isolation window width for each MS1 scan in the multiple MS1 scans can be increased with increasing MS1 isolation window center m / z to better isolate those ions having a desired charge state and / or chemical class, and to better attenuate ions having other undesirable charge states and / or chemical classes.

[0055] In certain embodiments, the MS1 isolation window is selected so that singly charged (1+) ions are excluded from the MS1 scan. Thus, the one or more trend lines based on which each MS1 isolation window is selected can include (only) trend lines for multiply charged ions (rather than trend lines for singly charged ions). This allows for more efficient use of the maximum charge capacity of the instrument.

[0056] In certain embodiments, the MS1 isolation window is selected so that ions having the same charge state and / or chemical class as the ions to be selected in the MS2 scan are selected in the MS1 scan. Thus, for example, an MS1 ​​isolation window for multiple MS1 mass analysis scans can be selected based on both a first trend line for a first (e.g., doubly charged) charge state and a second trend line for a different second (e.g., triply charged) charge state.

[0057] As described above, in this method the instrument performs multiple MS2 mass analysis scans during the second (and optionally third, fourth and / or additional) ion isolation scans of each cycle.

[0058] The number of MS2 mass analysis scans performed during the ion isolation scan will depend on the duration T relative to the MS1 mass analysis scan. MS1 The duration of the MS2 mass analysis scan is T MS2 , which can be selected as needed as mentioned above. Roughly M≈N×T MS1 / T MS2 Multiple MS2 mass analysis scans may be performed during an ion isolation scan. The multiple MS2 mass analysis scans performed during an ion isolation scan may together span substantially most or all of the duration T of the ion isolation scan. IMS , but this is not required.

[0059] In various embodiments of the DIA method, multiple MS2 mass analysis scans performed during an ion isolation scan can collectively span the m / z range of interest. Thus, in each MS2 mass analysis scan, the instrument can isolate and fragment ions within a sub-range of the m / z range of interest. Thus, the m / z range of interest can be divided into a plurality (M) of overlapping or non-overlapping m / z sub-ranges, wherein the plurality of sub-ranges span the entire m / z range of interest, and wherein each of the plurality of MS2 scans is configured to isolate and fragment ions having an m / z within a corresponding different sub-range of the sub-ranges.

[0060] To this end, each of the multiple MS2 mass analysis scans uses an MS2 isolation window from a plurality of different MS2 isolation windows. That is, the isolation window of the mass filter can be controlled to be different in each of the multiple MS2 mass analysis scans performed during the ion isolation scan.

[0061] The multiple MS2 isolation windows can differ from each other in their central m / z. That is, each MS2 scan in the multiple MS2 mass analysis scans can use an MS2 isolation window central m / z from a plurality of different MS2 isolation window central m / z (and the isolation window central m / z of the mass filter can be controlled to be different in each MS2 scan in the multiple MS2 mass analysis scans).

[0062] The multiple MS2 isolation windows may also differ from one another in their widths, although this is not required and, alternatively, each MS2 scan in the multiple MS2 mass analysis scans may use the same MS2 isolation window width.

[0063] The MS2 isolation window center m / z and width can be selected so that multiple MS2 scans together span the entire m / z range of interest.

[0064] In some embodiments, each MS2 isolation window is selected based on one or more trend lines, where (as described above) each trend line corresponds to (i) a particular charge state (e.g., singly, doubly, triply, etc.), or (ii) a particular combination of charge state and chemical class. As described above, each trend line can provide an approximate relationship between ion arrival times and m / z for ions having that charge state and, optionally, that chemical class. Each trend line can be used to determine an approximate expected ion arrival time for a sample ion, (i) depending on the m / z and charge state of the sample ion, or (ii) depending on the m / z, charge state, and chemical class of the sample ion.

[0065] By selecting an MS2 isolation window based on one or more trend lines during the second ion isolation scan (e.g., by controlling the isolation window of the mass filter to track one or more of the trend lines), it can be ensured that (only) sample ions of interest (e.g., having a specific charge and / or chemical class) are isolated and fragmented in the MS2 scan.

[0066] As described above, the method includes analyzing MS1 data acquired from one or more MS1 mass analysis scans, and configuring a plurality of MS2 isolation windows based on the analysis of the MS1 data. In particular, the first and / or second and / or third and / or further plurality of MS2 isolation windows can be configured by selecting a center m / z and / or width of one or more or each of the MS2 isolation windows based on the analysis of the MS1 data.

[0067] In some embodiments, configuring the multiple MS2 isolation windows can include selecting specific charge states and / or chemical classes of interest based on analysis of the MS1 data, where each MS2 isolation window is selected based on one or more trend lines. That is, based on the MS1 data, only some (but not all) trend lines can be selected and analyzed by one or more MS2 mass analysis scans in the multiple MS2 mass analysis scans.

[0068] In these embodiments, in the first and / or second and / or third and / or additional multiple MS2 mass analysis scans, each MS2 isolation window center m / z can be selected based on one or more trend lines in the trend lines. That is, the center m / z of the isolation window of the mass filter can be configured to track one or more trend lines in the trend lines. Thus, for example, where each MS2 isolation window center m / z is selected based on a single trend line, the isolation window center m / z of each MS2 scan can be (approximately) equal to the m / z value given by the single trend line at the center (average) arrival time of the MS2 scan. Where each MS2 isolation window center m / z is selected based on two or more trend lines, the isolation window center m / z of each MS2 scan can be (approximately) equal to the average m / z value given by those two or more trend lines at the center (average) arrival time of the MS2 scan.

[0069] Similarly, each MS2 isolation window width can also be selected based on one or more trend lines. Thus, for example, where each MS2 isolation window width is selected based on a single trend line, each MS2 isolation window width can be configured such that most or all ions having a charge state and / or chemical class associated with the single trend line are transmitted by the mass filter, and most or all ions having a charge state and / or chemical class other than the charge state and / or chemical class associated with the trend line are attenuated (not transmitted) by the mass filter. Where each MS2 isolation window width is selected based on two or more trend lines, each MS2 isolation window width can be configured such that most or all ions having one of the charge states and / or chemical classes associated with the two or more trend lines are transmitted by the mass filter, and most or all ions having a charge state and / or chemical class other than the charge state and / or chemical class associated with the two or more trend lines are attenuated (not transmitted) by the mass filter.

[0070] Thus, multiple MS2 isolation windows can be configured such that separated sample ions having charge states and / or chemical classes corresponding to charge states and / or chemical classes associated with one or more trend lines are isolated, while separated sample ions having charge states and / or chemical classes other than charge states and / or chemical classes associated with one or more trend lines are attenuated.

[0071] As described above, the MS2 isolation window width for each MS2 scan in the first and / or second and / or third and / or additional multiple MS2 scans can be increased with increasing MS2 isolation window center m / z to better isolate those ions having a desired charge state and / or chemical class, and to better attenuate ions having other undesirable charge states and / or chemical classes.

[0072] In these embodiments, the MS2 isolation window can be selected so that multiply charged ions having a singly charged state are selected, while singly charged (1+) ions are excluded from the MS2 scan. Thus, the one or more trend lines based on which each MS2 isolation window is selected can include a single trend line for multiply charged ions (e.g., doubly charged ions or triply charged ions).

[0073] In another embodiment, rather than having a single plurality of MS2 isolation windows track one or more trend lines, MS2 scans are performed using (i) a first plurality of MS2 mass analysis scans having a first plurality of MS2 isolation windows during a second ion isolation scan, (ii) a second plurality of MS2 mass analysis scans having a second plurality of MS2 isolation windows during a third ion isolation scan, (iii) optionally a third plurality of MS2 mass analysis scans having a third plurality of MS2 isolation windows during a fourth ion isolation scan, and (iv) optionally one or more additional plurality of MS2 mass analysis scans each having an additional plurality of MS2 isolation windows during additional ion isolation scans.

[0074] The first and / or second and / or third and / or additional multiple MS2 isolation windows can be selected so that the first and / or second and / or third and / or additional multiple MS2 mass analysis scans together cover a region in the ion arrival time-m / z space of interest (e.g., without leaving any gaps in the region). The region in the ion arrival time-m / z space of interest can correspond to one or more trend lines of interest, wherein (as described above) each trend line corresponds to an ion charge state and / or chemical class, and each trend line provides a relationship between ion arrival time and m / z for ions having that charge state and / or chemical class. For example, the region covered by multiple MS2 isolation windows can correspond to multiply charged ions.

[0075] In these embodiments, the plurality of MS1 isolation windows may include an integer number N of isolation windows, the first plurality of MS2 isolation windows may include an integer number M1 of isolation windows, the second plurality of MS2 isolation windows may include an integer number M2 of isolation windows, the third plurality of MS2 isolation windows may include an integer number M3 of isolation windows, and / or each additional plurality of MS2 isolation windows, if present, may include an integer number M f Isolation windows. M1, M2, M3 and M f can be equal or approximately equal, and can each be a multiple of N.

[0076] Each isolation window in the first plurality of MS2 isolation windows may correspond to a corresponding MS2 isolation window in the second and / or third and / or further pluralities of MS2 isolation windows, e.g., such that each isolation window in the first plurality of MS2 isolation windows is used at an ion arrival time in the second ion isolation scan that corresponds to an ion arrival time in the third and / or fourth and / or further ion isolation scan using a corresponding isolation window in the second and / or third and / or further pluralities of MS2 isolation windows. Similarly, each MS1 isolation window in the plurality of MS1 isolation windows may correspond to a corresponding set of MS2 isolation windows in the first and / or second and / or third and / or further pluralities of MS2 isolation windows, e.g., such that each MS1 isolation window in the plurality of MS1 isolation windows is used at an ion arrival time in the first ion isolation scan that corresponds to an ion arrival time in the second and / or third and / or fourth and / or further ion isolation scan using a corresponding set of MS2 isolation windows in the first plurality of MS2 isolation windows.

[0077] Thus, at each ion arrival time, there can be a corresponding set of MS2 isolation windows, wherein each isolation window in the set belongs to an ion isolation scan in the second and / or third and / or fourth and / or further ion isolation scans. Each MS2 isolation window in a set will have a different center m / z.

[0078] In some embodiments, all corresponding MS2 isolation windows in a group have approximately the same width. Then, configuring the first and / or second and / or third and / or additional multiple MS2 isolation windows based on the analysis of the MS1 data can include, for each group of corresponding MS2 isolation windows: assigning each MS2 isolation window in the group to an ion isolation scan in the second and / or third and / or fourth and / or additional ion isolation scan based on the ion abundance within each MS2 isolation window in the group. For example, the MS2 isolation windows in the group covering the highest abundance of ions can be assigned to the second ion isolation scan, the isolation windows in the group covering the second highest abundance of ions can be assigned to the third ion isolation scan, and so on. This will ensure that regions of the arrival time-m / z space of ions of interest with higher ion abundance are prioritized, i.e., are analyzed more quickly, for example, in the second ion isolation scan. However, the MS2 isolation windows can be configured so that regions of the arrival time-m / z space of ions of interest with lower ion abundance are prioritized.

[0079] Thus, the method may include:

[0080] For each MS2 isolation window in one or more or all of the first plurality of MS2 isolation windows: configuring the MS2 isolation window based on the MS1 data to include ions that are more abundant than a corresponding MS2 isolation window in the second plurality of MS2 isolation windows; and / or

[0081] For each MS2 isolation window in one or more or all of the second plurality of MS2 isolation windows: configuring the MS2 isolation window based on the MS1 data to include ions that are more abundant than a corresponding MS2 isolation window in the third plurality of MS2 isolation windows; and / or

[0082] For each MS2 isolation window in one or more or all of the third plurality of MS2 isolation windows: configuring the MS2 isolation window based on the MS1 data to include ions that are more abundant than a corresponding MS2 isolation window in any other plurality of MS2 isolation windows.

[0083] In other embodiments, instead of using the same width for all corresponding MS2 isolation windows in a group, the widths can be different. For example, the widths can be configured based on the MS1 data so that each isolation window in a group includes approximately the same ion abundance.

[0084] Thus, configuring the first and / or second and / or third and / or further multiple MS2 isolation windows may include:

[0085] For each MS2 isolation window in one or more or all of the second plurality of MS2 isolation windows: configuring the MS2 isolation window based on the MS1 data to include ions of the same or similar abundance as a corresponding MS2 isolation window in the first plurality of MS2 isolation windows; and / or

[0086] For each MS2 isolation window in one or more or all of the third plurality of MS2 isolation windows: configuring the MS2 isolation window based on the MS1 data to include ions of the same or similar abundance as a corresponding MS2 isolation window in the first plurality of MS2 isolation windows; and / or

[0087] For each MS2 isolation window in one or more or all of the additional plurality of MS2 isolation windows: configuring the MS2 isolation window based on the MS1 data to include ions of the same or similar abundance as a corresponding MS2 isolation window in the first plurality of MS2 isolation windows.

[0088] In these embodiments, the method may include:

[0089] determining a total ion current indicated by the MS1 data; and

[0090] for each MS2 isolation window in one or more or all of the second plurality of MS2 isolation windows: configuring the MS2 isolation window based on the MS1 data to include an equal or approximately equal portion of the total ion current as a corresponding MS2 isolation window in the first plurality of MS2 isolation windows; and / or

[0091] for each MS2 isolation window in one or more or all of the third plurality of MS2 isolation windows: configuring the MS2 isolation window based on the MS1 data to include an equal or approximately equal portion of the total ion current as a corresponding MS2 isolation window in the first plurality of MS2 isolation windows; and / or

[0092] For each MS2 isolation window in one or more or all of the additional plurality of MS2 isolation windows: configuring the MS2 isolation window based on the MS1 data to include an equal or approximately equal portion of the total ion current as a corresponding MS2 isolation window in the first plurality of MS2 isolation windows.

[0093] As described above, in some embodiments, in each MS2 mass analysis scan, ions are accumulated in the ion store for an accumulation time. In some further embodiments, the method includes determining a fill time for one or more or each MS2 scan based on analysis of the MS1 data.

[0094] According to a second aspect, there is provided a method of operating an analytical instrument, the method comprising:

[0095] ionizing the sample to produce sample ions;

[0096] (i) performing a first ion separation scan by separating sample ions according to a first physicochemical property, and analyzing the separated sample ions by performing one or more MS1 mass analysis scans; and

[0097] (ii) performing a second ion separation scan by separating the sample ions according to the first physicochemical property, and analyzing the separated sample ions by performing a plurality of MS2 mass analysis scans, wherein in each MS2 mass analysis scan, the ions are accumulated in the ion storage for an accumulation time;

[0098] The method further comprises:

[0099] analyzing MS1 data acquired from one or more MS1 mass analysis scans; and

[0100] The accumulation time for one or more or each MS2 scan is determined based on analysis of the MS1 data.

[0101] This aspect and the embodiments may, and in embodiments do, include any one or more or each of the optional features described herein.

[0102] In this aspect and these embodiments, determining the accumulation time of the MS2 scan may comprise:

[0103] using the one or more trend lines and the MS1 data to estimate ion abundances within an MS2 isolation window of the MS2 scan; and

[0104] The accumulation time for the MS2 scan was determined based on the estimated ion abundance.

[0105] Another aspect provides a non-transitory computer-readable storage medium storing computer software code that, when executed on a processor, performs the method described above.

[0106] Another aspect provides a control system for an analytical instrument, such as a mass spectrometer, the control system being configured to cause the analytical instrument to perform the method described above.

[0107] Another aspect provides an analytical instrument, such as a mass spectrometer, comprising a control system as described above.

[0108] Another aspect provides an analytical instrument, the analytical instrument comprising:

[0109] an ion source configured to ionize the sample to produce sample ions;

[0110] an ion separator configured to separate sample ions according to a first physicochemical property;

[0111] a mass filter configured to filter ions using an isolation window;

[0112] a fragmentation device configured to fragment the sample ions to produce fragment ions;

[0113] mass analyzer; and

[0114] A control system configured to:

[0115] (i) causing the instrument to perform a first ion separation scan by separating sample ions according to a first physicochemical property, and analyzing the separated sample ions by performing one or more MS1 mass analysis scans; and

[0116] (ii) causing the instrument to perform a second ion separation scan by separating the sample ions according to the first physicochemical property, and analyzing the separated sample ions by performing a plurality of MS2 mass analysis scans, wherein each MS2 scan in the plurality of MS2 mass analysis scans uses one MS2 isolation window of the plurality of MS2 isolation windows;

[0117] The control system is further configured as follows:

[0118] analyzing MS1 data acquired from one or more MS1 mass analysis scans; and

[0119] Multiple MS2 isolation windows are configured based on analysis of the MS1 data.

[0120] Another aspect provides an analytical instrument, the analytical instrument comprising:

[0121] an ion source configured to ionize the sample to produce sample ions;

[0122] an ion separator configured to separate sample ions according to a first physicochemical property;

[0123] a mass filter configured to filter ions using an isolation window;

[0124] a fragmentation device configured to fragment the sample ions to produce fragment ions;

[0125] mass analyzer; and

[0126] A control system configured to:

[0127] (i) causing the instrument to perform a first ion separation scan by separating sample ions according to a first physicochemical property, and analyzing the separated sample ions by performing one or more MS1 mass analysis scans; and

[0128] (ii) causing the instrument to perform a second ion separation scan by separating the sample ions according to the first physicochemical property, and analyzing the separated sample ions by performing a plurality of MS2 mass analysis scans, wherein in each MS2 mass analysis scan, the ions are accumulated in the ion storage for an accumulation time;

[0129] The control system is further configured as follows:

[0130] analyzing MS1 data acquired from one or more MS1 mass analysis scans; and

[0131] The accumulation time for one or more or each MS2 scan is determined based on analysis of the MS1 data.

[0132] These aspects and embodiments may be combined with, and in embodiments with, any one or more or each of the aspects, embodiments and / or optional features described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0133] Various embodiments will now be described in more detail with reference to the accompanying drawings, in which:

[0134] Figure 1 The conventional DIA workflow is schematically illustrated;

[0135] Figure 2 A mass spectrometer operable in accordance with embodiments is shown;

[0136] Figure 3 An example plot showing ion mobility arrival time versus m / z for peptide ions of different charge states;

[0137] Figure 4 Shown is a schematic diagram of the conventional DIA workflow. Figure 3 of peptide ions;

[0138] Figure 5 Schematically illustrates the sequence of MS1 scanning according to the embodiment;

[0139] Figure 6 Schematically illustrates the sequence of MS2 scanning according to the embodiment;

[0140] Figure 7 Schematically illustrates the sequence of MS2 scanning according to the embodiment;

[0141] Figure 8 A shows an example chromatographic elution peak, Figure 8 B schematically illustrates the sequence of MS1 scanning according to an embodiment, Figure 8 C schematically illustrates the sequence of MS2 scanning according to an embodiment, Figure 8 D schematically illustrates the sequence of MS1 scans according to an embodiment, and Figure 8 E schematically illustrates a sequence of MS2 scanning according to an embodiment;

[0142] Figure 9 A schematically illustrates the sequence of MS2 scanning according to an embodiment, and Figure 9 B schematically illustrates a sequence of MS2 scanning according to an embodiment;

[0143] Figure 10 schematically illustrates a calibration method according to an embodiment;

[0144] Figure 11Another example graph showing ion mobility arrival time versus m / z for peptide ions of different charge states;

[0145] Figure 12 Schematically illustrates the sequence of MS2 scanning according to the embodiment;

[0146] Figure 13 shows an example MS1 mass spectrum obtained according to an embodiment;

[0147] Figure 14 shows a 100 nm spherical lattice divided equally between four MS2 isolation windows. Figure 13 MS1 mass spectrum;

[0148] Figure 15 Shown Figure 14 Ion population in each MS2 isolation window of the MS2 isolation window;

[0149] Figure 16 shows the rearrangement into an order based on ion population Figure 14 Four MS2 isolation windows;

[0150] Figure 17 shows the partitioning between four MS2 isolation windows of different widths to achieve approximately equal ion populations. Figure 13 MS1 mass spectrum of

[0151] Figure 18 Shown Figure 17 The ion population of each MS2 isolation window is shown in Figure 5. DETAILED DESCRIPTION

[0152] Embodiments described herein relate to the application of mass spectrometry in proteomics and similar fields.Proteomics and similar methods are typically performed using two different data acquisition methods: data-dependent acquisition (DDA) and data-independent acquisition (DIA).

[0153] Figure 1 A typical DIA workflow is illustrated, where a sample (such as a protein digest) is separated by liquid chromatography (LC) and ionized into a mass spectrometer. The LC elution peaks (plotted on the y-axis) are superimposed on an illustration of a traditional DIA acquisition strategy. The mass spectrometer is configured to perform a single MS1 scan covering the entire m / z region of interest ( Figure 1 ), and this scan is then followed by a series of MS2 scans using various small mass filter m / z isolation windows ( Figure 1(Dark shaded box in the figure), this small mass filter m / z isolation window, when integrated across the entire series of MS2 scans, covers the m / z region analyzed by the initial MS1 scan. In other words, a single MS1 scan is collected for a wide isolation range, and then this analyzed m / z range is subdivided into MS2 windows, i.e., narrow m / z regions, which are isolated and subsequently fragmented.

[0154] This combination of an MS1 ​​scan and a subsequent MS2 scan is called a cycle. Figure 1 As shown, this cycle is repeated multiple times throughout the LC gradient. Since a single LC elution profile is on the order of seconds, and the cycle time (i.e., the time it takes the instrument to collect the MS1 scan and MS2 scan that constitute a single cycle) is typically 1-2 seconds, multiple cycles can occur along the elution profile, thereby facilitating the identification and quantification of sample ions (e.g., peptide ions).

[0155] Important to this workflow is the fact that the ion population does not change throughout the LC elution profile, i.e., the ions analyzed in the MS1 scan have the same compositional population as those analyzed in the MS2 scan. However, when such LC-MSDIA workflows incorporate additional separation techniques (such as ion mobility), this correlation between the MS1 and MS2 scans is no longer guaranteed.

[0156] Figure 2 Schematically illustrates an analytical instrument such as a mass spectrometer (MS) that can operate according to the embodiments. Figure 1 As shown, the instrument includes an ion source 10 , an ion separator 20 (such as an ion mobility (IM) separator), a mass filter 30 , a fragmentation device 40 and a mass analyzer 50 .

[0157] The ion source 10 is configured to generate ions from a sample. The ion source 10 can be any suitable continuous or pulsed ion source, such as an electrospray ionization (ESI) ion source, a MALDI ion source, an atmospheric pressure ionization (API) ion source, a plasma ion source, an electron ionization ion source, a chemical ionization ion source, and the like. More than one ion source can be provided and used. The ions can be any suitable type of ion to be analyzed, such as small and large organic molecules, biomolecules, DNA, RNA, proteins, peptides, fragments thereof, and the like.

[0158] The ion source 10 may be coupled to a separation device, such as a liquid chromatography separation device or a capillary electrophoresis separation device (not shown), so that the sample ionized in the ion source 10 is from the separation device.

[0159] The ion separator 20 is arranged downstream of the ion source 10 and is configured to receive ions from the ion source 10. The ion separator 20 is configured to separate the received ions according to a first physicochemical property. The first physicochemical property can be, for example, ion mobility, differential ion mobility, or mass-to-charge ratio (m / z). Various types of ion separators are described in more detail below.

[0160] In the case where ion separator 20 is an ion mobility separator, ion mobility separator can include the ion mobility separator of any suitable type. For example, an electric field such as a DC voltage gradient and / or a traveling DC voltage wave can be arranged to promote ions along the length of separator and make ions pass through gas so that ions are separated according to their ion mobility. Optionally, facing the countercurrent of gas or perpendicular to the countercurrent of gas promote ions. Alternatively, airflow can be arranged to promote ions along the length of separator, and an electric field such as a DC voltage gradient and / or a traveling DC voltage wave is arranged to be opposite to airflow so that ions are separated according to their ion mobility. Ion mobility separator 20 can be a linear separator or circulation (closed loop) separator with straight or folded path.

[0161] The mass filter 30 is arranged downstream of the ion separator 20 and is configured to receive ions from the ion source 10 (via the ion separator 20). The mass filter 30 is configured to filter the received ions according to the mass-to-charge ratio (m / z) of the received ions. The mass filter 30 can be configured so that the received ions with an m / z within the m / z transmission window of the mass filter are forwardly transmitted by the mass filter, while the received ions with an m / z outside the m / z transmission window are attenuated by the mass filter, for example, not forwardly transmitted by the mass filter. The width and / or center m / z of the transmission window are controllable (variable), for example, by appropriately controlling the RF voltage and DC voltage applied to the electrodes of the mass filter 30. Thus, for example, the mass filter 30 can be operated in a transmission mode of operation, whereby most or all ions within a relatively wide m / z window are transmitted forward by the mass filter 30, and can be operated in a filtering mode of operation, whereby only ions within a relatively narrow m / z window (centered about a desired m / z) are transmitted forward by the mass filter 30. The mass filter 30 can be any suitable type of mass filter, such as a quadrupole mass filter.

[0162] The fragmentation device 40 is arranged downstream of the mass filter 30 and is configured to receive most or all of the ions transmitted by the mass filter 30. The fragmentation device 40 can be configured to selectively fragment some or all of the ions received, i.e. so as to produce fragment ions. The fragmentation device 40 can be operated in a fragmentation operating mode, whereby most or all of the ions received are fragmented so as to produce fragment ions (which fragment ions can then be transmitted onward from the fragmentation device 40), and can be operated in a non-fragmentation operating mode, whereby most or all of the ions received are transmitted onward without being (intentionally) fragmented. It is also possible to achieve a non-fragmentation operating mode by causing the ions to bypass the fragmentation device 40. The fragmentation device 40 can also be operated in one or more intermediate operating modes, for example whereby the degree of fragmentation is controllable (variable).

[0163] The fragmentation device 40 may be any suitable type of fragmentation device, such as, for example, a collision induced dissociation (CID) fragmentation device, an electron induced dissociation (EID) fragmentation device, a photodissociation fragmentation device, etc. Many other types of fragmentation are possible.

[0164] The mass analyzer 50 is arranged downstream of the ion separator 20 and is configured to receive ions from the ion source 10 (via the ion separator 20 and the mass filter 30, and optionally via the fragmentation device 40). The mass analyzer 50 is configured to analyze the received ions in order to determine their mass-to-charge ratio and / or mass, i.e., to generate a mass spectrum of the ions. The mass analyzer 50 may be an ion trap mass analyzer, such as an electrostatic orbital trap mass analyzer, and more specifically an Orbitrap mass analyzer. TM FT mass analyzer.

[0165] Thus, the mass analyser 50 may comprise an inner electrode extending along the axis of the orbital trap and a pair of spaced apart outer electrodes, the pair of outer electrodes surrounding the inner electrode and defining a capture volume therebetween, in which ions are captured and oscillated by orbiting about the inner electrode, a capture voltage being applied to the inner electrode while oscillating back and forth along the axis of the trap. The pair of outer electrodes act as detection electrodes to detect image currents caused by the oscillations of the ions in the capture volume and thereby provide a detected signal. The outer electrodes are typically used as a differential detection electrode pair and are coupled to respective inputs of a differential amplifier which in turn forms part of a digital data acquisition system to receive the detected signal. The detected signal may be processed using a Fourier transform to obtain a mass spectrum of the ions within the trap.

[0166] It should be noted that Figure 2 This is illustrative only, and the apparatus can, and in embodiments does, include any number of one or more additional components.

[0167] For example, the instrument may include one or more ion transfer stages or ion trapping stages, e.g., arranged between the various illustrated devices. The one or more ion transfer stages may include, for example, an atmospheric pressure interface and / or one or more ion guides, lenses, and / or other ion optical devices configured to allow ions to be transferred between the various illustrated devices. The ion transfer stage may include any suitable number and configuration of ion optical devices, e.g., optionally including one or more RF ion guides and / or multipole ion guides, one or more ion guides for cooling ions, one or more mass selective ion guides, and the like.

[0168] like Figure 2 As also shown, the instrument is under the control of a control unit 60 (such as a suitably programmed computer), which controls the operation of the various components of the instrument. The control unit 60 can also receive and process data from the various components, including the analyzer 50. The control unit 60 is particularly configured to determine the settings for the ion separator 20, the mass filter 30, the fragmentation device 40, and the mass analyzer 50 for the analytical scan.

[0169] For example, the control system 60 may cause the instrument to perform one or more MS1 scans, wherein in each MS1 scan, the mass filter 30 is operated in its transmission mode or in its filtering mode, and ions are not (intentionally) fragmented, such that ions of a broad m / z range produced by the ion source 10 are mass analyzed by the mass analyzer 50. The control system 60 may also cause the instrument to perform one or more MS2 scans, wherein in each MS2 scan, the mass filter 30 is operated in its filtering mode and ions are fragmented in the fragmentation device 40, such that ions of a narrow m / z range produced by the ion source 10 are selected and fragmented, and the resulting fragment ions are mass analyzed by the mass analyzer 50.

[0170] As described above, using dispersed ion mobility (IM) separation techniques, ions are injected into the IM separator 20 and separated in time reflecting the mobility of the individual ion species. Figure 3 An example representation of eight peptide ions separated by IM-MS is shown. Figure 3 As shown, peptide ions represented by black ovals fall on different charge-dependent trend lines in arrival time-m / z space. Figure 3 Example trend lines are shown for singly (1+) ions, doubly (2+) ions, and triply (3+) ions. Thus, the result of the IM separation is a variety of peptide ions concentrated into charge-dependent trend lines in the arrival time-m / z space, which elute from the IM separator 20 in a distribution with a time width of tens of milliseconds throughout the IM separation time (typically 1-2 seconds).

[0171] Figure 4The same eight example peptides (black ovals) are shown superimposed onto a representation of a standard DIA acquisition scheme, where the lightly shaded boxes represent MS1 scans and the smaller darker shaded boxes represent MS2 scans. Figure 4 As can be seen, due to the separation in the IM domain, this acquisition strategy will not work, as the overlap between the analyzed m / z areas and arrival times does not exist. Furthermore, because the MS2 scan occurs at a longer arrival time than the preceding MS1 scan, the correlation between MS1 ​​and MS2 no longer exists.

[0172] Therefore, if Figure 4 As illustrated, when DIA is performed in an LC-IM-MS setting, conventional MS acquisition strategies no longer sample the same ion populations in both the MS1 and MS2 scans. That is, when the MS2 scan occurs, the ions present in the MS1 scan are no longer present due to their separation in the IM arrival time domain. This hinders the ability to identify and quantify peptide ions using this conventional acquisition approach. Therefore, new acquisition strategies are needed.

[0173] The embodiments described herein provide new data acquisition strategies to allow DIA measurements to be performed using an LC-IM-MS instrument configuration. As mentioned above, important for the success of DIA measurements is the correlation between the MS1 scan and the MS2 scan, i.e., these scans should analyze the same ion type. The acquisition strategies described herein ensure this correlation. Some embodiments optionally also effectively utilize IM separation to direct the mass spectrometer toward informative multiply charged ions.

[0174] Important to the operation of some embodiments is the fact that although in LC-IM-MS experiments (see Figure 4 ) during the ion population changes in time due to separation in the IM domain, but the IM separation does remain consistent for the same ion population across multiple IM injections across the eluting LC peak. Therefore, when a uniform ion population is eluted from a single chromatographic peak, the IM separation of these ions will be reproducible and can be used to correlate MS1 and MS2 scans.

[0175] Figure 5 The first step of the method according to the embodiment is illustrated. Multiple MS1 scans (dark grey boxes) are collected in a series, where the number of MS1 scans is limited by the maximum arrival time desired for analysis. Unlike standard DIA acquisition, all MS1 scans are collected sequentially during a single IM injection. Figure 5 In , the arrival time investigated by each MS1 scan is represented by the box height, and the mass filter isolation width is reflected by the box width. Figure 5In the example shown, the m / z region analyzed by each MS1 scan is derived from the 2+ trendline and the 3+ trendline; however, the mass analyzer isolation can be driven by a single charge state trendline and / or other charge states (e.g., 4+ or 5+), as required by the application. Figure 5 In the example shown, all peptide ion signals (black ovals) were analyzed in the MS1 scan.

[0176] After collecting this sequence of MS1 scans, in a second step another ion packet from the LC elution peak is injected into the IM separator 20 . Figure 6 One embodiment of this second step is illustrated. Figure 6 As shown, this method uses the previous MS1 scan boundary conditions ( Figure 6 The black outline in ) is used as the boundary of the MS2 scan ( Figure 6 In this example, the MS2 transient time allows eight MS2 scans to be obtained per MS1 scan window from the previous injection (although other numbers of MS2 scans are possible).

[0177] Therefore, MS1 scans are first acquired for a single IM injection, where the m / z region analyzed with each MS1 scan is determined by the charge state trend line of interest (which is in Figure 5 In the next IM injection, the method is then scanned by MS1 ( Figure 6 MS2 scans were collected within the m / z region analyzed (black frame in the figure) Figure 6 ). Figure 6 In the example shown, the MS2 scan is focused on the 2+ trend line, and the mass filter isolation of each scan is driven by the boundaries of that trend line; however, the MS2 scan may be focused on other and / or additional trend lines, as described further below.

[0178] Once this series of MS2 scans is acquired, the mass spectrometer will acquire a new set of MS1 scans upon the next IM injection.

[0179] Thus, using this acquisition strategy, a mass spectrometer cycle can consist of two IM injections, where the first IM injection is analyzed by N MS1 scans and the second IM injection is analyzed by M MS2 scans, where N is defined by Equation 1 and M is defined by Equation 2:

[0180]

[0181] This cycling scheme is applied continuously over the entire length of the LC gradient. Once the data is acquired, post-processing data analysis tools will require information from N MS1 scans and M MS2 scans to perform correlation. In the example shown, for a single cycle, peptides present in the first MS1 scan are fragmented in the first through eighth MS2 scans; peptides present in the second MS1 scan are fragmented in the ninth through sixteenth MS2 scans; and so on.

[0182] As mentioned above, in Figure 6 In the example shown, the 2+ trend line is analyzed exclusively; however, this is not required. For example, an MS2 scan can follow any charge state trend line or can overlay multiple trend lines, such as simultaneously overlaying 2+ and 3+ ions.

[0183] Figure 7 An example is shown where the MS2 scan is directed towards the 3+ trendline ion. Figure 7 As shown, the MS2 scan has a mass filter isolation window guided by the boundary conditions of this charge state.

[0184] Figure 8 This paper summarizes the various concepts of the method described in this paper. Figure 8 As shown in A, for the elution LC peak ( Figure 8 Black trace in A), multiple IM injections were performed across the peak curve ( Figure 8 The black dots in A, where the labels correspond to the IM injection numbers, were analyzed in an alternating fashion, where the acquisition scheme switched between MS1 ​​and MS2 scans. Figure 8 In B, the first IM injection includes multiple MS1 scans. Figure 8 In C, the second IM injection includes multiple MS2 scans. Figure 8 In D, the third IM injection includes multiple MS1 scans and Figure 8 In D, the fourth IM injection includes multiple MS2 scans.

[0185] exist Figure 8 In the example shown in FIG, the 2+ trend line and the 3+ trend line are targets of the MS1 scan and the MS2 scan. However, in general, the regions of the arrival time-m / z space targeted by the MS1 scan and the MS2 scan may vary.

[0186] In addition to embodiments comprising two IM injections per cycle (where the first IM injection collects MS1 scans and the second IM injection collects all MS2 scans), multiple MS2-focused IM injections can be integrated into each cycle. That is, the method cycle can be modified to have multiple IM injections collecting MS2 scans, for example, where each IM injection for MS2 analysis can be focused on a different charge state trendline.

[0187] Figure 9 The first IM injection where an MS1 ​​scan was collected is shown (see Figure 5 ) is followed by an example of two separate MS2 focused IM injections. In this example, the 2+ and 3+ ions are of most interest, resulting in the following acquisition cycle for the sequential IM injections: (i) MS1 scan, (ii) MS2 scan: 2+ focused, (iii) MS2 scan: 3+ focused. Thus, the first MS2 IM injection ( Figure 9 A) The mass filter is directed to the 2+ trend line and the second injection ( Figure 9 B) Direct the mass filter to the 3+ trend line. This cycle can be repeated for the entire LC gradient.

[0188] It will be appreciated that the above-described methods are illustrative of the general concepts described herein and that many alternative methods are possible depending on the implementation.

[0189] One limitation of any MS analysis is the upper limit on the number of ions that the mass spectrometer can store before space charging, i.e., the repulsion of like charges stored within the physical space, which leads to negative effects. TM In instruments, this storage limit is usually determined by the space charge capacity of the so-called "C-trap" from which the ion packets are injected into the Orbitrap TM in a mass analyzer for mass analysis.

[0190] In some embodiments, to ensure optimal sensitivity for MS analysis, the ions transferred to and stored in the C-trap should be "information-rich." Typically, in proteomics analysis, such "information-rich" ions are multiply charged, while background contaminant ions are typically singly charged. Therefore, it is analytically advantageous to fill the C-trap with multiply charged ions and release singly charged species that, if transferred, would contribute to the space charge capacity.

[0191] As mentioned above, the IM separation of the analytes resulted in a charge state dependent trend line (see Figure 3 ). These trend lines can be drawn using a calibration procedure, for example, as described in co-pending application US63 / 468,170. Thus, since there is a priori knowledge of the trend lines whose slope and intercept should be stable under consistent IM analyzer settings, this information can be used to focus the mass filter 30 of the mass spectrometer to eliminate singly charged ions and transmit multiply charged ions for each MS1 or MS2 scan. This feature is described above and Figures 5 to 9 is utilized in the example depicted in .

[0192] Figure 10 One possible such calibration scheme is schematically illustrated. Figure 10As shown, the calibration procedure uses a known set of analytes, e.g., P1, P2, P3, P4, separates these ions using IM, and analyzes the arrival time distribution using multiple MS1 scans (macro scans) within an IM cycle, where the MS1 scan in each subsequent macro scan is delayed by a known amount (Δt). In other words, in order to more accurately determine the arrival time of each peptide, the ion mobility separation is repeated multiple times, and the initiation of the MS1 scan, and therefore the effective arrival time bin being analyzed, is offset by a known delay Δt. This process is repeated until the MS1 scan transient window has reached the estimated maximum arrival time ("MS1 scan n") at the end of the macro scan. In other words, the MS1 scan is repeated a number of times with increasingly longer delays until the sliding transient window reaches the desired maximum arrival time "MS1 scan n."

[0193] After acquiring these mass spectra, the extracted ion chromatogram for each peptide can be determined as a function of the MS1 scan number. The scan number can be related to the effective arrival time, and the arrival time of each peptide can be determined.

[0194] The relative intensities of the resulting "calibrated" arrival time pairs P1, P2, P3, and P4 can then be determined. This information (arrival time as a function of m / z and charge state) can then be used to guide the mass filter center m / z during a DIA run in the manner described above. This calibration relies on the similarity of analytes of the same chemical class (e.g., tryptic peptides, proteome, lipidome, metabolome, etc.) because there is a correlation between arrival time and m / z, as well as a charge state dependency that forms a charge dependency trend line relating these two variables. Therefore, after applying the calibration procedure to a known analyte mixture, IM versus m / z and charge state trend lines in z space can be determined and used for unknown analytes of the same class separated under the same IM conditions.

[0195] While various specific embodiments have been described above, various alternative embodiments are possible.

[0196] For example, the trend lines analyzed during the MS2 scan can be varied for different applications, or multiple MS2 focused IM injections can be utilized.

[0197] In the above embodiment, the mass filter isolation window is guided by a uniform buffer around the trend line established by calibration, where for Figure 3 For each charge state in , a buffer region is shown by the shaded area centered around the dashed trend line. However, this buffer does not have to be uniform.

[0198] Figure 11An example is shown in which the buffer region (the shaded area encompassing each dashed trend line) that guides mass filter isolation during MS1 and MS2 scans is asymmetric. Figure 11 , the upper boundary of the state of charge buffer region is defined by the trend line slope of 107.5%, and the lower boundary is defined by the trend line slope of 85%. Other distributions may be used, and the buffer distribution may be tuned for a particular application, for example.

[0199] Furthermore, while in the above-described embodiments the mass filter isolation window becomes wider at longer arrival times as the upper and lower buffer regions extend away from the central trendline, a fixed quadrupole isolation width may instead be used along the trendline.

[0200] In some embodiments, the MS1 scan can be used intelligently to guide the MS2 scan, e.g., to prioritize or ignore the 3+ trend line due to the abundance of 3+ ions in the MS1 scan, and, for example, to make predictions related to ion numbers, e.g., for automatic gain control (AGC).

[0201] As will be appreciated from the foregoing, embodiments provide a novel acquisition order for MS1 and MS2 scans in a DIA workflow. As described above, in a conventional DIA workflow, a single broad m / z range MS1 scan is followed by several narrow m / z MS2 scans, and this cycle is repeated throughout the LC gradient. However, with the addition of ion mobility, this acquisition strategy cannot be used. To overcome this, embodiments collect all MS1 scans sequentially during a single IM injection. Then, during subsequent IM injections, MS2 scans are acquired.

[0202] In addition to this scan sequencing, some embodiments exploit the charge state separation provided by the IM to maximize the "information-rich" ions stored in an ion trap (e.g., a C-trap) prior to mass analysis by directing the mass filter to transmit only the m / z region as guided by the trend line of the multiply charged ions. That is, embodiments maximize the utilization of the spatial charge capacity of the ion trap (e.g., a C-trap) by transmitting only the region of the time-m / z space that has multiply charged "information-rich" ions.

[0203] When a DIA workflow is used with an LC-IM-MS instrument configuration, embodiments allow for the correlation of MS1 and MS2 scans. This instrument configuration is beneficial because the addition of IM increases the duty cycle and inherently improves sensitivity compared to non-IM MS workflows.

[0204] While the specific embodiments described above provide for isolating Figure 8) or follow the charge state-dependent trend line of ion arrival time ( Figure 9 ) of the above-described subset of ions, but further embodiments are provided.

[0205] It has been recognized that in some cases, methods involving the selection of all "informative" ions arriving at any given moment may perform poorly due to the high abundance of different peptides included within the isolation window. This can lead to overly complex fragmentation spectra that hinder interpretation by processing software.

[0206] Similarly, methods that select ions along individual charge-state-dependent trend lines can suffer from sample selection inhomogeneity, where some regions of the arrival time versus m / z space are sampled multiple times, while other regions are not sampled at all. This is because trend lines are just trend lines—not all ions of interest arrive exactly along them. In some cases, there is significant deviation, for example, from the point at which the entire region between the 2+ and 3+ trend lines can contain ions of interest.

[0207] Thus, various further embodiments provide a method that allows for complete coverage of the ions of interest without overloading the processing software with overly complex fragmentation spectra. To this end, by increasing the MS2DIA sampling window (e.g., Figure 8 The above method can be generalized by dividing the ion array (as shown) into any number of smaller isolated windows, where each set of windows is selected by subsequent ion implantation into the ion transport device.

[0208] Figure 12 An example of this method is shown, in which the MS2 isolation window is divided into three groups (referred to as isolation window groups MS2a, MS2b, and MS2c). Isolation window group MS2a can be selected during the first ion implantation into the ion mobility device, isolation window group MS2b can be selected during the second ion implantation, and isolation window group MS2c can be selected during the third ion implantation.

[0209] This approach provides complete coverage of the m / z space of interest by arrival time, but allows for smaller isolation windows to reduce the number of different peptides analyzed within any given scan. In other words, embodiments provide a method by which the number of peptides present in any given fragmentation scan is reduced, thereby allowing for successful deconvolution of the resulting MS2 spectrum.

[0210] Thus, in embodiments, multiple MS2-focused IM injections may be used after an initial MS1-focused IM injection, where the MS2 isolation window for a given arrival time is divided by the number of MS2-focused IM injections. For example, if an MS2 scan with arrival times of 250 ms-282 ms would isolate the m / z range of 300-400 m / z in a single MS2-focused IM injection strategy, the method may perform three separate MS2-focused IM injections, where the 300-400 m / z range is divided across three IM separations, i.e., for the first MS2-focused IM injection, the MS2 scan would isolate 300-333.3 m / z, the second MS2-focused IM injection would isolate 333.3-366.6 m / z, and the final MS2-focused IM injection would isolate 366.6 to 400 m / z.

[0211] This method can be generalized to any number of isolation windows.The above example describes the MS2 space being divided into three groups, but it can be any number of groups, such as two, four, or more than four.

[0212] In this method, the m / z width of each of the original MS2 windows can be divided equally among the multiple groups. Alternatively, the size of the smaller windows can be varied, for example to allow for different densities of different peptides in the arrival time versus m / z space. This variable window spacing can be performed based on calibration data collected using a calibration routine, such as described in US 63 / 468,170, so that regions of the arrival time versus m / z space with a higher density of peptides have narrower isolation windows, and regions with a lower density of peptides have wider windows.

[0213] While the above example describes collecting three sets of MS2 scans sequentially, this does not necessarily need to be the case, and other acquisition orders are possible. For example, the instrument can alternate between MS1 ​​scans and different sets of MS2 scans (e.g., MS1, MS2a, MS1, MS2b, MS1, MS2a, etc.), or, for example, for a larger number of sets, the instrument can collect multiple MS2 sets between each MS1 set (e.g., MS1, MS2a, MS2b, MS1, MS2c, MS2d, etc.).

[0214] In other embodiments, the spectral complexity (number of MS2 peaks) can be tuned by optimizing the number of groups and / or the m / z coverage window (variable or identical) of each of them. The maximum acceptable spectral complexity may be determined by the limitations of the data processing software that automatically extracts protein identifications and may vary depending on the specific algorithm / software package deployed.

[0215] The embodiments described thus far allow for good correlation between MS1 ​​and MS2 scans and effectively utilize both LC and IM separations prior to MS analysis. Additional embodiments do so while also utilizing the data in real time, for example, by utilizing information obtained during the MS1 scan to drive the MS2 scans or the order in which they are collected.

[0216] In some embodiments, the IM-MSDIA method is driven by the estimated boundaries of the charge state trend line to guide the quadrupole m / z isolation area. However, in some cases, ions may be unevenly distributed in this m / z space. There may be a narrow m / z region with many precursor ions and a wide m / z region with relatively few precursor ions. This information is not known until the arrival time distribution is measured by MS1 scanning.

[0217] Various additional embodiments provide a method that utilizes this MS1 scan information to intelligently drive the MS2 scan order to maximize ion utilization and minimize the amount of instrument time wasted on sparse m / z regions as LC peaks elute to the MS.

[0218] In these embodiments, as MS1 scans arrive, they are analyzed, for example, by the instrument's control system (internal PC) to determine metrics (not inclusive lists) about the distribution of precursor ions as a function of m / z, charge state, and intensity. This information is then used to drive the acquisition strategy for the subsequent MS2 focused IM injection. One example of such an "intelligent" acquisition strategy is to determine the number of ions in various m / z isolation bins.

[0219] Figure 13 An example of a spectrum obtained from an MS1 ​​scan with an area of ​​450-1000 m / z for a specific arrival time window is shown. A broad distribution of precursor ions can be seen within the m / z region of interest, 450-1000 m / z. However, the precursor ions are not evenly distributed in this m / z space. That is, there is a denser distribution of ions, and therefore a denser distribution of precursors, in the 550-850 m / z range compared to the areas of 450-550 m / z and 850-1000 m / z.

[0220] If the total area is analyzed by four consecutive MS2 focused IM injections (e.g., as above for Figure 12 As described above), the range of 450-1000 m / z will be divided equally among four separate IM injections, as Figure 14 (where "MS2 Area" describes the quadrupole isolation window for that particular IM injection). Figure 14 Shown superimposed on Figure 13Figure 1 shows various quadrupole isolation areas on an MS1 ​​spectrum that will be analyzed using a four MS2 focused IM injection strategy. With this strategy, the quadrupole isolation window is gradually shifted across the entire m / z range with each incremental IM injection.

[0221] Figure 15 Shown as Figure 14 Ion population as a function of the MS2 area of ​​the scheme. It can be seen that the area covered by the scheme IM injection 2 has the largest ion signal density, followed by the area for IM injection 3, while the areas for injection 1 and injection 4 have smaller signals of smaller magnitude. Therefore, Figure 14 The acquisition method shown in results in an asymmetric ion loading distribution of four separate IM injections.

[0222] In an embodiment, using this data (which may be determined in real time, e.g., as the MS1 scan arrives at the internal instrument PC), the method prioritizes areas based on the cumulative signal intensity present in the MS1 scan for that quadrupole isolation area. Figure 14 and Figure 15 In the example, this would result in Figure 16 IM acquisition order shown.

[0223] Therefore, using the ion loading per quadrupole isolation region determined in real time from the MS1 scan, the regions can be sorted from most to least populated (e.g., similar to a "Top-N" approach using DDA) and then acquired in that order. This ensures that the regions with the most information are acquired first. In the case of peptide distributions that elute almost completely from the LC column, this can ensure that instrument time is used wisely.

[0224] It should be noted that a collection scheme from most abundant to least abundant is not the only possible strategy. For example, for some applications, such as where low abundance analytes are of particular interest, it may be beneficial to collect the least populated to the most populated.

[0225] Further exploitation of the MS1 data can be achieved by determining the isolation window required to have equal plasma loading in each of n IM implants, which in this example case is four IM implants.

[0226] To do this, the total ion current (TIC) of the MS1 scan can be calculated, and this TIC can be divided by the number n of MS2 focused IM injections established for the DIA method (e.g., as selected by the user). Then, based on the MS1-specific m / z distribution, the isolation window that achieves the number of ions that achieve TIC / n can be calculated. The quadrupole isolation windows can be calculated in real time to establish this distribution by varying the width of each.

[0227] for Figure 13The MS1 scan shown, TIC is 1 × 10 9 , for n = 4 MS2 focused IM injections, this would result in a target ion loading per isolation area in the IM injection of 2.5 × 10 8 .

[0228] Calculating the window that produces this ion loading distribution yields Figure 17 Here, the windows are not equally spaced, but each isolation area has an equal amount of ion loading, as shown in Figure 18 As illustrated. Figure 18 As can be seen in Figure 2, by calculating the varying window size, the ion load is evenly distributed across the four IM injections. This approach avoids overfilling one MS2 scan while underfilling others.

[0229] In addition, if Figure 8 As shown in , the mass resolutions typically used for MS1 and MS2 scans are different, which results in different arrival time distributions analyzed by a single MS1 and MS2 scan. Figure 8 In the example presented in Figure 2, there are eight MS2 scans analyzing the same arrival time range as a single MS1 scan. The data contained in the MS1 scan covers the arrival times that will be analyzed by multiple MS2 scans in subsequent IM injections due to differences in transient lengths.

[0230] Since the available data will be from the MS1 level and therefore a combination of all arrival times in that region, the charge state dependent trend line data (e.g., as determined by a calibration method) can be used in conjunction with the charge state of the precursor and the m / z of the precursor to determine which arrival time bin, i.e., which of the eight MS2 scans, it will be expected to arrive in. This can be done while executing either of the two real-time decision options described above. These calculations can also be used to implement AGC control for the various MS2 scans by calculating the expected ion population and / or adjusting the IM fill time and ion injection time in the MS.

[0231] It should be appreciated that embodiments utilize real-time decisions made from collected MS1 levels to drive how MS2 scans are acquired. Rather than being driven solely by a predetermined MS1-independent m / z window order, embodiments can more efficiently utilize instrument time to guide the instrument's focus on regions of high ion load first. Alternatively, embodiments can directly balance the ion loads of various IM injections to avoid underfilling a single IM injection or overfilling another IM injection. Furthermore, embodiments can be used to determine AGC control for MS2 scans on subsequent IM injections.

[0232] Although various embodiments have been described above specifically in terms of ion mobility separation, it is possible to perform the method using any type of ion separation technique in which ions are separated according to a ("first") physicochemical property. For example, the method can be performed using ion mobility separation, differential ion mobility separation, or mass-to-charge ratio (m / z) separation. Suitable types of ion separators include, but are not limited to, the following examples:

[0233] 1. An ion separation apparatus configured to sort ions by m / z, for example, as described in U.S. Patent No. 10,256,088. An RF voltage is applied across each electrode of a first array of uniformly spaced, parallel, and coplanar electrodes and its corresponding electrode of a second array of uniformly spaced, parallel, and coplanar electrodes. The amplitude of the RF voltage varies according to an RF voltage amplitude gradient. The RF voltage generates an array of different quadrupole RF electric fields in a uniform gap between the first array and the second array. A DC voltage is superimposed on each electrode of the first array and its corresponding electrode of the second array. The DC voltage varies according to the DC voltage gradient to generate a DC electric field in the uniform gap. When ions are introduced into the uniform gap, as the RF voltage amplitude increases, the DC electric field causes the ions to drift toward the quadrupole RF electric field, where the ions are trapped according to their m / z.

[0234] 2. Ion traps or ion trap arrays, for example, as described in US Pat. No. 9,111,741.

[0235] The ion storage device can be configured to separate ions by mass-to-charge ratio. The ion trap can include at least two or more rows of parallel electrode arrays, wherein each electrode array includes at least two or more parallel strip electrodes. By applying alternating voltages of different phases to different strip electrodes to generate an alternating electric field in the space between two parallel electrodes in different rows of the electrode array, multiple linear ion trapping fields are formed in the space between different rows of the electrode array, and the electrode array is open adjacent to each other without an actual potential barrier.

[0236] 3. Separation devices using the "Zeno Pulse" technology, for example, as described in the article "A W-

[0237] Geometry Ortho-TOF MS with High Resolution and Up to 100% Duty Cycle for MS / MS", J.Am.Soc.Mass Spectrom., 2009, 20, pp. 1342-13.

[0238] 4. Stacked-well ion traps, such as those described in U.S. Patent No. 7,872,228. Multiple electrodes can be positioned and driven by RF potentials to form multiple adjacent pseudopotential wells. Ions can be manipulated, reacted, analyzed, and ejected from the device in a manner similar to conventional ion traps. Furthermore, selected ions or groups of ions can be transferred from one pseudopotential well to another without ion loss due to physical barriers.

[0239] 5. A positive-current ion trap array, such as that described in U.S. Patent No. 10,651,025. The ion separation device may include: a plurality of electrodes arranged in a two-dimensional grid; a gas supply configured to provide a gas flow in a first direction; and an ion inlet configured to receive ions. The plurality of electrodes may be configured to generate one or more pseudopotential barriers whose magnitude increases along the first direction. The drag force exerted on the ions by the gas flow is opposite to the pseudopotential gradient of the plurality of electrodes.

[0240] 6. An annular trap, for example, as described in U.S. patent application No. US18 / 090,730. A system for sorting ions has: a set of multipole electrodes configured to form an ion trap; and an ion guide adjacent to the set of multipole electrodes. An RF voltage is then applied to the set of multipole electrodes using an RF and DC voltage device to generate a pseudo-barrier configured to confine one or more ions. The RF and DC voltage device is also used to apply a DC voltage to generate an axial field opposite to the pseudo-barrier at the exit of the trap. Depending on the usage, the RF voltage or DC voltage is then ramped up or down so that the at least one ion is eluted across the pseudo-barrier.

[0241] 7. An ion centrifuge ion separation apparatus, for example as described in European Patent Application No. 4,020,524. The ion separation apparatus may comprise: (a) a first ion blanket and a second ion blanket, each comprising: a substrate having a first face and a second face; and a set of electrodes disposed on or below the first face, wherein a first plurality of electrodes in the set of electrodes are arranged to define at least one set of circular sectors; (b) an ion exit aperture passing through one of the ion blankets; and (c) one or more power supplies configured to provide a radio frequency voltage to a first subset of electrodes of each ion blanket, provide a potential difference across the electrodes of the first subset of electrodes of each ion blanket, and provide a time-varying voltage to the first plurality of electrodes of each ion blanket migrating through the sector as a traveling wave, wherein the ion blankets are arranged parallel to each other with a gap therebetween, with the first faces facing each other across the gap.

[0242] 8. An ion mobility separation device using a lens array, for example, as described in U.S. Patent No. 11,119,070. The mobility separator includes a two-dimensional electrode grid spanning the passage between the first wall and the second wall. The first wall and the second wall include an inlet aperture and a plurality of outlet apertures, respectively. The two-dimensional electrode grid is configured to generate an electric field within the passage. A plurality of ion channels are arranged adjacent to the plurality of outlet apertures. The ion movement between the inlet aperture and the plurality of outlet apertures is controlled by the electric field and the airflow through the passage between the first wall and the second wall, so that the ions are classified based on the corresponding mobility of the ions and are directed to different passages.

[0243] 9. Trapped ion mobility spectrometry (TIMS) devices, for example, as described in the following article:

[0244] Meier, F. et al., Parallel Accumulation-Serial Fragmentation (PASEF):

[0245] Multiplying Sequencing Speed ​​and Sensitivity by Synchronized Scans in a Trapped Ion Mobility Device; Journal of Proteome Research 2015,12,5378-5387.

[0246] While the invention has been described with reference to various embodiments, it should be understood that various changes can be made without departing from the scope of the invention as set forth in the following claims.

Claims

1. A method of operating an analytical instrument, the method comprising: ionizing the sample to produce sample ions; (i) performing a first ion separation scan by separating sample ions according to a first physicochemical property, and analyzing the separated sample ions by performing one or more MS1 mass analysis scans; as well as (ii) performing a second ion isolation scan by separating the sample ions according to the first physicochemical property, and analyzing the separated sample ions by performing a plurality of MS2 mass analysis scans, wherein each MS2 scan in the plurality of MS2 mass analysis scans uses one MS2 isolation window from a plurality of MS2 isolation windows; The method further comprises: analyzing MS1 data acquired from the one or more MS1 mass analysis scans; as well as The plurality of MS2 isolation windows are configured based on the analysis of the MS1 data.

2. The method according to claim 1, wherein: The plurality of MS2 mass analysis scans is a first plurality of MS2 mass analysis scans, and the plurality of MS2 isolation windows is a first plurality of MS2 isolation windows; and The method further comprises: configuring a second plurality of MS2 isolation windows based on said analysis of said MS1 data; as well as (iii) performing a third ion separation scan by separating the sample ions according to the first physicochemical property, and analyzing the separated sample ions by performing a second plurality of MS2 mass analysis scans, wherein each MS2 scan in the second plurality of MS2 mass analysis scans uses one MS2 isolation window of the second plurality of MS2 isolation windows.

3. The method according to claim 2, wherein the method further comprises: configuring a third plurality of MS2 isolation windows based on said analysis of said MS1 data; as well as (iv) performing a fourth ion separation scan by separating the sample ions according to the first physicochemical property, and analyzing the separated sample ions by performing a third plurality of MS2 mass analysis scans, wherein each MS2 scan in the third plurality of MS2 mass analysis scans uses one MS2 isolation window of the third plurality of MS2 isolation windows.

4. The method according to claim 2 or 3, wherein configuring the MS2 isolation window comprises: For each MS2 isolation window of one or more or all of the first plurality of MS2 isolation windows: configuring the MS2 isolation window based on the MS1 data to include ions that are more abundant than a corresponding MS2 isolation window of the second plurality of MS2 isolation windows; and / or For each MS2 isolation window in one or more or all of the second plurality of MS2 isolation windows: configuring the MS2 isolation window based on the MS1 data to include ions that are more abundant than a corresponding MS2 isolation window in the third plurality of MS2 isolation windows.

5. The method according to claim 4, further comprising: For each MS2 isolation window in one or more or all of the second plurality of MS2 isolation windows: configuring the MS2 isolation window to have a width equal to or approximately equal to a width of a corresponding MS2 isolation window in the first plurality of MS2 isolation windows; and / or For each MS2 isolation window in one or more or all of the third plurality of MS2 isolation windows: configuring the MS2 isolation window to have a width equal to or approximately equal to the width of the corresponding MS2 isolation window in the first plurality of MS2 isolation windows.

6. The method according to claim 2 or 3, wherein configuring the MS2 isolation window comprises: For each MS2 isolation window of one or more or all of the second plurality of MS2 isolation windows: configuring the MS2 isolation window based on the MS1 data to include ions of the same or similar abundance as a corresponding MS2 isolation window of the first plurality of MS2 isolation windows; and / or For each MS2 isolation window in one or more or all of the third plurality of MS2 isolation windows: configuring the MS2 isolation window based on the MS1 data to include ions of the same or similar abundance as a corresponding MS2 isolation window in the first plurality of MS2 isolation windows.

7. The method of claim 6, wherein analyzing the MS1 data and configuring the MS2 isolation window comprises: determining a total ion current indicated by the MS1 data; as well as for each MS2 isolation window in one or more or all of the second plurality of MS2 isolation windows: configuring the MS2 isolation window based on the MS1 data to include an equal or approximately equal portion of the total ion current as a corresponding MS2 isolation window in the first plurality of MS2 isolation windows; and / or For each MS2 isolation window in one or more or all of the third plurality of MS2 isolation windows: configuring the MS2 isolation window based on the MS1 data to include an equal or approximately equal portion of the total ion current as a corresponding MS2 isolation window in the first plurality of MS2 isolation windows.

8. A method according to any one of the preceding claims, wherein: The MS2 isolation window is configured such that the MS2 mass analysis scans together cover a region of ion arrival time-m / z space of interest; and The region of ion arrival time-m / z space of interest corresponds to one or more trend lines of interest, wherein each trend line corresponds to an ion charge state and / or chemical class, and wherein each trend line provides a relationship between ion arrival time and m / z for ions having the charge state and / or the chemical class.

9. The method according to any one of claims 1 to 7, wherein: each MS2 isolation window is configured based on one or more trend lines, wherein each trend line corresponds to an ion charge state and / or chemical class of interest, and wherein each trend line provides a relationship between ion arrival time and m / z for ions having said charge state and / or said chemical class; and Configuring the plurality of MS2 isolation windows includes selecting the charge state and / or chemical class of interest based on the analysis of the MS1 data.

10. The method according to any one of the preceding claims, wherein: During each MS2 mass analysis scan, ions are accumulated in the ion store for the accumulation time; and The method further comprises determining the accumulation time for one or more or each MS2 scan based on the analysis of the MS1 data.

11. A method of operating an analytical instrument, the method comprising: ionizing the sample to produce sample ions; (i) performing a first ion separation scan by separating sample ions according to a first physicochemical property, and analyzing the separated sample ions by performing one or more MS1 mass analysis scans; as well as (ii) performing a second ion separation scan by separating the sample ions according to the first physicochemical property, and analyzing the separated sample ions by performing a plurality of MS2 mass analysis scans, wherein in each MS2 mass analysis scan, the ions are accumulated in the ion storage for an accumulation time; The method further comprises: analyzing MS1 data acquired from the one or more MS1 mass analysis scans; as well as The accumulation time for one or more or each MS2 scan is determined based on analysis of the MS1 data.

12. A method according to any one of the preceding claims, wherein the method comprises performing a plurality of MS1 mass analysis scans during the first ion isolation scan, and wherein: In each of the multiple MS1 mass analysis scans, isolating the separated sample ions using an MS1 ​​isolation window, and performing mass analysis on the isolated sample ions; and Each MS1 scan in the plurality of MS1 mass analysis scans uses one MS1 isolation window from a plurality of MS1 isolation windows, optionally wherein each MS1 isolation window is selected based on one or more trend lines.

13. A method according to claim 12 when appended to claim 10 or 11, wherein determining the accumulation time of an MS2 scan comprises: estimating ion abundance within the MS2 isolation window of the MS2 scan using the one or more trend lines and the MS1 data; as well as The accumulation time of the MS2 scan is determined based on the estimated ion abundance.

14. The method according to any one of the preceding claims, wherein the first physicochemical property is ion mobility, differential ion mobility or mass-to-charge ratio (m / z).

15. A method according to any one of the preceding claims, wherein the method comprises the analytical instrument performing a plurality of repeated cycles, wherein in each cycle the analytical instrument performs steps (i) and (ii).

16. A method according to claim 15 when appended to claim 2 or 3, wherein: In each cycle, the analytical instrument performs steps (i), (ii) and (iii); or In each cycle, the analytical instrument performs steps (i), (ii), (iii) and (iv).

17. The method according to claim 15 or 16, wherein: The sample is provided from a chromatographic separation device; and The method includes the analytical instrument continuously performing repeated cycles during chromatographic separation in the chromatographic separation device.

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

19. A control system for an analytical instrument, such as a mass spectrometer, the control system being configured to cause the analytical instrument to perform the method according to any one of claims 1 to 17.

20. An analytical instrument comprising the control system according to claim 19.

21. An analytical instrument, comprising: an ion source configured to ionize the sample to produce sample ions; an ion separator configured to separate sample ions according to a first physicochemical property; a mass filter configured to filter ions using an isolation window; a fragmentation device configured to fragment sample ions to produce fragment ions; mass analyzer; and A control system configured to: (i) causing the instrument to perform a first ion separation scan by separating sample ions according to the first physicochemical property, and analyzing the separated sample ions by performing one or more MS1 mass analysis scans; as well as (ii) causing the instrument to perform a second ion isolation scan by separating sample ions according to the first physicochemical property, and analyzing the separated sample ions by performing a plurality of MS2 mass analysis scans, wherein each MS2 scan in the plurality of MS2 mass analysis scans uses one MS2 isolation window of a plurality of MS2 isolation windows; The control system is further configured to: analyzing MS1 data acquired from the one or more MS1 mass analysis scans; and The plurality of MS2 isolation windows are configured based on the analysis of the MS1 data.

22. An analytical instrument, comprising: an ion source configured to ionize the sample to produce sample ions; an ion separator configured to separate sample ions according to a first physicochemical property; a mass filter configured to filter ions using an isolation window; a fragmentation device configured to fragment sample ions to produce fragment ions; mass analyzer; and A control system configured to: (i) causing the instrument to perform a first ion separation scan by separating sample ions according to the first physicochemical property, and analyzing the separated sample ions by performing one or more MS1 mass analysis scans; as well as (ii) causing the instrument to perform a second ion separation scan by separating sample ions according to the first physicochemical property, and analyzing the separated sample ions by performing a plurality of MS2 mass analysis scans, wherein in each MS2 mass analysis scan, ions are accumulated in the ion storage for an accumulation time; The control system is further configured to: analyzing MS1 data acquired from the one or more MS1 mass analysis scans; and The accumulation time for one or more or each MS2 scan is determined based on analysis of the MS1 data.

23. An analytical instrument according to claim 20, 21 or 22, wherein: The ion separation device is an ion mobility separator, and the first physicochemical property is ion mobility; The ion separation device is a differential ion mobility separator, and the first physicochemical property is differential ion mobility; or The ion separation device is a device configured to separate ions according to their mass-to-charge ratio (m / z), and the first physicochemical property is the mass-to-charge ratio (m / z) of the ions.

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