Mass spectrometry methods, mass spectrometers, and computer software

Abstract

To provide methods of mass spectrometry.SOLUTION: The method comprises, for each of multiple sub-ranges selected from an overall m / z range: accumulating in an ion storage a sample of precursor ions to be analyzed, the precursor ions having m / z values within the sub-range; accumulating in the ion storage a sample of fragmented precursor ions to be analyzed, the fragmented precursor ions being formed from fragmentation of the precursor ions having m / z values within the sub-range; and simultaneously analyzing the combined samples of the precursor ions and the fragmented precursor ions in a mass analyzer.SELECTED DRAWING: Figure 1

Description

[Technical Field]

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

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

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

[0004] In the case of Data Independent Acquisition (DIA) methods, the MS1 scan is optional and may be skipped to allow time to generate additional MS2 spectra. The list of m / z targets for each of the multiple MS2 scans may be a stepwise increasing / decreasing list of m / z across the m / z range of interest. An example of this is described in European Patent No. 3,410,463, which is incorporated herein by reference. The resulting precursor information can be used for quantification, while the fragment information can be used for identification.

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

[0006] To mass analyze ions, ions (fragment ions or precursor ions) are typically first stored in an ion trap, and then the stored ions are released as packets into a mass analyzer for mass analysis. Storage improves instrument sensitivity, but requires careful control of the filling time of ions into the ion trap (so-called "Automatic Gain Control" (AGC)) to avoid deleterious space charge effects.

[0007] One problem with existing methods is the limited dynamic range of MS1 spectra. Digested peptide concentrations can vary by up to 10 orders of magnitude, but the dynamic range of single-shot spectra in, for example, an electrostatic orbital trap mass analyzer (such as the Orbitrap™ FT mass analyzer manufactured by Thermo Fisher Scientific™) can be limited to about 4 orders of magnitude. Furthermore, the number of ions that can be injected into an orbital trap mass analyzer is limited to approximately 10 orders of magnitude by the capacity of the storage C trap. 5As a result, low-intensity peptide precursor signals can be overwhelmed. For DDA experiments, these problems are significant and can result in precursor targets being missed. For DIA experiments, the lack of good precursor data hinders identification and quantification.

[0008] One method for improving the dynamic range of MS1 spectra is the "boxcar" method (described in Meier et al., Nature Methods, 2018, 15, 440-448) and the high dynamic range (HDR) method described in UK Patent Application No. 2211790.7, which are incorporated herein by reference. In these approaches, the broad mass range of the analysis is subdivided into several narrower isolation windows. For each isolation window, a separate injection into the C-trap is performed with a different fill time depending on the ion current. This method attenuates dense m / z regions and amplifies sparse m / z regions. This improves detection sensitivity for relatively weak peaks and increases the effective dynamic range of the scan. However, the use of these methods significantly increases the time required to accumulate ions, especially when a significant number of isolation windows are required. Due to the long fill times for low-level windows and the time required to switch quadrupole and ion source voltages, HDR scans can require a significant amount of additional time, which can affect the speed at which MS2 scans can be performed. These methods can therefore reduce the time available for MS2 scans in fast LC-MS methods that are limited by the time it takes for the sample to elute from the chromatographic column.

[0009] Multi-window HDR also suffers from the drawback of discarding a significant number of ions due to quadrupole isolation. Ion loss can be addressed by a pre-accumulation process. For example, a trapped ion mobility device can be used to pre-accumulate ions before the quadrupole and release them into a mass filter in a synchronized mass / mobility-dependent manner, significantly reducing ion loss (as described in Meier et al., Molecular & Cellular Proteomics, 2018, 17, 2524-2545).

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

[0011] In DIA experiments, MS2 spectra may retain a proportion of unfragmented precursor ions (in quantities insufficient to be analytically useful). Some Orbitrap instruments offer an optional feature called "stepped collision energy," described in U.S. Patent No. 9,536,717. This feature provides multiple separate injections into the collision cell (also called the "fragmentation chamber") at a range of collision energies; the summed populations of ions are then transferred to the C-trap / Orbitrap mass analyzer and analyzed together. This variation in energy improves the probability that one of the energies used is optimal for fragmenting the precursor ions. However, this approach also uses many collision energies that are not optimal for fragmenting the precursor ions, with the associated cost of ion beam time. The actual action of changing collision energy and performing a second or third injection into the collision cell may not be excessively time-consuming, possibly adding 1-3 ms of overhead to a scan cycle of over 40 ms.

[0012] U.S. Patent No. 8,686,350 describes a method in which different types of ions can be accumulated in an ion trap before being released into a mass analyzer. In one example, a combination of two types of ions having the same narrow mass range is injected into the ion trap, one fragmented and one remaining as an intact precursor. This can increase confidence that the precursor ion can be detected and accurately mass-measured. However, quantification can be compromised by the proportion of unfragmented precursor residues from the MS2 injection. Summary of the Invention

[0013] A method of mass spectrometry is provided, comprising: for each of a plurality of subranges selected from the overall m / z range: storing a sample of precursor ions to be analyzed in an ion store, the precursor ions having m / z values ​​within a subrange; storing a sample of fragmented precursor ions to be analyzed in an ion store, the fragmented precursor ions being formed from fragmentation of precursor ions having m / z values ​​within a subrange; and analyzing the combined sample of precursor ions and fragmented precursor ions simultaneously in a mass analyzer.

[0014] In this method, multiple MS2 scans are performed for a list of m / z target subranges (in a DDA experiment, or more preferably in a DIA experiment). For at least some (preferably all) of the MS2 scans, precursor ion information is acquired along with regular fragment ion information. Each set of precursor and fragment information is acquired simultaneously or parallel in time without changing the quadrupole mass filter settings. The precursor information for each MS2 scan is for a narrow m / z subrange (and thus contains information equivalent to a so-called "selected ion monitoring" (SIM) scan).

[0015] The precursor information from all subrange scans can be combined to effectively create a complete (at least in DIA experiments where the list of m / z targets spans a wide m / z range of interest) or otherwise partially complete MS1 spectrum. Therefore, the present method performs SIM and MS2 scans in parallel to improve quantification of accumulated ions.

[0016] By constructing an MS1 ​​spectrum from multiple narrow m / z subranges, each m / z subrange can be acquired using a tailored fill time, thereby improving the dynamic range of the overall MS1 spectrum (in the same manner as the "boxcar" or HDR methods). However, unlike the "boxcar" or HDR methods, the methods disclosed herein are performed in a manner that reduces the additional time required to acquire MS1 data (and are therefore more compatible with high-speed LC-MS experiments). This is achieved, at least in part, because the sequence of the quadrupole mass filter is not altered from the sequence it follows with respect to the list of m / z targets for multiple MS2 scans.

[0017] In this first method, each set of precursor and fragment information is preferably acquired in one combined scan using a method such as that described in US Pat. No. 8,686,350.

[0018] In this first method, SIM and MS2 ions are combined in a single scan, thus reducing the time required to acquire MS1 and MS2 data.

[0019] Additionally, a special DIA method may be provided to minimize source / quadrupole transition times by dividing the overall m / z range into multiple subranges, which may optionally be stepped through sequentially so that small adjustments to the ion filter are made between subranges.

[0020] This combination of features is not known from any of the prior art mentioned above.

[0021] An additional advantage of the present method is the construction of high-quality HDR scans, or equivalent precursor data from many scans, suitable for quantifying a wide dynamic range of analyte ions. Performing such scans would typically involve long delays to switch ion sources and quadrupoles to scan through the mass range independently of the DIA cycle. Such processes consume a significant percentage of ion beam time due to the large number of ions processed for HDR scans. Therefore, the methods described herein can realize significant time savings and achieve improved efficiency. When available time is limited (such as in LC-MS), the methods described herein may enable greater resolution within the available time.

[0022] The method may further include estimating the amount of unfragmented precursor ions stored in the ion store during accumulation of the fragmented precursor ions.

[0023] In the first method, unfragmented precursor ions may be present among the MS2 fragment ions. These unfragmented ions are mixed with the SIM precursor ions in the ion trap. This can obscure the accurate quantitative information provided by the SIM precursor ions. This problem can be addressed by predicting (and then correcting for) the amount of unfragmented precursor ions present among the MS2 fragment ions.

[0024] By accounting for residual precursor ions from the MS2 injection (e.g., using post-identification AI tools), data from the SIM portion of the scan can be used for accurate quantification.

[0025] The SIM data from each subrange (corrected to account for residual precursor ions from the MS2 injection) can be combined to provide a high-resolution MS1 ​​scan for the entire m / z range (if the subranges are contiguous and completely cover the entire range).

[0026] The abundance of unfragmented precursor ions can be predicted using data analysis tools such as the CHIMERYS™ search engine by MSAID™.

[0027] The method may further include obtaining scan data from the simultaneous analysis of the combined samples.

[0028] Estimating the amount of unfragmented precursor ions can be performed using software configured to deconvolute a plurality of fragment spectra from the scan data, the plurality of fragment spectra being selected from a database of fragment spectra.

[0029] Estimating the amount of unfragmented precursor ions can be performed using a neural network configured to deconvolute a plurality of fragment spectra from the scan data, the plurality of fragment spectra corresponding to a plurality of precursor ion species.

[0030] Estimating the amount of unfragmented precursor ions can be performed using a neural network configured to predict the fragment spectrum of the precursor ions, including the intensities of the m / z peaks.

[0031] The method may further include configuring the ion filter to transmit precursor ions having m / z values ​​within the sub-range.

[0032] Configuring the ion filter may include setting a transmission window of the ion filter. The transmission window may be adjusted between each of a plurality of subranges. For each subrange, the transmission window for accumulating the sample of precursor ions may be the same as the transmission window for accumulating the sample of fragmented precursor ions. In other words, the transmission window may not be adjusted between these steps.

[0033] The method may further include configuring the ion mobility separator to transmit precursor ions having m / z values ​​within the sub-range to the ion filter.

[0034] The method may further include controlling the ion mobility separator such that precursor ions transmitted to the ion filter correspond to transmission windows of the ion filter for each of a plurality of subranges within the overall m / z range.

[0035] The mass analyzer may be a time-of-flight (ToF) analyzer, such as a multi-reflection time-of-flight (MR-ToF) analyzer.

[0036] The mass analyzer can be a Fourier transform mass analyzer (eg, an Orbitrap™ mass analyzer).

[0037] Accumulating a sample of precursor ions can include controlling the fill time for precursor ions based on the relative abundance of precursor ion species within corresponding subranges, which can improve the dynamic range of MS1 scans generated by combining SIM scans.

[0038] Fragmented precursor ions may be formed from fragmentation of precursor ions having sub-ranges of m / z values ​​at multiple different collision energies, and a separate ion storage step may be performed for each collision energy to store all of the fragments in the ion store.

[0039] Multiple subranges may be contiguous (and may be combined to make up the full m / z range).

[0040] The method may further include acquiring scan data for the precursor ions for each subrange. The method may further include combining the scan data for the precursor ions for each subrange to form a high-resolution scan for the entire m / z range.

[0041] Combining SIM scan data (for precursor ions of each subrange) can be particularly useful when the subranges are contiguous and / or completely cover the entire m / z range, in which case the combined SIM scan data can be used to form a high-resolution scan for the entire m / z range.

[0042] The overall m / z range can include a second plurality of sub-ranges that do not overlap with the plurality of sub-ranges. The method can further include, for each of the second plurality of sub-ranges, analyzing in the mass analyzer a sample of fragmented precursor ions formed from fragmentation of precursor ions having m / z values ​​within the sub-range.

[0043] In other words, SIM scans may not be required for some of the subranges, and MS2-only scans may be performed for these subranges, which may be subranges of the overall m / z range that includes the high-intensity precursor peak in the MS1 scan.

[0044] In some variations of the method, it may be preferable not to perform SIM scans for m / z subranges of the overall precursor mass range that are already abundant. A full MS scan can be used to determine which m / z subranges of the overall precursor mass range are already abundant. A full MS scan should be able to collect sufficient data for such regions by itself. Omitting SIM scans for these subranges can further reduce the time required for the overall method. HDR MS1 scans can be obtained by combining a full MS1 scan with a SIM scan.

[0045] Each of the multiple subranges may have the same width. In other words, the m / z range of each subrange may be equal. The width of the subrange may be preset before scanning. When the subranges are predetermined, this method may be referred to as a data independent acquisition (DIA) method.

[0046] The method may further include analyzing the sample of precursor ions having m / z values ​​from the global m / z range (ie, not mass filtered) in a mass analyzer.

[0047] Analyzing a sample of precursor ions having m / z values ​​from the entire m / z range can include acquiring scan data for the entire m / z range.

[0048] The method may further include acquiring scan data for precursor ions for each subrange, and expanding the scan data for the entire m / z range using the scan data for precursor ions for each subrange to form a high-resolution scan for the entire m / z range.

[0049] Extending the scan data for the entire m / z range can be useful when the subranges are not contiguous and / or do not completely cover the entire m / z range. By extending a specific region of the MS1 scan with SIM data, an HDR scan can be obtained.

[0050] The method may further include acquiring scan data for precursor ions for each subrange, comparing the scan data for the overall m / z range with the scan data for precursor ions for each subrange, and adjusting one of the scan data for the overall m / z range or the scan data for precursor ions for each subrange based on the other of the scan data for the overall m / z range or the scan data for precursor ions for each subrange.

[0051] In other words, the SIM data is compared to the full scan data, and one of the data sets is corrected based on the other. The SIM scanned precursor ions may be compared to the full MS and used as an internal calibration, which is particularly useful in hybrid instruments where the SIM scan is performed by the jitter-prone MR-ToF analyzer and the full MS is performed by the more stable Orbitrap analyzer.

[0052] The method may further include determining a plurality of subranges from the overall m / z range based on scan data acquired from analyzing a sample of precursor ions having m / z values ​​from the overall m / z range. Where the subranges are based on MS1 ​​data, the method may be a DDA method.

[0053] The plurality of subranges may include a first subrange and a second subrange.

[0054] The step of analyzing the combined sample of precursor ions and fragmented precursor ions from the first subrange may at least partially overlap (in time) with the step of accumulating a sample of precursor ions having m / z values ​​within the second subrange.

[0055] The step of analyzing the combined sample of precursor ions and fragmented precursor ions from the first subrange may at least partially overlap (in time) with the step of accumulating a sample of fragmented precursor ions formed from fragmentation of precursor ions having m / z values ​​within the second subrange.

[0056] Some steps for the next subrange may be performed while other steps from the previous subrange are still in progress. In other words, parallel processing can be used to reduce the overall time it takes to perform the scan.

[0057] There may also be an overlap (in time) between steps within a subrange, for example injection of precursor ions into the ion store may be carried out in parallel with fragmentation of the precursor ions.

[0058] For each subrange, the step of accumulating a sample of precursor ions may be performed before the step of accumulating a sample of fragmented precursor ions.

[0059] Alternatively, for each subrange, the step of accumulating a sample of fragmented precursor ions is performed before the step of accumulating a sample of precursor ions.

[0060] In other words, the order of ion injection is either precursor ions followed by fragment ions, or fragment ions followed by precursor ions.

[0061] The method may further include adjusting the collision energy used for fragmenting the precursor ions between the steps of accumulating a sample of precursor ions and accumulating a sample of fragmented precursor ions.

[0062] The method may further comprise cooling the fragmented precursor ions, which applies in particular when the step of storing a sample of fragmented precursor ions is performed before the step of storing a sample of precursor ions.

[0063] The method may be performed within a period of time based on the width of the chromatographic peak of the sample as it elutes from the chromatographic system.

[0064] The method may further include ionizing the sample to produce precursor ions.

[0065] The method may further include fragmenting the precursor ions to produce fragmented precursor ions.

[0066] Precursor ions may be fragmented in a collision cell, such as an ion routing multipole, IRM, collision cell, etc.

[0067] The method may further include performing quantification of the precursor ions based on scan data for the precursor ions. The quantification may be performed using data for the MS1 domain.

[0068] The method may further include identifying the precursor ion based on scan data for the fragmented precursor ion, wherein the identification may be performed using data associated with the MS2 domain.

[0069] A second method of mass spectrometry is provided. The second method comprises: For each of a plurality of subranges selected from the overall m / z range, configuring an ion filter to transmit precursor ions having m / z values ​​within a subrange; analyzing the sample of precursor ions received from the configured ion filter in a mass analyzer; analyzing in a mass analyzer a sample of fragment ions produced by fragmenting the precursor ions received from the configured ion filter.

[0070] In an alternative method, similar to the first method described above, multiple MS2 scans are performed (in a DDA experiment, or more preferably, in a DIA experiment) for a list of m / z target subranges. Precursor ion information is acquired for at least some (preferably all) of the MS2 scans, along with normal fragment ion information. However, unlike the first method, which uses a single scan for MS2 and SIM ions within each subrange (with multiple injections for each scan), the second method performs multiple scans for each subrange.

[0071] The fragment ions and precursor ions are analyzed sequentially in time within the same mass analyzer without changing the quadrupole mass filter settings.

[0072] The precursor information for each MS2 scan is for a narrow m / z subrange (thus containing information equivalent to a so-called "selected ion monitoring" (SIM) scan).

[0073] As with the first method described above, precursor information from all of the subrange scans acquired in the second method can be combined to effectively create a complete (at least in DIA experiments where the list of m / z targets spans a wide m / z range of interest) or otherwise partially complete MS1 spectrum. Thus, the method may perform SIM and MS2 scans in parallel to improve quantification of accumulated ions.

[0074] As with the first method, MS1 spectra can be constructed from multiple m / z subranges. Each m / z subrange can be acquired using a tailored fill time, thereby improving the dynamic range of the overall MS1 spectrum. The overall time required to acquire MS1 data can be reduced by performing SIM scans in parallel with MS2 scans. In this way, the quadrupole mass filter sequence remains unchanged from the sequence it follows for the list of m / z targets for multiple MS2 scans. This method is therefore compatible with high-speed LC-MS experiments.

[0075] The second method involves separate steps of analyzing a sample of precursor ions and separate steps of analyzing a sample of fragment ions, in other words, precursor and fragment information is acquired in separate scans.

[0076] The mass analyzer used to perform the scan may be a time-of-flight, ToF, analyzer, such as a multi-reflecting time-of-flight, MR-ToF, analyzer.

[0077] Analyzing the sample of precursor ions can include passing the precursor ions multiple times through a ToF analyzer. SIM scans can be acquired in the ToF analyzer via a process known as "zoom mode," in which a mass subrange is passed multiple times through the ToF analyzer to produce higher resolution.

[0078] Alternatively, the mass analyzer may be a Fourier transform mass analyzer (eg, an Orbitrap™ mass analyzer).

[0079] Analyzing the sample of precursor ions may generate a time-varying transient signal, and the method may further include generating a mass spectrum from the time-varying transient signal using a phase-restricted spectral deconvolution method (ΦSDM), optionally limited to the precursor frequency range.

[0080] The method may further include storing a sample of precursor ions in an ion store. Storing the sample of precursor ions in the ion store may include controlling a fill time for the precursor ions based on the relative abundance of the precursor ion species within the corresponding subrange.

[0081] The method may further include storing a sample of the fragmented precursor ions in an ion store. The fragment ions and precursor ions may be stored sequentially in the same ion store.

[0082] A front-end storage device, e.g., an ion mobility separator such as a trapping ion mobility separator (as incorporated into the Bruker TIMS-ToF series of mass spectrometers), may be used (and would be advantageous for the methods described herein). Such a device can release ions within an m / z range synchronized to the quadrupole isolation window. As a result, ion transmission is significantly enhanced. A further consequence is that the required injection time can be reduced (because the isolated ion beam is brighter). This reduction in injection time can be used to offset the additional time overhead of SIM injection.

[0083] The method may further include configuring the ion mobility separator to transmit precursor ions having m / z values ​​within the sub-range to the ion filter.

[0084] The method may further include controlling the ion mobility separator such that precursor ions transmitted to the ion filter correspond to transmission windows of the ion filter for each of a plurality of subranges within the overall m / z range.

[0085] Fragmented precursor ions can be formed from fragmentation of precursor ions having m / z values ​​within a sub-range at a number of different collision energies.

[0086] There may be a separate accumulation step for each collision energy.

[0087] The method includes, for each of a plurality of different collision energies: fragmenting the precursor ions at each collision energy to generate a sample portion of fragmented precursor ions; accumulating a portion of the sample of fragmented precursor ions in an ion store.

[0088] In other words, the method may include analyzing (in a mass analyzer) a sample of fragment ions produced by fragmenting precursor ions received from an ion filter configured at a plurality of different collision energies, wherein the fragments produced at the different collision energies may be stored together in an ion store.

[0089] The subranges may be contiguous. The subranges may be combinable to create an overall m / z range.

[0090] The method may further include acquiring scan data for the precursor ions for each subrange. The method may further include combining the scan data for the precursor ions for each subrange to form a high-resolution scan for the entire m / z range.

[0091] Combining SIM scan data (for precursor ions of each subrange) can be particularly useful when the subranges are contiguous and / or completely cover the entire m / z range, in which case the combined SIM scan data can be used to form a high-resolution scan for the entire m / z range.

[0092] The overall m / z range can include a second plurality of sub-ranges that do not overlap with the plurality of sub-ranges. The method can further include, for each of the second plurality of sub-ranges, analyzing in the mass analyzer a sample of fragmented precursor ions formed from fragmentation of precursor ions having m / z values ​​within the sub-range.

[0093] In other words, SIM scans may not be required for some of the subranges, and MS2-only scans may be performed for these subranges, which may be subranges of the overall m / z range that includes the high-intensity precursor peak in the MS1 scan.

[0094] In some variations of the method, it may be preferable not to perform SIM scans for m / z subranges of the overall precursor mass range that are already abundant. A full MS scan can be used to determine which m / z subranges of the overall precursor mass range are already abundant. A full MS scan should be able to collect sufficient data for such regions by itself. Omitting SIM scans for these subranges can further reduce the time required for the overall method. HDR MS1 scans can be obtained by combining a full MS1 scan with a SIM scan.

[0095] Each of the plurality of subranges may have the same width. The width may be set before scanning. The method may be a DIA method.

[0096] The method may further include analyzing the sample of precursor ions having m / z values ​​from the global m / z range (ie, not mass filtered) in a mass analyzer.

[0097] Analyzing a sample of precursor ions having m / z values ​​from the entire m / z range can include acquiring scan data for the entire m / z range.

[0098] The method may further include acquiring scan data for precursor ions for each subrange, and expanding the scan data for the entire m / z range using the scan data for precursor ions for each subrange to form a high-resolution scan for the entire m / z range.

[0099] Extending the scan data for the entire m / z range can be useful when the subranges are not contiguous and / or do not completely cover the entire m / z range. By extending a specific region of the MS1 scan with SIM data, an HDR scan can be obtained.

[0100] The method may further include acquiring scan data for precursor ions for each subrange, comparing the scan data for the overall m / z range with the scan data for precursor ions for each subrange, and adjusting one of the scan data for the overall m / z range or the scan data for precursor ions for each subrange based on the other of the scan data for the overall m / z range or the scan data for precursor ions for each subrange.

[0101] In other words, the SIM data is compared to the full scan data, and one of the data sets is corrected based on the other. The SIM scanned precursor ions may be compared to the full MS and used as an internal calibration, which is particularly useful in hybrid instruments where the SIM scan is performed by the jitter-prone MR-ToF analyzer and the full MS is performed by the more stable Orbitrap analyzer.

[0102] The method may further include determining a plurality of subranges from the overall m / z range based on scan data acquired from analyzing a sample of precursor ions having m / z values ​​from the overall m / z range. Where the subranges are based on MS1 ​​data, the method may be a DDA method.

[0103] The method may further include adjusting the collision energy of a fragmentation chamber used to fragment the precursor ions. Precursor ions from the ion filter may pass through the fragmentation chamber before being stored in the ion store. The collision energy of the fragmentation chamber may be adjusted between storing a sample of precursor ions in the ion store and storing a sample of fragmented ions in the ion store. Before storing a sample of precursor ions in the ion store, precursor ions from the ion filter may pass through the fragmentation chamber at a collision energy setting below a threshold or at a zero collision energy setting.

[0104] Alternatively, precursor ions may be passed from the ion filter to the ion store, bypassing the fragmentation chamber, before storing a sample of precursor ions in the ion store.

[0105] The method may be performed within a period of time based on the width of the chromatographic peak of the sample as it elutes from the chromatographic system.

[0106] The method may further include ionizing the sample to produce precursor ions.

[0107] The method may further include fragmenting the precursor ions to produce fragmented precursor ions.

[0108] Precursor ions may be fragmented in a collision cell, such as an ion routing multipole, IRM, collision cell, etc.

[0109] The method may further include performing quantification of the precursor ions based on scan data for the precursor ions. The quantification may be performed using data for the MS1 domain.

[0110] A third method of mass spectrometry is provided, the method comprising: analyzing in a mass analyzer a sample of precursor ions having m / z values ​​from an overall m / z range, wherein analyzing a sample of precursor ions having m / z values ​​from an overall m / z range comprises acquiring scan data for the overall m / z range; identifying one or more precursor ion species for further analysis based on the scan data; For each of the one or more identified precursor ion species, storing a sample of fragmented precursor ions in an ion store, the sample of fragmented precursor ions comprising ions formed from fragmentation of precursor ions of the identified precursor ion species; identifying a precursor ion species of interest and storing a sample of precursor ions of the precursor ion species in an ion store; and analyzing the combined sample of precursor ions and fragmented precursor ions simultaneously in a mass analyzer.

[0111] A sample of fragmented precursor ions and a sample of precursor ions may be combined in an ion store.

[0112] The method may further include identifying a first m / z sub-range for each of the one or more precursor ion species, the m / z values ​​of the precursor ion species being within the first sub-range.

[0113] The sample of fragmented precursor ions may include ions formed from fragmentation of precursor ions having m / z values ​​within the first sub-range, and may include precursor ions of the identified precursor ion species.

[0114] Identifying the associated precursor ion species may include identifying a second m / z sub-range, wherein the m / z values ​​of the associated precursor ion species are within the second sub-range.

[0115] Storing the sample of fragmented precursor ions in the ion store may include configuring an ion filter to transmit precursor ions having m / z values ​​within a first sub-range, the sample of fragmented precursor ions including ions formed from fragmentation of precursor ions received from the configured ion filter.

[0116] Accumulating the sample of precursor ions in the ion store may include configuring an ion filter to transmit precursor ions having m / z values ​​within a second sub-range, the sample of precursor ions including precursor ions received from the configured ion filter.

[0117] Configuring the ion filter may include setting a transmission window of the ion filter, the transmission window being adjusted between each accumulation.

[0118] Storing the sample of precursor ions in the ion store can include configuring the ion mobility separator to transfer precursor ions having m / z values ​​within a second sub-range to the ion filter.

[0119] Storing the sample of fragmented precursor ions in the ion store may include configuring the ion mobility separator to transfer precursor ions having m / z values ​​within a first sub-range to the ion filter.

[0120] The ion mobility separator may be controlled so that the precursor ions transmitted to the ion filter correspond to a transmission window of the ion filter.

[0121] For each of the one or more identified precursor ion species, the transmission window for accumulating a sample of precursor ions may be different from the transmission window for accumulating a sample of fragmented precursor ions.

[0122] The first subrange and the second subrange may not overlap.

[0123] Advantageously, if the transmission windows (also called "isolation windows") do not overlap, the peak for the relevant precursor ion is separate from the peak for the remaining unfragmented precursor ion in the injection of the fragmented precursor ion.

[0124] The associated precursor ion species may be an isotope of the identified precursor ion species.

[0125] The associated precursor ion species may be alternative charge states of the identified precursor ion species.

[0126] Analyzing the combined sample of precursor ions and fragmented precursor ions in a mass analyzer can include obtaining SIM / fragment scan data from simultaneous analysis of the combined sample.

[0127] The method may further include estimating the amount of unfragmented precursor ions accumulated in the ion store during accumulation of the fragmented precursor ions by comparing the intensity of peaks in the SIM / fragment scan data corresponding to the unfragmented precursor ions with the intensity of peaks in the SIM / fragment scan data corresponding to a sample of precursor ions (related precursors).

[0128] Estimating the amount of unfragmented precursor ions is software configured to deconvolute a plurality of fragment spectra from the SIM / fragment scan data, the plurality of fragment spectra being selected from a database of fragment spectra; a neural network configured to deconvolute a plurality of fragment spectra from the SIM / fragment scan data, the plurality of fragment spectra corresponding to a plurality of precursor ion species; and a neural network configured to predict the fragment spectrum of the precursor ion, including the intensities of the m / z peaks.

[0129] Accumulating a sample of precursor ions may include controlling the fill time of the precursor ions based on the relative abundance of associated precursor ion species.

[0130] The method may further include adjusting the collision energy used for fragmenting the precursor ions between the steps of accumulating a sample of precursor ions and accumulating a sample of fragmented precursor ions.

[0131] The step of accumulating a sample of fragmented precursor ions may be performed before the step of accumulating a sample of precursor ions. The method may further include cooling the sample of fragmented precursor ions.

[0132] Fragmented precursor ions may be formed from fragmentation of precursor ions having m / z values ​​within a first sub-range at a plurality of different collision energies.

[0133] The method may further include identifying the precursor ion based on scan data for the fragmented precursor ion, wherein the identification may be performed using data associated with the MS2 domain.

[0134] A mass spectrometer configured to perform any of the above-described methods is also provided.

[0135] Computer software is also provided, which comprises instructions that, when executed on a processor of a computer, cause the computer to perform any of the methods described above.

[0136] The invention may be carried out in a number of ways and specific embodiments will now be described, by way of example only, and with reference to the following drawings: [Brief explanation of the drawings]

[0137] [Figure 1]1 shows a schematic diagram of a mass spectrometer suitable for carrying out methods according to embodiments of the present invention; [Figure 2] An illustrative combined SIM / MS2 spectrum is shown showing both the intense precursor ion population and the fragment distribution. [Figure 3] 1 shows a first exemplary DIA scan sequence that mixes SIM injections into all MS2 scans. [Figure 4] 1 illustrates an exemplary timing diagram for implementing an exemplary scanning sequence. [Figure 5] 1 illustrates a second exemplary DIA scan sequence in which each SIM and MS2 injection has its own analytical scan. [Figure 6] 1 illustrates an exemplary DIA scan sequence incorporating SIM injection and graded collision energy injection into each MS2 scan. [Figure 7] 1 illustrates an exemplary DDA scan sequence that incorporates SIM injections of relevant precursors into an MS2 scan. DETAILED DESCRIPTION OF THE INVENTION

[0138] 1 shows a schematic diagram of a mass spectrometer 10 suitable for carrying out methods according to embodiments of the present invention. The mass spectrometer 10 may be a Hybrid Orbitrap multi-reflecting time-of-flight mass spectrometer (MR-ToF) as described in U.S. Patent No. 10,699,888, which is incorporated by reference. Details of the mass analyzer are described in U.S. Patent No. 9,136,101, which is incorporated by reference herein.

[0139] In Figure 1, a sample to be analyzed is delivered (e.g., from an autosampler) to a chromatography device (not shown in Figure 1), such as a liquid chromatography (LC) column. One such example of an LC column is the Thermo Fisher Scientific, Inc. ProSwift monolithic column, which provides high-performance liquid chromatography (HPLC) by forcing the sample through a stationary phase of irregular or spherical particles under high pressure in a mobile phase. In an HPLC column, sample molecules elute at different rates depending on the extent of their interaction with the stationary phase.

[0140] A detector (e.g., a mass spectrometer) can be used to measure the amount of sample molecules eluting from the HPLC column over time to generate a chromatogram. Sample molecules eluting from the HPLC column are detected as peaks above the baseline measurement of the chromatogram. If different sample molecules have different elution rates, multiple peaks on the chromatogram may be detected. Preferably, individual sample peaks are separated in time from other peaks in the chromatogram so that different sample molecules do not interfere with each other.

[0141] In chromatography, the presence of a chromatographic peak corresponds to the time period during which the sample molecules are present in the detector. Therefore, the width of a chromatographic peak corresponds to the time period during which the sample molecules are present in the detector. Preferably, the chromatographic peak has a Gaussian-shaped profile or can be assumed to have a Gaussian-shaped profile. Therefore, the width of a chromatographic peak can be determined based on several standard deviations calculated from the peak. For example, the peak width can be calculated based on four standard deviations of the chromatographic peak. Alternatively, the peak width can be calculated based on the width at half the maximum height of the peak. Other methods for determining peak width known in the art may also be suitable.

[0142] The sample molecules thus separated by liquid chromatography are then ionized using an electrospray ionization source (ESI source) 20 at atmospheric pressure.

[0143] The sample ions then enter the vacuum chamber of the mass spectrometer 10 and are guided by a capillary 25 into an RF-only S-lens 30 (also called an ion funnel). The S-lens 30 focuses the ions into an injection flatapole 40 (also called a quadrupole prefilter), which injects the ions into a bent flatapole 50 with an axial field. The bent flatapole 50 guides (charged) ions along a curved path through it, while unwanted neutral molecules, such as entrained solvent molecules, are not guided along the curved path and are lost. The curved path may be, for example, a 90-degree bend or an S-shaped meander.

[0144] The TK lens 60 was located at the distal end of the bent flatapole 50. Ions pass from the bent flatapole 50 to a downstream mass selector in the form of a quadrupole mass filter 70. The TK lens acts as a fringe field corrector for the quadrupole mass filter 70. The quadrupole mass filter 70 is typically, but not necessarily, segmented and functions as a bandpass filter, allowing passage of selected mass numbers or a limited mass range while rejecting ions of other mass-to-charge ratios (m / z). The mass filter can also be operated in an RF-only mode, which is not mass-selective, i.e., transmits substantially all m / z ions. For example, the quadrupole mass filter 70 may be controlled by a controller to select a range of mass-to-charge ratios for the passage of precursor ions, while other ions in the precursor ion stream are filtered (attenuated). Alternatively, the S-lens 30 may operate as an ion gate, and the ion gate (TK lens) 60 may be an electrostatic lens.

[0145] Although a quadrupole mass filter is shown in Figure 1, those skilled in the art will understand that other types of mass selection devices may also be suitable for selecting precursor ions within a mass range of interest, such as an ion separator as described in U.S. Patent Application Publication No. 2015287585(A), an ion trap as described in WO 2013076307(A), an ion mobility separator as described in U.S. Patent Application Publication No. 2012256083(A), an ion gate mass selection device as described in WO 2012175517(A), or a charged particle trap as described in U.S. Patent No. 799223, which are incorporated herein by reference. Those skilled in the art will understand that other methods of selecting precursor ions according to ion mobility, differential mobility, and / or transverse modulation may also be suitable.

[0146] Isolation of multiple ions of different masses or mass ranges may also be performed using a method known as Synchronous Precursor Scanning (SPS) in an ion trap. Furthermore, in some embodiments, more than one ion or mass selection device may be provided. For example, a further mass selection device may be provided downstream of the fragmentation chamber 120. In this way, multiple ions of different masses or mass ranges may be selected, if desired. 3 Scanning or MS n Scanning can be performed (typically using a ToF mass analyzer for mass analysis).

[0147] The ions then pass, optionally via a charge detector (not shown), through a quadrupole exit lens / split lens arrangement 80, which acts as an ion gate to control the passage of ions into a first transfer multipole 90. The first transfer multipole 90 directs mass-filtered ions from the quadrupole mass filter 70 into a curved linear ion trap (C-trap) 100. The C-trap (first ion trap) 100 has longitudinally extending curved electrodes supplied with RF voltage and end caps supplied with DC voltage. This results in a potential well extending along the curved longitudinal axis of the C-trap 100. In a first mode of operation, a DC end cap voltage is set on the C-trap so that ions arriving from the first transfer multipole 90 are trapped in the potential well of the C-trap 100 and cooled there. The injection time (IT) of ions into the C-trap determines the number of ions (ion population) subsequently ejected from the C-trap into the mass analyzer.

[0148] The cooled ions accumulate in a cloud toward the bottom of the potential well and are then ejected orthogonally from the C-trap toward the first mass analyzer 110. As shown in FIG. 1 , the first mass analyzer is an orbital trap mass analyzer 110, such as an Orbitrap® mass analyzer sold by Thermo Fisher Scientific. The orbital trap mass analyzer 110 has an off-center injection aperture through which ions are injected as coherent packets into the orbital trap mass analyzer 110. The ions are then trapped within the orbital trap mass analyzer by a hyperlogarithmic electric field and undergo a longitudinal back-and-forth motion as they orbit around the inner electrode.

[0149] The axial (z) component of motion of an ion packet in an orbital trap mass analyzer is defined as (more or less) simple harmonic motion, with the angular frequency in the z direction related to the square root of the mass-to-charge ratio of a given ion species. Thus, ions separate over time according to their mass-to-charge ratio.

[0150] Ions in an orbital trap mass analyzer are detected using an imaging current detector (not shown), which produces a "transient" in the time domain containing information about all ion species as they pass through the imaging current detector. The transient is then subjected to a Fast Fourier Transform (FFT), which produces a series of peaks in the frequency domain. From these peaks, a mass spectrum can be produced that represents the presence / intensity of ions versus m / z.

[0151] In the above configuration, sample ions (more specifically, a mass range segment of sample ions within a mass range of interest selected by quadrupole mass filter 70) are analyzed without fragmentation by orbital trap mass analyzer 110. The resulting mass spectrum is denoted MS1.

[0152] Although an orbital trap mass analyzer 110 is shown in Figure 1, other mass analyzers, including other Fourier transform mass analyzers, may be used instead. For example, a Fourier transform ion cyclotron resonance (FTICR) mass analyzer may be utilized as the mass analyzer for MS1 scanning. Mass analyzers such as orbital trap mass analyzers and ion cyclotron resonance mass analyzers may also be used in the present invention, even if other types of signal processing besides Fourier transform are used to obtain mass spectral information from the transient signals (see, e.g., WO 2013 / 171313, Thermo Fisher Scientific).

[0153] In a second operating mode of the C-trap 100, ions that enter the C-trap 100 through the quadrupole exit lens arrangement / split lens arrangement 80 and the first transfer multipole 90 may continue their path through the C-trap and into the fragmentation chamber 120, which may be an “Ion Routing Multipole” (IRM) collision cell. Thus, in the second operating mode, the C-trap effectively operates as an ion guide. Alternatively, cooled ions in the C-trap 100 may be axially ejected from the C-trap into the fragmentation chamber 120. The fragmentation chamber 120 is a high-energy collisional dissociation (HCD) device supplied with collision gas in the mass spectrometer 10 of FIG. 1 . Precursor ions arriving at the fragmentation chamber 120 collide with collision gas molecules, resulting in the precursor ions being fragmented into fragment ions.

[0154] 1, other fragmentation devices using methods such as collision induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), and photodissociation can be used instead. Additionally, ion fragmentation can be performed in the high pressure region of the extraction trap 140.

[0155] The fragmented ions may be ejected from the fragmentation chamber 120 at the opposite axial end to the C-trap 100. The ejected fragmented ions pass into a second transfer multipole 130, which directs the fragmented ions from the fragmentation chamber 120 into an extraction trap (second ion trap) 140. The extraction trap 140 is a radio frequency voltage controlled trap containing a buffer gas. For example, a suitable buffer gas is 5×10-4 mBar~1×10 -2 The second ion trap is typically argon at a pressure in the mBar range. The extraction trap has the ability to quickly switch off the applied RF voltage and apply a DC voltage to extract the trapped ions. A suitable planar extraction trap, also referred to as a linear ion trap, is further described in U.S. Pat. No. 9,548,195, which is incorporated herein by reference. Alternatively, a C trap may also be suitable for use as the second ion trap.

[0156] An extraction trap 140 is provided to form ion packets of fragmented ions prior to injection into the time-of-flight mass analyzer 150. The extraction trap 140 accumulates the fragmented ions prior to injection into the time-of-flight mass analyzer 150.

[0157] Although an extraction trap (ion trap) is shown in the embodiment of Figure 1, those skilled in the art will appreciate that other methods of forming ion packets of fragmented ions are equally suitable for the present invention. For example, relatively slow movement of ions through a multipole can be used to affect bunching of ions, which can then be emitted as a single packet into a ToF mass analyzer. Alternatively, orthogonal displacement of ions may be used to form packets. Further details of these alternatives are provided in U.S. Patent Application Publication No. 2003 / 0001088, which describes a traveling wave ion bunching method, and is incorporated herein by reference.

[0158] In FIG. 1, the time-of-flight mass analyzer 150 shown is a multi-reflecting time-of-flight mass analyzer (MR-ToF) 150. The MR-ToF 150 is built around two opposing ion mirrors 160, 162 that are elongated in the drift direction. The mirrors face each other in a direction perpendicular to the drift direction. An extraction trap 140 injects ions into the first mirror 160, which then oscillates between the two mirrors 160, 162. The angle of ion ejection from the extraction trap 140 and additional deflectors 170, 172 allows for control of the ion's energy in the drift direction, so that as the ions oscillate, they are directed down the length of the mirrors 160, 162, creating a zigzag trajectory. The mirrors 160, 162 themselves are tilted relative to each other, creating a potential gradient that slows the ion's drift velocity and reflects the ions in the drift dimension, focusing them onto a detector 180. Tilting the opposing mirrors typically has the negative side effect of changing the period of ion oscillation as it travels through the drift dimension. This is compensated for by stripe electrodes 190 (acting as compensation electrodes) that vary the length of the opposing mirrors 160, 162 downward to alter the flight potential over a portion of the inter-mirror space. The combination of the varying width of the stripe electrodes 190 and the varying distance between the mirrors 160, 162 allows for maintaining good temporal focusing as well as reflection and spatial focusing of ions onto the detector 180. MR-ToF 150s suitable for use in the present invention are further described in U.S. Patent Application Publication No. 2015028197(A1), which is incorporated herein by reference.

[0159] In one example, an MS1 ​​scan may be performed by a first mass analyzer (e.g., the orbital trap mass analyzer 110). In a second example, precursor ions may be fragmented and an MS2 scan performed by either the first mass analyzer (the orbital trap mass analyzer 110) or the second mass analyzer (the time-of-flight mass analyzer), depending on whether the fragmentation chamber is controlled to eject ions backward toward the C-trap 100 or forward toward the second transfer multipole 130. In a further mode of operation, the second mass analyzer (the time-of-flight mass analyzer 150) may perform an MS1 ​​scan of ions. In this mode of operation, ions are directed axially through the C-trap 100 into the fragmentation chamber, but no fragmentation gas is input, and the ions are guided to the second transfer multipole 130 without fragmentation. The ions may then be accumulated into packets in the extraction trap 140, as described above.

[0160] Ions accumulated in the extraction trap are injected into the MR-ToF analyzer 150 as ion packets once a predetermined number of ions have accumulated in the extraction trap. By ensuring that each packet of ions injected into the MR-ToF 150 has at least a predetermined (minimum) number of ions, the resulting packets of ions reaching the detector will represent the entire mass range of interest for the MS1 or MS2 spectrum. A single packet of precursor ions or fragmented ions is sufficient to acquire an MS1 ​​or MS2 spectrum for each ion. For MS2, this represents increased sensitivity compared to traditional acquisition of time-of-flight spectra, in which multiple spectra are typically acquired and summed for each given mass range segment. Preferably, a minimum total ion current (TIC) within each mass window is accumulated in the extraction trap before release into the time-of-flight mass analyzer. In some examples, at least N spectra (scans) per second in the MS2 domain are acquired by the time-of-flight mass analyzer, with N=50, or more preferably 100, 200, or more.

[0161] Preferably, at least X% of the MS2 scans contain more than Y ions (X=30, or 50, or 70, or most preferably 90 or more, and Y=200, or 500, or 1000, or 2000, or 3000, or 5000 or more). Most preferably, at least 90% of the MS2 scans contain more than 500 ions, or more preferably more than 1000 ions, and ideally more than 5000 ions. This provides an increased dynamic range of the MS2 spectra. The desired number of ions for each MS2 scan may be provided by adjusting the number of ions contained in each packet of fragmented ions. For example, in the embodiment of FIG. 1, the accumulation time of the extraction trap may be adjusted to ensure that a sufficient number of ions have accumulated. Thus, the controller may be configured to determine that a suitable packet of fragmented ions has been formed when either a predetermined number of ions are present in the extraction trap or a predetermined period of time has elapsed. The predetermined period may be specified to ensure that the time-of-flight mass analyzer operates at a desired frequency when the current of ions into the extraction trap is relatively low.

[0162] The mass spectrometer 10 is under the control of a controller configured to, for example, control the timing of release of trapped components, set appropriate potentials on electrodes such as the quadrupole to focus and filter ions, acquire mass spectral data from the orbital trap device 110, control the sequence of MS1 and MS2 scans, etc. to acquire mass spectral data from the MR-ToF 150. It will be appreciated that the controller may comprise a computer operable according to a computer program containing instructions to cause the mass spectrometer to perform steps of a method according to the present invention.

[0163] It should be understood that the particular arrangement of components shown in Figure 1 is not required for the methods described below. Indeed, other configurations for carrying out the methods of embodiments of the present invention are suitable. In some examples, all scans (MS1, MS2, and / or SIM) are performed by an MR-ToF analyzer, which is faster than the orbital trap analyzer.

[0164] A front-end storage device, such as an ion mobility separator (e.g., a trapping ion mobility separator, TIMS), can be configured to release ions within the m / z range corresponding to the quadrupole isolation window. This results in improved ion transmission through the quadrupole filter. A further consequence of the ion mobility separator is that the required injection time can be reduced (due to the brighter isolated ion beam). This reduction in injection time can be used to at least partially offset the additional time overhead of SIM injection.

[0165] The ion mobility separator may comprise a stacked ring ion guide, which applies a DC gradient to push ions in one direction against a gas wind in the opposite direction.

[0166] One example of an ion mobility separator uses electric field barriers within a gas flow to block ions according to their ion mobility. A decrease in the field barrier releases ions with increasing ion mobility.

[0167] TIMS are described in detail in U.S. Patent No. 7,838,826, U.S. Patent No. 9,891,194, and Meier et al., 2018, Molecular & Cellular Proteomics 17, 2534-2545, which are incorporated herein by reference.

[0168] The extended ion funnel consists of a number of segmented electrodes assembled around a common axis. The extended ion guide is divided into three sections: entrance focusing section, Mobility analysis section, and It can be treated as an exit focusing section.

[0169] In the focusing section, the distance between adjacent electrodes is approximately equal to the thickness of the electrode. The diameter of the opening in the electrode is a function of the electrode's position in the ion funnel assembly. For example, the segment electrode with the largest opening is at the entrance end of the ion funnel, and the segment electrode with the smallest opening is at the exit end of the ion funnel.

[0170] In some examples, the aperture diameter may be a linear function of the position of the segmented electrodes. In other examples, this function may be nonlinear. The angle formed between the common axis and the inner boundary of the ion funnel (i.e., formed by the inner rims of the segmented electrodes) may be approximately 19°. However, any angle between 0° and 90° may be used.

[0171] In the mobility analysis section of the ion funnel, the segmented electrodes may all have the same inner diameter. The space between adjacent electrodes may be filled with a dielectric or electrically resistive gasket. The thickness of the segmented electrodes must be less than their inner diameter, and the spacing between electrodes must be less than the thickness of the segmented electrodes to maintain a uniform RF field, so that the axial DC field is uniform near the axis.

[0172] The gaskets or O-rings between the electrodes form a substantially gas-tight seal, so that the openings in the electrodes form gas-tight channels through which gas can flow. The gas enters the channel in the inlet focusing section, forms a uniform laminar flow through the mobility analysis section, is constricted through the outlet focusing section, and then exits through the opening in the final electrode. The openings are substantially cylindrically symmetric to maintain a cylindrically symmetric flow profile. During operation, the symmetric laminar flow of gas means that all ions of a given type at a given position along the axis experience a given force due to the gas flow, substantially independent of their lateral position relative to the axis.

[0173] A quadrupole ion filter consists of four rods equally spaced at a predetermined radius around a central axis. A radio frequency (RF) potential (e.g., a 1 MHz sine wave) is applied between the rods. The potentials of adjacent rods are 180° out of phase. The rods on either side of the quadrupole axis are electrically connected so that the quadrupole is formed as two pairs of rods. Ions travel along the quadrupole axis and exit the quadrupole through an aperture. The RF potential applied between the rods tends to confine ions radially. When only RF is applied between the rods, virtually all ions are transmitted through the quadrupole. By applying DC and RF potentials between the rod pairs, only ions within a limited mass range are transmitted through the quadrupole. Ions outside this mass range are filtered out and do not reach the exit end.

[0174] The DC electric field strength varies as a function of position along the axis. However, at some position within the analytical section, the field strength reaches a maximum, forming a barrier that ions must overcome to reach the exit end of the funnel. Near this position of maximum field strength, this is the point where ions are selected based on their mobility, so DC field uniformity is important. Therefore, the DC field should be cylindrically symmetric.

[0175] An exemplary method of operation is: forming a DC barrier in the analysis section; applying an RF field to focus ions towards an axis; Producing ions in an ion source; introducing ions in a carrier gas into an extended ion funnel; introducing ions into the focusing section by applying a potential to electrodes and / or deflection electrodes of the focusing section; transferring ions to the analysis section by applying a DC potential to electrodes in the focusing section; Optionally, preventing additional ions from entering the analysis section by applying a DC potential to the deflection electrodes and / or electrodes of the focusing section; directing a carrier gas flow through the channel using a pump downstream from the outlet end of the funnel; gradually reducing the DC barrier in the analytical section to allow the carrier gas flow to push ions from the ion population over the DC barrier in order of ion mobility; and focusing the ions through an opening in the exit electrode.

[0176] Another example uses a DC gradient to push ions against a gas wind: increasing the DC potential releases ions in order of mobility.

[0177] In one exemplary method, a full mass scan is performed by the Orbitrap mass analyzer 110 with a long acquisition transient to produce a high-resolution MS1 ​​spectrum. In parallel, the MR-ToF 150 analyzer performs a series of MS2 acquisitions at very fast scan rates and high sensitivity.

[0178] An exemplary method for combined ion implantation and analysis is described in US Pat. No. 8,686,350, which is incorporated herein by reference.

[0179] The exemplary method described herein accumulates and combines different types of ions in the ion trap using two injections: the first injection of ions that are fragmented, and the second injection of ions that remain intact as precursor ions (the first and second ion injections can be performed in either order). The ions may come from the same ion source and have the same quadrupole isolation window, but may have different fragmentation energies (the precursor injection has a collision energy of zero). The combined ions are then analyzed in a mass analyzer to provide an analytical scan, as depicted in FIG. 2. Such a scan provides precursor information (illustrated as SIM injections in FIG. 2) in addition to fragment spectra (illustrated as MS2 injections / sec in FIG. 2). Spectra can be recorded quickly because there is no additional quadrupole switching time and the additional injection time of the precursor ion (SIM) component should be shorter than for fragmentation injections. Depending on whether fragmentation or SIM injections are performed first, some delay is required to cool the fragments or switch to a higher collision energy.

[0180] In some embodiments of the DIA method according to the present invention, for each injection performed with fragmentation, there is an additional injection with the same quadrupole isolation window but with reduced or no collision energy. The target mass is scanned through a predetermined range and isolation step size, as is typical for DIA. Figure 3 shows such a scan sequence from m / z 300 to 900 Th with a 5 Th isolation window, as might be used for "bottom-up" measurements of a digested protein sample. An optional full MS1 scan is also performed cycle-by-cycle through the entire mass range. In the hybrid Orbitrap / MR-ToF instrument of Figure 1, this MS1 scan is performed by the Orbitrap™ mass analyzer, and the SIM / MS2 scan is performed by the MR-ToF mass analyzer. Overlapping all SIM injection spectra creates an extremely HDR MS1 scan. As will be appreciated by those skilled in the art, actual overlapping of the spectra is not necessary. For data processing, it is sufficient that data from all SIM injection spectra are available.

[0181] The method of Figure 3 can be performed on a combined Orbitrap mass analyzer and MR-ToF instrument as illustrated in Figure 1. Alternatively, the method can be performed (which may take longer) on a single mass analyzer instrument, such as a dedicated ToF instrument or a dedicated Orbitrap mass analyzer instrument.

[0182] Figure 4 illustrates an exemplary timing diagram for implementing the method illustrated in Figure 3 on a combined Orbitrap mass analyzer and MR-ToF instrument such as that illustrated in Figure 1. As can be seen in Figure 4, the timing of different operations may be configured such that certain operations are performed in parallel to reduce overall turnaround time. The Orbitrap / MR-ToF instrument illustrated in Figure 1 may be capable of performing a single injection at approximately 200 Hz, with a 3 ms injection time (also referred to as "fill time") and 2 ms overhead. The additional overhead depletes the duty cycle and reduces instrument sensitivity.

[0183] Schematic timing for the steps of ion injection and transport through the components of a mass spectrometer is illustrated in Figure 4. Ions are transported from the ion source to the analyzer. The vertical displacement in Figure 4 provides an indication of parallel processing, as multiple separate ion packets can be processed simultaneously in different components.

[0184] As illustrated in FIG. 4 , additional SIM / MS2 injections can be alternated depending on whether fragmentation is performed in the high-pressure region of the IRM 120 or the extraction trap 140. Additional injections (also referred to as “filling”) may be performed into the ion store (also referred to as the “extraction trap”) in a manner that minimizes additional overhead. The additional injections may involve precursor ions from an ion filter with the same m / z characteristics. During operation according to some exemplary methods, the collision energy of the fragmentation chamber may be adjusted. The fragmentation energy may be adjusted from a preset MS2 level used to generate a sample of fragmented precursor ions to a reduced level, such as zero, so that the precursor ions pass through the fragmentation chamber unfragmented.

[0185] Ions from different injections should not be mixed before the fragmentation step. If fragmentation is performed in the IRM collision cell 120, the second injection follows the first after a short delay required to change the IRM offset and cool the ions from the first injection. Both packets of ions can then be mixed and transported to the extraction trap 100, 140. However, if fragmentation is performed by the high-pressure region of the extraction trap 140, the IRM collision cell 120 must clear the first ion packet before accepting the second ion packet. This effectively adds an extra transport step, slowing instrument operation. Nevertheless, because the second injection has the same m / z range as the first injection, there is less need to completely clear the ion region between injections. Therefore, the delay times for the transport and cooling steps can be shortened to compensate for some of the lost duty cycle (and reduce the required time).

[0186] It is desirable to account for any remaining unfragmented precursor from the MS2 injection. Otherwise, a fixed amount from the SIM injection will be added to the unknown amount of unfragmented precursor, which reduces the reliability of the SIM data for quantification purposes. For a single precursor species, this can be achieved by summing all ions in the spectrum. However, in the case of DIA and wide-window DDA, MS2 spectra are typically chimeric and contain more than one precursor ion. In this case, there are two possible methods for estimating the amount of unfragmented precursor from the MS2 injection, which are described in more detail below.

[0187] In the first method, prediction of the residual amount of unfragmented precursor from MS2 injection is based on the known fragmentation pattern of the identified precursor. Bioinformatics solutions such as the Chimerys™ AI-based search engine (MSAID GmbH, Germany) can predict both the fragmentation pattern and relative intensity of the fragments and the residual precursor. For example, if 3 ms is stored for MS2 and 1 ms for SIM, only a 1.6-fold correction is required. However, if the fraction of residual precursor is actually one-fourth (e.g., 15% instead of 20%) due to some experimental error, this would result in an overall error of less than 10% in this example (1.45-fold instead of 1.6-fold), i.e., still much better than without SIM.

[0188] In the second method, the quadrupole isolation window can be shifted to an adjacent mass range during scan storage. For example, MS2 can be performed in the mass range 300-305 Th, and SIM can be performed in the mass range 305-310 Th, and then acquired in addition to scan 2. However, complex interruptions of ion flow may be required to achieve this. Furthermore, there may be dead time during adjustment of the quadrupole isolation window. One advantage of this method is that the intensity of peaks in the SIM scan does not require correction due to the fact that the m / z window immediately above the precursor m / z value is usually completely empty of ions.

[0189] An alternative DIA method is illustrated in Figure 5. In this second method, instead of accumulating multiple injections per mass spectrum, each MS2 and SIM injection has its own analytical scan. This is particularly suitable when one or both of the scans are performed on an MR-ToF analyzer, which is significantly faster than an Orbitrap mass analyzer. The time overhead is also greater in an Orbitrap mass analyzer, and as a result, the MS2 and SIM scan generation frequency can be limited to approximately 50 Hz using Orbitrap instruments. Two scans (MS2 and SIM) can be summed to generate a spectrum similar to that generated by the method of Figure 3, but this is not necessary for the analysis.

[0190] One advantage of performing separate scans is that SIM scans can be recorded via "zoom mode," in which a narrow mass range is passed through the MR-ToF analyzer multiple times to generate higher resolution. This method is described in UK Patent Application No. 2300355.1, which is incorporated herein by reference. Another form of zoom mode, described in UK Patent Application No. 2208939.5 (incorporated herein by reference), involves selecting only a narrow mass range and applying zoom mode to it, while allowing the remaining ions to fly normally. In principle, this method is suitable for multiple injection spectra, where high-resolution precursor information is desired, but unambiguous m / z assignments and maximum sensitivity for fragments are also desired. With respect to Orbitrap mass analyzer scans, the closest comparable method to achieve higher resolution across a narrow mass band is to apply phi-SDM analysis of transients limited to the precursor frequency range (Bekker-Jenson et al., Mol. Cell. Proteomics, 2020, 14, 716-729).

[0191] Each mass spectrometry procedure may generate a time-varying transient signal. A mass spectrum may be generated from each time-varying transient signal by deconvolving the transient signal using a deconvolution technique. In certain embodiments, the deconvolution technique is a high-resolution deconvolution technique such as "phase-constrained spectral deconvolution method" (also known as ΦSDM), i.e., as described in Grinfeld, et al., "Phase-constrained spectrum deconvolution for Fourier transform mass spectrometry," Anal. Chem, 89(2):1202-1211 (2017), and also in European Patent Application No. 3,086,354, the entire contents of which are incorporated herein by reference.

[0192] As described in European Patent Application No. 3,086,354, in these embodiments, a Fourier transform of the transient signal is performed to generate a first set of complex amplitudes, each corresponding to a respective frequency of the first set of frequencies. The first set of frequencies may be equally spaced in frequency. A second set of complex amplitudes is created, each corresponding to a respective frequency of the second set of frequencies. The second set of frequencies may be equally spaced in frequency. The second set of frequencies may have a spacing (or minimum spacing) smaller than that of the first set of frequencies. The second set of frequencies may have a spacing (or minimum spacing) smaller than the reciprocal of the duration of the transient signal. The second set of complex amplitudes may cover (or span or correspond to) the same frequency range as the first set of complex amplitudes, and therefore the second set may include more complex amplitudes than the first set. Thus, the second set of complex amplitudes may provide greater resolution.

[0193] The second set of complex amplitudes may be optimized to produce an improved second set of complex amplitudes. At least some of the complex amplitudes from the improved second set may be used to produce a mass spectrum. The improved second set of complex amplitudes may provide a mass spectrum of better quality.

[0194] Optimizing the second set of complex amplitudes may include varying at least one of the complex amplitudes of the second set based on (or depending on) the objective function. For example, at least one complex amplitude may be varied with the aim of obtaining a substantially extremum of the objective function. Optionally, all of the complex amplitudes from the second set may be varied as part of the optimization step, or a subset may be optimized as part of the optimization step.

[0195] The optimization may be performed subject to constraints. That is, for at least some of the complex amplitudes in the second set, a constraint may be imposed on the phase of each of at least some of the complex amplitudes relative to one or more expected phases. The expected phases may be frequency-dependent. The objective function may depend on one or more of the complex amplitudes in the first set and one or more of the complex amplitudes in the second set. The objective function may relate, for each frequency in the first set of frequencies, one or more complex amplitudes in the second set to a respective complex amplitude from the first set (e.g., by making the objective function a function of one or more complex amplitudes in the second set and a respective complex amplitude from the first set). The constraints may apply to all complex amplitudes in the second set that are being varied as part of the optimization step, or to a subset of those complex amplitudes.

[0196] By creating and optimizing the second set of complex amplitudes, transient phenomena can be thought of as being resolved onto a finer frequency grid. Because the second set of complex amplitudes is not combined with the first set of complex amplitudes as a linear combination of these amplitudes, resolution increases as the grid spacing of the second set of frequencies decreases. This significantly improves the accuracy of the resulting mass spectrum. In other words, the ΦSDM method can be thought of as operating on two sets of frequencies. The first set of frequencies may include frequencies with a minimum separation of 1 / T, where T is the duration of the transient signal. The second set of frequencies may include frequencies with a minimum separation of less than 1 / T. The second set of frequencies may include the first set as a subset. Because the minimum spacing of the second set of frequencies is smaller than the minimum spacing of the first set, the second set of complex amplitudes may provide greater resolution.

[0197] It will be appreciated that "complex number" should be understood as relating to a number that can be expressed with a real part and an imaginary part, where the imaginary part may be zero (i.e., complex numbers as used herein cover real numbers).

[0198] One advantage of the ΦSDM method is the integrability of the mass spectra generated. In other words, the intensities of all peaks, both resolved and unresolved, are preserved. Thus, the suppression effect of conventional Fourier transform approaches caused by interference of adjacent peaks is avoided. The ΦSDM method is therefore particularly beneficial when highly accurate intensity information is desired. Furthermore, calculations can be performed for shorter transient events, increasing the speed and throughput of the instrument.

[0199] In some embodiments, performing a Fourier transform includes windowing the Fourier transformed transient signal in the frequency domain, with the first set of complex amplitudes corresponding to the windowed Fourier transformed transient signal. The windowing may include applying a windowing function to the first set of complex amplitudes. Typically, applying the windowing function includes scaling each complex amplitude of the first set of complex amplitudes by a value of the windowing function at the respective frequency. Additionally or alternatively, the windowing may include discarding complex amplitudes whose respective frequencies lie outside one or more predefined ranges. For example, complex amplitudes of the first set whose respective frequencies are above the Nyquist frequency of the transient signal may be discarded and / or set to zero.

[0200] Advantageously, this may allow for increased processing speed and reduced computational load, as subsequent processing may be limited to only regions of interest: for sufficiently sparse spectra or sufficiently sparse segments of interest, calculations may be performed only within windows of the spectrum that encapsulate these regions.

[0201] A further modification of the DIA process of Figure 3 is illustrated in Figure 6. This method is a variation of the first method, as SIM ions are mixed with fragment ions in the ion store and analyzed together in a single scan. In this method, in addition to the SIM injection, a graded fragmentation energy is also applied to each MS2 scan. In other words, MS2 ions are applied to the ion store in multiple injections, each injection having a different fragmentation energy.

[0202] In some variations of the method, it may be preferable not to perform SIM scans for m / z subranges of the overall precursor mass range that are already abundant. This can be determined from a full MS scan, which should be able to collect sufficient data for such regions on its own. Omitting SIM scans for these subranges can further reduce the time required for the overall method. HDR MS1 scans can be obtained by combining a full MS1 scan with a SIM scan.

[0203] The SIM scanned precursor ions may be compared to the full MS and used as an internal calibration, which is particularly useful in hybrid instruments where the SIM scan is performed by the jitter-prone MR-ToF analyzer and the full MS is performed by the more stable Orbitrap analyzer.

[0204] A further modification of the process of Figure 3 is illustrated in Figure 7. In this method, SIM scanned precursor ions are acquired from a specific isolation window and used as an internal calibration in the DDA method. This method is a variation of the first method, as the SIM ions are mixed with the fragment ions in the ion store and analyzed together in a single scan.

[0205] In the alternative method of Figure 7, fragments of the main precursor are stored in the ion reservoir and combined with a sample of precursor ions related to the main precursor. The related precursor ions are stored in a zero-collision energy injection. The presence of the related precursor ions in the scan data can be used to quantify isotopes of the main precursor or different charge states of the main precursor (as typically detected in a full MS scan). As a result, convolution between the quantified precursor ions (from a zero-collision energy injection) and overlapping unfragmented precursors from fragment injections (injections with non-zero collision energy) can be avoided. As can be seen in Figure 7, the SIM injection is separate from the unfragmented precursor.

[0206] The difference in isotope ratios can be calculated from theory, and the charge state still responds to the relative behavior.

[0207] In contrast to the previously described method, the transmission window may be adjusted between injections.

[0208] One reason for this is to exclude related precursors (e.g., isotopes or different charge state ions) from implants with non-zero collision energies, and another reason is to exclude the main precursor from implants with zero collision energy.

[0209] If the relevant precursor is an isotope of the main precursor, the transmission window can be shifted by a very short distance between injections. Advantageously, this can cause a negligible time delay.

[0210] The isolation window for the related precursor may be small to limit overlap between the two implants (especially if the window is close, such as when the related precursor is an isotope of the main precursor). In some instances, the related precursor ion species may be the only precursor ion species transmitted by the ion filter in a precursor ion implant.

[0211] If the relevant precursor is of a different charge than the main precursor, the transmission window can be shifted by a larger distance and the scan speed can be affected.

[0212] The isolation window for the relevant precursor can be selected based on the full MS scan data. Therefore, the proposed method can be a DDA method.

[0213] If the related precursor is an isotope of the main precursor, the isolation window may be precisely selected based on the scan data.

[0214] If the related precursors are different charge states of the main precursor, the scan data can be used to identify where to direct the SIM scan to acquire the different charge states and set the isolation window accordingly.

[0215] If the relevant precursor is of a different charge state than the main precursor, the higher charge state can be chosen to avoid contamination from the charge-removed precursor.

[0216] As used herein, the term mass may be used to refer to mass-to-charge ratio, m / z. The resolution of a mass analyzer should be understood to refer to the resolution of the mass analyzer as determined by the mass-to-charge ratio 200, unless otherwise specified.

[0217] In the description of the invention herein, unless otherwise understood or stated, implicitly or explicitly, it is understood that words appearing in the singular include their plural equivalents, and words appearing in the plural include their singular equivalents. Furthermore, unless otherwise understood or stated, implicitly or explicitly, it is understood that for any given component or embodiment described herein, any of the possible candidates or alternatives listed for that component may generally be used individually or in combination with each other. Furthermore, it should be understood that the figures illustrated herein are not necessarily drawn to scale, and that some of the elements may be drawn merely to clarify the invention. Also, reference numerals may be repeated among various figures to indicate corresponding or similar elements. Additionally, unless otherwise understood or stated, it will be understood that any listing of such candidates or alternatives is merely illustrative and not limiting.

[0218] Unless otherwise defined, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the case of conflict, the present specification, including definitions, shall control. It will be understood that there is an implicit "about" before quantitative terms referred to in this disclosure so that insignificant deviations are within the scope of the present teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" is not intended to be limiting. As used herein, "a" or "an" may refer to "at least one" or "one or more." Also, the use of "or" is inclusive, so that the phrase "A or B" is true when "A" is true, when "B" is true, or when both "A" and "B" are true.

[0219] As used herein, the term "scan," when used as a noun, refers to a mass spectrum, regardless of the type of mass analyzer used to create or acquire the mass spectrum. As used herein as a verb, the term "scan" refers to creating or acquiring a mass spectrum by a method of mass spectrometry, regardless of the type of mass analyzer or mass spectrometry used to create or acquire the mass spectrum. As used herein, the term "full scan" refers to a mass spectrum that encompasses a range of mass-to-charge (m / z) values ​​that includes multiple mass spectral peaks.

[0220] As used herein, the terms "liquid chromatography" and "liquid chromatography" (both abbreviated as "LC") and "liquid chromatography mass spectrometry" (abbreviated as "LC-MS") are each intended to apply to any type of liquid separation system capable of separating a liquid sample bearing multiple analytes into various "fractions" or "separates," where the chemical composition of each such "fraction" or "separate" differs from the chemical composition of other such fractions or separates, and the term "chemical composition" refers to the number, concentration, and / or identity of the various analytes in the fraction or separate. Thus, the terms "liquid chromatography," "liquid chromatography," "liquid chromatography mass spectrometry," "LC," and "LC-MS" are intended to include, but are not limited to, liquid chromatographs, high-performance liquid chromatographs, ultra-high-performance liquid chromatographs, size-exclusion chromatographs, and capillary electrophoresis devices.

[0221] Instead of an LC device, any other separation device can be interfaced to the mass spectrometer, including an ion mobility device, HPLC, GC, or ion chromatography, and any known fragmentation method (including collision-activated dissociation, photon-induced dissociation, electron capture, or electron transfer dissociation) will generate data suitable for use in the present invention.

Claims

1. 1. A method of mass spectrometry comprising: For each of a plurality of subranges selected from the overall m / z range, storing a sample of precursor ions to be analyzed in an ion store, the precursor ions having m / z values ​​within the subrange, and storing the sample of precursor ions includes controlling a fill time for the precursor ions based on the relative abundance of precursor ion species within a corresponding subrange; storing a sample of fragmented precursor ions to be analyzed in the ion store, the fragmented precursor ions being formed from fragmentation of precursor ions having m / z values ​​within the sub-range; and simultaneously analyzing the combined sample of the precursor ions and the fragmented precursor ions in a mass analyzer, said method comprising: acquiring scan data for the precursor ions for each subrange; combining the scan data for the precursor ions for each subrange.

2. The method of claim 1 , further comprising estimating the amount of unfragmented precursor ions stored in the ion store during accumulation of fragmented precursor ions.

3. The method further includes acquiring scan data from a simultaneous analysis of the combined sample, wherein estimating the amount of unfragmented precursor ions comprises: software configured to deconvolute a plurality of fragment spectra from the scan data, the plurality of fragment spectra being selected from a database of fragment spectra; a neural network configured to deconvolute a plurality of fragment spectra from the scan data, the plurality of fragment spectra corresponding to a plurality of precursor ion species; and and a neural network configured to predict a fragment spectrum of the precursor ion, including intensities of m / z peaks.

4. 2. The method of claim 1, further comprising configuring an ion filter for each of the plurality of subranges to transmit precursor ions having m / z values ​​within the subrange, wherein configuring the ion filter comprises setting a transmission window of the ion filter, the transmission window being adjusted within each of the plurality of subranges, and wherein, for each subrange, the transmission window for accumulating a sample of the precursor ions is the same as the transmission window for accumulating a sample of the fragmented precursor ions.

5. the plurality of subranges includes a first subrange and a second subrange, and analyzing a sample including the precursor ions and the fragmented precursor ions from the first subrange; accumulating a sample of said precursor ions having m / z values ​​within said second sub-range; and / or 2. The method of claim 1, wherein said step of at least partially overlaps with accumulating a sample of said fragmented precursor ions formed from fragmenting precursor ions having m / z values ​​within said second sub-range.

6. 10. The method of claim 1, further comprising adjusting the collision energy used for fragmenting the precursor ions between storing the sample of precursor ions and storing the sample of fragmented precursor ions.

7. 2. The method of claim 1 , wherein for each subrange, accumulating a sample of the fragmented precursor ions is performed before accumulating a sample of the precursor ions, and the method further comprises cooling the fragmented precursor ions.

8. 1. A method of mass spectrometry comprising: For each of a plurality of subranges selected from the overall m / z range, configuring an ion filter to transmit precursor ions having m / z values ​​within said subrange; storing a sample of the precursor ions received from the configured ion filter in an ion store for analysis in a mass analyzer, wherein storing the sample of the precursor ions in an ion store includes controlling a fill time for the precursor ions based on the relative abundance of precursor ion species within a corresponding subrange; and and analyzing in the mass analyzer a sample of fragment ions produced by fragmenting precursor ions received from the ion filter configured, the method comprising: acquiring scan data for the precursor ions for each subrange; combining the scan data for the precursor ions for each subrange.

9. 9. The method of claim 8, wherein configuring the ion filter includes setting a transmission window of the ion filter, the transmission window being adjusted within each of the plurality of subranges, and for each subrange, the transmission window for receiving the sample of precursor ions from the configured ion filter is the same as the transmission window for receiving precursor ions that are fragmented to produce the sample of fragment ions from the configured ion filter.

10. 9. The method of claim 1 or 8, wherein the mass analyzer is a multi-reflecting time-of-flight (MR-ToF) analyzer, and analyzing the sample of precursor ions comprises passing the precursor ions through the MR-ToF analyzer multiple times.

11. 9. The method of claim 4 or 8, further comprising configuring an ion mobility separator to transmit precursor ions having m / z values ​​within the sub-ranges to the ion filter, and further comprising controlling the ion mobility separator such that the precursor ions transmitted to the ion filter correspond to a transmission window of the ion filter for each of the plurality of sub-ranges within the overall m / z range.

12. 10. The method of claim 1 or 8, wherein the fragmented precursor ions are formed from fragmentation of precursor ions having m / z values ​​within the sub-range at a plurality of different collision energies.

13. the plurality of subranges are contiguous, and the method comprises: The method of claim 1 or 8, further comprising combining the scan data for the precursor ions for each subrange to form a high resolution scan for the entire m / z range.

14. the overall m / z range includes a second plurality of subranges that do not overlap with the plurality of subranges, and the method further comprises: For each of the second plurality of subranges:

10. The method of claim 1 or 8, further comprising analyzing in a mass analyzer a sample of fragmented precursor ions formed from fragmenting precursor ions having m / z values ​​within the sub-range.

15. analyzing in a mass analyzer a sample of precursor ions having m / z values ​​from the entire m / z range, wherein analyzing the sample of precursor ions having m / z values ​​from the entire m / z range comprises acquiring scan data for the entire m / z range; acquiring scan data for the precursor ions for each subrange; using the scan data for the precursor ions for each subrange to expand the scan data for the entire m / z range to form a high resolution scan for the entire m / z range; or 9. The method of claim 1, further comprising: comparing the scan data for the overall m / z range with the scan data for the precursor ions for each subrange; and adjusting one of the scan data for the overall m / z range or the scan data for the precursor ions for each subrange based on the other of the scan data for the overall m / z range or the scan data for the precursor ions for each subrange.

16. analyzing in a mass analyzer a sample of precursor ions having m / z values ​​from said global m / z range; 9. The method of claim 1 or 8, further comprising: determining the plurality of sub-ranges from the overall m / z range based on scan data obtained from an analysis of a sample of the precursor ions having m / z values ​​from the overall m / z range.

17. 1. A method of mass spectrometry comprising: analyzing in a mass analyzer a sample of precursor ions having m / z values ​​from an overall m / z range, wherein analyzing the sample of precursor ions having m / z values ​​from the overall m / z range comprises acquiring scan data for the overall m / z range; identifying one or more precursor ion species for further analysis based on the scan data; For each of the one or more identified precursor ion species, storing a sample of fragmented precursor ions in an ion store, the sample of fragmented precursor ions comprising ions formed from fragmentation of precursor ions of the identified precursor ion species; identifying a precursor ion species of interest and storing a sample of precursor ions of the precursor ion species of interest in the ion store; simultaneously analyzing the combined sample of said precursor ions and said fragmented precursor ions in a mass analyzer.

18. 20. The method of claim 1, 8, or 17, wherein the method is performed within a time period based on the width of a chromatographic peak of the sample as it elutes from a chromatographic system.

19. 20. A mass spectrometer configured to carry out the method of any one of claims 1, 8 or 17.

20. 20. Computer software comprising instructions which, when executed by a processor of a computer, cause the computer to perform the method of any one of claims 1, 8 or 17.

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