Data independent acquisition (DIA) using ion separation

Abstract

A method of operating an analytical instrument comprises ionizing a 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; and (ii) performing 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. Each MS2 scan of the plurality of MS2 mass analysis scans uses one MS2 isolation window of a plurality of MS2 isolation windows. The method further comprises 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

FIELD OF THE INVENTION

[0001] The present invention relates to the field of mass spectrometry and, more particularly, to mass spectrometry incorporating ion separation, such as ion mobility separation. STATE OF THE ART

[0002] The application of mass spectrometry to proteomics and related fields is typically performed using two different data acquisition methodologies: data-dependent acquisition (DDA) and data-independent acquisition (DIA).

[0003] Fig. Figure 1 schematically illustrates a typical DIA workflow in which a sample is separated by liquid chromatography (LC) and ionized into a mass spectrometer (MS). The mass spectrometer is configured to perform a single MS 1 scan (lightly shaded boxes in Fig. 1) covering an entire m / z region of interest, and this scan is followed by a series of MS2 scans (dark hatched boxes in Fig. 1) using various small mass filter m / z isolation windows that, when integrated across the entire series of MS2 scans, cover the m / z region analyzed by the initial MS1 scan. This combination of an MS1 ​​scan and subsequent MS2 scans is referred to as a cycle, and this cycle is repeated many times across the entire LC gradient. Since a single LC elution profile is on the order of several seconds and the cycle time is typically 1-2 seconds, multiple cycles can occur throughout the elution profile, facilitating the identification and quantification of sample ions.

[0004] It is believed that there is still room for improvement in mass spectrometry methods and equipment. SUMMARY

[0005] A first aspect provides a method of operating an analytical instrument, comprising: Ionizing a 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; and (ii) performing 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 each MS2 scan of the plurality of MS2 mass analysis scans uses an MS2 isolation window of a plurality of MS2 isolation windows.

[0006] The method may further comprise 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.

[0007] Embodiments are directed to methods of operating an analytical instrument, such as a mass spectrometer. The instrument may include an ion source configured to generate ions from a sample, an ion separator disposed downstream of the ion source and configured to separate received ions according to a first physicochemical property, a mass filter disposed downstream of the ion separator and configured to filter received ions according to their mass-to-charge ratio (m / z) (i.e.using an isolation window having an average mass-to-charge ratio (m / z) and a width), a fragmentation device located downstream of the mass filter and configured to selectively fragment received ions, and a mass analyzer located downstream of the mass filter and / or the fragmentation device and configured to subject received ions to mass analysis. The incorporation of an ion separation device (such as an ion mobility separator (IM separator), a differential ion mobility separator, or a device configured to separate ions according to their mass-to-charge ratio (m / z)) is advantageous because it improves the duty cycle and sensitivity of the instrument.

[0008] The inventors have recognized that the conventional DIA workflow no longer works for analytical instruments that incorporate ion separation, such as ion mobility (IM) separation. As described in more detail below, this is because the prior correlation between MS1 ​​and MS2 scans is no longer present, so the sample ions of interest are typically not present in the MS2 scans. The embodiments described herein provide a new data acquisition strategy that facilitates DIA methods using instruments with an ion separation device, such as IM-MS instruments.

[0009] In the 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 separation scan, followed by performing a plurality of MS2 mass analysis scans during a second, e.g., immediately subsequent, ion separation scan. The instrument can perform one or more further MS2 mass analysis scans during one or more (immediately) subsequent ion separation scans. These steps can be repeated, e.g., such that the analytical instrument repeatedly switches between performing MS1 mass analysis scans and MS2 mass analysis scans.

[0010] In the method, each MS2 scan of the plurality of MS2 mass analysis scans uses a different MS2 isolation window of one of the plurality of MS2 isolation windows. That is, the isolation window of the mass filter is controlled to be different for each MS2 scan of the plurality of MS2 mass analysis scans performed during the second (or third, or fourth, etc.) ion separation scan.

[0011] Furthermore, each MS2 isolation window is configured based at least in part on the analysis of MS1 data acquired from the one or more MS1 mass analysis scans. As further described below, the sensitivity and / or duty cycle of the instrument can be significantly improved by configuring the plurality of MS2 isolation windows based on the analysis of the MS1 data. Thus, the method provides an improved DIA workflow for an instrument including an ion separator.

[0012] The analytical instrument may be a mass spectrometer, for example, including an ion source. Ions may be generated from a sample in the ion source. The ion source may be coupled to a chromatographic separation device, such as a liquid chromatography (LC) separator, a gas chromatography (GC) separator, etc., such that the sample to be ionized comes from the chromatographic separation device.

[0013] The analytical instrument may comprise an ion separator disposed downstream of the ion source and configured to perform ion separation scans to separate ions received from the ion source according to the first physicochemical property. The ion separator may be an ion mobility separator; in this case, each ion separation scan is an ion mobility separation scan, and the first physicochemical property is ion mobility. Alternatively, the ion separator may be a differential ion mobility separator; in this case, each ion separation scan is a differential ion mobility separation scan, and the first physicochemical property is differential ion mobility.Alternatively, the ion separator may be a device configured to separate ions according to their mass-to-charge ratio (m / z); in this case, each ion separation scan is a mass-to-charge ratio (m / z) separation scan, and the first physicochemical property is the mass-to-charge ratio (m / z). Further details on the different possible types of ion separators are provided below.

[0014] The ion separator can be operated in a cyclic manner, i.e., to perform repeated ion separation scans. During each ion separation scan, the ion separator can receive ions from the ion source and, for example, accumulate an ion packet in an accumulation region. Alternatively, ion packets can be accumulated in an ion trap upstream of the ion separator. The ion separator can then separate the ion packet according to the ions' first physicochemical property, e.g., by passing the ion packet through an ion separation region. Ions with a higher value of the first physicochemical property reach the end of the ion separation region (and exit the separator) before ions with a lower value of the first physicochemical property (or vice versa).

[0015] Each ion separation scan can have a duration T IMS In other words, the ion separator can have a cycle time T IMSThe duration T IMS may include the time required to accumulate an ion packet together with the time required to separate ions. Alternatively, the duration T IMS also only correspond to the time required to separate ions, whereby the accumulation of an ion packet is carried out in parallel with the separation of a previously accumulated ion packet. The duration T IMS can be in the order of hundreds or a few thousand milliseconds. The duration T IMS can be constant within any given experiment, but can also be varied between experiments by appropriate control of the instrument.

[0016] The analytical instrument may include a mass filter disposed downstream of the ion separator and configured to receive separated ions from the ion separator. The mass filter may be any suitable mass filter operable to filter ions according to their m / z, such as a quadrupole mass filter. The mass filter may be configured such that received ions having an m / z within an m / z isolation window are isolated and passed through the mass filter, while received ions having an m / z outside the m / z isolation window are attenuated by the mass filter, e.g., not passed through the mass filter. The width and / or mean m / z of the isolation window may be controllable (variable), e.g., by appropriate control of RF and / or DC voltage(s) applied to the mass filter.Thus, for example, the mass filter may be operable in a transmitting mode of operation, where most or all ions within a relatively wide m / z isolation window are passed through the mass filter, and a filtering mode of operation, where only ions within a relatively narrow m / z isolation window (centered at a desired m / z) are isolated and passed through the mass filter.

[0017] The analytical instrument may comprise a fragmentation device arranged downstream of the mass filter and configured to receive ions transmitted through the mass filter. The fragmentation device may be configured to selectively fragment some or all of the received ions, i.e., to produce fragment ions. The fragmentation device may be operable in a fragmentation mode of operation, in which most or all of the received ions are fragmented to produce fragment ions (which can then be passed on by the fragmentation device), and a non-fragmentation mode of operation, in which most or all of the received ions are passed on without being (intentionally) fragmented. It would also be possible to implement a non-fragmentation mode of operation by causing ions to bypass the fragmentation device.

[0018] The analytical instrument may comprise a mass analyzer located downstream of the fragmentation device and configured to perform mass analysis scans to determine the mass-to-charge ratio (m / z) of the received ions. The mass analyzer may be operable in a cyclic manner, i.e., to perform mass analysis scans repeatedly. During each mass analysis scan, the mass analyzer receives ions and subjects them to mass analysis. The mass analyzer may be an ion trap mass analyzer, such as an electrostatic orbital trap, and more specifically, an Orbitrap™ FT mass analyzer. Other mass analyzer types are possible. For example, the mass analyzer may be a time-of-flight (ToF) mass analyzer, such as a multi-reflection time-of-flight (MR-ToF) mass analyzer.

[0019] It would be possible for the instrument to be configured such that ions can be delivered to the mass analyzer in the form of an ion beam, e.g., without having been accumulated before being delivered to the mass analyzer. Thus, in embodiments, ions are accumulated directly within the mass analyzer. In these embodiments, the number of ions accumulated within the mass analyzer can be controlled by controlling an accumulation time (e.g., filling time) of ions into the mass analyzer.This, in turn, can be controlled by operating a gate or lens of the mass analyzer and / or a gate or lens within the instrument upstream of the mass analyzer (between the ion source and the mass analyzer) in an open (transmitting) mode of operation for a desired period of time (and otherwise operating the gate or lens in a closed (non-transmitting) mode of operation).

[0020] However, in particular embodiments, ions are delivered to the mass analyzer from an ion trap located upstream of the mass analyzer. The ions may initially accumulate within the ion trap and then be delivered to the mass analyzer, e.g., in the form of an ion packet. 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), as described, e.g., 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 instead (in an MS 2-mode) can be used as the fragmentation device to fragment ions before the fragment ions are injected into the mass analyzer. Multiple ion traps can also be used.

[0021] In these embodiments, the number of ions accumulated within the mass analyzer can be controlled by controlling an accumulation time (e.g., fill time) of ions into the ion trap. This, in turn, can be controlled by operating a gate or lens of the ion trap and / or a gate 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 period of time (and otherwise operating the gate or lens in a closed (non-transmitting) mode of operation).

[0022] Each mass analysis scan has a duration T MAIn other words, the mass analyzer has a cycle time T MA on. T MA can include all overheads associated with the operation of the mass analyzer. The duration T MA may include the time required to accumulate an ion packet (and optionally to cool and / or fragment these ions and optionally to inject these ions into the mass analyzer), along with the time required for the mass analyses of these ions. Alternatively, the duration T MA only the time required to subject an ion packet to mass analysis, or only the time required to accumulate an ion packet (and optionally to cool and / or fragment those ions), where the accumulation of an ion packet is performed in parallel with the mass analysis of a previously accumulated ion packet.

[0023] In particular embodiments, the mass analysis scan time T MA less than the ion separation scan time T IMS , i.e. T MA < T IMS . In embodiments, the mass analyzer is of a type in which the mass analysis scan time T MA of the mass analyzer is relatively long, e.g., such that two or more, several dozen, or a few hundred mass analysis scans can be performed during each ion separation scan. The duration T MA can be in the order of several dozen or hundreds of milliseconds. The duration T MA can be constant within any given experiment, but can also be varied between experiments by appropriate control of the instrument.

[0024] The analytical instrument may be operable in at least one MS1 operating mode in which the instrument performs one or more MS1 mass analysis scans, and one MS2 operating mode in which the instrument performs one or more MS2 mass analysis scans.

[0025] In each MS 1 mass analysis scan, the mass filter is operated in its transmit mode or in its filtering mode with a relatively wide isolation window width (e.g., on the order of hundreds or thousands of Th), and ions are not (intentionally) fragmented, so that a relatively wide m / z range of ions produced by the ion source are subjected to mass analysis by the mass analyzer.

[0026] During each MS2 mass analysis scan, the mass filter is operated in its filtering mode with a relatively narrow isolation window width (e.g., on the order of one or ten Th) to isolate ions, and the isolated ions are fragmented in the fragmentation device such that a relatively narrow m / z range of ions produced by the ion source is isolated and fragmented, and the resulting fragment ions are subjected to mass analysis by the mass analyzer.

[0027] Each MS1 mass analysis scan can have a duration T MS1 and each MS2 mass analysis scan can have a duration T MS2 As a rule, T MS1 > T MS2, because high-resolution data is relatively more important for the MS1 scans, while high speed is relatively more important for the MS2 scans (e.g., so that a much larger number of MS2 scans can be acquired per unit time). If the instrument is operated in a cyclic manner, T MS2 usually as a fraction of T MS1 be set, e.g. T MS2 / T MS1 = ½, ¼, ½, 1 / 16 etc.

[0028] In the method of various embodiments, the ion source ionizes a sample (e.g., received from the (LC) separation device) to produce sample ions, and the ion separator performs a plurality of repeated ion separation scans, wherein the ion separator receives sample ions in each ion separation scan and separates them according to the first physicochemical property. Simultaneously, the mass analyzer performs a plurality of mass analysis scans, wherein the mass analyzer receives separated sample ions or fragmented ions derived from separated sample ions in each mass analysis scan and mass analyzes the received ions.

[0029] In particular, the analytical instrument may (i) perform one or more MS1 mass analysis scans during a first ion separation scan and (ii) perform a plurality of MS2 mass analysis scans during a second ion separation scan. The second ion separation scan may, in the plurality of repeated ion separation scans, immediately follow the first ion separation scan, but this is not necessary. Steps (i) and (ii) may be performed repeatedly, e.g., such that the analytical instrument repeatedly switches between performing MS1 mass analysis scan(s) in one ion separation scan and performing MS2 mass analysis scans in the subsequent ion separation scan (followed by performing MS1 mass analysis scan(s) in the subsequent ion separation scan, and so on).

[0030] In further embodiments, each cycle includes more than two ion separation scans. Thus, for example, the method may comprise the analytical instrument performing a plurality of repeated cycles, each cycle comprising any number, such as three, four, five, or more, of the plurality of repeated ion separation scans. Each ion separation scan in a cycle may be directed to either an MS1 ​​analysis or an MS2 analysis. In each cycle, there may be one or more (e.g., two, three, four, etc.) MS1-directed ion separation scans and one or more (e.g., two, three, four, etc.) MS2-directed ion separation scans. The timing of the mass analysis scans and the isolation windows used for each mass analysis scan can be configured so that each ion separation scan in a cycle is directed to a different region of the ion arrival time m / z space (i.e., the first physicochemical property m / z space).However, it would also be possible that a region of the ion arrival time-m / z space is analyzed (reisolated) more than once during a cycle.

[0031] In some embodiments, each cycle includes two or more ion separation scans, and MS1 mass analysis scan(s) are performed during the earlier ion separation scan(s) of each cycle and MS2 mass analysis scans are performed during the later ion separation scan(s) of each cycle. For example, each cycle may comprise the analytical instrument (i) performing one or more MS1 mass analysis scans during a first ion separation scan, (ii) performing a first plurality of MS2 mass analysis scans during a second (e.g., immediately subsequent) ion separation scan, (iii) performing a second plurality of MS2 mass analysis scans during a third (e.g., immediately subsequent) ion separation scan, (iv) optionally performing a third plurality of MS2 mass analysis scans during a fourth (e.g., immediately subsequent) ion separation scan, and (v) optionally performing one or more further (e.g.,immediately following) ion separation scan(s) one or more further pluralities of MS2 mass analysis scans.

[0032] Other sequences of MS1 scans and MS2 scans in each cycle would be possible. For example, one or more additional MS1-guided ion separation scans may be performed in each cycle, e.g., after the first ion separation scan and before the MS2-guided ion separation scan(s). Although in some embodiments the "second" ion separation scan immediately follows the "first" ion separation scan, in further embodiments, there may be one or more ion separation scan(s) between the "first" and "second" ion separation scans in each cycle (and similarly for the "third" and "fourth" ion separation scans).

[0033] The analytical instrument can perform the plurality of repeated cycles during an entire chromatographic separation run of the chromatographic (LC) separation device.

[0034] During the first ion separation scan of each cycle (and / or during any subsequent MS1-directed ion separation scans), 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 subsequent MS1) ion separation scans of a cycle may together approximately cover most or all of the duration T IMS of the first ion separation scan. Thus, if only a single MS1 mass analysis scan is performed during the first (and / or subsequent MS1) ion separation scans, T MS1 ≈ T IMS . If a large number of MS1 mass analysis scans are performed during the first (and / or subsequent MS1) ion separation scans, approximately N ≈ T IMS / T MS1MS1 mass analysis scans are performed during the first (and / or subsequent MS1) ion separation scans.

[0035] The one or more MS1 mass analysis scans performed during the first (and / or subsequent MS1) ion separation scans of a cycle can collectively span an m / z range of interest. The m / z range of interest can be any suitable m / z range, such as between about 100 and 2000 or similar. Thus, if only a single MS1 mass analysis scan is performed during the first (and / or subsequent MS1) ion separation scans, the mass filter can be operated in its transmitting mode or in its filtering mode with a wide isolation window width encompassing the m / z range of interest such that the MS1 mass analysis scan can determine the mass-to-charge ratio (m / z) of ions over the entire m / z range of interest.

[0036] If a plurality of MS1 mass analysis scans are performed during the first (and / or subsequent MS1) ion separation 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 of (N) 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 of 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.

[0037] For this purpose, each MS1 scan of the plurality of MS1 mass analysis scans can use one MS1 isolation window from a plurality of different MS1 isolation windows. That is, the mass filter isolation window can be controlled to be different for each MS1 scan of the plurality of MS1 mass analysis scans performed during the first (and / or subsequent MS1) ion separation scan.

[0038] The plurality of MS1 isolation windows may differ from each other in terms of their mean m / z values. That is, each MS1 scan of the plurality of MS1 mass analysis scans may use a mean m / z of the MS1 isolation window from a plurality of different mean m / z of the MS1 isolation window (and the mean m / z of the mass filter isolation window may be controlled to be different in each MS1 scan of the plurality of MS1 mass analysis scans).

[0039] The plurality of MS1 isolation windows may also differ from each other in terms of their width, although this is not necessary and it would instead be possible to use the same MS1 isolation window width for each MS1 scan of the plurality of MS1 mass analysis scans.

[0040] The mean m / z values ​​and widths of the MS1 isolation window can be selected so that the multiple MS1 scans together span the entire m / z range of interest. Furthermore, each MS1 isolation window can be selected based on one or more trend lines.

[0041] In some embodiments, each trend line corresponds to an ion charge state, and each trend line provides a relationship between the ion arrival time and the m / z for ions having that charge state. For example, each trend line may provide a relationship between the ion mobility arrival time and the m / z for ions having that charge state.

[0042] It has been recognized that there is a relationship between ion mobility arrival time and m / z for ions with various different charge states (e.g., singly charged, doubly charged, triply charged, etc.). These relationships take the form of a "trend line" with respect to each different charge state. The relationship between ion mobility arrival time and m / z can be linear for ions with a particular charge state and can therefore be described as a trend line, e.g., in the form of a slope and intercept. However, depending on the nature of the sample ions, nonlinear trend lines are also possible. In practice, there will be some scatter in arrival times for ions with a particular charge and m / z, but this scatter is usually sufficiently small to allow ions of different charge states to be distinguished over most of the ion mobility arrival time-m / z space.

[0043] In some embodiments, each trend line, as well as the charge state, or instead, corresponds to a particular chemical class, with each trend line providing a relationship between the ion arrival time and m / z for ions of that chemical class, and optionally with that charge state. That is, different chemical classes may have different (charge-dependent) trend lines. Examples of a chemical class include peptides derived from the tryptic digestion of a protein; a group or mixture of proteins; lipids or a group of lipids; metabolites or a group of metabolites; nucleotides, and so on.

[0044] Each trend line can thus correspond to (i) a particular charge state (e.g., singly charged, doubly charged, triply charged, 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 the ion arrival time and m / z for ions having that charge state and / or chemical class. Each trend line can be used to determine an approximate expected ion arrival time for sample ions (i) dependent on the m / z and charge state of the sample ions, (ii) dependent on the m / z and chemical class of the sample ions, or (iii) dependent on the m / z, charge state, and chemical class of the sample ions.

[0045] It would also be possible to define and use a trend line based on another property of the sample ions.

[0046] As further described below and more fully in co-pending application US63 / 468,170, the entire contents of which are hereby incorporated by reference, the one or more trend lines may be determined by performing a calibration for the instrument.

[0047] In embodiments, each MS1 isolation window of the plurality of MS1 isolation windows is selected based on one or more of these trend lines. By selecting the MS1 isolation windows based on the one or more trend lines (e.g., by controlling the mass filter's isolation window to track one or more of the trend lines) during the first (and / or subsequent MS1) ion separation scans, it can be ensured that (only) sample ions of interest (e.g., with a particular charge state or multiple particular charge states and / or a particular chemical class of interest) are present in the MS1 scans.

[0048] Thus, each mean m / z of the MS1 isolation window can be selected based on one or more of the trendlines. That is, the mean m / z of the mass filter isolation window can be configured to track one or more trendlines. Thus, for example, if each mean MS1 ​​isolation window m / z is selected based on a single trendline, the mean isolation window m / z of each MS1 scan can be (approximately) equal to the m / z value given by the single trendline at the mean (average) arrival time at which that MS1 scan is performed.If each mean MS1 ​​isolation window m / z is selected based on two or more trendlines, the mean isolation window m / z of each MS1 scan can be (approximately) equal to the average m / z value given by these two or more trendlines at the mean (average) arrival time at which this MS1 scan is performed.

[0049] Likewise, the width of each MS1 isolation window can be selected based on the one or more trend lines. Thus, for example, if the width of each MS1 isolation window is selected based on a single trend line, the width of each MS1 isolation window can be configured such that most or all ions with the 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 with a different charge state and / or chemical class than the charge state and / or chemical class associated with the single trend line are attenuated (not transmitted) by the mass filter.If each MS 1 isolation window width is selected based on two or more trend lines, each MS 1 isolation window width can be configured such that most or all ions with one of the charge states and / or chemical class associated with the two or more trend lines are transmitted through the mass filter, and such that most or all ions with a different charge state and / or chemical class than one of the charge states and / or chemical class associated with the two or more trend lines are attenuated (not transmitted) through the mass filter.

[0050] Thus, the plurality of MS 1 isolation windows may generally be configured to isolate separate sample ions having a charge state and / or chemical class corresponding to a charge state and / or chemical class associated with one or more trend lines, while attenuating separate sample ions having a different charge state and / or chemical class than a charge state and / or chemical class associated with one or more trend lines.

[0051] The width of the MS1 isolation window used for each MS1 scan in the plurality of MS1 scans may increase with increasing mean m / z of the MS1 isolation window to better isolate those ions with the desired charge state and / or chemical class and to better attenuate ions with other undesired charge states and / or other chemical classes.

[0052] In particular embodiments, the MS1 isolation windows are selected to exclude singly charged (1+) ions from the MS1 scans. Thus, the one or more trend lines on the basis of which each MS1 isolation window is selected can include (only) trend lines for multiply charged ions (and no trend lines for singly charged ions). This allows the instrument's maximum charge capacity to be used more efficiently.

[0053] In particular embodiments, the MS1 isolation windows are selected such that ions with the same charge state(s) and / or chemical class as the ions to be selected in the MS2 scans are selected in the MS1 scans. Thus, for example, the MS1 isolation windows for the plurality of MS1 mass analysis scans may 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 second, different (e.g., triply charged) charge state.

[0054] As described above, in the method, the instrument performs a plurality of MS2 mass analysis scans during a second (and optionally third, fourth and / or subsequent) ion separation scan of each cycle.

[0055] The number of MS2 mass analysis scans performed during an ion separation scan depends on the duration T MS2 of the MS2 mass analysis scans relative to the duration T MS1 of the MS1 mass analysis scans, which can be selected as desired as described above. Approximately M ≈ N × T MS1 / T MS2 MS2 mass analysis scans can be performed during an ion separation scan. The multitude of MS2 mass analysis scans performed during an ion separation scan can together approximately account for most or all of the duration T IMS this ion separation scan, but this is not necessary.

[0056] In the DIA method of various embodiments, the plurality of MS2 mass analysis scans performed during an ion separation scan may together span the m / z range of interest. Thus, in each MS2 mass analysis scan, the instrument may isolate and fragment ions within a sub-range of the m / z range of interest. Thus, the m / z range of interest may be divided into a plurality of (M) 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 MS2 scan of the plurality of MS2 scans is configured to isolate and fragment ions having an m / z within a respective different one of the sub-ranges.

[0057] For this purpose, each MS2 scan of the multiple MS2 mass analysis scans uses one MS2 isolation window from a plurality of different MS2 isolation windows. This means that the mass filter's isolation window can be controlled to be different for each MS2 scan of the multiple MS2 mass analysis scans performed during an ion separation scan.

[0058] The plurality of MS2 isolation windows may differ from each other in terms of their mean m / z values. That is, each MS2 scan of the plurality of MS2 mass analysis scans may use a mean m / z of the MS2 isolation window from a plurality of different mean m / z of the MS2 isolation window (and the mean m / z of the mass filter isolation window may be controlled to be different in each MS2 scan of the plurality of MS2 mass analysis scans).

[0059] The plurality of MS2 isolation windows may also differ from each other in terms of their width, although this is not necessary and it would instead be possible to use the same MS2 isolation window width for each MS2 scan of the plurality of MS2 mass analysis scans.

[0060] The mean m / z and widths of the MS2 isolation window can be selected so that the multitude of MS2 scans together span the entire m / z range of interest.

[0061] 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 charged, doubly charged, triply charged, 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 time 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 sample ions (i) depending on the m / z and charge state of the sample ions, or (ii) depending on the m / z, charge state, and chemical class of the sample ions.

[0062] By selecting the MS2 isolation windows based on the one or more trend lines (e.g., by controlling the mass filter isolation window to track one or more of the trend lines) during the second ion separation scan, it can be ensured that (only) sample ions of interest (e.g., with a particular charge and / or chemical class) are isolated and fragmented in the MS2 scans.

[0063] As described above, the method comprises 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. In particular, the first and / or second and / or third and / or further pluralities of MS2 isolation windows may be configured by selecting the mean m / z and / or width of one or more or each of the MS2 isolation windows based on the analysis of the MS1 data.

[0064] In some embodiments where each MS2 isolation window is selected based on one or more trend lines, configuring the plurality of MS2 isolation windows may include selecting the particular charge state(s) and / or chemical class of interest based on the analysis of the MS1 data. That is, depending on the MS1 data, only some (and not all) of the trend lines may be selected and analyzed by one or more pluralities of MS2 mass analysis scans.

[0065] In these embodiments, in the first and / or second and / or third and / or further plurality of MS2 mass analysis scans, each mean MS2 isolation window m / z may be selected based on the one or more trend lines. That is, the mean m / z of the mass filter's isolation window may be configured to track one or more trend lines. Thus, for example, if each mean MS2 isolation window m / z is selected based on a single trend line, the mean isolation window m / z of each MS2 scan may be (approximately) equal to the m / z value given by the single trend line at the mean (average) arrival time at which that MS2 scan is performed.If each mean MS2 isolation window m / z is selected based on two or more trendlines, the mean isolation window m / z of each MS2 scan can be (approximately) equal to the average m / z value given by these two or more trendlines at the mean (average) arrival time at which this MS2 scan is performed.

[0066] Similarly, the width of each MS2 isolation window can also be selected based on the one or more trend lines. Thus, for example, if the width of each MS2 isolation window is selected based on a single trend line, the width of each MS2 isolation window can be configured such that most or all ions with the 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 with a different charge state and / or chemical class than the charge state and / or chemical class associated with the trend line are attenuated (not transmitted) by the mass filter.If 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 with one of the charge states and / or chemical class associated with the two or more trend lines are transmitted through the mass filter, and such that most or all ions with a different charge state and / or chemical class than one of the charge states and / or chemical class associated with the two or more trend lines are attenuated (not transmitted) through the mass filter.

[0067] Thus, the plurality of MS2 isolation windows may be configured to isolate separate sample ions having a charge state and / or chemical class corresponding to a charge state and / or chemical class associated with one or more trend lines, while attenuating separate sample ions having a different charge state and / or chemical class than a charge state and / or chemical class associated with one or more trend lines.

[0068] As described above, the width of the MS2 isolation window used for each MS2 scan in the first and / or second and / or third and / or further plurality of MS2 scans may increase with increasing mean m / z of the MS2 isolation window to better isolate those ions having the desired charge state and / or chemical class and to better attenuate ions having further undesired charge states and / or chemical classes.

[0069] In these embodiments, the MS2 isolation windows can be selected to select multiply charged ions of a single charge state, while excluding singly charged (1+) ions from the MS2 scans. Thus, the one or more trend lines 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).

[0070] In further embodiments, the MS2 scans are not performed using a single plurality of MS2 isolation windows tracing one or more trend lines, but using (i) a first plurality of MS2 mass analysis scans having a first plurality of MS2 isolation windows during the second ion separation scan, (ii) a second plurality of MS2 mass analysis scans having a second plurality of MS2 isolation windows during a third ion separation scan, (iii) optionally a third plurality of MS2 mass analysis scans having a third plurality of MS2 isolation windows during a fourth ion separation scan, and (iv) optionally one or more further pluralities of MS2 mass analysis scans, each having a further plurality of MS2 isolation windows during a further ion separation scan.

[0071] The first and / or second and / or third and / or further pluralities of MS2 isolation windows may be selected such that the first and / or second and / or third and / or further pluralities of MS2 mass analysis scans together cover a region of the ion arrival time-m / z space of interest (e.g., without leaving any gaps in the region). The region of interest of the ion arrival time-m / z space may correspond to one or more trend lines of interest, where (as described above) each trend line corresponds to a charge state and / or chemical class, and each trend line provides a relationship between the ion arrival time and m / z for ions having that charge state and / or chemical class. For example, the region covered by the pluralities of MS2 isolation windows may correspond to multiply charged ions.

[0072] In these embodiments, the plurality of MS1 isolation windows may comprise an integer number N of isolation windows, the first plurality of MS2 isolation windows may comprise an integer number M1 of isolation windows, the second plurality of MS2 isolation windows may comprise an integer number M2 of isolation windows, the third plurality of MS2 isolation windows may comprise an integer number M3 of isolation windows, and / or, if present, each further plurality of MS2 isolation windows may comprise an integer number M f of insulation windows. M1, M2, M3 and M f can be equal or approximately equal and can each be a multiple of N.

[0073] Each isolation window of the first plurality of MS2 isolation windows may correspond to a respective one of the second and / or third and / or further plurality of MS2 isolation windows, e.g., such that each isolation window of the first plurality of MS2 isolation windows is used at an ion arrival time in the second ion separation scan that corresponds to an ion arrival time in the third and / or fourth and / or further ion separation scan using the corresponding isolation window of the second and / or third and / or further plurality of MS2 isolation windows. Similarly, each MS1 isolation window of the plurality of MS1 isolation windows may correspond to a respective group of MS2 isolation windows of the first and / or second and / or third and / or further plurality of MS2 isolation windows, e.g.,such that each MS1 isolation window of the plurality of MS1 isolation windows is used at an ion arrival time in the first ion separation scan that corresponds to an ion arrival time in the second and / or third and / or fourth and / or further ion separation scan in which the corresponding group of MS2 isolation windows of the first plurality of MS2 isolation windows is used.

[0074] Thus, for each ion arrival time, there can be a set of corresponding MS2 isolation windows, with each isolation window of the set corresponding to one of the second and / or third and / or fourth and / or subsequent ion separation scans. Each MS2 isolation window of a set has a different mean m / z.

[0075] In some embodiments, all of the corresponding MS2 isolation windows in a set have approximately the same width. Then, configuring the first and / or second and / or third and / or further pluralities of MS2 isolation windows based on the analysis of the MS1 data for each set of corresponding MS2 isolation windows may comprise: assigning each MS2 isolation window of the set to one of the second and / or third and / or fourth and / or further ion separation scans based on an ion abundance within each MS2 isolation window of the set. For example, the MS2 isolation window of the set encompassing the highest abundance of ions may be assigned to the second ion separation scan, the isolation window of the set encompassing the second highest abundance of ions may be assigned to the third ion separation scan, and so on.This ensures that the regions of the ion arrival time m / z space of interest that exhibit higher ion abundances are prioritized, i.e., analyzed earlier, e.g., in the second ion separation scan. However, it would be possible to configure the MS2 isolation windows to prioritize regions of the ion arrival time m / z space of interest that exhibit lower ion abundances.

[0076] Thus, the procedure may include: for each of one or more or all of the first plurality of MS2 isolation windows: configuring that MS2 isolation window based on the MS1 data to include a higher abundance of ions than a corresponding one of the second plurality of MS2 isolation windows; and / or for each of one or more or all of the second plurality of MS2 isolation windows: configuring that MS2 isolation window based on the MS1 data to include a higher abundance of ions than a corresponding one of the third plurality of MS2 isolation windows; and / or for each of one or more or all of the third plurality of MS2 isolation windows: configuring that MS2 isolation window based on the MS1 data to include a higher abundance of ions than a corresponding one of any further plurality of MS2 isolation windows.

[0077] In further embodiments, the widths may be different, rather than using the same width for all of the corresponding MS2 isolation windows in a set. For example, the widths may be configured based on the MS1 data so that each isolation window of a set encompasses approximately the same ion abundance.

[0078] Thus, configuring the first and / or second and / or third and / or further pluralities of MS2 isolation windows may include: for each of one or more or all of the second plurality of MS2 isolation windows: configuring that MS2 isolation window based on the MS1 data to include an equal or similar abundance of ions to a corresponding one of the first plurality of MS2 isolation windows; and / or for each of one or more or all of the third plurality of MS2 isolation windows: configuring that MS2 isolation window based on the MS1 data to include an equal or similar abundance of ions to a corresponding one of the first plurality of MS2 isolation windows; and / or for each of one or more or all of the further plurality of MS2 isolation windows: configuring that MS2 isolation window based on the MS1 data to include an equal or similar abundance of ions to a corresponding one of the first plurality of MS2 isolation windows.

[0079] In these embodiments, the method may include: Determining a total ion current indicated by the MS1 data; and for each of one or more or all of the second plurality of MS2 isolation windows: configuring that MS2 isolation window based on the MS1 data to include an equal or approximately equal portion of the total ion current as a corresponding one of the first plurality of MS2 isolation windows; and / or for each of one or more or all of the third plurality of MS2 isolation windows: configuring that MS2 isolation window based on the MS1 data to include an equal or approximately equal proportion of the total ion current as a corresponding one of the first plurality of MS2 isolation windows; and / or for each of one or more or all of the further plurality of MS2 isolation windows: configuring that MS2 isolation window based on the MS1 data to include an equal or approximately equal proportion of the total ion current as a corresponding one of the first plurality of MS2 isolation windows.

[0080] As described above, in some embodiments, during each MS2 mass analysis scan, ions are accumulated in an ion reservoir for an accumulation time. In some further embodiments, the method includes determining the fill time for one or more or each MS2 scan based on the analysis of the MS1 data.

[0081] According to a second aspect, there is provided a method of operating an analytical instrument comprising: Ionizing a 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; and (ii) performing 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 an ion storage for an accumulation time; the method further comprising: Analyzing MS1 data acquired from the one or more MS1 mass analysis scans; and Determine the accumulation time for one or more or each MS2 scan based on the analysis of the MS1 data.

[0082] This aspect and these embodiments may, and in embodiments do, include any one or more of any of the optional features described herein.

[0083] From this perspective and in these embodiments, determining the accumulation time for an MS2 scan may include: Using the one or more trend lines and the MS1 data to estimate an ion abundance within the MS2 isolation window for the MS2 scan; and Determine the accumulation time for the MS2 scan based on the estimated ion abundance.

[0084] Another aspect provides a non-transitory computer-readable storage medium storing computer software code that, when executed on a processor, performs the method(s) described above.

[0085] Another aspect provides a control system for an analytical instrument, such as a mass spectrometer, wherein the control system is configured to cause the analytical instrument to perform the method(s) described above.

[0086] Another aspect provides an analytical instrument, such as a mass spectrometer, comprising the control system described above.

[0087] Another aspect provides an analysis tool that includes: an ion source configured to ionize a sample to produce sample ions; an ion separator configured to separate the 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; a 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; and (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, each MS2 scan of the plurality of MS2 mass analysis scans using one MS2 isolation window of a plurality of MS2 isolation windows; wherein the control system is further configured to: Analyzing MS1 data acquired from one or more MS1 mass analysis scans; and Configure the multiple MS2 isolation windows based on the analysis of the MS1 data.

[0088] Another aspect provides an analysis tool that includes: an ion source configured to ionize a sample to produce sample ions; an ion separator configured to separate the 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; a 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; and (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 each MS2 mass analysis scan accumulates ions in an ion storage for an accumulation time; wherein the control system is further configured to: Analyzing MS1 data acquired from one or more MS1 mass analysis scans; and Determine the accumulation time for one or more or each MS2 scan based on the analysis of the MS1 data.

[0089] These aspects and embodiments may, and in some embodiments do, be combined with any one or more of each of the aspects, embodiments, and / or optional features described herein. DESCRIPTION OF THE DRAWINGS

[0090] Various embodiments will now be described in more detail with reference to the accompanying figures, in which: Fig. Figure 1 schematically illustrates a conventional DIA workflow; Fig. 2 shows a mass spectrometer that can be operated according to embodiments; Fig. Figure 3 shows an example plot of ion mobility arrival time versus m / z for peptide ions of different charge states; Fig. 4 the peptide ions from Fig. 3 shows a schematic illustration of a conventional DIA workflow superimposed on it; Fig. 5 schematically illustrates a sequence of MS1 scans according to embodiments; Fig. 6 schematically illustrates a sequence of MS2 scans according to embodiments; Fig. 7 schematically illustrates a sequence of MS2 scans according to embodiments; Fig. 8A shows a chromatographic example elution peak, Fig. 8B schematically illustrates a sequence of MS1 scans according to embodiments, Fig. 8C schematically illustrates a sequence of MS2 scans according to embodiments, Fig. 8D schematically illustrates a sequence of MS1 scans according to embodiments and Fig. 8E schematically illustrates a sequence of MS2 scans according to embodiments; Fig. 9A schematically illustrates a sequence of MS2 scans according to embodiments and Fig. 9B schematically illustrates a sequence of MS2 scans according to embodiments; Fig. 10 schematically illustrates a calibration method according to embodiments; Fig. Figure 11 shows another example plot of ion mobility arrival time versus m / z for peptide ions of different charge states; Fig. 12 schematically illustrates a sequence of MS2 scans according to embodiments; Fig. 13 shows an example MS1 mass spectrum obtained according to embodiments; Fig. 14 the MS 1 mass spectrum of Fig. 13 shows, equally divided between four MS2 isolation windows; Fig. 15 the ion population of each of the MS2 isolation windows of Fig. 14 shows; Fig. 16 the four MS2 isolation windows of Fig. 14, rearranged into an order based on the ion population; Fig. 17 the MS 1 mass spectrum of Fig. 13 shows, divided between four MS2 isolation windows of varying width to achieve approximately equal ion populations; and Fig. 18 the ion population of each of the MS2 isolation windows of Fig. 17 shows. DETAILED DESCRIPTION

[0091] The embodiments described herein relate to the application of mass spectrometry to proteomics and related fields. Proteomics and related methods are typically performed using two distinct data acquisition methodologies: data-dependent acquisition (DDA) and data-independent acquisition (DIA).

[0092] Fig. Figure 1 illustrates a typical DIA workflow in which a sample, such as a proteome digest, is separated using liquid chromatography (LC) and ionized into a mass spectrometer. An LC elution peak (plotted on the Y-axis) is overlaid on an illustration of the traditional DIA acquisition strategy. The mass spectrometer is configured to acquire a single MS 1 scan (light-hatched boxes in Fig. 1) covering an entire m / z region of interest, and this scan is then followed by a series of MS2 scans (dark hatched boxes in Fig. 1) using various small mass filter m / z isolation windows that, when integrated across the entire series of MS2 scans, cover the m / z region analyzed by the initial MS1 scan. In other words, a single MS1 scan is collected for a broad isolation range, and this analyzed m / z range is then divided into MS2 windows, i.e., narrow m / z regions that are isolated and subsequently fragmented.

[0093] This combination of an MS 1 scan and the subsequent MS2 scans is called a cycle. As in Fig. As shown in Figure 1, this cycle is repeated many times throughout the entire LC gradient. Since a single LC elution profile is on the order of several seconds and the cycle time—the time required for the instrument to collect the MS1 and MS2 scans of a single cycle—is typically 1-2 seconds, multiple cycles can occur throughout the elution profile, facilitating the identification and quantification of sample ions, such as peptide ions.

[0094] Important for this workflow is the fact that the ion population does not change across the LC elution profile, meaning that the ions analyzed in an MS1 ​​scan have the same compositional population as those analyzed in the MS2 scans. However, when such LC-MS-DIA workflows incorporate an additional separation technique such as ion mobility, this correlation between MS1 ​​and MS2 scans is no longer guaranteed.

[0095] Fig. Figure 2 schematically illustrates an analytical instrument, such as a mass spectrometer (MS), that may be operated according to embodiments. As in Fig. 1, the instrument includes an ion source 10, an ion separator 20 such as an ion mobility separator (IM separator), a mass filter 30, a fragmentation device 40, and a mass analyzer 50.

[0096] 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 so on. More than one ion source can be provided and used. The ions can be any suitable type of ion to be analyzed, e.g., small and large organic molecules, biomolecules, DNA, RNA, proteins, peptides, fragments thereof, and the like.

[0097] 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), such that the sample ionized in the ion source 10 comes out of the separation device.

[0098] The ion separator 20 is arranged downstream of the ion trap 10 and is configured to receive ions from the ion trap 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). Different types of ion separators are described in more detail below.

[0099] If the ion separator 20 is an ion mobility separator, the ion mobility separator may comprise any suitable type of ion mobility separator. For example, an electric field, such as a DC voltage gradient and / or a traveling DC voltage wave, may be arranged to drive ions along the length of the separator and through a gas, such that the ions are separated according to their ion mobility. The ions may optionally be driven against or perpendicular to a countercurrent gas flow. Alternatively, a gas flow may be arranged to drive the ions along the length of the separator, while an electric field, such as a DC voltage gradient and / or a traveling DC voltage wave, may be arranged to oppose the gas flow, such that the ions are separated according to their ion mobility.The ion mobility separator 20 may be a linear separator with a straight or folded path or a cyclic separator (with a closed loop).

[0100] 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 their mass-to-charge ratio (m / z). The mass filter 30 can be configured such that received ions having an m / z within an m / z transmission window of the mass filter are passed through the mass filter, while received ions having an m / z outside the m / z transmission window are attenuated by the mass filter, e.g., are not passed through the mass filter. The width and / or mean m / z of the transmission window can be controllable (variable), e.g., by appropriately controlling RF and / or DC voltage(s) applied to the mass filter 30.Thus, for example, the mass filter 30 may be operable in a transmitting mode of operation, where most or all ions within a relatively wide m / z window are passed through the mass filter 30, and in a filtering mode of operation, where only ions within a relatively narrow m / z window (centered on a desired m / z) are passed through the mass filter 30. The mass filter 30 may be any suitable type of mass filter, such as a quadrupole mass filter.

[0101] The fragmentation device 40 is arranged downstream of the mass filter 30 and is configured to receive most or all of the ions transmitted through the mass filter 30. The fragmentation device 40 may be configured to selectively fragment some or all of the received ions, i.e., to produce fragment ions. The fragmentation device 40 may be operable in a fragmentation mode of operation, in which most or all of the received ions are fragmented to produce fragment ions (which may then be forwarded by the fragmentation device 40), and in a non-fragmentation mode of operation, in which most or all of the received ions are forwarded without being (intentionally) fragmented. It would also be possible for a non-fragmentation mode of operation to be implemented by causing ions to bypass the fragmentation device 40.The fragmentation device 40 may also be operable in one or more intermediate operating modes, e.g., in which the degree of fragmentation is controllable (variable).

[0102] The fragmentation device 40 may be any suitable type of fragmentation device, such as a collision-induced dissociation (CID) fragmentation device, an electron-induced dissociation (EID) fragmentation device, a photodissociation fragmentation device, and so on. Numerous other types of fragmentation are possible.

[0103] The mass analyzer 50 is located 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 to determine their mass-to-charge ratio and / or mass, i.e., to produce 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™ FT mass analyzer.

[0104] Thus, the mass analyzer 50 may comprise an inner electrode elongated along the orbital trap axis and a split pair of outer electrodes surrounding the inner electrode and defining therebetween a trapping volume in which ions are trapped and oscillated by orbiting around the inner electrode, to which a trapping voltage is applied, while oscillating back and forth along the trap axis. The pair of outer electrodes function as detection electrodes to detect an image current induced by the oscillation of the ions in the trapping volume, thereby providing a detected signal. The outer electrodes typically function as a differential pair of detection electrodes 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 can be processed using a Fourier transform to obtain a mass spectrum of ions within the trap.

[0105] It should be noted that Fig. 2 is merely schematic and that the instrument may, and in embodiments does, include any number of one or more additional components.

[0106] For example, the instrument may include one or more ion transfer or capture stages, e.g., disposed between the various illustrated devices. The one or more ion transfer stages may include, e.g., an atmospheric pressure interface and / or one or more ion guides, lenses, and / or other ion optical devices configured to transfer ions between the various illustrated devices. The ion transfer stage(s) may include any suitable number and configuration of ion optical devices, e.g., optionally including one or more RF and / or multipole ion guides, one or more ion guides for cooling ions, one or more mass-selective ion guides, and so on.

[0107] As in Fig. As shown in Figure 2, the instrument is under the control of a control unit 60, such as an appropriately programmed computer, which controls the operation of various components of the instrument. The control unit 60 can also receive and process data from various components, including the analyzer 50. The control unit 60 is configured, among other things, to determine the settings for the ion separator 20, the mass filter 30, the fragmentation device 40, and the mass analyzer 50 for analytical scans.

[0108] For example, the control system 60 may cause the instrument to perform one or more MS1 scans, wherein during each MS1 scan, the mass filter 30 is operated in its transmit mode or in its filtering mode and ions are not (intentionally) fragmented, so that a broad m / z range of ions 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 during each MS2 scan, the mass filter 30 is operated in its filtering mode and ions are fragmented in the fragmentation device 40, so that a narrow m / z range of the ions produced by the ion source 10 are selected and fragmented, and the resulting fragment ions are mass analyzed by the mass analyzer 50.

[0109] As described above, in separation techniques using dispersive ion mobility (IM separation techniques), ions are injected into the IM separator 20 and separate in a time-representative manner for the mobility of the individual ion species. Fig. Figure 3 shows an example representation of eight peptide ions separated by IM-MS. As Fig. As illustrated in Figure 3, the peptide ions represented as black ellipses fall on clear, charge-dependent trend lines in the arrival time-m / z space. Fig. Figure 3 shows example trend lines for singly charged (1+) ions, doubly charged (2+) ions, and triply charged (3+) ions. The result of the IM separation is thus various peptide ions concentrated in charge-dependent trend lines in the arrival time m / z space, which elute from the IM separator 20 in distributions with time widths of several tens of milliseconds over the entire IM separation time (typically 1-2 seconds).

[0110] Fig. Figure 4 shows the same eight example peptides (black ellipses) overlaid on a representation of a standard DIA acquisition scheme, with the light-shaded boxes representing MS1 scans and the smaller, dark-shaded boxes representing MS2 scans. As can be seen from Fig. As can be seen in Figure 4, this acquisition strategy fails due to the separation in the IM domain, as there is no overlap between the analyzed m / z range and the arrival time. Furthermore, since the MS2 scans occur at a longer arrival time than the preceding MS1 scan, the correlation between MS1 ​​and MS2 is no longer present.

[0111] When DIA is performed in an LC-IM-MS setup, the traditional MS acquisition strategy, as described in Fig. As illustrated in Figure 4, the same ion populations are no longer sampled in the MS1 and MS2 scans. That is, the ions present in the MS1 scan would no longer be present when the MS2 scans take place due to their separation in the IM arrival time domain. This blocks the ability to identify and quantify the peptide ions using this traditional detection methodology. Therefore, a new detection strategy is required.

[0112] The embodiments described herein provide new data acquisition strategies to enable DIA measurements with LC-IM-MS instrument configurations. As described above, the correlation between the MS1 and MS2 scans is important for the success of a DIA measurement; that is, these scans should analyze the same ion species. The acquisition strategies described herein ensure this correlation. Some embodiments also optionally effectively utilize IM separation to steer the mass spectrometer toward informative, multiply charged ions.

[0113] For the operation of some embodiments, it is important that while the ion population during an LC-IM-MS experiment (see Fig. 4) due to the separation in the IM domain, this IM separation for the same ion population remains consistent across an elution LC peak for multiple IM injections. Since a uniform population of ions elutes from a single chromatographic peak, the IM separation for these ions is therefore reproducible and can be used to correlate MS1 and MS2 scans.

[0114] Fig. Figure 5 illustrates a first step of a method according to embodiments. Multiple MS1 scans (dark gray boxes) are collected in a series, where the number of MS1 scans is defined by the maximum arrival time desired to be analyzed. Unlike a standard DIA acquisition, all MS1 scans during a single IM injection are collected in a sequence. In Fig. 5, the arrival time examined by each MS 1 scan is represented by the box height and the isolation width of the mass filter is reflected by the box width. In the example of Fig. 5, the m / z regions analyzed by each MS1 scan are derived from the 2+ and 3+ trend lines; however, this mass analyzer isolation can be controlled by a single charge state trend line and / or additional charge states, e.g., 4+ or 5+, as required by the application. It can be seen that in the example of Fig. 5 all the peptide ion signals (black ellipses) are analyzed in one MS1 scan.

[0115] After this sequence of MS 1 scans has been collected, in a second step another ion packet from the LC elution peak is injected into the IM separator 20. Fig. Figure 6 illustrates an embodiment of this second step. As in Fig. 6, the method uses the previous boundary conditions of the MS1 scan (black outlines in Fig. 6) as boundaries for the MS2 scans (dark grey boxes in Fig. 6). In this example, the MS2 transition time allows for eight MS2 scans to be obtained per MS1 scan window from the previous injection (other numbers of MS2 scans are also possible).

[0116] Thus, MS1 scans are first acquired for a single IM injection, with the m / z regions analyzed with each MS1 scan being dictated by the charge state trend lines of interest (which are shown in Fig. 5 are the charge states 2+ and 3+). During the next IM injection, the method then acquires MS2 scans (dark grey boxes in Fig. 6) within the m / z region analyzed by the MS1 scans (black box frames in Fig. 6). In the Fig. In the example shown in Figure 6, the MS2 scans are focused on the 2+ trendline, and the mass filter isolation of each scan is controlled by the boundaries of this trendline; however, the MS2 scans can focus on further and / or additional trendlines, as described below.

[0117] Once this series of MS2 scans is acquired, the mass spectrometer acquires a new set of MS1 scans during the next IM injection.

[0118] With this acquisition strategy, the mass spectrometer cycle can thus consist of two IM injections, where the first IM injection is analyzed by a plurality N of MS1 scans and the second IM injection is analyzed by a plurality M of MS2 scans, where N is defined by Equation 1 and M is defined by Equation 2: N=MaximumArrivalTimeIMTransientLengthMS1 M=N×TransientLengthMS1TransientLengthMS2

[0119] This cycling scheme is applied continuously across the entire LC gradient length. Once the data is acquired, the post-processing data analysis tools require information from N MS1 scans and M MS2 scans to perform the correlation. In the example shown, for a single cycle, the peptides present in the first MS1 scan are fragmented in the first through eighth MS2 scans; the peptides present in the second MS1 scan are fragmented in the ninth through sixteenth MS2 scans; and so on.

[0120] As described above, in the Fig. In the example shown in Figure 6, only the 2+ trend line is analyzed; however, this is not required. For example, the MS2 scans can follow any charge state trend line or can cover multiple trend lines, e.g., both the 2+ and 3+ ions simultaneously.

[0121] Fig. Figure 7 illustrates an example where the MS2 scans are directed towards the 3+ trend line ions. As in Fig. As shown in Figure 7, the MS2 scans exhibit mass filter isolation windows that are governed by the boundary conditions of this charge state.

[0122] Fig. Figure 8 summarizes various concepts of the methods described herein. As in Fig. 8A, are used for an eluting LC peak (black trace in Fig. 8A) multiple IM injections were performed over the peak profile (black dots in Fig. 8A, where the label corresponds to the IM injection number). These IM injections are analyzed in an alternating manner, with the acquisition scheme alternating between MS1 ​​and MS2 scans. Thus, in Fig. 8B the first IM injection multiple MS 1 scans, in Fig. 8C, the second IM injection includes multiple MS2 scans, in Fig. 8D, the third IM injection includes multiple MS 1 scans and in Fig. 8D, the fourth IM injection includes multiple MS2 scans.

[0123] In the example of Fig. 8, the 2+ and 3+ trend lines are targeted by both the MS 1 and MS2 scans. In general, however, the region of arrival time-m / z space targeted by the MS 1 and MS2 scans can vary.

[0124] In addition to embodiments that include 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 incorporated into each cycle. That is, the process cycle can be modified to include multiple IM injections that collect MS2 scans, where, for example, each IM injection can focus on a different charge state trend line for MS2 analysis.

[0125] Fig. Figure 9 illustrates an example where the first IM injection where MS1 scans are collected (see Fig. 5), followed by 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 sequential IM injections: (i) MS1 scans, (ii) MS2 scans: 2+-focused, (iii) MS2 scans: 3+-focused. Thus, the first MS2 IM injection ( Fig. 9A) the mass filter to the 2+ trend line and the second injection ( Fig. 9B) directs the mass filter to the 3+ trend line. This cycle can be repeated for the entire LC gradient.

[0126] It is understood that the methods described above are illustrative of the general concepts described herein, and that many alternative methods are possible according to embodiments.

[0127] A limitation of any MS analysis is the upper limit of the number of ions a mass spectrometer can store before space charge, i.e., the repulsion of like charges stored within a physical space, which leads to adverse effects. In Orbitrap™ instruments, this storage limit is typically dictated by the space charge capacity of the so-called "C-trap," from which ion packets are injected into the Orbitrap™ mass analyzer for mass analysis. To ensure optimal sensitivity of an MS analysis, in some embodiments, the ions transferred to and stored in the C-trap should be "information-rich." Typically, such "information-rich" ions are multiply charged in proteomic analyses, whereas contaminating background ions are often singly charged.Therefore, it may be analytically advantageous to fill the C-trap with multiply charged ions and discharge singly charged species that would contribute to the space charge capacity if transferred.

[0128] As described above, IM separation of analytes results in charge state-dependent trend lines (see Fig. 3). These trend lines can be mapped using a calibration procedure, such 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 spectrometer's mass filter 30 to eliminate singly charged ions and transmit multiply charged ions for each MS1 or MS2 scan. This feature is utilized in the techniques described above and in Fig. 5 to 9 examples are used throughout.

[0129] Fig. Figure 10 schematically illustrates such a calibration scheme. As in Fig. As shown in Figure 10, the calibration procedure takes a known set of analytes, e.g., P1, P2, P3, P4, separates these ions with IM, and analyzes the arrival time distribution with multiple MS1 scans over the IM cycle (macroscan), with the MS1 scans being delayed by a known amount (Δt) in each subsequent macroscan. In other words, to more accurately determine the arrival time of each peptide, the ion mobility separation is repeated several times, and the start of the MS1 scan, and thus the effective arrival time bin being analyzed, is shifted by a known delay Δt. This process is repeated until the transient window of the MS1 scan reaches the estimated maximum arrival time (“MS1-scan n”) at the end of the macroscan. In other words, the number of MS1 scans is repeated with increasingly longer delays until the sliding transient window reaches the desired maximum arrival time “MS1-scan n.”

[0130] After acquiring these mass spectra, the extracted ion chromatogram of each peptide can be determined as a function of the MS1 scan number. The scan numbers can be correlated with the effective arrival time, and the arrival time of each peptide can be determined.

[0131] The resulting "calibrated" arrival time versus relative intensity for P1, P2, P3, and P4 can then be determined. This information, the arrival time as a function of m / z and charge state, can then be used to guide the mean m / z over the course of 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, groups of proteins, groups of lipids, groups of metabolites, etc., as there is a correlation between arrival time and m / z, as well as charge state dependencies that form charge-dependent trend lines between these two variables. Thus, after applying the calibration procedure to a known analyte mixture, the charge state trend lines in the space of IM versus m / z and z can be determined and used for an unknown analyte of the same class separated using the same IM conditions.

[0132] Although various particular embodiments have been described above, various alternative embodiments are possible.

[0133] For example, the trend line(s) analyzed during MS2 scans can be varied with different applications, or multiple MS2-focused IM injections can be used.

[0134] In the embodiments described above, the mass filter isolation windows are controlled by a uniform buffer around the trend line established by the calibration, the buffer region being shown by the shaded area shown in Fig. 3 is centered around the dashed trend line for each charge state. However, this buffer need not be uniform.

[0135] Fig. Figure 11 shows an example in which the buffered regions (the shaded region enclosing each dashed trend line) that guide the mass filter isolation during the MS1 and MS2 scans are asymmetric. In particular, Fig. 11, the upper limit of a buffered charge state region is defined by 107.5% of the trend line slope, and the lower limit is defined by 85% of the trend line slope. Other distributions can be used, and this buffer distribution can be adapted, for example, for specific applications.

[0136] Furthermore, while in the embodiments described above the mass filter isolation windows become wider at longer arrival times as the upper and lower buffer regions expand away from the center trend line, a fixed quadrupole isolation width along the trend line(s) may be used instead.

[0137] In some embodiments, the MS1 scans may be intelligently used to steer the MS2 scans, e.g., to steer MS2 scans to either prioritize or ignore the 3+ trend line based on the abundance of 3+ ions in the MS1 scans, and e.g., to make predictions regarding the number of ions, e.g., for automatic gain control (AGC).

[0138] From the foregoing, it is understood that embodiments provide a new acquisition sequence for MS1 and MS2 scans in a DIA workflow. As described above, in a conventional DIA workflow, a single broad m / z MS1 scan is followed by multiple narrow m / z MS2 scans, and this cycle is repeated throughout the LC gradient. However, with the addition of ion mobility, such an acquisition strategy cannot be used. To overcome this, embodiments collect all MS1 scans in a sequential manner during a single IM injection. Then, during the subsequent IM injection(s), the MS2 scans are acquired.

[0139] In addition to this scan order, some embodiments utilize the charge-state separation enabled by IM to maximize the "information-rich" ions stored in the ion trap (e.g., C-trap) prior to mass analysis by instructing the mass filter to transmit only m / z regions as governed by the trend lines of multiply charged ions. That is, embodiments maximize the utilization of the space charge capacity of the ion trap (e.g., C-trap) by transmitting only regions of arrival-time m / z space that contain multiply charged, "information-rich" ions.

[0140] Embodiments allow the correlation of MS1 and MS2 scans when using a DIA workflow with an LC-IM-MS instrument configuration. This instrument configuration is advantageous because the addition of IM increases the duty cycle and inherently increases the sensitivity compared to MS workflows without IM.

[0141] Although the particular embodiments described above provide methods that either detect all the “information-rich” ions arriving at a given time ( Fig. 8), or isolate a subset of them by following charge-state-dependent trend lines of ion arrival times ( Fig. 9), further embodiments are provided.

[0142] It has been recognized that the approach involving the selection of all "information-rich" ions arriving at a given moment can, under certain circumstances, yield poor performance due to an overabundance of different peptides contained within the isolation windows. This can result in overly complicated fragmentation spectra, complicating interpretation by the processing software.

[0143] Similarly, the approach that selects ions along individual, charge-state-dependent trend lines can suffer from sample selection inconsistency, with some regions of the arrival time-versus-m / z space being sampled multiple times and additional regions not being sampled at all. This is because the trend lines are merely trend lines—not all ions of interest arrive exactly on the trend line. In some cases, there is significant deviation, for example, to the point where the entire region between the 2+ trend line and the 3+ trend line may contain ions of interest.

[0144] Various further embodiments thus provide a method that allows complete coverage of the ions of interest without overloading the processing software with overly complicated fragmentation spectra. To this end, the method described above is generalized by using the MS2-DIA sampling windows, e.g., as in Fig. 8, into an arbitrary number of smaller isolation windows, with each set of windows being selected by subsequent injections of ions into the ion mobility device.

[0145] Fig. Figure 12 shows an example of such a method, where the MS2 isolation windows are divided into three sets (referred to as isolation window sets MS2a, MS2b, and MS2c). Isolation window set MS2a can be selected during a first ion injection into the ion mobility device, isolation window set MS2b can be selected during a second ion injection, and isolation window set MS2c can be selected during a third ion injection.

[0146] This approach provides complete coverage of the arrival time versus the m / z space of interest, but allows for smaller isolation windows to reduce the number of different peptides analyzed within a given scan. In other words, embodiments provide a method for reducing the number of peptides present in any given fragmentation scan, allowing for successful deconvolution of the resulting MS2 spectra.

[0147] Thus, in embodiments, multiple MS2-focused IM injections may be used after the 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 the MS2 scan for an arrival time of 250-282 ms would isolate the m / z range of 300-400 m / z in a single MS2-focused IM injection strategy, the method can perform three separate MS2-focused IM injections, splitting this 300-400 m / z range across the 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 last MS2-focused IM injection would isolate 366.6 to 400 m / z.

[0148] This method can be generalized to any number of isolation windows. The above example describes dividing the MS2 space into three sets, but it could be any number of sets, such as two sets, four sets, or more than four sets.

[0149] In this method, the m / z width of each of the original MS2 windows can be equally divided among the multiple sets. Alternatively, the size of the smaller windows can be varied, for example, to allow for a varying density of different peptides in the arrival time-versus-m / z space. This variable spacing of the windows can be performed based on calibration data collected using the calibration routine, e.g., as described in US63 / 468,170, such that regions of the arrival time-versus-m / z space with a higher density of incoming peptides have narrower isolation windows, and regions with a lower density of incoming peptides have wider windows.

[0150] Although the above example describes three sets of MS2 scans collected sequentially, this is not necessarily the case, and other acquisition sequences 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, and so on), or, for a larger number of sets, it can collect a variety of MS2 sets between each MS1 set (e.g., MS1, MS2a, MS2b, MS1, MS2c, MS2d, and so on).

[0151] In further embodiments, the spectral complexity (number of MS2 peaks) can be adjusted by optimizing the number of sets and / or the m / z coverage window (variable or identical) for each of them. The maximum acceptable spectral complexity may be determined by limitations of the data processing software used to automatically extract protein identifications and may vary depending on the particular algorithms / software package used.

[0152] The embodiments described so far allow for good correlation between MS1 ​​and MS2 scans and effectively utilize both LC and IM separation prior to MS analysis. Further embodiments do this while also exploiting the data in real time, e.g., by using the information obtained during the MS1 scans to guide the MS2 scans or the order in which they are collected.

[0153] In some embodiments, the IM-MS-DIA methods are guided by the estimated boundaries of the charge-state trend lines to regulate the quadrupole m / z isolation region. However, in some cases, the ions may not be uniformly distributed across this m / z space. There may be narrow m / z regions with many precursor ions and broad m / z regions with relatively few precursor ions. This information is unknown until the arrival time distribution is measured by an MS1 ​​scan.

[0154] Various further embodiments provide a method that utilizes this MS1 scan information to intelligently control the MS2 scan order to maximize the utility of the ions and minimize the amount of instrument time wasted on sparse m / z regions while the LC peak elutes into the MS.

[0155] In these embodiments, the MS1 scans are analyzed as they arrive, e.g., by the instrument's control system (internal PC), to determine metrics for the precursor ion distribution as a function of m / z, charge state, and intensity (non-exhaustive list). This information is then used to control the acquisition scheme of subsequent MS2-focused IM injections. One example of this "smart" acquisition scheme is the determination of the number of ions in the various m / z isolation compartments.

[0156] Fig. Figure 13 shows an example of a spectrum obtained from an MS1 ​​scan with a region of 450–1000 m / z for a specific arrival time window. Within the m / z region of interest (450–1000 m / z), a broad distribution of precursor ions is evident. However, the precursor ions are not evenly distributed across this m / z space. In the range from 550 to 850 m / z, there is a much denser distribution of ions, and therefore precursors, compared to the regions from 450–550 m / z and 850–1000 m / z.

[0157] If the area as a whole were analyzed by four consecutive MS2-focused IM injections (e.g. as described above with respect to Fig. 12), the range of 450-1000 m / z would be divided equally between the four separate IM injections, as in Fig. 14 (where “MS2 region” describes the quadrupole isolation window for this particular IM injection). Fig. 14 thus shows, over the MS1 spectrum of Fig. 13, the different quadrupole isolation regions that would be analyzed using a strategy of four MS2-focused IM injections. With this strategy, the quadrupole isolation window is gradually shifted across the entire m / z range with each incremental IM injection.

[0158] Fig. Figure 15 shows ion populations as a function of MS2 areas for the scheme of Fig. 14. It can be seen that the area planned to be covered by IM injection 2 has the most ion signal density, followed by the area for IM injection 3, while the areas of injection 1 and 4 have one magnitude less signal. Thus, the result in Fig. 14 shown detection methods in an asymmetric ion loading distribution in the four individual IM injections.

[0159] In embodiments, using this data (which may be determined in real time, e.g., when the MS1 scan arrives at the internal instrument PC), the method prioritizes the regions based on the cumulative signal intensity present in the MS1 scan for that quadrupole isolation region. In the example of Fig. 14 and Fig. 15 this would be in the Fig. 16 shown IM acquisition sequence.

[0160] Thus, using the ion loading per quadrupole isolation region determined in real time from the MS1 scan, the regions can be ranked from most populated to least populated (e.g., similar to a "top-N" approach using DDA) and then acquired in that order. This ensures that the region with the most information is acquired first. In the case of a peptide distribution that is almost completely eluted from the LC column, this can ensure that instrument time is used effectively.

[0161] It should be noted that a "most abundant to least abundant" coverage scheme is not the only possible strategy. For example, for some applications, it may be advantageous to cover from least populated to most populated, e.g., when low-abundance analytes are of particular interest.

[0162] A further use of the MS1 data can be realized by determining the isolation windows required to have an equal ion loading in each of the n-number IM injections, which in this example case is four IM injections.

[0163] For this purpose, the total ion current (TIC) for an MS1 ​​scan can be calculated and this TIC divided by the number, n, of MS2-focused IM injections established for the DIA procedure (e.g., as selected by the user). Then, based on the MS1-specific m / z distribution, isolation windows that achieve the TIC / n number of ions can be calculated. The quadrupole isolation windows can be calculated in real time to establish this distribution by varying the width of each.

[0164] For the Fig. In the MS1 scan shown in Figure 13, the TIC is 1 × 10 9, which for n = 4 MS2-focused IM injections with one IM injection per isolation region at a target ion loading of 2.5 × 10 8 would result.

[0165] Calculating windows that allow this distribution of ion loading to arise results in the isolation windows that are in Fig. 17. Here, the windows are not evenly spaced, but each isolation region has an equal amount of ion loading, as Fig. 18. From Fig. Figure 18 shows that by calculating varying window sizes, the ion loading is evenly distributed across the four IM injections. This approach can potentially avoid overfilling a particular MS2 scan while leaving other MS2 scans unfilled.

[0166] In addition, as Fig. 8, the mass resolutions used for MS 1 and MS2 scans are often different, resulting in different arrival time distributions being analyzed by a single MS 1 and MS2 scan. In the Fig. In the example presented in Figure 8, there are eight MS2 scans analyzing the same arrival time range as the single MS1 scan. The data contained in the MS1 scan covers arrival times that would be analyzed by multiple MS2 scans due to differences in transition lengths during subsequent IM injections.

[0167] Since the available data originates from the MS1 plane and is thus the combination of all arrival times within this region, the charge-state-dependent trendline data (as determined, for example, by the calibration procedure) can be used in combination with the charge state of a precursor and the m / z of that precursor to determine in which arrival time bin, i.e., in which of the eight MS2 scans, it would be expected to arrive. This can be done while performing one of the two real-time decision-making options described above. These calculations can also be used to implement AGC control for the various MS2 scans by calculating the expected ion populations and / or adjusting the IM fill time and also the ion injection times in the MS.

[0168] It is understood that embodiments utilize real-time decision-making from the collected MS1 level to control how the MS2 scans are acquired. Embodiments may more effectively utilize instrument time to instruct the instrument to focus on regions of high ion loading first, rather than being solely controlled by a predetermined, MS1-independent m / z window order. Alternatively, embodiments may directly balance the ion loading of different IM injections to avoid underfilling a single IM injection or overfilling another. Furthermore, embodiments may be utilized to determine AGC control for MS2 scans during subsequent IM injections.

[0169] Although various embodiments have been described above, particularly with regard to ion mobility separation, it would be possible to carry out the process using any type of ion separation technology in which ions are separated according to a ("first") physicochemical property. For example, the process may be carried out 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: 1. Ion separation devices configured to sort ions by m / z, such as described in US Patent No. 10,256,088. An RF voltage is applied to each electrode of a first array of uniformly spaced, parallel, and coplanar electrodes and to its corresponding electrode of a second array of uniformly spaced, parallel, and coplanar electrodes. The RF voltage varies in amplitude according to an amplitude gradient of the RF voltage. The RF voltage produces an array of distinct 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 a DC voltage gradient to produce a DC electric field in the uniform gap.When ions are introduced into the uniform gap, the DC electric field causes the ions to migrate toward quadrupole RF electric fields with increasing RF voltage amplitude, where the ions are trapped according to their m / z. 2. Ion traps or ion trap arrays, as described, for example, in US Patent No. 9,111,741. An ion trap device can be configured to separate ions according to their mass-to-charge ratio. An ion trap can comprise at least two or more rows of parallel electrode arrays, each electrode array including at least two or more parallel rod-shaped electrodes. By applying different phase AC voltages to different rod electrodes to generate AC electric fields within the space between two parallel electrodes of different rows of electrode arrays, multiple linear ion-trapping fields are formed in the space between the different rows of electrode arrays, which are open adjacent to each other without a true barrier. 3. Separation devices using Zeno-pulsing techniques, such as those described in the article "A W-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. 4. Stacked-well ion traps, such as those described in US Patent No. 7,872,228. A plurality of electrodes can be positioned and controlled by RF potentials to form a plurality of adjacent pseudopotential wells. The ions can be manipulated, converted, analyzed, and ejected from the device in a manner similar to conventional ion traps. In addition, selected ions or groups of ions can be directed from one pseudopotential well to another without ion losses due to physical obstructions. 5. Orthogonal flow ion trap arrays, such as those described in US 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 along the first direction, and an ion inlet arranged to receive ions. The plurality of electrodes may be configured to create one or more pseudopotential barriers of increasing magnitude along a first direction. An air drag force may be applied to the ions by the gas flow, opposing a pseudopotential gradient of the plurality of electrodes. 6. Annular traps, such as those described in U.S. Patent Application No. US18 / 090,730. A system for sorting ions includes an array of multipole electrodes configured to form an ion trap and an ion conductor adjacent to the array of multipole electrodes. An RF and DC voltage device is used to apply an RF voltage to the array of multipole electrodes, creating a pseudopotential barrier configured to confine one or more ions. The RF and DC voltage device is also used to apply a DC voltage that creates an axial field opposing the pseudopotential barrier at the trap exit. The RF or DC voltage is then ramped up or down, depending on the application, to cause at least one ion to elute across the pseudopotential barrier. 7. Ion centrifuge ion separation devices, such as those described in European Patent Application No. EP 4,020,524. An ion separation device may comprise: (a) first and second ion carpets, each comprising: a substrate having first and second surfaces; and a set of electrodes mounted on or below the first surface, wherein a configuration of a first plurality of the set of electrodes defines at least one group of circular sectors; (b) an ion exit aperture traversing an ion carpet;and (c) one or more power supplies configured to provide radio frequency voltages to a first subset of the electrodes of each ion carpet, to provide electrical potential differences across electrodes of the first subset of electrodes of each ion carpet, and to provide time-varying voltages to the first plurality of electrodes of each ion carpet migrating as a traveling wave through the sectors, wherein the ion carpets are mounted parallel to each other with a gap therebetween, the first surfaces facing each other across the gap; 8. Ion mobility separation devices utilizing a lens array, such as described in U.S. Patent No. 11,119,070. A mobility separator includes a two-dimensional electrode grid spanning a passageway between first and second walls. The first and second walls each include an inlet port and a plurality of exit ports. The two-dimensional electrode grid is configured to generate an electric field within the passageway. The plurality of ion channels are disposed adjacent to the plurality of exit ports. The movement of ions between the inlet port and the plurality of exit ports is governed by the electric field and a gas flow through the passageway between the first and second walls such that the ions are sorted and directed into different channels based on their respective mobility. 9. Trapped ion mobility separation (TIMS) devices, as described, for example, in the article by Meier, F. et al. "Parallel Accumulation-Serial Fragmentation (PASEF): Multiplying Sequencing Speed ​​and Sensitivity by Synchronized Scans in a Trapped Ion Mobility Device." Journal of Proteome Research 2015, 12, 5378-5387.

[0170] Although the present invention has been described with reference to various embodiments, it will be understood that various changes may be made without departing from the scope of the invention as set forth in the appended claims. QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions. Cited patent literature

[0000] WO 2008 / 081334

[0020] US 63 / 468,170 [0046, 0128] US 63 / 468,170

[0149] US 10,256,088

[0169] US 9,111,741

[0169] US 7,872,228

[0169] US 10,651,025

[0169] US 18 / 090,730

[0169] EP 4,020,524

[0169] US 11,119,070

[0169] Cited non-patent literature

[0000] Zeno-Pulsing“-Techniken verwenden, wie z. B. beschrieben in dem Artikel „A W-Geometry Ortho-TOF MS with High Resolution and Up to 100 % Duty Cycle for MS / MS“, J. Am. Soc. Mass Spectrom., 2009, 20, S. 1342-13

[0169] Meier, F. et al. Parallel Accumulation-Serial Fragmentation (PASEF) beschrieben: Multiplying Sequencing Speed and Sensitivity by Synchronized Scans in a Trapped Ion Mobility Device. Journal of Proteome Research 2015, 12, 5378-5387

[0169]

Claims

[1] A method of operating an analytical instrument, comprising: Ionizing a 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; and (ii) performing 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, each MS2 scan of the plurality of MS2 mass analysis scans using an MS2 isolation window of a plurality of MS2 isolation windows; the method further comprising: Analyzing MS1 data acquired from the one or more MS1 mass analysis scans; and Configure the multiple MS2 isolation windows based on the analysis of the MS1 data. [2] The method of 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 comprising: Configuring a second plurality of MS2 isolation windows based on the analysis of the MS1 data; and (iii) performing a third ion separation scan by separating 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 of the second plurality of MS2 mass analysis scans uses an MS2 isolation window of the second plurality of MS2 isolation windows. [3] The method of claim 2, wherein the method further comprises: Configuring a third plurality of MS2 isolation windows based on the analysis of the MS1 data; and (iv) performing a fourth ion separation scan by separating 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 of the third plurality of MS2 mass analysis scans uses an MS2 isolation window of the third plurality of MS2 isolation windows. [4] The method of claim 2 or 3, wherein configuring the MS2 isolation windows comprises: for each of one or more or all of the first plurality of MS2 isolation windows: configuring that MS2 isolation window based on the MS1 data to include a higher abundance of ions than a corresponding one of the second plurality of MS2 isolation windows; and / or for each of one or more or all of the second plurality of MS2 isolation windows: configuring that MS2 isolation window based on the MS1 data to include a higher abundance of ions than a corresponding one of the third plurality of MS2 isolation windows. [5] The method of claim 4, further comprising: for each of one or more or all of the second plurality of MS2 isolation windows: configuring that MS2 isolation window to have a width equal to or approximately equal to the width of the corresponding one of the first plurality of MS2 isolation windows; and / or for each of one or more or all of the third plurality of MS2 isolation windows: configuring that MS2 isolation window to have a width equal to or approximately equal to the width of the corresponding one of the first plurality of MS2 isolation windows. [6] The method of claim 2 or 3, wherein configuring the MS2 isolation windows comprises: for each of one or more or all of the second plurality of MS2 isolation windows: configuring that MS2 isolation window based on the MS1 data to include an equal or similar abundance of ions to a corresponding one of the first plurality of MS2 isolation windows; and / or for each of one or more or all of the third plurality of MS2 isolation windows: configuring that MS2 isolation window based on the MS1 data to include an equal or similar abundance of ions to a corresponding one of the first plurality of MS2 isolation windows. [7] The method of claim 6, wherein analyzing the MS1 data and configuring the MS2 isolation windows comprises: Determining a total ion current indicated by the MS 1 data; and for each of one or more or all of the second plurality of MS2 isolation windows: configuring that MS2 isolation window based on the MS1 data to include an equal or approximately equal proportion of the total ion current as a corresponding one of the first plurality of MS2 isolation windows; and / or for each of one or more or all of the third plurality of MS2 isolation windows: configuring that MS2 isolation window based on the MS1 data to include an equal or approximately equal portion of the total ion current to a corresponding one of the first plurality of MS2 isolation windows. [8] Method according to one of the preceding claims, wherein: the MS2 isolation windows are configured such that the MS2 mass analysis scans together cover a region of the ion arrival time-m / z space of interest; and the region of interest of the ion arrival time-m / z space corresponds to one or more trend lines of interest, each trend line corresponding to an ion charge state and / or chemical class, and each trend line providing a relationship between the ion arrival time and the m / z for ions having that charge state and / or chemical class. [9] A method according to any one of claims 1 to 7, wherein: each MS2 isolation window is configured based on one or more trend lines, each trend line corresponding to an ion charge state and / or chemical class, and each trend line providing a relationship between the ion arrival time and m / z for ions having that charge state and / or chemical class. and configuring the plurality of MS2 isolation windows comprises selecting the charge state(s) and / or chemical class of interest based on the analysis of the MS1 data. [10] Method according to one of the preceding claims, wherein: During each MS2 mass analysis scan, ions are accumulated in an ion storage device for an 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, comprising: Ionizing a 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; and (ii) performing 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 an ion storage for an accumulation time; the method further comprising: Analyzing MS1 data acquired from the one or more MS1 mass analysis scans; and Determine the accumulation time for one or more or each MS2 scan based on the 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 separation scan, and wherein: in each MS1 mass analysis scan of the plurality of MS1 mass analysis scans, separate sample ions are isolated using an MS1 ​​isolation window, and the isolated sample ions are subjected to mass analysis; and each MS1 scan of the plurality of MS1 mass analysis scans uses one MS1 isolation window of a plurality of MS1 isolation windows, optionally selecting each MS1 isolation window based on one or more trend lines. [13] The method of claim 12 when dependent on claim 10 or 11, wherein determining the accumulation time for an MS2 scan comprises: Using the one or more trend lines and the MS1 data to estimate an ion abundance within the MS2 isolation window for the MS2 scan; and Determine the accumulation time for the MS2 scan based on the estimated ion abundance. [14] A 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 performing a plurality of repeated cycles by the analytical instrument, wherein the analytical instrument performs steps (i) and (ii) in each cycle. [16] A method according to claim 15 when dependent on claim 2 or 3, wherein: the analytical instrument performs steps (i), (ii) and (iii) in each cycle; or the analysis instrument performs steps (i), (ii), (iii) and (iv) in each cycle. [17] A method according to claim 15 or 16, wherein: the sample is provided by a chromatographic separation device; and the method comprises the analytical instrument continuously performing repeated cycles of the chromatographic separation device during the course of a chromatographic separation. [18] A non-transitory computer-readable storage medium storing computer program code which, when executed on a processor, performs the method of any preceding claim. [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 1-17. [20] An analysis instrument comprising the control system of claim 19. [21] Analysis tool comprising: an ion source configured to ionize a sample to produce sample ions; an ion separator configured to separate the 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; a 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 MS 1 mass analysis scans; and (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, each MS2 scan of the plurality of MS2 mass analysis scans using one MS2 isolation window of a plurality of MS2 isolation windows; wherein the control system is further configured to: Analyzing MS1 data acquired from one or more MS1 mass analysis scans; and Configure the multiple MS2 isolation windows based on the analysis of the MS1 data. [22] Analysis tool, comprising: an ion source configured to ionize a sample to produce sample ions; an ion separator configured to separate the 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; a 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; and (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 each MS2 mass analysis scan accumulates ions in an ion storage for an accumulation time; wherein the control system is further configured to: Analyzing MS1 data acquired from one or more MS1 mass analysis scans; and Determine the accumulation time for one or more or each MS2 scan based on the 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 the 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 their mass-to-charge ratio (m / z).

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