Systems and methods for simultaneous analysis of multiple charged particles using a charge detection mass spectrometer

Abstract

Systems and methods are provided for recovering charged particle measurement information during operation of a charge detection mass spectrometer, in which multiple ions are captured and simultaneously measured. The recovered charged particle measurement information exemplarily includes charged particle measurement information for at least some ions whose oscillation frequencies overlap with the oscillation frequencies of other ions among the multiple captured ions. One example charged particle recovery process operates on charged particle measurement information in which the overlapping oscillation frequencies are distinguishable from each other, while another example charged particle recovery process operates on charged particle measurement information in which the overlapping oscillation frequencies are indistinguishable from each other. The charge detection mass spectrometer can be operated using any one or both of the said charged particle recovery processes.

Description

[0001] Cross-reference to related applications This application claims priority to U.S. Provisional Application No. 63 / 605025, filed December 1, 2023, and U.S. Provisional Application No. 63 / 651607, filed May 24, 2024, pursuant to 35 USC §119(e), the disclosures of which are expressly incorporated herein by reference in their entirety.

[0002] Government rights This invention was made with government funding under GM131100 granted by the National Institutes of Health (NIH). The U.S. government holds certain rights to this invention. Technical Field

[0003] This disclosure generally relates to charge detection mass spectrometry (CDMS), and more specifically to systems and techniques for the simultaneous analysis of multiple charged particles. Background Technology

[0004] Charge detection mass spectrometry (CDMS) is a charged particle analysis technique in which the mass of each charged particle is determined by simultaneously measuring its mass-to-charge ratio (m / z) and charge (z). Charged particles are trapped in an electrostatic linear ion trap or orbital trap, and the charge induced on the charge detector by the charged particles is measured as they oscillate back and forth through or around the charge detector during the duration of the trapping event. Simultaneous trapping of multiple charged particles can significantly improve CDMS throughput, thereby reducing analysis time. However, due to errors in determining the charge and / or oscillation frequency caused by charged particles with overlapping oscillation frequencies, this method may result in more charged particle measurements being discarded from the analytical results compared to single-particle analysis techniques. Summary of the Invention

[0005] This disclosure may include one or more of the features recited in the appended claims, and / or one or more of the following features and combinations thereof. In one aspect, a method of operating a charge detection mass spectrometer comprising an electrostatic linear ion trap (ELIT) or an orbital trap may include: (i) trapping a plurality of ions generated from a sample in the ELIT or orbital trap such that, during an ion trapping event, the plurality of trapped ions oscillate back and forth through or around a charge detector of the ELIT or orbital trap; (ii) determining a set of oscillation frequency (OFR) and charge quantity (CM) pairs, each pair corresponding to a different ion among the plurality of trapped ions; and (iii) forming a filter file and a recovery file from the set of OFR and CM pairs, the filter file including the OFR and CM pairs from which the spectral distribution of the sample from which it is generated, and the recovery file including the oscillation frequency having the same oscillation frequency as other OFR and CM pairs. (iv) For at least one OFR and CM pair in the recovery file, (a) the charge of one of the OFR and CM pairs in the filter file is modified depending on the charge of one of the OFR and CM pairs in the filter file, wherein the oscillation frequency of one of the OFR and CM pairs in the ELIT or orbital trap is within the frequency window of the oscillation frequency of the OFR and CM pair in the ELIT or orbital trap, and (b) the filter file is updated by adding the OFR and the modified CM pair to the filter file, and (v) the spectral distribution is generated from the updated filter file of the OFR and CM pairs.

[0006] The second aspect includes the features of the first aspect and may further include: performing (i)-(iv) multiple times, and then performing (v) using the updated filter file containing OFR and CM pairs for all of the multiple executions of (i)-(iv).

[0007] The third aspect includes the features of the first or second aspect, and may further include: during the ion trapping event, collecting charge detection data derived from the detection of charges induced on the charge detector by the plurality of ions. Wherein, (ii) includes: analyzing the collected charge data to determine the set of oscillation frequency (OFR) and charge quantity (CM) pairs, and determining the charge quantity standard deviation for each of the OFR and CM pairs.

[0008] The fourth aspect includes the features of the third aspect, and wherein, (iv) further includes: requiring the OFR and CM pairs in the filter file to have a charge standard deviation less than a first threshold, and requiring the OFR and CM pairs in the recovery file to have a charge standard deviation greater than the first threshold.

[0009] The fifth aspect includes the features of the first or second aspect, and may further include: performing (iv) for each of the OFR and CM pairs in the recovery file.

[0010] The sixth aspect includes features of any one of the first to fifth aspects, and wherein, (iv) includes: collecting the charge amount of all OFR and CM pairs in the filter file whose oscillation frequency in the ELIT or orbital trap is within the frequency window of the oscillation frequency of the OFR and CM pairs in the recovery file in the ELIT or orbital trap, randomly selecting one of the collected charge amounts, and modifying the charge amount of the OFR and CM pairs in the recovery file according to the randomly selected charge amount.

[0011] The seventh aspect includes the features of the sixth aspect, and wherein modifying the charge quantity includes: modifying the charge quantity by adding a noise value to a charge quantity randomly selected from the collected charge quantities, and replacing the charge quantity of the OFR and CM pair in the recovery file with the modified charge quantity.

[0012] The eighth aspect includes the features of any one of the first to seventh aspects, and wherein, (ii) further includes: excluding from the set of OFR and CM pairs all OFR and CM value pairs of ions that were not captured in the ELIT or orbital trap during the entire duration of the ion capture event.

[0013] The ninth aspect includes the features of any one of the third to eighth aspects, and wherein, (ii) further includes: determining the oscillation frequency standard deviation for each of the OFR and CM pairs, and wherein, (iii) further includes: excluding from each of the filter file and the recovery file all OFR and CM pairs whose oscillation frequency standard deviation is greater than a second threshold.

[0014] The tenth aspect includes the features of the ninth aspect, and wherein, (ii) further includes: calculating a series of short-time overlap Fourier transforms (STFTs) sequentially through the collected charge detection data, and determining from the STFTs the set of OFR and CM pairs, and the standard deviation of the charge quantity and the standard deviation of the oscillation frequency for each of the OFR and CM pairs.

[0015] The eleventh aspect includes the features of the first aspect and may further include between (i) and (ii): during the ion capture event, collecting charge detection data derived from the detection of charges sensed on the charge detector by the plurality of ions, and calculating a series of short-time overlapped Fourier transforms (STFTs) sequentially stepping through the collected charge detection data, the STFTs including STFT frequency values ​​and STFT charge values, and wherein, (ii) includes: determining the set of OFR and CM pairs from the STFT frequency values ​​and the STFT charge values.

[0016] The twelfth aspect includes the features of the eleventh aspect, and wherein the recovery file includes a first recovery file, and wherein (iii) further includes: forming a second recovery file from the set of OFR and CM pairs, the second recovery file including OFR and CM pairs having an oscillation frequency that overlaps with the oscillation frequencies of other OFR and CM pairs, wherein the overlapping oscillation frequencies in the second recovery file are indistinguishable from each other, and wherein the method further includes the steps between (iv) and (v): (vi) for at least one OFR and CM pair in the second recovery file, (a) determining first and second OFR values ​​based on the STFT charge and frequency values ​​of the OFR and CM pair, and (b) modifying the second recovery file by replacing the OFR and CM pair in the second recovery file with the first and second OFR and CM pairs having the determined first OFR value and the charge of the OFR and CM pair, and the second OFR and CM pair having the determined second OFR value and the charge of the OFR and CM pair.

[0017] The thirteenth aspect includes the features of the twelfth aspect and may further include: performing (vi) on each of the OFR and CM pairs in the second recovery file.

[0018] The fourteenth aspect includes the features of the twelfth or thirteenth aspect, and may further include the following steps after (vi) and between (iv) and (v): (viii) for each OFR and CM pair in the modified second recovery file, (a) modifying the charge of one of the OFR and CM pairs in the filter file based on the charge of the OFR and CM pair having an oscillation frequency in the ELIT or orbital trap that is within a frequency window of the oscillation frequency of the OFR and CM pair in the ELIT or orbital trap, and (b) updating the filter file by adding the OFR and CM pair having the modified charge to the filter file.

[0019] The fifteenth aspect includes the features of any one of the twelfth to fourteenth aspects, and wherein, (vi) includes determining the first and second OFR values ​​by depending on the STFT charge and frequency values ​​of the selected OFR and CM pair in the following manner: (1) determining a center frequency from the mode of the STFT frequency values ​​of the selected OFR and CM pair, (2) fitting a sine function to the STFT charge values ​​of the selected OFR and CM pair to determine a frequency difference, (3) adding half of the frequency difference to the center frequency to determine the first OFR value, and (4) subtracting half of the frequency difference from the center frequency to determine the second OFR value.

[0020] The sixteenth aspect includes the features of any one of aspects twelve to fifteen, and wherein forming the second recovery file further includes processing the STFT, and the second recovery file contains only OFR and CM pairs where: the change in the corresponding STFT charge value across the STFT exceeds a first percentage, the change in the corresponding STFT charge value across the STFT is non-linear in shape, and less than a second percentage of the corresponding STFT frequencies across the STFT are within a frequency threshold of the oscillation frequency of the corresponding OFR and CM pair.

[0021] The seventeenth aspect includes the features of any one of aspects twelve through sixteen, and may further include: performing (iv) for each of the OFR and CM pairs in the first recovery file.

[0022] The eighteenth aspect includes the features of the fourteenth aspect and may further include: performing (i)-(iv), (vi) and (vii) multiple times, and then performing (v) using the updated filtered file containing OFR and CM pairs for all of the multiple executions of (i)-(iv), (vi) and (vii).

[0023] The nineteenth aspect includes the features of any one of the first to eighteenth aspects, and wherein (v) includes: determining at least one of the mass-to-charge ratio and mass of the corresponding ion for each OFR and CM pair in the updated filter file, and including one or any combination of the corresponding mass, mass-to-charge ratio and charge (CM) for each OFR and CM pair in the updated filter file in the spectral distribution.

[0024] In a twentieth aspect, a method of operating a charge detection mass spectrometer comprising an electrostatic linear ion trap (ELIT) or an orbital trap may include: (i) trapping a plurality of ions generated from a sample in the ELIT or orbital trap such that, during an ion trapping event, the plurality of trapped ions oscillate back and forth through or around a charge detector of the ELIT or orbital trap; (ii) during the ion trapping event, collecting charge detection data derived from the detection of charges induced on the charge detector by the plurality of ions; (iii) determining a set of oscillation frequency (OFR) and charge quantity (CM) pairs from the collected charge detection data, each pair corresponding to a different ion among the plurality of trapped ions; (iv) forming a filter file and a recovery file from the set of OFR and CM pairs, the filter file comprising OFR and CM pairs from which the spectral distribution of the sample from which it is generated, and the recovery file comprising OFR and CM pairs having oscillation frequencies overlapping with the oscillation frequencies of other OFR and CM pairs, wherein the overlapping oscillation frequencies in the recovery file are indistinguishable from each other; and (v) for the OFR and CM in the recovery file... For at least one OFR and CM pair, (a) determining first and second OFR values ​​for the OFR and CM pair from the collected charge detection data, and (b) modifying the recovery file by replacing the OFR and CM pair in the recovery file with the first and second OFR and CM pairs having the determined first OFR value and the charge amount of the OFR and CM pair, and the second OFR and CM pair having the determined second OFR value and the charge amount of the OFR and CM pair, (vi) for each OFR and CM pair in the modified recovery file, (a) modifying the charge amount of the OFR and CM pair based on the charge amount of one of the OFR and CM pairs in the filter file, wherein the oscillation frequency of one of the OFR and CM pairs in the ELIT or orbital trap is within a frequency window of the oscillation frequency of the OFR and CM pair in the ELIT or orbital trap, and (b) updating the filter file by adding the OFR and CM pair having the modified charge amount, and (vii) generating the spectral distribution of the sample from the updated filter file.

[0025] The twenty-first aspect includes the features of the twentieth aspect and may further include: multiple executions of (i)-(vi), followed by execution of (vii) using the updated filter file containing both OFR and CM pairs for all multiple executions of (i)-(vi).

[0026] The twenty-second aspect includes the features of the twentieth or twenty-first aspect, and wherein (v)(a) includes: collecting the charge amounts of all OFR and CM pairs in the filter file whose oscillation frequencies in the ELIT or orbital trap are within the frequency window of the oscillation frequencies of the OFR and CM pairs in the recovery file in the ELIT or orbital trap, randomly selecting one of the collected charge amounts, and modifying the charge amounts of the OFR and CM pairs in the recovery file according to the randomly selected charge amount.

[0027] The twenty-third aspect includes the features of the twenty-second aspect, and wherein modifying the charge quantity includes: modifying the charge quantity by adding a noise value to a charge quantity randomly selected from the collected charge quantities, and replacing the charge quantity of the corresponding OFR and CM pair with the modified charge quantity.

[0028] The twenty-fourth aspect includes the features of any one of the twenty to twenty-third aspects, and wherein, (iii) further includes: excluding from the set of OFR and CM pairs all OFR and CM value pairs of ions that were not captured in the ELIT or orbital trap during the entire duration of the ion capture event.

[0029] The twenty-fifth aspect includes the features of any one of the twentieth to twenty-fourth aspects, and wherein, (iii) further includes: calculating a series of short-time overlapped Fourier transforms (STFTs) sequentially through the collected charge detection data, the STFTs including STFT charge values ​​and STFT frequency values, and determining the standard deviation of the oscillation frequency of each of the OFR and CM pairs from the STFT charge values ​​and frequency values.

[0030] The twenty-sixth aspect includes the features of the twenty-fifth aspect, and wherein, (iv) further includes: excluding from the filter file and from the recovery file all OFR and CM pairs whose oscillation frequency standard deviation is greater than a second threshold.

[0031] The twenty-seventh aspect includes the features of the twenty-fifth or twenty-sixth aspect, and wherein, (iii) further includes: calculating a series of short-time overlapped Fourier transforms (STFTs) sequentially stepping through the collected charge detection data, the STFTs including STFT charge values ​​and STFT frequency values, and determining the charge standard deviation of each of the OFR and CM pairs from the STFT charge values ​​and frequency values, and wherein, (iv) further includes: requiring the OFR and CM pairs in the filtered file to have a charge standard deviation less than a first threshold, and requiring the OFR and CM pairs in the recovered file to have a charge standard deviation greater than the first threshold.

[0032] The twenty-eighth aspect includes the features of any one of aspects 20 to 27, and may further include: performing (vi) on each of the OFR and CM pairs in the recovery file.

[0033] The twenty-ninth aspect includes the features of any one of the twenty-fifth to twenty-eighth aspects, and wherein, (iii) includes: determining the set of OFR and CM pairs from the STFT charge value and frequency value, and wherein, (v)(a) includes: determining the first and second OFR values ​​of the OFR and CM pair depending on the STFT charge value and STFT frequency value.

[0034] The thirtieth aspect includes the features of any one of the twenty-fifth to twenty-ninth aspects, and wherein (v)(a) includes determining the first and second OFR values ​​by depending on the STFT charge and frequency values ​​of the selected OFR and CM pair in the following manner: (1) determining a center frequency from the mode of the STFT frequency values ​​of the selected OFR and CM pair, (2) fitting a sine function to the STFT charge values ​​of the selected OFR and CM pair to determine a frequency difference, (3) adding half of the frequency difference to the center frequency to determine the first OFR value, and (4) subtracting half of the frequency difference from the center frequency to determine the second OFR value.

[0035] The thirty-first aspect includes the features of the twenty-ninth or thirtieth aspect, and wherein forming the recovery file further includes processing the STFT, and the recovery file contains only OFR and CM pairs where: the change in the corresponding STFT charge value across the STFT exceeds a first percentage, the change in the corresponding STFT charge value across the STFT is non-linear in shape, and less than a second percentage of the corresponding STFT frequencies across the STFT are within a frequency threshold of the oscillation frequency of the corresponding OFR and CM pair.

[0036] The thirty-second aspect includes the features of any one of aspects twenty to thirty-one, and wherein, (vii) includes: determining at least one of the mass-to-charge ratio and mass of the corresponding ion for each OFR and CM pair in the updated filter file, and including one or any combination of the corresponding mass, mass-to-charge ratio and charge (CM) for each OFR and CM pair in the updated filter file in the spectral distribution.

[0037] In a thirty-third aspect, a method of operating a charge detection mass spectrometer comprising an electrostatic linear ion trap (ELIT) or an orbital trap may include: (i) trapping a plurality of ions generated from a sample in the ELIT or orbital trap such that, during an ion trapping event, the plurality of trapped ions oscillate back and forth through or around a charge detector of the ELIT or orbital trap; (ii) during the ion trapping event, collecting charge detection data derived from the detection of charges induced on the charge detector by the plurality of ions; (iii) determining a set of oscillation frequency (OFR) and charge quantity (CM) pairs from the collected charge detection data, each pair corresponding to a different ion among the plurality of trapped ions; and (iv) from the set of OF... R and CM pairs form a filter file and first and second recovery files, the filter file including OFR and CM pairs from which the spectral distribution of the sample is generated, and the first and second recovery files each including OFR and CM pairs having oscillation frequencies that overlap with the oscillation frequencies of other OFR and CM pairs, wherein the overlapping oscillation frequencies in the first recovery file are distinguishable from each other, and the overlapping oscillation frequencies in the second recovery file are indistinguishable from each other, (v) for at least one OFR and CM pair in the first recovery file, (a) the charge of the OFR and CM pair is modified depending on the charge of one of the OFR and CM pairs in the filter file, one of the OFR and CM pairs in the ELI The oscillation frequency in the T or orbital trap is within the frequency window of the oscillation frequency of the OFR and CM pair in the ELIT or orbital trap, and (b) the filter file is updated by adding the OFR and the modified CM pair to the filter file, and (vi) for at least one OFR and CM pair in the second recovery file, (a) the first and second OFR values ​​of the OFR and CM pair are determined from the collected charge detection data, and (b) the second recovery file is modified by replacing the OFR and CM pair in the second recovery file with the first and second OFR and CM pairs, the first OFR and CM pair having the determined first OFR value and the charge amount of the OFR and CM pair. The second OFR and CM pair has a determined second OFR value and a charge amount of the OFR and CM pair. (vii) For each OFR and CM pair in the modified second recovery file, (a) the charge amount of the OFR and CM pair is modified depending on the charge amount of one of the OFR and CM pairs in the filter file, wherein the oscillation frequency of one of the OFR and CM pairs in the ELIT or orbital trap is within a frequency window of the oscillation frequency of the OFR and CM pair in the ELIT or orbital trap, and (b) the filter file is updated by adding the OFR and CM pair with the modified charge amount to the filter file, and (viii) the spectral distribution of the sample is generated from the updated filter file.

[0038] The thirty-fourth aspect includes the features of the thirty-third aspect and may further include: performing (i)-(vii) multiple times, and then performing (viii) using the updated filter file containing both OFR and CM pairs for the multiple executions of (i)-(vii).

[0039] The thirty-fifth aspect includes the features of the thirty-third or thirty-fourth aspect, and may further include: performing (v) for each of the OFR and CM pairs in the first recovery file.

[0040] The thirty-sixth aspect includes the features of any one of the thirty-third to thirty-fifth aspects, and may further include: performing (vi) on each of the OFR and CM pairs in the second recovery file.

[0041] The thirty-seventh aspect includes the features of any one of the thirty-third to thirty-sixth aspects, and wherein, (iii) further includes: calculating a series of short-time overlapped Fourier transforms (STFTs) sequentially stepping through the collected charge detection data, the STFTs including STFT charge values ​​and STFT frequency values, and determining the charge standard deviation of each of the OFR and CM pairs from the STFT charge values ​​and frequency values, and wherein, (iv) further includes: requiring the OFR and CM pairs in the filtered file to have a charge standard deviation less than a first threshold, and requiring the OFR and CM pairs in each of the first and second recovery files to have a charge standard deviation greater than the first threshold.

[0042] The thirty-eighth aspect includes the features of any one of the thirty-third to thirty-seventh aspects, and wherein, (iv) further includes: excluding from the set of OFR and CM pairs all OFR and CM value pairs of ions that were not captured in the ELIT or orbital trap during the entire duration of the ion capture event.

[0043] The thirty-ninth aspect includes the features of any one of aspects thirty-three to thirty-eight, and wherein, (iii) further includes: calculating a series of short-time overlapped Fourier transforms (STFTs) sequentially stepping through the collected charge detection data, the STFTs including STFT charge values ​​and STFT frequency values, and determining the oscillation frequency standard deviation of each of the OFR and CM pairs from the STFT charge values ​​and frequency values, and wherein, (iv) further includes: excluding all OFR and CM pairs whose oscillation frequency standard deviation is greater than a second threshold from the filter file and from each of the first and second recovery files.

[0044] The fortieth aspect includes features of any one of aspects thirty-three to thirty-nine, and wherein (v)(a) includes: collecting the charge amounts of all OFR and CM pairs in the filter file whose oscillation frequencies in the ELIT or orbital trap are within the frequency window of the oscillation frequencies of the OFR and CM pairs in the first recovery file in the ELIT or orbital trap; randomly selecting one of the collected charge amounts; and modifying the charge amounts of the OFR and CM pairs in the first recovery file based on the randomly selected charge amount.

[0045] The forty-first aspect includes the features of the fortieth aspect, wherein modifying the charge quantity comprises: modifying the charge quantity by adding a noise value to a charge quantity randomly selected from the collected charge quantities, and replacing the charge quantity of the OFR and CM pair in the first recovery file with the modified charge quantity.

[0046] The forty-second aspect includes the features of any one of the thirty-third to forty-first aspects, and wherein, (iii) includes: calculating a series of short-time overlapped Fourier transforms (STFTs) sequentially through the collected charge detection data, the STFTs including STFT charge values ​​and STFT frequency values, and determining the set of OFR and CM pairs from the STFT charge values ​​and frequency values.

[0047] The forty-third aspect includes the features of the forty-second aspect, and wherein, (vi)(a) includes determining the first and second OFR values ​​by depending on the STFT charge and frequency values ​​of the selected OFR and CM pair in the following manner: (1) determining a center frequency from the mode of the STFT frequency values ​​of the selected OFR and CM pair, (2) fitting a sine function to the STFT charge values ​​of the selected OFR and CM pair to determine a frequency difference, (3) adding half of the frequency difference to the center frequency to determine the first OFR value, and (4) subtracting half of the frequency difference from the center frequency to determine the second OFR value.

[0048] The forty-fourth aspect includes the features of the forty-second or forty-third aspect, and wherein forming the second recovery file further includes processing the STFT, and the second recovery file contains only OFR and CM pairs where: the change in the corresponding STFT charge value across the STFT exceeds a first percentage, the change in the corresponding STFT charge value across the STFT is non-linear in shape, and less than a second percentage of the corresponding STFT frequencies across the STFT are within a frequency threshold of the oscillation frequency of the corresponding OFR and CM pair.

[0049] The forty-fifth aspect includes the features of any one of the thirty-third to forty-fourth aspects, and wherein, (vii)(a) includes: collecting the charge amount of all OFR and CM pairs in the filter file whose oscillation frequency in the ELIT or orbital trap is within the frequency window of the oscillation frequency of the OFR and CM pairs in the modified second recovery file in the ELIT or orbital trap, randomly selecting one of the collected charge amounts, and modifying the charge amount of the OFR and CM pairs in the modified second recovery file according to the randomly selected charge amount.

[0050] The forty-sixth aspect includes the features of the forty-fifth aspect, and wherein modifying the charge quantity includes: modifying the charge quantity by adding a noise value to a charge quantity randomly selected from the collected charge quantities, and replacing the charge quantity of the OFR and CM pair in the modified second recovery file with the modified charge quantity.

[0051] The forty-seventh aspect includes the features of any one of aspects thirty-three to forty-six, and wherein, (viii) includes determining at least one of the mass-to-charge ratio and mass of the corresponding ion for each OFR and CM pair in the updated filter file, and includes one or any combination of the corresponding mass, mass-to-charge ratio and charge (CM) for each OFR and CM pair in the updated filter file in the spectral distribution. Attached Figure Description

[0052] Figure 1 This is a simplified side view of a charge detection mass spectrometer (CDMS) that includes an embodiment of an electrostatic linear ion trap (ELIT) along with control and measurement components coupled thereto.

[0053] Figure 2 yes Figure 1 A simplified side view of an example embodiment of the ion source for CDMS shown.

[0054] Figure 3 yes Figure 1 A simplified diagram of an embodiment of the processor shown.

[0055] Figures 4A-4C yes Figure 1 A simplified side view of the ELIT shows the sequential control and operation of the ion mirrors to trap ions within the ELIT and cause the ions to oscillate back and forth between the ion mirrors each time they pass through the charge detection cylinder, during which charge detection data of the ions are measured and recorded during the duration of the ion trapping event.

[0056] Figure 5 It shows the process used in Figure 1A flowchart illustrating an example process for obtaining time-based charge detection data (CDD) by simultaneously capturing and measuring multiple charged particles in the ELIT of CDMS.

[0057] Figure 6 It shows the use of by Figure 5 The flowchart shows an example process for quality checking of the oscillation frequency and charge quantity values ​​determined by the process.

[0058] Figure 7 It is shown in Figure 5 The flowchart is an example of an embodiment of the charged particle recovery process performed in step 120 in some embodiments of the process.

[0059] Figure 8 Including panels 8a), 8b), 8c), and 8d), and showing the entire event FFT and corresponding STFT (panels 8a) and 8b) for ions with oscillation frequencies 50 Hz apart, and the entire event FFT and corresponding STFT (panels 8c) and 8d) for ions with oscillation frequencies 10 Hz apart.

[0060] Figure 9 Includes panels 9a), 9b), 9c), and 9d), and shows two waveforms that are 180° out of phase with each other (panels 9a and 9b, respectively), two superimposed waveforms (panel 9c), and two combined waveforms (panel 9d).

[0061] Figure 10 It is the amount of charge across Figure 9 A graph of the STFT window for time-domain data stepping is shown. Figure 9 The frequency beat patterns are derived from the in-phase and out-of-phase shifts of the two signals in a) and 9b).

[0062] Figure 11 It is shown in Figure 5 The flowchart is an example of an embodiment of the charged particle recovery process performed in step 122, which is one of the embodiments of the process.

[0063] Figure 12 Includes panels 12a), 12b), 12c), and 12d), and shows L-glutamate dehydrogenase (GDH) samples with and without [the enzyme / product]. Figure 7 The process of charged particle recovery and the mass distribution at different measurement rates.

[0064] Figure 13 In having and not having Figure 7 A graph showing the relative intensities of GDH monomers, dimers, and trimers versus the measured rate during the charged particle recovery process.

[0065] Figure 14Includes panels 14a), 14b), 14c), and 14d), and shows Qβ virus-like particle samples with and without... Figure 7 The process of charged particle recovery and the mass distribution at different measurement rates.

[0066] Figure 15 Including panels 15a) and 15b), and shown in Figure 7 Before the charged particle recovery process ( Figure 15 a) and afterwards ( Figure 15 b) The relative abundance of AAV8 ions in different mass bands.

[0067] Figure 16 Includes panels 16a) and 16b). Panel 16a) compares L-glutamate dehydrogenase (GDH) samples in the absence of any charged particle recovery process, with Figure 7 The process of charged particle recovery, and having Figure 11 The mass distribution during the recovery process of charged particles. Panel 16d) compares the mass distribution at different measurement rates using... Figure 11 The relative intensity measured during the recovery process of charged particles. Detailed Implementation

[0068] To facilitate understanding of the principles of this disclosure, reference will now be made to some exemplary embodiments shown in the accompanying drawings, which will be described using specific language.

[0069] This disclosure relates to systems and methods for the simultaneous analysis of multiple charged particles using charge detection mass spectrometry (CDMS), and particularly to determining the charge quantity and / or oscillation frequency of trapped charged particles having overlapping oscillation frequencies in such systems. The charge quantity and oscillation frequency of some such trapped charged particles, which might otherwise be intentionally omitted from the analytical results due to measurement inaccuracies related to the overlap of the oscillation frequencies of two or more trapped charged particles, can now be included in the analytical results as described below to provide improved spectral distribution accuracy. For the purposes of this disclosure, the phrase "charged particle detection event" is defined as the detection of a charge induced on a charge detector by a charged particle that passes through or around an electrostatic linear ion trap (ELIT) or orbital trap in a single pass. In CDMS instruments in which multiple charged particles are trapped for simultaneous analysis in an ELIT or orbital trap, as is the case with the CDMS instruments disclosed herein, a charged particle detection event, as just defined, occurs for each of the multiple trapped charged particles. The phrase "charged particle measurement event" is defined as a set of charged particle detection events resulting from the controlled oscillation of multiple charged particles through or around a charge detector a selected number of times or for a selected time period. Since this oscillation of charged particles is achieved by the controlled capture of multiple charged particles within an ELIT or orbital trap, as will be described in detail below, the phrase "charged particle measurement event" may alternatively be referred to herein as a "charged particle capture event" or simply "ion capture event" or "capture event," and the phrases "charged particle measurement event," "charged particle capture event," "ion capture event," "capture event," and variations thereof should be understood to be synonymous with each other. For the purposes of this disclosure, the terms "ion" and "charged particle," and variations thereof, will be understood to be synonymous. In any of the definitions above, the term "ion" may therefore replace the term "charged particle."

[0070] refer to Figure 1 This illustration shows an embodiment of a charge detection mass spectrometer (CDMS) 10, which exemplarily includes an electrostatic linear ion trap (ELIT) 14 as a charged particle detector. In some alternative embodiments, a conventional orbital trap may be used instead of the ELIT 14 as the charged particle detector, and a non-limiting example of such an orbital trap is disclosed in WO2020 / 106310A1, the disclosure of which is expressly incorporated herein by reference in its entirety. In any case, during the operation of the CDMS 10, as will be described in detail below, multiple charged particles are simultaneously captured in the ELIT 14 (or orbital trap) and analyzed by it simultaneously.

[0071] Figure 1The CDMS 10 illustrated in the example further includes a charged particle source 12 operatively coupled to an ELIT 14, wherein the charged particle source 12 and the ELIT 14 together exemplarily define a longitudinal axis 20 extending therethrough. The charged particle source 12 exemplarily includes any conventional means or apparatus for generating charged particles from a sample, and may further include one or more means and / or instruments for guiding, separating, collecting, filtering, controlling / setting energy, fragmenting, and / or normalizing or converting the charge state of the generated charged particles according to one or more molecular properties. As an example (and not to be considered as limiting in any way), the means or apparatus for generating charged particles from a sample may be or include conventional electrospray ionization sources, matrix-assisted laser desorption / ionization (MALDI) sources, etc. In some embodiments, the charged particle source 12 may further include a conventional mass spectrometer having a charged particle inlet configured to receive the generated charged particles. If included, the mass spectrometer can be of any conventional design, including, for example, but not limited to, time-of-flight (TOF) mass spectrometers, orthogonal accelerator TOF mass spectrometers, reflectance mass spectrometers, Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, quadrupole mass spectrometers, triple quadrupole mass spectrometers, magnetic sector mass spectrometers, orbital traps, etc. In such embodiments, the charged particle source 12 may further include one or more devices and / or instruments between the apparatus or device for generating charged particles and the mass spectrometer and / or between the mass spectrometer and the ELIT 14, for guiding, separating, collecting, filtering, controlling / setting energy, fragmenting, and / or normalizing or converting the charge state of the generated charged particles according to one or more molecular properties. In embodiments excluding the mass spectrometer, the charged particle source 12 may include one or more devices and / or instruments between the apparatus or device for generating charged particles and the ELIT 14 for guiding, separating, collecting, filtering, controlling / setting energy, fragmenting, and / or normalizing or converting the charge state of the generated charged particles according to one or more molecular properties. In any case, the sample from which charged particles are generated can be or include any biological material or other material or combination of materials.

[0072] Now for reference Figure 2 The diagram illustrates an example embodiment of a CDMS 10', which includes an ion source 12 operatively coupled to an ELIT 14, and thereby represents... Figure 1 The CDMS 10 shown is a non-limiting example embodiment. Figure 2In the example embodiment shown, the ion source 12 exemplary includes a conventional electrospray ionization source 24 having a charged particle outlet positioned to supply charged particles to a charged particle inlet of a conventional charged particle interface 26, which is configured to guide charged particles from a high-pressure (e.g., atmospheric pressure) region where the ionization source 24 is located to a lower-pressure (e.g., vacuum condition) region of a downstream stage 28 of the ion source 12. Non-limiting examples of such charged particle interfaces 26 are disclosed in WO2019 / 236139A1 and WO2019 / 236572A1, both of which are expressly incorporated herein by reference in their entirety.

[0073] exist Figure 2 In the embodiment depicted, stage 28 of ion source 12 is exemplary implemented as a conventional RF-only hexapole. The RF-only hexapole 28 has a charged particle inlet operatively coupled to a charged particle outlet of charged particle interface 26, and a charged particle outlet operatively coupled to a charged particle inlet of downstream stage 30. The operation of the RF-only hexapole 28 is conventional and serves to guide charged particles axially from the charged particle inlet through the hexapole 28 to its charged particle outlet, while also radially focusing and constraining charged particles to arrive at and around a longitudinal axis 20 defined by the center of ion source 12 (see [link to documentation]). Figure 1 ).

[0074] Figure 2 The stage 30 of the ion source 12 is exemplarily implemented in the form of a conventional RF-only quadrupole. The RF-only quadrupole 30 has a charged particle inlet operably coupled to the charged particle outlet of the hexapole 28, and a charged particle outlet operably coupled to the charged particle inlet of the downstream stage 32. The operation of the RF-only quadrupole 30 is conventional and is used to guide charged particles axially from the charged particle inlet through the quadrupole 30 to its charged particle outlet, while also radially focusing and constraining the charged particles around a longitudinal axis 20 defined by the center of the ion source 12 (see [link to relevant documentation]). Figure 1 In some, but not necessarily all, embodiments, the RF-only quadrupole 30 may be controlled as described in co-pending international application No. PCT / US2023 / 073710 (whose disclosure is fully and expressly incorporated herein by reference) to eliminate or at least reduce the “nodding” effect associated with other modes of normal operation of the RF-only quadrupole, wherein the term “nodding” is used in... Figure 2 In the context of this, it refers to charged particles being ejected off-axis from the charged particle exit of the quadrupole 30 alone, causing at least some of the charged particles leaving the charged particle exit of the quadrupole 30 to deviate angularly from the central longitudinal axis 20 as the charged particles move away from the charged particle exit of the quadrupole 30.

[0075] Figure 2The stage 32 of the ion source 12 is exemplarily implemented in the form of a conventional charged particle focusing optics. Charged particles passing through the optics 32 are focused into the charged particle inlet of a conventional dual-hemispherical energy analyzer 34, which has a charged particle outlet operatively coupled to or aligned with the charged particle inlet of the ELIT 14. The energy analyzer 34 is exemplarily operated to deliver only charged particles having ion energy within a narrow band of ion energy to the ELIT 14. In one embodiment in which the ELIT 14 is provided in the form described in the example below, the energy analyzer 34 is configured to deliver only charged particles with ion energy within a narrow energy band centered at approximately 130 eV / z; however, it will be understood that in other embodiments, the energy analyzer 34 may be configured to deliver charged particles centered at an energy band or window of any desired size and / or at other charged particle energy values ​​(i.e., different from 130 eV / z).

[0076] exist Figure 2 In the illustrated embodiment, multiple charged particles are fed directly from the focusing optics 32 into the ELIT 14 via an energy analyzer 34. In some alternative embodiments, a mass spectrometer 36 may be interposed between the focusing optics 32 and the energy analyzer 34 to provide separation of charged particles based on their mass-to-charge ratio (m / z) before entering the ELIT 14, such as... Figure 2 The dashed lines in the diagram indicate what is depicted. In one example embodiment, mass spectrometer 36 may be a conventional orthogonal accelerated TOF mass spectrometer; however, in other embodiments including it, mass spectrometer 36 may take other conventional forms, as described above. Figure 1 Some examples of these are described.

[0077] Refer again Figure 1The ELIT 14, as illustrated by example, exemplarily includes a charge detector CD surrounded by a grounded cavity or cylinder GC and operatively coupled to opposing ion mirrors M1, M2, respectively located at opposite ends of the charge detector CD. Ion mirrors M1, M2 may alternatively be referred to herein as “end caps” or “end caps,” and it should be understood that, for the purposes of this disclosure, the terms ion mirror and end cap (or end cap) are synonymous. Ion mirror M1 is operatively located between the charged particle outlet of the charged particle source 12 and one end of the charge detector CD, and ion mirror M2 is operatively located at the opposite end of the charge detector CD. Each ion mirror M1, M2 defines a corresponding ion mirror region R1, R2 therein. The regions R1 and R2 of ion mirrors M1 and M2, the charge detector CD, and the spatial axial alignment between the charge detector CD and ion mirrors M1 and M2, together define a longitudinal axis 20 centered therethrough, which, as will be described in more detail below, exemplarily represents the ideal ion travel path through ELIT 14 and between ion mirrors M1 and M2. The axially defined region between the opposing inner surfaces of ion mirrors M1 and M2 (i.e., the region where the charge detector CD is located) exemplarily defines a field-free region FFR, i.e., no electric field is established in the FFR during ELIT 14 operation.

[0078] In the illustrated embodiment, voltage sources V1 and V2 are electrically coupled to ion mirrors M1 and M2, respectively. Each voltage source V1 and V2 exemplarily includes one or more switchable DC voltage sources that can be controlled or programmed to selectively generate a programmable or controllable voltage of number N, where N can be any positive integer. As will be described in detail below, these voltages can be exemplarily programmed to establish each of two different operating modes for each ion mirror M1 and M2. In any case, ELIT 14 is designed such that charged particles move within ELIT 14 near the longitudinal axis 20 under the influence of the electric fields selectively established by voltage sources V1 and V2 in ion mirrors M1 and M2.

[0079] Voltage sources V1 and V2 are exemplarily shown as being electrically connected to a conventional processor 16 via a number of signal paths of P. The processor 16 includes a memory 18 storing instructions that, when executed by the processor 16, cause the processor 16 to control the voltage sources V1 and V2 to generate desired DC output voltages, so as to selectively establish ion transport and ion reflection electric fields T and R, respectively, within regions R1 and R2 of the respective ion mirrors M1 and M2 (see example...). Figures 4A-4CP can be any positive integer. In some alternative embodiments, either or both of the voltage sources V1 and V2 may be alternatively or additionally programmable to selectively generate one or more constant output voltages. In other alternative embodiments, either or both of the voltage sources V1 and V2 may be configured to generate one or more time-varying output voltages of any desired shape. It will be understood that in alternative embodiments, more or fewer voltage sources may be electrically connected to mirrors M1 and M2. In any case, in some embodiments, ion mirrors M1 and M2 are composed of a plurality of axially spaced conductive electrodes or rings.

[0080] The voltage output of voltage source V1 is electrically connected to the corresponding conductive electrode in the conductive electrode of ion mirror M1, and the voltage output of voltage source V2 is electrically connected to the corresponding conductive electrode in the conductive electrode of ion mirror M2. Various voltage outputs of voltage sources V1 and V2 are controlled in a conventional manner to selectively establish ion transport and ion reflection electric fields (T, R) in corresponding regions R1 and R2 of ion mirrors M1 and M2, respectively. Each ion mirror M1 and M2 can be exemplarily controlled and switched between ion transport mode and ion reflection mode by selectively applying the voltages generated by voltage sources V1 and V2. In ion transport mode, the voltages generated by the corresponding voltage sources V1 and V2 establish an ion transport electric field (T) in their respective regions R1 and R2, while in ion reflection mode, the voltages generated by the corresponding voltage sources V1 and V2 establish an ion reflection electric field (R) in their respective regions R1 and R2.

[0081] The charge detector CD is exemplarily provided in the form of a conductive cylinder, exemplarily referred to herein as a charge detection cylinder, which is electrically connected to the signal input of a charge-sensitive preamplifier CP, and the signal output of the charge-sensitive preamplifier CP is electrically coupled to the processor 16. In embodiments where the charged particle detector is implemented in the form of an orbital trap, as described above, the charge detector CD may be exemplarily provided in the form of a conductive mandrel assembly around which ions oscillate in a conventional manner. In any case, refer again to Figure 1The voltage sources V1 and V2 are exemplarily controlled in such a way that multiple charged particles entering the ELIT 14 are selectively captured within the ELIT 14, and each of the captured charged particles oscillates back and forth within the ELIT 14 between the ion mirrors M1 and M2, each time axially passing through the charge detection cylinder CD. For example, the ion transport electric field T established in the ion mirrors M1 and M2 is exemplarily operated to focus the charged particles toward the longitudinal axis 20 of the ELIT 14 as they pass through the ion mirrors M1 and M2. Conversely, the ion reflection electric field R established in the ion mirrors M1 and M2 is exemplarily used to decelerate and stop the charged particles entering the ion mirrors M1 and M2 from the charge detection cylinder CD, and then accelerate the stopped charged particles back to the corresponding end of the charge detection cylinder CD in the opposite direction, focusing the charged particles toward the central longitudinal axis 20. By selectively establishing ion transport and ion reflection electric fields (T and R, respectively) in ion mirrors M1 and M2, multiple ions can be trapped in ELIT 14 and oscillated back and forth between ion mirrors M1 and M2, each time passing through the charge detection cylinder CD and inducing a corresponding charge on the charge detection cylinder CD, which is detected by the charge-sensitive preamplifier CP. Therefore, by trapping multiple charged particles within ELIT 14 and oscillating back and forth between ion mirrors M1 and M2, the charge-sensitive preamplifier CP is exemplarily operable in a conventional manner to detect the charge (CH) induced on the charge detection cylinder CD as each of the multiple charged particles repeatedly passes through the charge detection cylinder CD between ion mirrors M1 and M2, and to generate a corresponding charge detection signal (CHD). Further details regarding the structure and operation of exemplary embodiments of ion mirrors M1, M2, and ELIT 14 are disclosed in WO2019 / 140233, the disclosure of which is fully and expressly incorporated herein by reference.

[0082] The charge detection signal CHD is exemplarily periodic and recorded in the form of amplitude and period values, whereby each amplitude and period pair represents ion measurement information for a charge detection event in which a corresponding one of a plurality of charged particles is passing through the charge detection cylinder CD. The amplitude is the magnitude of the charge induced on the charge detection cylinder by the charged particle as it passes through, and the period value is the duration of time the charged particle passes through the charge detection cylinder. Multiple such amplitude and period values ​​are measured and recorded during the respective charged particle measurement event (i.e., during a charged particle capture event), and the multiple recorded values ​​obtained for the charged particle measurement event (i.e., the set of recorded charged particle measurement information) are processed to determine the mass-to-charge ratio (m / z) and charge quantity of each of the plurality of charged particles, as described below. The charged particle mass value is then calculated based on the m / z and the corresponding charge quantity value. Multiple charged particle measurement events can be processed in this manner, and the mass-to-charge ratio and / or mass and / or charge spectrum of a sample can be exemplarily constructed from them in a conventional manner.

[0083] Now for reference Figure 3 , showing Figure 1 An embodiment of the processor 16 shown is illustrated. In the illustrated embodiment, the processor 16 includes a conventional amplifier circuit 40 having an input terminal that receives a charge detection signal CHD generated by a charge-sensitive preamplifier CP and an output terminal electrically connected to the input terminal of a conventional analog-to-digital (A / D) converter 42. The output terminal of the A / D converter 42 is electrically connected to the processor 50 (P1). The amplifier 40 is conventionally operable to amplify the charge detection signal CHD generated by the charge-sensitive preamplifier CP, and the A / D converter 42 is in turn conventionally operable to convert the amplified charge detection signal into a digital charge detection signal CDS.

[0084] Figure 3The processor 16 shown further exemplarily includes a conventional comparator 44 having a first input receiving a charge detection signal CHD generated by a charge-sensitive preamplifier CP, a second input receiving a threshold voltage CTH generated by a threshold voltage generator (TG) 46, and an output electrically connected to the processor 50. The comparator 44 is conventionally operable to generate a trigger signal TR at its output, which depends on the amount of the charge detection signal CHD relative to the threshold voltage CTH. In one embodiment, for example, as long as CHD is less than CTH, the comparator 44 is operable to generate an “inactive” trigger signal TR at or near a reference voltage (e.g., ground potential), and when CHD reaches or exceeds CTH, it is operable to generate an “active” TR signal at or near the power supply voltage of circuits 40, 42, 44, 46, 50, or otherwise distinguishable from the inactive TR signal. In an alternative embodiment, comparator 44 may be operable to generate an "inactive" trigger signal TR at or near the supply voltage as long as CHD is less than CTH, and operable to generate an "active" trigger signal TR at or near the reference potential when CHD reaches or exceeds CTH. Those skilled in the art will recognize that other different trigger signal quantities and / or different trigger signal polarities may be used to establish the "inactive" and "active" states of the trigger signal TR, provided that these different trigger signal quantities and / or different trigger signal polarities can be distinguished by processor 50, and will understand that any such other different trigger signal quantities and / or different trigger signal polarities are intended to fall within the scope of this disclosure. In any case, comparator 44 may additionally be designed in a conventional manner to include a desired amount of hysteresis to prevent rapid switching of the output between the reference voltage and the supply voltage.

[0085] Processor 50 is exemplarily operable to generate a threshold voltage control signal THC and provide THC to threshold generator 46 to control its operation. In some embodiments, processor 50 is programmed or programmable to control the generation of the threshold voltage control signal THC in a manner that controls threshold voltage generator 46 to generate CTH with a desired amount and / or polarity. In other embodiments, a user may, for example, provide instructions to processor 50 in real time via a downstream processor (e.g., via virtual control and visualization unit 56) to control the generation of the threshold voltage control signal THC in a manner that controls threshold voltage generator 46 to generate CTH with a desired amount and / or polarity. In any case, threshold voltage generator 46 is exemplarily implemented in some embodiments as a conventional controllable DC voltage source configured to generate an analog threshold voltage CTH having a polarity and amount defined by the digital threshold control signal THC in response to the threshold control signal THC in digital form (e.g., in the form of a single serial digital signal or multiple parallel digital signals). In some alternative embodiments, the threshold voltage generator 46 may be provided in the form of a conventional digital-to-analog converter (D / A) that generates an analog threshold voltage CTH having a quantity (and polarity) defined by a digital threshold control signal THC in response to a serial or parallel digital threshold voltage TCH. In some such embodiments, the D / A converter may form part of the processor 50. Those skilled in the art will recognize other conventional circuitry and techniques for selectively generating a threshold voltage CTH of a desired quantity and / or polarity in response to one or more control signals THC in digital and / or analog form, and will understand that any such other conventional circuitry and / or techniques are intended to fall within the scope of this disclosure.

[0086] In addition to the functions performed by processor 50 as described above, processor 50 is further operable as described above regarding Figure 1 The control voltage sources V1 and V2 selectively establish ion transport and reflection fields (T and R, respectively) within regions R1 and R2 of ion mirrors M1 and M2, respectively. In some embodiments, processor 50 is programmed or programmable to control voltage sources V1 and V2. In other embodiments, one or more voltage sources V1 and / or V2 may be programmed in real time by a user, for example, via a downstream processor 52 (e.g., via a virtual control and visualization unit) or otherwise controlled. In any case, in one embodiment, processor 50 is provided, exemplarily, in the form of a field-programmable gate array (FPGA), which is programmed or otherwise instructed by a user to collect and store charge detection signals CDS for charge detection events and ion measurement events, generate one or more threshold control signals TCH from which the amount and / or polarity of threshold voltage CTH is determined or derived, and control voltage sources V1 and V2. In this embodiment, regarding Figure 1The described memory 18 is integrated into the programming of the FPGA and forms part of it. In an alternative embodiment, the processor 50 may be provided as one or more conventional microprocessors or controllers and one or more accompanying memory cells storing instructions that, when executed by the one or more microprocessors or controllers, cause the one or more microprocessors or controllers to operate as just described. In other alternative embodiments, the processing circuitry 50 may be implemented purely as one or more conventional hardware circuits designed to operate as described above, or may be implemented as a combination of such one or more hardware circuits and at least one microprocessor or controller operable to execute instructions stored in memory to operate as described above.

[0087] Figure 3 An embodiment of processor 16 depicted in the example further exemplarily includes a second processor 52 operatively coupled to the first processor 50 and also coupled to at least one memory unit 54. In some embodiments, processor 52 may include one or more peripheral devices, such as a display monitor, one or more input and / or output devices, etc.; however, in other embodiments, processor 52 may not include any such peripheral devices. In any case, processor 52 is exemplarily configured (i.e., programmed) to perform at least one process for analyzing ion measurement events. Time-based charge detection data (CDD), received by processor 50 via charge detection signal CDD and taking the form of charge quantity and charge timing data (i.e., the timing detection of the charge induced by ions on the charge detection cylinder), is exemplarily transmitted directly from processor 50 to processor 52 for processing and analysis after each ion measurement event.

[0088] In some embodiments, the processor 52 is exemplarily provided as a high-speed server operable to perform both the collection / storage and analysis of such data. In such embodiments, one or more high-speed memory units 54 may be coupled to the processor 52 and operable to store data received and analyzed by the processor 52. In one embodiment, the one or more memory units 54 exemplarily include at least one local memory unit for storing data that the processor 52 is using or will use, and at least one persistent storage memory unit for long-term storage of data. In one such embodiment, the processor 52 is exemplarily provided as a Linux® server (e.g., OpenSuse Leap 42.1) with four Intel® Xeon™ processors (e.g., E5-465L v2, 12 cores, 2.4 GHz). In this embodiment, an average analysis time of more than 100x is achieved compared to a conventional Windows® PC (e.g., i5-2500K, 4 cores, 3.3 GHz). Similarly, the processor 52 of this embodiment, together with (one or more) high-speed / high-performance memory units 54, exemplarily provides an improvement of more than 100x in data storage speed. Those skilled in the art will recognize that one or more other high-speed data processing and analysis systems can be implemented as processor 52, and will understand that any such one or more other high-speed data processing and analysis systems are intended to fall within the scope of this disclosure. In an alternative embodiment, processor 52 may be provided in the form of one or more conventional microprocessors or controllers and one or more accompanying memory units storing instructions that, when executed by the one or more microprocessors or controllers, cause the one or more microprocessors or controllers to operate as described herein.

[0089] In the illustrated embodiment, memory unit 54 exemplary stores instructions executable by processor 52 to analyze ion measurement event data generated by ELIT 14 to determine the ion spectral distribution of the analyzed sample, i.e., ion mass-to-charge ratio (m / z), ion charge, ion mass, etc. In one embodiment, processor 52 is operable to receive from processor 50 ion measurement event data in the form of charge quantity and charge detection timing information measured during each of a plurality of "charge detection events" (as defined above) constituting an "ion measurement event" (as defined above), and to process such charge detection events constituting such ion measurement events to determine ion charge and mass-to-charge ratio data, and then determine ion mass data therefrom. The mass spectral distribution of the analyzed sample can be created in this way from ion measurement events that capture multiple charged particles in ELIT 14 as described above. Multiple ion measurement events can also be processed in a similar manner to create the mass spectral distribution of the analyzed sample.

[0090] In some embodiments, the CDMS 10 described above can be directly managed in real time by and from the processor 52, wherein, for example, the operating parameters of the CDMS system 10, particularly the ELIT 14, can be selected in real time or at any time, and output file management and display can be managed. In other embodiments, the processor 16 may include, for example, Figure 3The example shown is a separate processor 56 coupled to processor 52. In such embodiments, processor 56 is exemplary a conventional processor or processing system for which drawing utilities and data processing programs widely known and used are available. In one example embodiment, processor 56 is implemented in the form of a conventional Windows®-based personal computer (PC) containing one or more such drawing utilities and data processing programs installed thereon. Those skilled in the art will recognize other conventional processors or processing systems that may be suitable as processor 56, and will understand that any such other conventional processors or processing systems are intended to fall within the scope of this disclosure. In any case, in embodiments including processor 56, a graphical user interface (GUI) (e.g., an RTA GUI) may be included to provide a user-friendly and real-time controlled GUI accessible via processor 56. In one embodiment, the real-time controlled GUI is stored in memory 54 and executed by processor 52, and processor 56 is used to access the user GUI from processor 52, for example, via a secure connection between the two processors 52, 56. In an alternative embodiment, the real-time controlled GUI may be stored on processor 56 and executed thereon. In either case, the processor 56 exemplarily acts as a virtual control and visualization unit, which allows a user to visualize and control one or more aspects of the real-time operation and real-time analysis process of the CDMS 10 via a real-time control GUI. Whether used for controlling the real-time operation of the CDMS or not, the processor 56 can be exemplarily used to visualize the output data and spectral distribution information generated by the CDMS instrument 10.

[0091] As briefly described above, voltage sources V1 and V2 are exemplarily controlled by processor 16 (e.g., via processor 50) in such a way that ion transport and ion reflection electric fields (T and R, respectively) are selectively established in region R1 of ion mirror M1 and region R2 of ion mirror M2 to guide charged particles from charged particle source 12 into ELIT 14, thereby selectively capturing and confining multiple charged particles within ELIT 14, such that the multiple captured charged particles repeatedly pass through charge detection cylinder CD as they oscillate back and forth between M1 and M2. Reference Figures 4A-4C , showing Figure 1 A simplified diagram of ELIT 14 illustrates an example of this sequential control and operation of the ion mirrors M1 and M2 of ELIT 14. In the following example, processor 50 will be described as controlling the operation of voltage sources V1 and V2 according to its programming; however, it will be understood that the operation of voltage source V1 and / or voltage source V2 may alternatively be at least partially controlled by processor 52.

[0092] like Figure 4AAs shown, the ELIT control sequence begins with the processor 50 controlling voltage source V1 to control ion mirror M1 in ion transport operation mode (T) by establishing an ion transport field within region R1 of ion mirror M1, and also controlling voltage source V2 to control ion mirror M2 in ion transport operation mode (T) by similarly establishing an ion transport field within region R2 of ion mirror M2. As a result, charged particles generated by charged particle source 12 enter ion mirror M1, and as they enter charge detection cylinder CD, they are directed towards the central longitudinal axis 20 by the ion transport field established in region R1. Figure 1 The charged particles are then focused. They pass through the charge detection cylinder CD and enter the ion mirror M2. An ion transport field established within region R2 of M2 focuses the charged particles toward the longitudinal axis 20, causing them to... Figure 4A The ion trajectory depicted in Figure 60 is shown passing through M2.

[0093] Now for reference Figure 4B After both ion mirrors M1 and M2 have been operating in ion transport mode for a selected time period and / or until successful ion transport has been achieved during this period (e.g., by monitoring the charge detection signal CDS to determine the presence of charged particles passing through the charge detection cylinder CD), processor 50 is exemplarily operable to control voltage source V2 to control ion mirror M2 in ion reflection mode (R) by establishing an ion reflection field in region R2 of ion mirror M2, while simultaneously maintaining ion mirror M1 in ion transport mode (T) as shown. As a result, charged particles generated by charged particle source 12 enter ion mirror M1 and are focused towards the central longitudinal axis 20 by the ion transport field T established in region R1, such that the charged particles are as just shown about... Figure 4A The charged particles pass through the ion mirror M1 and enter the charge detection cylinder CD. Then, the charged particles pass through the charge detection cylinder CD and enter the ion mirror M2. An ion reflection field R established in region R2 of M2 reflects the charged particles, causing them to travel in the opposite direction and return to the charge detection cylinder CD. Figure 4B The ion trajectory in 62 is shown.

[0094] Now for reference Figure 4CAfter an ion reflection electric field has been established in region R2 of ion mirror M2, processor 50 is operable to control voltage source V1 to control ion mirror M1 to be in ion reflection operation mode (R) by establishing an ion reflection field in region R1 of ion mirror M1, while simultaneously maintaining ion mirror M2 in ion reflection operation mode (R) to capture multiple charged particles within ELIT 14. Between the time when the ion reflection electric field (R) is established in region R1 of ion mirror M1 and the time when the ion reflection electric field (R) is established in region R1 of ion mirror M2, multiple charged particles enter and are captured within ELIT 14. By controlling both ion mirrors M1 and M2 to ion reflection operation mode (R), the relative ion reflection fields established in regions R1 and R2 of ion mirrors M1 and M2 respectively cause multiple captured charged particles to be captured each time (e.g., Figure 4C As shown in Figure 64, the ion trajectory oscillates back and forth between ion mirrors M1 and M2 as it passes through the charge detection cylinder CD (as described above). In one embodiment, the processor 50 is operable to maintain... Figure 4C The operating state shown continues until the multiple captured charged particles pass through the charge detection cylinder CD a selected number of times. In an alternative embodiment, the processor 50 is operable to maintain this state for a selected time period after the multiple ions have been captured in ELIT 14. Figure 4C The operating state is shown in the figure.

[0095] In any embodiment, Figure 4C The number of cycles or time spent in the state shown can be programmed, for example, via instructions stored in memory 18, or controlled via a user interface, and in any case, ion detection event information (i.e., in the form of time-based charge detection data (CDD) as described above) obtained by each of the multiple captured ions passing through the charge detection cylinder CD each time is temporarily stored in processor 50, for example, in the form of an ion measurement file. When the multiple captured charged particles have passed through the charge detection cylinder CD a selected number of times or oscillated back and forth between ion mirrors M1 and M2 for a selected time period, the total number of charged particle detection events stored in processor 50 represents the charge detection data (CDD) for the charged particle measurement events (i.e., "ion capture events") for the multiple captured charged particles as described above, and after the charged particle measurement event is completed, the charged particle detection data (CDD) for that charged particle measurement event is stored in a charge detection file (CDF), and then the file is passed to processor 52 or retrieved by processor 52. Then, Figures 4A-4C The sequence shown returns to Figure 4AThe sequence shown is described above, wherein control voltage sources V1 and V2 control ion mirrors M1 and M2 to be in ion transport operation mode (T) by establishing ion transport fields in regions R1 and R2 of ion mirrors M1 and M2, respectively. The sequence shown is then repeated many times as needed.

[0096] The following text is about Figure 5-7 As will be described in more detail, in the frequency domain, a sequential step-by-step fast Fourier transform (FFT) algorithm is used to analyze time-based charge detection data (CDD) in a charge detection file (CDF). In such an implementation, an oscillation frequency and charge quantity pair is determined for each of the plurality of charged particles captured in ELIT 14 based on the fundamental frequency (f0) of the series of time-overlapping FFTs, wherein the oscillation frequency and charge quantity of each of the plurality of charged particles correspond, respectively, to the frequency and quantity of the fundamental frequency f0 of that charged particle after averaging over the series of short-time-overlapping FFTs, as further described below. Finally, i.e., after processing the CDD of one or more ion capture events, the mass-to-charge ratio (m / z) of the charged particles is determined according to Equation 1 below based on the fundamental frequency f0 and the calibration constant (C), and then the mass of the charged particles is determined as the product of the corresponding m / z and charge quantity for each charged particle.

[0097] Equation 1: Now for reference Figure 5 The diagram shows a flowchart of process 100, which processes time-based charge detection data (CDD) derived from measurements of the charge induced on a charge detection cylinder CD by a charge-sensitive preamplifier CP during one or more capture events as described above, as multiple captured charged particles oscillate back and forth between ion mirrors M1 and M2 and pass through the charge detection cylinder CD, and is used to ultimately determine the mass-to-charge ratio and charge quantity of such charged particles. Process 100 further exemplarily includes at least one process for recovering charged particle data that has been rejected (i.e., omitted from the analysis results) due to measurement inaccuracies associated with charged particles having overlapping oscillation frequencies. This is achieved through one or more iterations of process 100 (including...). Figure 6 The embedding process shown in the example and Figure 7 and Figure 11The result of any one or more of the embedding processes (exemplified in any one or both of the examples) is a charged particle spectrum distribution for each of a plurality of captured charged particles in one or more capture events, including one or a combination of mass-to-charge ratio (m / z), mass (m), and charge (z). Process 100 is exemplarily stored in and executed by the processor 50 described above, stored in memory 54 and executed by processor 52, stored in and executed by processor 56, partially stored in processor 50 and partially stored in memory 54 and partially executed by processor 50 and partially executed by processor 52, or partially stored in processor 56 and partially stored in memory 54 and partially executed by processor 56 and partially executed by processor 52. In this respect, the phrase "50, 52, and / or 56" refers to one of processors 50, 52, 56 or any sub-combination or combination thereof. In any case, process 100 begins at step 102, wherein time-based charge detection data (CDD) for ion capture events (ITE) (alternatively, "ion measurement events" as described above) are collected and stored in a charge detection file (CDF), as described above. Figures 4A-4C As illustrated in the example. In one embodiment, charge detection data (CDD) for the first time window of the ITE is discarded, and CDD for the remaining duration is collected and stored in the CDF. In a non-limiting example of this embodiment, CDD for the first 2 milliseconds (ms) of the ITE is discarded, and the next 104.8 ms (corresponding to 2...) is collected. 18 (1 point) and store it in the CDF. In an alternative embodiment, more or fewer (or no) initial or front-end CDDs may be discarded, and the ITE can continue for any desired duration to collect any number of data points during the ITE duration.

[0098] Following step 102, process 100 proceeds to step 104, where processors 50, 52, and / or 56 are operable to calculate the “whole event” Fourier transform (FEFT) of all charge detection data in the CDF. In one embodiment, processors 50, 52, and / or 56 are operable to calculate the FEFT using conventional Fast Fourier Transform (FFT) techniques; however, in alternative embodiments, processors 50, 52, and / or 56 may alternatively or additionally use any conventional Fourier transform technique. Following step 104, processors 50, 52, and / or 56 are operable in step 106 to determine, based on the FEFT, whether no ions were captured in ELIT 14 during the ITE, i.e., whether the ion capture event failed to capture any charged particles in ELIT 14. For example, if the FEFT in step 104 fails to produce any frequency peaks, capturing only noise peaks, etc., making it impossible to determine a valid oscillation frequency and charge quantity pair from the FEFT, then processors 50, 52, and / or 56 determine that no charged particles were captured in ELIT 14 during the ion capture event (ITE). If so, the process proceeds to step 108, where processors 50, 52, and / or 56 are operable to discard the charge detection data (CDD) in the charge detection file (CDF), and process 100 loops back to step 102. In some alternative embodiments, step 108 may perform a process where processors 50, 52, and / or 56 are operable to analyze the CDD and / or FEFT in the CDF in more detail (before discarding the CDD) to determine whether the charge detection data (CDD) actually contains some identifiable charged particle measurement information. If such analysis concludes that the CDD does indeed contain identifiable charged particle measurement information, then, as indicated by the dashed arrow extending between steps 108 and 110, the corresponding FEFT is passed to step 110 for further processing; otherwise, process 100 loops back to step 102 as described above. A non-limiting example of this more detailed process for analyzing the CDD and / or FEFT in the CDF in step 108 is disclosed in WO2019 / 236140, the disclosure of which is expressly incorporated herein by reference in its entirety.

[0099] In any case, after step 108 or after the "No" branch of step 106, processors 50, 52, and / or 56 are operable in step 110 to analyze the FEFT, thereby determining and identifying each of the multiple ions captured (and measured) during ELIT 14 during ITE in a conventional manner, for example, from the frequency peaks in the FEFT. Subsequently, in step 112, processors 50, 52, and / or 56 are operable to (e.g., using FFT or other conventional Fourier transform determination techniques) calculate a series of short-time overlapped Fourier transforms (STFTs) of the charge detection data (CDD) in the sequentially stepped-through charge detection file (CDF) (i.e., the time-dependent sequentially stepped-through CDD). The number and size (i.e., width) of the STFTs can vary and can depend on one or more factors, examples of which may include, but are not limited to, the size of the CDF file (i.e., the duration of time), the size of the time-based CDD signal (i.e., the amplitude of (one or more) signals), the number of captured charged particles determined in step 110, and so on. In some embodiments, the STFTs are zero-padded to produce a file size equivalent to the file sizes of the CDD and FEFT.

[0100] Following step 112, process 100 proceeds to step 114, where processors 50, 52, and / or 56 are operable to process STFTs to track each of the plurality of captured charged particles throughout the ion capture event and determine the oscillation frequency (OFR) and charge quantity (CM) for each of the plurality of captured charged particles. For example, for each of the plurality of captured charged particles, at least some STFTs throughout the ITE will include the frequency peak of that charged particle at its fundamental frequency f0 within ELIT 14. For each of these plurality of captured charged particles, processors 50, 52, and / or 56 are exemplary operable in step 114 to determine the oscillation frequency (OFR) of that charged particle as the average of the fundamental frequency f0 of that charged particle across all STFTs containing f0 of that charged particle, and to determine the charge quantity (CM) as the average of the peak quantities of all such fundamental frequency f0. Processors 50, 52, and / or 56 are further exemplarily operable in step 114 to calculate, for each of the plurality of charged particles, the standard deviation of the oscillation frequency (SDOF) and the standard deviation of the charge (SDC) relative to all fundamental and kurtosis values ​​used to calculate the respective averages. Thus, step 114 yields the oscillation frequency (OFR) and charge (CM) pairs, along with their standard deviations (SDOF) and (SDC), for each of the plurality of captured charged particles. It will be understood that, in alternative embodiments, the analysis and / or calculations performed in steps 110-114 may alternatively be performed using other frequency domain analysis techniques, and in other still embodiments, such analysis and / or calculations may be performed using time domain analysis techniques. In any case, the result of step 114 will be the generation of the OFR, CM, SDOF, and SDC for each of the plurality of captured charged particles, and in step 114, processors 50, 52, and / or 56 exemplarily store the OFR, CM, SDOF, and SDC values ​​for each of these charged particles in an ion processing file (IPF).

[0101] Following step 114, process 100 proceeds to step 116, wherein processors 50, 52, and / or 56 are operable to perform a quality check on the values ​​of the oscillation frequency and charge quantity pairs (OFR and CM) stored in the ion processing file IPF. OFR and CM pairs deemed acceptable according to the quality check are exemplarily stored in the filter file FF, while OFR and CM pairs deemed unacceptable according to the quality check are exemplarily stored in the rejection file RF. In some embodiments, the standard deviation values ​​SDOF and SDC associated with each OFR and CM pair are also stored in the corresponding filter file FF or rejection file RF.

[0102] An example implementation of the quality check performed at step 116 is provided by Figure 6 The example shows, Figure 6A flowchart illustrating an example of a quality check is provided. In the illustrated embodiment, quality check 116 exemplarily begins at step 150, where processors 50, 52, and / or 56 select one of the OFR and CM pairs stored in the ion processing file IPF. Subsequently, at step 152, processors 50, 52, and / or 56 are operable to store the selected OFR and CM pair in the rejection file RF if the following conditions are met: (1) the ion is lost before the end of the ion capture event, and (2) the SDC (associated with the selected OFR and CM pair) is greater than a threshold standard deviation value TH1, or the SDOF (also associated with the selected OFR and CM pair) is greater than another threshold standard deviation value TH2. The filtering condition (1) is exemplarily performed for each charged particle identified in step 110 by tracking the corresponding charged particle across a series of short-time overlap FFTs and determining whether the frequency data of the charged particle exists in the entire ITE. In one embodiment, a “whole” capture event is exemplarily defined as follows, wherein charged particles are captured within ELIT 14 for at least a set amount of the total capture time of the capture event ITE (e.g., at least 90% of the total capture time of the capture event ITE). In other embodiments, the set time amount may be greater than or less than 90% of the ITE, and in still other embodiments, the set time amount may be 100% of the ITE; that is, a “whole” capture event corresponds to a complete ITE. Exemplarily, the filter condition (1) analysis may be performed by processors 50, 52 and / or 56 in step 152 by analyzing STFT data for the selected OFR and CM pairs as just described. Alternatively, the filter condition (1) analysis may be performed by processors 50, 52 and / or 56 in Figure 5 The process 100 is performed during step 114 of the STFT processing step. In some such embodiments, processors 50, 52, and / or 56 may be operable to set status indicators, such as flags or other indicators, having some type of pass / fail value, such that if the STFT data of the charged particle in question is present in a complete ITE, the flag or other indicator is set to "pass" (or other value), otherwise the flag or other indicator is set to "fail" (or other value). Figure 6 Step 152 of the quality check 116 can be performed simply by checking the status of the flags or other indicators associated with the selected OFR, CM pair to perform the filter condition (1) analysis.

[0103] Regarding filtering condition (2), trapped charged particles with similar oscillation frequencies may have overlapping f0 values ​​in the frequency domain, and the amount or degree of frequency overlap will affect the charge standard deviation value SDC. Because the charge of an individual charged particle in this group of trapped charged particles is determined in the frequency domain as the average of the peaks corresponding to the fundamental frequency value f0 of a series of STFTs, this charge determination becomes less reliable as the overlap between the oscillation frequencies of the trapped charged particles increases due to frequency peak congestion. Moreover, the greater the amount or degree of frequency overlap, for example, the larger the corresponding SDC value will be. In this regard, processors 50, 52, and / or 56 in step 156 consider charged particles with a charge standard deviation SDC greater than a threshold standard deviation value TH1 to have charge values ​​that cannot be precisely determined, and therefore the OFR, CM pairs of these charged particles are deemed unacceptable. The selection of the threshold TH1 will generally depend on several factors, including the expected accuracy of the charge value and / or other considerations.

[0104] Regarding the filtering condition (3), captured charged particles with more stable travel paths within ELIT 14 will generally have a lower standard deviation in their corresponding f0 (or OFR) than those with less stable travel paths. For example, in ELIT 14, highly stable charged particles will generally oscillate back and forth between ion mirrors M1 and M2 at a substantially constant oscillation frequency, while less stable and unstable charged particles may deviate from their originally constant oscillation frequency by deviating from their flight paths on or near the longitudinal axis 20 within ion mirrors M1, M2, and / or the charge detection cylinder CD. In this regard, processors 50, 52, and / or 56 consider charged particles with a standard deviation SDOF greater than a threshold standard deviation value TH2 to be unstable and therefore unacceptable in step 152. The selection of the threshold TH2 will generally depend on several factors related to the specific structure and operation of ELIT 14 and will generally be determined empirically.

[0105] In any case, OFR / CM pairs lost before the end of the ITE process, with a charge standard deviation SDC greater than TH1, or an oscillation frequency standard deviation SDO greater than TH2, are stored in the rejection file RF by processors 50, 52, and / or 56 in step 152, while all other OFR / CM pairs are stored in the filter file FF by processors 50, 52, and / or 56 in step 154. After step 154, processors 50, 52, and / or 56 are operable in step 156 to cycle back to step 150 until all OFR / CM pairs in the ion processing file IPF have been processed. Then, the quality check process 116 returns to... Figure 5 The process 100.

[0106] When charge detection information of multiple charged particles is captured and simultaneously measured in CDMS 10 as described above, the OFR, CM pairs included in the filter file FF after step 116 are those that are routinely used by processors 50, 52, and / or 56 (e.g., for one or more iterations of process 100) to calculate and represent the charged particle spectral distribution (i.e., ion mass-to-charge ratio (m / z), ion charge, ion mass, etc.) for the analyzed sample in step 124. However, in Figure 5 In an embodiment of process 100 shown, additional steps 118-122 are exemplary included after step 116, wherein processors 50, 52, and / or 56 are operable to perform any one or both of two different charged particle recovery processes, wherein certain OFR,CM pairs in the rejection file RF are processed and modified as described below, and then added to the filter file FF to provide a more complete and accurate representation of the charged particle spectral distribution of the analyzed sample. In particular, each process operation in these charged particle recovery processes involves OFR,CM pairs rejected in step 152 due to the filtering condition (2). In particular, for charged particles with similar oscillation frequencies and associated frequency overlap in the frequency domain, recovering any such previously rejected charged particles will yield a more accurate ion spectral distribution.

[0107] Now for reference Figure 5 The charged particle recovery steps 118-120 of process 100 shown herein include step 118, a preparation step in which OFR and CM pairs in the rejection file RF that meet certain criteria are selected for further processing by any one or both of two different charged particle recovery processes. For example, some embodiments of process 100 may include only charged particle recovery process 1 in step 120, and in such embodiments, preparation step 118 exemplaryly includes only the portion of step 118 in which the charged particle recovery file REC1 is filled from the rejection file RF filled in step 116. As another example, an alternative embodiment of process 100 may include only charged particle recovery process 2 in step 122, and in such embodiments, preparation step 118 exemplaryly includes only the portion of step 118 in which the charged particle recovery file REC2 is filled from the rejection file RF. Further embodiments of process 100 may include both charged particle recovery process 1 in step 120 and charged particle recovery process 2 in step 122, and in such embodiments, preparation step 118 exemplaryly fills both REC1 and REC2 from the rejection file RF. An exemplary embodiment of the charged particle recovery process 1 is as follows: Figure 7 An exemplary embodiment of the charged particle recovery process 2 is also shown in the form of a flowchart depicted in the example. Figure 11 The examples depicted are shown in the form of flowcharts, and both examples will be described in detail below.

[0108] As briefly described above, charged particle recovery process 1 and charged particle recovery process 2 operate on OFR, CM pairs in the rejected file RF due to the filtering condition (2); that is, SDC > TH1, corresponding to trapped charged particles having similar oscillation frequencies (possibly having overlapping f0 values in the frequency domain), i.e., having overlapping oscillation frequencies. In an embodiment of process 100 including step 120, the processors 50, 52, and / or 56 are exemplary operable in step 118 to store each OFR and CM pair in the rejected file RF that satisfies the following conditions in REC1: (1) the ion was trapped throughout the ITE, (2) SDC > TH1, (3) SDOF < TH2, and (4) F1 < OFR < F2. Conditions (1) and (3) are exemplary opposite to conditions (1) and (3) of step 152 of step 116 described above, while condition (2) is the same as condition (2) of step 116. Condition (1) of step 118 exemplary ensures that the data in the recovery file REC1 only includes OFR, CM pairs of those charged particles that were not lost during the ion trapping event (ITE), i.e., the charged particles trapped in the ELIT 14 during the "entire" trapping event. Condition (3) of step 118 exemplary ensures that the data in the recovery file REC only includes OFR, CM pairs of stable charged particles, the stable charged particles being those with a frequency standard deviation SDOF less than TH2, where TH2 exemplary has the same value as the value used in step 152 of process 116.

[0109] The condition (4) of step 118 exemplarily provides the selection of a frequency window that includes OFR, CM pairs whose oscillation frequencies OFR overlap with those of other charged particles in the recovery file REC1 within that window. In this regard, F2 exemplarily represents an oscillation frequency at which overlap between the oscillation frequencies of charged particles begins to occur. In one example implementation (which should not be considered as limiting in any way), F2 can be approximately 50 Hz; however, it will be understood that in other implementations, F2 can be greater than or less than 50 Hz. On the other hand, F1 exemplarily represents an oscillation frequency at which the oscillation frequencies of two charged particles overlap, but where the oscillation frequencies of these two overlapping charged particles are indistinguishable from each other, i.e., the oscillation frequencies are indistinguishable relative to each other. In one example implementation (which should not be considered as limiting in any way), F1 can be approximately 10 Hz; however, it will be understood that in other implementations, F1 can be greater than or less than 10 Hz. In one embodiment, processors 50, 52 and / or 56 may be operable in step 118 to include each OFR,CM pair having SDC>TH1 (and conditions (1) and (3)) and OFR within 50 Hz or less (F2) of another OFR,CM pair (the OFR value of which is distinguishable from the OFR value of the first OFR,CM pair) in REC1, and to exclude from REC1 each OFR,CM pair having SDC>TH1 (and conditions (1) and (3)) and OFR not within 50 Hz or less (F2) of another OFR,CM pair (the OFR value of which is distinguishable from the OFR value (F1) of the first OFR,CM pair).

[0110] In an embodiment of process 100 including step 122, processors 50, 52, and / or 56 are exemplarily operable in step 118 to store each OFR,CM pair in the rejection file RF that also satisfies conditions (1)-(3) of steps 118 but has an OFR value less than F1 in REC2. In one embodiment, processors 50, 52, and / or 56 may be operable in step 118 to include each OFR,CM pair having SDC>TH1 (and conditions (1) and (3)) and whose OFR is not within 50 Hz or less (F2) of another OFR,CM pair (whose OFR value is distinguishable from the OFR value of the first OFR,CM pair (F1)), as described above. It will be understood that processors 50, 52, and / or 56 may alternatively use other processing techniques to populate REC1 and / or REC2 in step 118. In any case, any procedure used to fill REC1 from the rejection file RF will store OFR,CM pairs in REC1 that satisfy conditions (1) and (3) of step 118, have oscillation frequencies that overlap with the oscillation frequencies of other OFR,CM pairs, and wherein the overlapping oscillation frequencies are distinguishable from each other. Any procedure used to fill REC2 from the rejection file RF will store OFR,CM pairs in REC2 that satisfy conditions (1) and (3) of step 118, have oscillation frequencies that overlap with the oscillation frequencies of other OFR,CM pairs, but wherein the overlapping oscillation frequencies are indistinguishable from each other.

[0111] Following the first execution of step 122 (in embodiments including it), or starting from the first execution of step 120 (in embodiments excluding step 122), the filter file FF is populated with the OFR,CM values ​​of an ion capture event (ITE). In some embodiments, process 100 proceeds directly from this point to step 124. In other embodiments, process 100 loops back to step 102 to process another ion capture event (ITE). In this way, the filter file FF may be exemplarily populated with the OFR,CM values ​​of one or any number of ion capture events (ITE) before proceeding to step 124. In any case, when process 100 proceeds to step 124, processors 50, 52, and / or 56 are exemplarily operable to determine and generate a spectral distribution from the OFR,CM pairs in the most recently updated filter file FF. As described above, the OFR values ​​of the OFR,CM pairs can be converted to charged particle mass-to-charge ratio values ​​(m / z) using Equation 1 above (where f0 = OFR). Alternatively or additionally, the mass value (m) of a charged particle can be calculated using the m / z value and the corresponding charge value CM.

[0112] Now for reference Figure 7 , showing Figure 5The flowchart of an example embodiment of the ion recovery process 1, step 120 of process 100. Figure 7 The example depicts the ion recovery process 120 and Figure 5 The process 100 described herein is similar and can be executed by processors 50, 52 and / or 56, as executively. Figure 7 The charged particle recovery process 120 shown begins at step 200, wherein processors 50, 52 and / or 56 are operable to select the OFR, CM pair (OFR and CM, respectively) in the charged particle recovery file REC1. R and CM R Subsequently, in step 202, processors 50, 52, and / or 56 are operable to collect the charge values ​​(i.e., charge magnitude values ​​CM) of all such OFR,CM pairs in the filtered file FF, where the OFR,CM pairs have an oscillation frequency OFR selected in step 200. R CM R The oscillation frequency OFR R Frequency window (e.g., F) W Within ) . In one embodiment, the frequency window F W An exemplary case is the same oscillation frequency at which the overlap of the oscillation frequencies of charged particles begins to occur, and such overlap does actually occur at frequencies below that, for example, 50 Hz; however, in alternative embodiments, F W It can have higher or lower values.

[0113] In any case, process 120 proceeds from step 202 to step 204, wherein processors 50, 52, and / or 56 are operable to randomly select a charge value CV from the charge quantity CM in a set of charge quantity values ​​collected in step 202. Subsequently, in step 206, processors 50, 52, and / or 56 are operable to modify the selected charge value CV to account for fluctuations caused by electrical noise. In the illustrated embodiment, for example, processors 50, 52, and / or 56 are operable in step 206 to account for such fluctuations by adding a noise value NV to the selected charge value CV; that is, CV = CV + NV. In one embodiment, NV is determined by processors 50, 52, and / or 56 from a Gaussian distribution whose standard deviation corresponds to the standard deviation of the charge value CV selected in step 204. Exemplarily, the noise value NV is a fraction of the elementary charge e and, in some embodiments, constitutes a small proportion. In an alternative embodiment, processors 50, 52, and / or 56 may be operable in step 206 to calculate the noise value NV according to other conventional noise analysis techniques, and / or modify the selected charge value CV using one or more other or additional mathematical operators, equations, or models. In any case, process 120 proceeds from step 206 to step 208.

[0114] In steps 208 and 210, processors 50, 52, and / or 56 are exemplarily operable to perform checks or analyses to ensure that the modified charge value CV determined in step 206 is reasonable. For example, in the OFR and CM data stored in the filter file FF, there may be different subfills of charged particles with similar or identical OFR oscillation frequency values ​​but different charge CM fillings, and in steps 208 and 210, processors 50, 52, and / or 56 exemplarily evaluate (in step 206) the modified charge value CV relative to the OFR selected in step 200. R CM R The charge CM R The rationality of this. In the illustrated embodiment, for example, processors 50, 52 and / or 56 are operable in step 208 to combine the modified CV (in step 206) with the CM. R The comparison is performed, and then in step 210, if CV and CM... R The difference between them does not exceed the charge threshold C TH If so, then accepting the modified CV is reasonable. For example, selecting C... TH This ensures that the modified charge value CV must match the charge value CM. R There are substantial differences to satisfy the inequality and proceed from the "yes" branch of step 210 to step 212. In an example implementation (which should not be considered restrictive in any way), C is chosen. TH For CM R Standard deviation (SDC) R twice that of C, i.e. TH =+ / -(2*SDC R However, it will be understood that in the alternative implementation, C... TH It can be greater than or less than CM R Standard deviation SDC R twice, and / or in C TH In the values, "+ / -" can be replaced by either "+" only or "-" only.

[0115] From the "Yes" branch in step 210, process 120 proceeds to step 212, where processors 50, 52, and / or 56 are operable to determine whether the OFR selected in step 200 has been exceeded. R CM RThe maximum number of attempts to assign a modified charge value CV. If not, process 120 loops back to step 204, where processors 50, 52, and / or 56 are operable to randomly select another charge value CV from the set of charge values ​​collected in step 202. Exemplarily, the maximum number of attempts to assign a charge value to a rejected charged particle will depend on the application and may depend on factors such as, for example, the total number of charge values ​​collected in step 202, the maximum expected analysis time for each OFR in the REC1 file, the maximum number of CM pairs, etc. In any case, if processors 50, 52, and / or 56 determine in step 212 that looping back to step 204 would exceed the maximum number of attempts, process 120 loops back to step 200, where the attempt to assign a charge value to the selected OFR is abandoned. R CM R The process of assigning charge values ​​and selecting different OFRs from the recovery file REC1. R CM R Yes, and it should be handled as just described.

[0116] If in step 210, the modified charge value CV and CM R The difference between them is less than C TH Then process 120 proceeds to step 214, wherein processors 50, 52 and / or 56 are operable to select the OFR in step 200. R CM R Correct CM R Replace with the modified charge value CV, and then obtain the updated OFR. R CM R (i.e., where CM) R The updated charge value (CV) is added to the filter file FF. After step 214, the process proceeds to step 216, where processors 50, 52, and / or 56 are operable to determine all OFRs in REC1. R CM R Check whether all have been processed. If not, process 120 loops back to step 200. If, in step 214, processors 50, 52, and / or 56 determine all OFRs in REC1... R CM R If all have been processed, then process 120 returns to... Figure 5 The process 100.

[0117] The updated filter file FF obtained from each execution of process 120 contains OFR and CM pairs for the initially acceptable charged particles (i.e., in Figure 5 and 6The steps of process 100 shown are those that are accepted and stored in the filter file FF in step 116, as well as the OFR, CM pairs that were initially rejected in REC1 that now have successfully assigned charge values.

[0118] The process 120 described above produces the desired result for ions with overlapping oscillation frequencies, where the oscillation frequency of each overlapping ion can be directly observed, i.e., where the oscillation frequencies of overlapping charged particles are distinguishable from each other (e.g., overlapping oscillation frequencies of approximately >10 Hz). However, for ions with overlapping oscillation frequencies, where the oscillation frequencies of each overlapping ion are too close to be directly observed (e.g., overlapping oscillation frequencies of approximately ≤10 Hz), process 120 cannot recover both the OFR and CM of such ions because it cannot distinguish their OFR values. Figure 8 This is illustrated by example, where, Figure 8 Figure a) shows the entire event FFT of two ions with an oscillation frequency interval of 50 Hz, resulting in two clearly distinguishable peaks 250 and 252, centered at approximately 1 kHz and 1.05 kHz, respectively. Figure 8 In b), the STFT of the time-domain signal is shown, where the same two ions produce corresponding peaks 256 and 258, also centered at approximately 1 kHz and 1.05 kHz, respectively; however, they are no longer baseline-distinguished (showing the beginning of oscillation frequency overlap). As the frequency interval between the two ions decreases, the peak overlap becomes more severe until, at a certain point, only a single peak is observed. Figure 8 This is illustrated exemplarily in c) and 8d), where the entire event FFT and corresponding STFT of two ions with oscillation frequencies spaced 10 Hz are shown. Figure 8 c) FFT and Figure 8 In the STFT of d), only single peaks 254 and 260 were observed, thus masking the fact that there are actually two ion peaks.

[0119] Overlap frequencies occur more frequently for features with high abundance in a mass distribution; therefore, discarding ions with overlap frequencies from the spectral distribution leads to distortion of relative abundance. Figure 5 Step 120 is illustrated in the example and in Figure 7 Process 120 in the example also illustrates ion recovery process 1, rescuing these ions by reassigning some of the discarded ions with charges using statistical methods, which in turn mitigates some distortion of the relative abundance. However, as the frequency difference between two adjacent ions decreases to and below the point where the oscillation frequencies of the two ions are indistinguishable from each other (e.g., ≤10 Hz), as... Figure 8The ion frequency peaks 254 and 260 in c) and 8d) are exemplarily shown, and neither the overall event FFT nor the STFT can distinguish the oscillation frequencies of these two ions. Figure 8 Between the frequency difference of a) and 8c), the respective frequency peaks 250 and 252 of the two ions have begun to merge, and this merging occurs as the difference between the frequency peaks decreases (e.g., decreases to and falls below 10 Hz). Figure 8 c) The FFT of a single frequency peak 254 is exemplarily depicted, with the center frequency shifted to a value similar to the mean of both. If the STFT is calculated for this data, as... Figure 8 Peak 260 in d) is exemplarily depicted, and the peak begins to broaden. Therefore, it is impossible to reliably determine from this data whether two ions with very similar frequencies were indeed captured. In this case, Figure 7 The exemplary process 120 shown may only recover a single ion, thus reducing the total number of ions. However, as described in detail below, the case where such ions produce two indistinguishable peaks can be identified by analyzing the beat frequency patterns derived from two ions moving in and out of phase with similar oscillation frequencies. A process employing this beat frequency pattern analysis can then be developed to recover charged particles that would otherwise be indistinguishable, having overlapping peak frequencies, for example, ≤10 Hz.

[0120] Now for reference Figure 9 (a) and (b) the two simulated ion signals 270 and 272 have slightly different frequencies and are initially 180° out of phase with each other. In the example shown, ion signal 270 has a frequency of 1 kHz and ion signal 272 has a frequency of 1.01 kHz, that is, they are 10 Hz out of phase. Figure 9 c) shows signal pattern 274 representing two signals 270 and 272 superimposed on each other, while Figure 9 d) shows the superimposed summation of two signals 270 and 272 to form a composite signal 276. If we assume the two ions carry the same charge (equal to 1 unit of charge), the detected signal will be 2 when both ions are simultaneously in the detection cylinder. The signal will be 1 when one ion is inside the cylinder and the other is outside, and 0 when both ions are outside. The signal moves in phase or out of phase relative to each other at a frequency equal to the difference in frequencies between the two ions. However, the actual signal has electrical noise, which masks the signal... Figure 9 The behavior shown in d) and, in order to overcome this noise, a series of STFTs can be derived as described above to reveal the interference between the two signals.

[0121] Figure 10 This shows the steps taken by the STFT window as it progresses. Figure 9A graph of the charge amount determined from the time-domain data, as described above. Point 278 is the STFT result varying with the STFT window number (only a selection of points is shown for clarity), and line 280 passing through point 278 represents the fit to the expected behavior (i.e., the absolute value of the sine function). The variation in charge indicates the beat frequency pattern resulting from the in-phase and out-of-phase shifts of the signals. At STFT window number 0, the two signals are completely in phase, resulting in the maximum charge, while for the minimum value (near STFT window number 39), the two signals are completely out of phase. The fit 280 of the expected behavior (the absolute value of the sine function) to the STFT value reveals the frequency difference between the two signals. For Figure 9 In the example shown, two frequencies, 1000 Hz and 1010 Hz, produce a beat frequency of 10 Hz. Using this method, it is possible to identify ions with oscillation frequencies that are close enough to be indistinguishable in the FFT frequency domain (e.g., ≤10 Hz) in order to accurately determine the difference between these frequencies.

[0122] If the two ions are initially in phase at the start of the 100 ms capture period, a frequency difference of 10 Hz will occur. Figure 10 The beat frequency pattern shown is 280. However, for actual signals, the two ions are not necessarily in phase at the start of the capture period. Therefore, Figure 10 The beat frequency pattern will be offset by an amount that reflects the phase difference at the start of the acquisition period. Furthermore, for frequency differences less than 10 Hz, only [the difference] will be visible during the acquisition period. Figure 10 This is part of the mid-beat frequency mode. Therefore, in order to identify cases where two trapped ions have overlapping frequencies that are indistinguishable in frequency space, it is necessary to identify the STFT charge values ​​that follow... Figure 10 The case of at least a portion (offset by an arbitrary amount) of the beat frequency pattern shown.

[0123] Now for reference Figure 11 , showing Figure 5 The flowchart of an example embodiment of the ion recovery process 2, step 122 of process 100. Figure 11 The example depicts the ion recovery process 122 and Figure 5 The process 100 described herein is similar and can be executably performed by processors 50, 52, and / or 56. Process 122 exemplarily uses the aforementioned beat frequency analysis to recover charged particles with overlapping oscillation frequencies, such that the oscillation frequency intervals are close enough to each other that direct observation of each oscillation frequency is impossible. Therefore, the resulting filtered file FF will include the original (i.e., without using...) Figure 5 and 11The charged particle recovery process in step 122 of process 100 will remove charged particle data from the filter file FF. The resulting filter file FF will also include data that is limited to single use due to the above-mentioned limitations. Figure 7 Additional data on charged particles that cannot be recovered by restoring ion recovery 120.

[0124] Figure 11 The process 122 illustrated in the example begins at step 300, wherein processors 50, 52, and / or 56 are operable to initially perform some tests on each OFR,CM pair stored in the charged particle recovery file REC2 to identify pairs with indistinguishable frequencies, and OFR,CM pairs that fail or otherwise satisfy these tests are removed from REC2. The OFR and CM pairs stored in the charged particle recovery file REC2 are those OFR and CM pairs from the rejection file RF whose oscillation frequency intervals are sufficiently close to each other to prevent direct observation of the oscillation frequency of each, and those that satisfy the above regarding... Figure 5 The other standard OFR and CM pairs described in step 118 of process 100. In the illustrated embodiment, the test in step 300 includes the following two tests on the charge quantity of the STFT to identify OFR / CM pairs with indistinguishable oscillation frequencies: 1) the STFT charge quantity value should change by at least X% as the STFT window sweeps through the time-domain data; and 2) the change in the STFT charge quantity value across ion capture events should exhibit parabolic (i.e., nonlinear) behavior. Tests 1) and 2) can be performed, exemplarily, in step 300 by reanalyzing the STFT data from step 112, or alternatively, can be performed during the STFT processing step in step 114 and retrieved from memory in step 300. In one embodiment, X is exemplarily 90; however, in alternative embodiments, X may be greater than or less than 90.

[0125] In any case, for any OFR,CM pair in REC2 via either 1) or 2), the following third test (3) is performed by the processors 50, 52, and / or 56 on the frequency of the STFT: if the frequency greater than Y% is within Z% of the OFR (or within Z Hz), then the OFR,CM pair is considered to originate from a single ion. This mainly occurs under the following conditions: the frequency difference between the two ions is less than 1 Hz, or more than two ions exist in the same small frequency range (exemplarily, in Figure 11In process 122 shown, only two ions with similar frequencies are considered. In one embodiment, Y is 90 Hz and Z is 0.75 Hz; however, it will be understood that in alternative embodiments, other values ​​may be used for either or both of Y and Z. In any case, if less than 90% of the STFT frequency for the OFR,CM pair is within 0.75 Hz of the Z Hz of the OFR, such that all three criteria of step 300 of process 122 are met, then the OFR,CM pair is identified as potentially having an oscillation frequency within 0.75 Hz. W Two ions within a certain range (e.g., approximately 1-10 Hz) are retained in the charged particle recovery file REC2. All OFR,CM pairs in the charged particle recovery file REC2 are processed in a similar manner, and all OFR,CM pairs retained in REC2 are assumed to represent two ions with indistinguishable oscillation frequencies as described above, and are further processed in steps 302-316 of process 122 as described below.

[0126] Process 122 proceeds from step 300 to step 302, wherein processors 50, 52, and / or 56 are operable to select one of the OFR / CM pairs retained in the charged particle recovery file REC2 in step 300. Subsequently, in step 304, processors 50, 52, and / or 56 are operable to determine the center frequencies of the two ions in the selected OFR / CM pair from the mode of the STFT frequency values ​​of the selected OFR / CM pair. Then, in step 306, processors 50, 52, and / or 56 are operable to fit a sine function to the STFT charge values ​​of the selected OFR / CM pair to determine the beat frequency mode frequency; that is, to determine the frequency difference between the two ions. The fitting performed in step 306 exemplarily requires the following quantities: the charge and frequency of the two ions, the phase difference, and the duration of the capture event (because sometimes ions may not survive the entire capture event). For small frequency differences, the charge STFT does not include the entire beat frequency mode. Assuming that the charges of the two ions are not significantly different from each other, and that the STFT charge data are exemplary in that they are normalized by optimizing the phase and frequency difference to minimize the root mean square bias before fitting a sinusoidal function, this method can reliably determine the frequency difference between two ions down to 1 Hz.

[0127] Following step 306, processors 50, 52, and / or 56 are operable in step 308 to halve the frequency difference determined in step 30, add the halved value to the center frequency (determined in step 304), and subtract the halved value from the center frequency to determine the corresponding oscillation frequencies OFR1 and OFR2 for each of the two ions. The two ions have the same charge CM, which is determined using process 100 as described above. In step 310, processors 50, 52, and / or 56 are operable to assign the charge CM to each of the two ions to form two oscillation frequency and charge pairs, OFR1, CM and OFR2, CM, and then replace the original OFR, CM pair (selected in step 302) in the charged particle recovery file REC2 with OFR1, CM and OFR2, CM. Subsequently, in step 312, processors 50, 52, and / or 56 are operable to determine whether all OFR, CM pairs retained in the charged particle recovery file REC2 in step 300 have been analyzed. If not, process 300 loops back to step 302 to select another OFR,CM pair; otherwise, process 300 proceeds from step 312 along the "yes" branch.

[0128] After step 312, the charge measurements of each pair of ions with the newly assigned oscillation frequencies remain poor, unchanged by steps 302-312 of process 122. However, with the oscillation frequency of each ion now newly assigned in the charged particle recovery file REC2, this provides a basis for using... Figure 7 The charged particle recovery process 120 performs post-analysis on such ions to provide sufficient information to correctly assign the charge amount to each OFR,CM pair in REC2. In some embodiments, process 122 therefore includes step 314 following the "yes" branch of step 312, wherein processors 50, 52 and / or 56 are operable to re-execute. Figure 7 Process 120 is used to restore the correct charge amount for each OFR, CM pair in file REC2 to the charged particles. When process 120 is re-executed in step 314, it will be understood that... Figure 7 Step 200 of process 120 will be modified to apply to REC2 (instead of as...) Figure 7 Each OFR in REC1 (described at step 200) R CM R The operation is performed and the OFR is updated by process 120. R CM R The filter file is added as described above in step 214 of process 120.

[0129] In an embodiment of process 100 including step 120, charged particles having overlapping oscillation frequencies differing by, for example, 10-50 Hz (otherwise rejected from the filter file FF due to high charge standard deviation) are now recovered and added to the filter file FF by performing ion recovery process 1 alone. In an embodiment of process 100 including step 122, charged particles having overlapping oscillation frequencies differing by, for example, 1-10 Hz (otherwise rejected from the filter file FF due to high charge standard deviation) are now recovered and added to the filter file FF by performing ion recovery process 2 alone. In an embodiment of process 100 including both steps 120 and 122, charged particles having overlapping oscillation frequencies differing by, for example, 1-50 Hz (otherwise rejected from the filter file FF due to high charge standard deviation) are now recovered and added to the filter file FF by performing both ion recovery processes 1 and 2. Therefore, performing either or both of ion recovery processes 1 and 2 improves the accuracy of the spectral distribution.

[0130] Example Example 1 Figure 7 One objective of the charged particle recovery process 158 illustrated in the example is to recover previously rejected and discarded charged particles by assigning appropriate charge values ​​to charged particles with overlapping oscillation frequencies, provided that the oscillation frequencies of the overlapping charged particles are distinguishable from each other. As a result, relative abundance can be quantified more accurately regardless of signal strength. L-glutamate dehydrogenase (GDH) was chosen as the first test case because it can be prepared in high concentrations, where higher-order oligomers can be distinguished and detected by CD-MS. Representative results are shown in... Figure 12 middle. Figure 12 In a), 12c), and 12e), spectra 400, 404, and 408 respectively represent failures. Figure 6 and 7 The process 158 processes the spectrum, that is, they represent the spectrum processed during execution. Figure 6 Step 156 follows the spectrum of data from the filtered file FF, and Figure 12 Spectra 402, 406, and 410 in 12d) and 12f) represent spectra processed by charged particle recovery process 158, respectively. Spectra are shown for average measurement rates of 16, 60, and 93 ions / second. Figure 12(From top to bottom). The measurement rate was defined as the total number of ions in the spectrum after the charged particle recovery process 158 divided by the measurement time. At 16 ions / s (both panels 12a and 12b), peaks at M, D, and T were present at 335 kDa, 670 kDa, and 1005 kDa, respectively, which were attributed to GDH hexamer (GDH6), dodecamer (GDH6)2, and octadecamer (GDH6)3. (GDH6) was the dominant substance in the spectrum, while the intensity of the GDH6 oligomer decreased monotonically. At the concentrations used here, the relative abundance of hexamers GDH6, (GDH6)2, and (GDH6)3 was approximately 4:2:1. For an average measurement rate of 16 ions / s, the implementation of the charged particle recovery process 158 resulted in an 8% increase in the number of ions and had a minor effect on the relative abundance.

[0131] At an average measurement rate of 60 ions / sec (panels 12c and 12d), the relative abundance in the uncorrected spectrum (panel 12c) was significantly affected, with the relative abundance of GDH6 decreasing relative to (GDH6)2. This illustrates the problem of simultaneously capturing many ions; the most abundant substances are most susceptible to frequency overlap and undercounting. After applying the charged particle recovery process 158, the corrected spectrum (panel 12d) contained 45% more ions than the uncorrected spectrum, and the oligomer ratio was restored to approximately 4:2:1.

[0132] At a measurement rate of 93 ions / sec, the uncorrected spectrum (panel 12e) shows that the relative intensities are strongly affected by frequency overlap. The relative intensities of the monomer, dimer, and trimer peaks are similar. After applying the charged particle recovery process 158, the number of ions in the spectrum (panel 12f) increases by 113%, and the relative intensities are similar to those in the spectrum measured at 16 ions / sec.

[0133] Figure 13 Showing for Figure 12 The dataset contains graphs of the relative intensities of monomers, dimers, and trimers against the measurement rates. Monomers, dimers, and trimers 450, 460, and 470 from the uncorrected file (i.e., the original filtered file after performing step 156) are compared to monomers, dimers, and trimers 452, 462, and 472 from the corrected file (i.e., after performing charged particle recovery process 158). These results indicate that after applying charged particle recovery process 158, the relative intensity of monomer 452 still decreases slightly with increasing measurement rate, while the relative intensities of dimers 462 and trimers 472 increase slightly. However, these changes are much smaller than those observed before applying charged particle recovery process 158.

[0134] For typical spectra, we usually collect 5000 ions. For CD-MS measurements capturing only a single ion, this would take approximately 25 minutes. At a measurement rate of 92 ions / second, spectra of 5000 ions can be collected in 1 minute. This is the fastest ion trap CD-MS measurement rate reported to date.

[0135] Example 2 Qβ virus-like particles (VLPs) have emerged as a versatile platform for vaccine development and drug delivery. Qβ is a single-stranded RNA phage containing major and minor capsid proteins (CPs), as well as a single mature protein bound to a 4217 nt genomic RNA (gRNA). The assembly of CPs around the gRNA is mediated by packaging signals, forming a pseudo-icosahedron. T =3 capsid. Like many viruses, CP can also assemble into VLPs. When CP is overexpressed, alternative morphologies are frequently observed, as is the case with Qβ, in addition to the typical icosahedral form. T In addition to the 3-capsid (with 180 CP), oblate and elongated spherical geometries were also observed in cryo-electron microscopy studies. The oblate form (150 CP) was obtained by... T =3 The geometric shape was generated by removing 5 hexamers, while the elongated spherical shape (210 CP) was generated by adding 5 hexamers. A smaller elongated spherical shape (132 CP) was also observed, and this was observed in a different study. T =1 particle (60 CP).

[0136] VLP was chosen to demonstrate the utility of charged particle recovery process 158 as an assembly intermediate for VLP. Uncorrected spectra 480 and 490 (i.e., without charged particle recovery process 158) were respectively... Figure 14 The spectra are depicted in a) and 14c), while the corrected spectra 482 and 492 (i.e., using the charged particle recovery process 158) are respectively in Figure 14 Depicted in b) and 14d). The Qβ VLP has a material distribution ranging from 1–3 MDa, exhibiting the previously measured range of heterogeneity. Figure 14a) Low ion flux measurements of the Qβ VLP are shown. Qβ is divided into five main ranges: 0–1.37, 1.37–1.87, 1.87–2.33, 2.33–2.83, and 2.83+. These values ​​are depicted in the table below. Even low ion fluxes can be reliably measured at 18 ions / s due to the increasing size and heterogeneity of the Qβ VLP, still far exceeding levels achievable with single-ion capture. As shown in the table, the 1.87–2.33 and 2.83–10.0 ranges are primarily affected, jumping from approximately 28% to 15% and from 11% to 25%, respectively, with increasing ion flux. These fluctuations are significantly mitigated after charged particle recovery correction, decreasing only to 26% for the 1.87–2.33 range from 30% and to 14% for the 2.83–10.0 range.

[0137] In this case, the charged particle recovery process 158 again showed little change in the relative abundance of other ranges and spectra, but produced 367% more ions in the final output, demonstrating how quickly measurements can be performed.

[0138] As expected, for high-throughput measurements, the intensity of the most prominent and homogeneous material decreased, and the sample trajectory only recovered to the abundance of low-throughput samples after applying a charged particle recovery process. It is also noteworthy that the total number of ions increased significantly, leading to... Figure 14 The ion flux of 120 ions / second shown in d) is expected. If a substance of this size (megadaltons) were to be separated by liquid chromatography, a similar ion flux would be anticipated, and based on these results, this signal intensity could be assessed in real time using multi-ion CD-MS.

[0139] Example 3 Recombinant adeno-associated virus (AAV) is a gene therapy vector that has received FDA approval for three therapeutic applications. CD-MS has proven to be a useful tool for characterizing AAV formulations. The relative abundance of empty AAV particles, particles containing partial genomes, and particles containing complete genomes (empty / partial / complete ratio) is a key quality attribute. Therefore, applying the charged particle recovery process 158 to high-throughput analysis of AAV is particularly important.

[0140] Spectra were measured at four different rates (8, 15, 27, and 43 ions / sec), and the ion count was integrated over five mass bands: empty 3.4 to 3.8 MDa (empty), 3.8–4.16 MDa (partial), 4.16–4.55 MDa (whole), 4.55–4.85 MDa (intermediate), and 4.85–5.6 MDa (overpacked). Figure 15a) shows that the relative abundance of the five bands was plotted as a function of the measurement rate before treatment by the charged particle recovery process 158; Figure 15 The mass bands 500, 504, 508, 512, and 516 identified in a) correspond to mass bands of 4.16–4.55, 4.85–5.6, 4.55–4.85, 3.4–3.8, and 3.8–4.16 MDa, respectively. A significant systematic decrease in the relative abundance of the most abundant substance (the entire peak at 4.3 MDa) is observed. The relative abundance of empty and overpacked peaks increases with measurement rate. However, the relative abundance of the partial and intermediate bands (between the three peaks) shows less variability. Figure 15 b) A graph showing the relative abundance control measurement rate after treatment via charged particle recovery process 158. Figure 15 In b), mass bands 502, 506, 510, 514, and 518 correspond to mass bands of 4.16–4.55, 4.85–5.6, 4.55–4.85, 3.4–3.8, and 3.8–4.16 MDa, respectively. The variation in relative abundance is far less than... Figure 15 It is not as obvious in the unprocessed data of a).

[0141] Example 4 In order to evaluate Figure 11 The performance of the charged particle recovery process 300 shown in the figure was re-analyzed using process 300. Figure 7 The charged particle recovery process 158 shown is an L-glutamate dehydrogenase (GDH) analyzed in Example 1. Figure 16 a) Shows the situation where there is no charged particle recovery process 158 or charged particle recovery process 300 (peaks 600, 602, and 604), with Figure 7 The charged particle recovery process 158 shown (peaks 700, 702, and 704), and the cases with Figure 11 The comparison shows CDMS data analyzed under the conditions of charged particle recovery process 300 (peaks 800, 802, and 804). Charged particle recovery process 300 increases the total number of ions in the spectrum by 64%, which in some embodiments increases the measurement rate from approximately 90 ions / sec to nearly 150 ions / sec. This means that in some embodiments, the time required to measure a typical spectrum of 5000 ions is reduced from 56 seconds to 34 seconds.

[0142] The original spectra 600, 602, and 604 show the distinctions that occur at high count rates. It should be noted that the most abundant substances in the original spectra are the least abundant relative to the spectra 800, 802, and 804 corresponding to process 300; therefore, the three mass peaks 600, 602, and 604 have approximately the same abundance in the original spectra. Applying charged particle recovery process 158 significantly increases the abundance of the two lowest mass peaks 700 and 702, while applying charged particle recovery process 300 further increases the relative abundance of the lowest mass peaks 800 and 802. To verify that charged particle recovery process 300 recovers the relative abundance ratios measured at low count rates, Figure 16 b) shows a comparison of the spectra measured at 150 ions / sec (peaks 900, 902, 904) with those measured at a much lower count rate, 16 ions / sec, for the same sample (peaks 906, 908, 910). The spectra measured at 16 ions / sec... Figure 16 b) is offset for easier comparison. The relative abundances are very similar.

[0143] While this disclosure has been illustrated and described in detail in the foregoing drawings and description, this should be considered illustrative rather than restrictive. It should be understood that exemplary embodiments thereof are shown and described only, and it is intended to protect all changes and modifications falling within the spirit of this disclosure. For example, it will be understood that the ELIT14 shown in the drawings and described herein is provided by way of example only, and the above concepts, structures, and techniques can be directly implemented in various alternative ELIT designs. Any such alternative ELIT design may, for example, include any one or a combination of two or more ELIT regions, more, fewer, and / or different ion mirror electrodes, more or fewer voltage sources, more or fewer voltage signals generated by one or more voltage sources, one or more ion mirrors defining an additional electric field region, and so on.

Claims

1. A method for operating a charge detection mass spectrometer, the charge detection mass spectrometer comprising an electrostatic linear ion trap (ELIT) or an orbital trap, the method comprising: (i) A plurality of ions generated from the sample are captured in the ELIT or orbital trap, such that during an ion capture event, the plurality of captured ions oscillate back and forth through or around a charge detector of the ELIT or orbital trap. (ii) Determine a set of oscillation frequency (OFR) and charge quantity (CM) pairs, each pair corresponding to a different ion among the plurality of captured ions. (iii) A filter file and a recovery file are formed from the set of OFR and CM pairs, the filter file comprising OFR and CM pairs from which the spectral distribution of the sample is derived, and the recovery file comprising OFR and CM pairs having oscillation frequencies that overlap with the oscillation frequencies of other OFR and CM pairs, wherein the overlapping oscillation frequencies in the recovery file are distinguishable from each other. (iv) For at least one OFR and CM pair in the recovery file, (a) the charge of the OFR and CM pair is modified based on the charge of one of the OFR and CM pairs in the filter file, wherein the oscillation frequency of the OFR and CM pair in the ELIT or orbital trap is within a frequency window of the oscillation frequency of the OFR and CM pair in the ELIT or orbital trap, and (b) the filter file is updated by adding the OFR and the modified CM pair to the filter file. (v) The spectral distribution is generated from the updated filtered file of OFR and CM pairs.

2. The method according to claim 1, further comprising: Execute (i)-(iv) multiple times, and then execute (v) using the updated filter file containing both OFR and CM pairs from the multiple executions of (i)-(iv).

3. The method according to claim 1 or claim 2, further comprising: During the ion capture event, charge detection data is collected from the detection of charges induced on the charge detector by the plurality of ions. Wherein, (ii) includes: analyzing the collected charge data to determine the set of oscillation frequency (OFR) and charge quantity (CM) pairs, and determining the charge quantity standard deviation for each of the OFR and CM pairs.

4. The method according to claim 3, wherein, (iv) Further includes: requiring the OFR and CM pairs in the filter file to have a charge standard deviation less than a first threshold, and requiring the OFR and CM pairs in the recovery file to have a charge standard deviation greater than the first threshold.

5. The method according to claim 1 or claim 2, further comprising: Perform (iv) for each of the OFR and CM pairs in the recovery file.

6. The method according to any one of claims 1 to 5, wherein, (iv) Includes: Collect the charge amount of all OFR and CM pairs in the filtered file whose oscillation frequencies in the ELIT or orbital trap are within the frequency window of the OFR and CM pairs in the recovered file, which are located within the oscillation frequencies in the ELIT or orbital trap. Randomly select one of the collected charge quantities, and The charge amount of the OFR and CM pair in the recovery file is modified depending on the randomly selected charge amount.

7. The method according to claim 6, wherein, Modifying the charge amount includes: The charge quantity is modified by adding a noise value to one of the randomly selected charge quantities from the collected charge quantities, and The modified charge values ​​are used to replace the charge values ​​of the OFR and CM pairs in the recovery file.

8. The method according to any one of claims 1 to 7, wherein, (ii) Further includes: excluding from the set of OFR and CM pairs all OFR and CM value pairs of ions that were not captured in the ELIT or orbital trap during the entire duration of the ion capture event.

9. The method according to any one of claims 3 to 8, wherein, (ii) Further includes: determining the standard deviation of the oscillation frequency for each of the OFR and CM pairs, Furthermore, (iii) further includes: excluding from each of the filtered file and the recovered file all OFR and CM pairs whose oscillation frequency standard deviation is greater than a second threshold.

10. The method according to any one of claims 9, wherein, (ii) further includes: The calculation sequentially steps through a series of short-time overlapped Fourier transforms (STFTs) of the collected charge detection data, and The standard deviation of the charge and the standard deviation of the oscillation frequency of the set of OFR and CM pairs, as well as the standard deviation of the charge of each pair of OFR and CM pairs, are determined from the STFT.

11. The method of claim 1, further comprising between (i) and (ii): During the ion trapping event, charge detection data is collected from the detection of charges induced on the charge detector by the plurality of ions, and The calculation sequentially steps through a series of short-time overlapped Fourier transforms (STFTs) of the collected charge detection data, wherein the STFTs include STFT frequency values ​​and STFT charge values. And among them, (ii) includes: determining the set of OFR and CM pairs from the STFT frequency value and the STFT charge value.

12. The method according to claim 11, wherein, The recovery files include a first recovery file. Furthermore, (iii) further includes: forming a second recovery file from the set of OFR and CM pairs, the second recovery file including OFR and CM pairs having oscillation frequencies that overlap with the oscillation frequencies of other OFR and CM pairs, wherein the overlapping oscillation frequencies in the second recovery file are indistinguishable from each other. Furthermore, the method further includes the following steps between (iv) and (v): (vi) For at least one OFR and CM pair in the second recovery file, (a) first and second OFR values ​​are determined based on the STFT charge and frequency values ​​of the OFR and CM pair, and (b) the second recovery file is modified by replacing the OFR and CM pair in the second recovery file with the first and second OFR and CM pairs, the first OFR and CM pair having the determined first OFR value and the charge of the OFR and CM pair, and the second OFR and CM pair having the determined second OFR value and the charge of the OFR and CM pair.

13. The method of claim 12, further comprising: Execute (vi) for each of the OFR and CM pairs in the second recovery file.

14. The method according to claim 12 or claim 13, further comprising the following steps after (vi) and between (iv) and (v): (viii) For each OFR and CM pair in the modified second recovery file, (a) the charge of the OFR and CM pair is modified based on the charge of one of the OFR and CM pairs in the filter file, wherein the oscillation frequency of the OFR and CM pair in the ELIT or orbital trap is within the frequency window of the oscillation frequency of the OFR and CM pair in the ELIT or orbital trap, and (b) the filter file is updated by adding the OFR and CM pair with the modified charge to the filter file.

15. The method according to any one of claims 12 to 14, wherein, (vi) This includes determining the first and second OFR values ​​based on the STFT charge and frequency values ​​of the selected OFR and CM pair: (1) Determine the center frequency from the mode of the STFT frequency values ​​of the selected OFR and CM pairs. (2) Fit a sine function to the STFT charge values ​​of the selected OFR and CM pairs to determine the frequency difference. (3) Add half the frequency difference to the center frequency to determine the first OFR value, and (4) Subtract half the frequency difference from the center frequency to determine the second OFR value.

16. The method according to any one of claims 12 to 15, wherein, Forming the second recovery file further includes processing the STFT, and the second recovery file contains only the following OFR and CM pairs: The change in the corresponding STFT charge value across the STFT exceeds a first percentage. The change in the corresponding STFT charge value across the STFT is non-linear in shape, and Less than a second percentage of the corresponding STFT frequencies across the STFT are within the frequency threshold of the oscillation frequency of the corresponding OFR and CM pair.

17. The method according to any one of claims 12 to 16, further comprising: Perform (iv) for each of the OFR and CM pairs in the first recovery file.

18. The method of claim 14, further comprising: Execute (i)-(iv), (vi) and (vii) multiple times, and then execute (v) using the updated filtered file containing OFR and CM pairs from the multiple executions of (i)-(iv), (vi) and (vii).

19. The method according to any one of claims 1 to 18, wherein, (v) includes: determining at least one of the mass-to-charge ratio and mass of the corresponding ion for each OFR and CM pair in the updated filter file, and including one or any combination of the corresponding mass, mass-to-charge ratio and charge (CM) for each OFR and CM pair in the updated filter file in the spectral distribution.

20. A method of operating a charge detection mass spectrometer, the charge detection mass spectrometer comprising an electrostatic linear ion trap (ELIT) or an orbital trap, the method comprising: (i) A plurality of ions generated from the sample are captured in the ELIT or orbital trap, such that during an ion capture event, the plurality of captured ions oscillate back and forth through or around a charge detector of the ELIT or orbital trap. (ii) During the ion trapping event, charge detection data is collected from the detection of charges induced on the charge detector by the plurality of ions. (iii) Determine a set of oscillation frequency (OFR) and charge quantity (CM) pairs from the collected charge detection data, each pair corresponding to a different ion among the plurality of captured ions. (iv) From the set of OFR and CM pairs, a filter file and a recovery file are formed, the filter file comprising OFR and CM pairs from which the spectral distribution of the sample is derived, and the recovery file comprising OFR and CM pairs having oscillation frequencies that overlap with the oscillation frequencies of other OFR and CM pairs, wherein the overlapping oscillation frequencies in the recovery file are indistinguishable from each other. (v) For at least one OFR and CM pair in the recovered file, (a) determine first and second OFR values ​​for the OFR and CM pair from the collected charge detection data, and (b) modify the recovered file by replacing the OFR and CM pair in the recovered file with the first and second OFR and CM pairs, the first OFR and CM pair having the determined first OFR value and the charge amount of the OFR and CM pair, and the second OFR and CM pair having the determined second OFR value and the charge amount of the OFR and CM pair. (vi) For each OFR and CM pair in the modified recovery file, (a) the charge of the OFR and CM pair is modified based on the charge of one of the OFR and CM pairs in the filter file, wherein the oscillation frequency of the OFR and CM pair in the ELIT or orbital trap is within a frequency window of the oscillation frequency of the OFR and CM pair in the ELIT or orbital trap, and (b) the filter file is updated by adding the OFR and CM pair with the modified charge to the filter file, and (vii) Generate the spectral distribution of the sample from the updated filter file.

21. The method of claim 20, further comprising: Execute (i)-(vi) multiple times, and then execute (vii) using the updated filtered file containing both OFR and CM pairs from the multiple executions of (i)-(vi).

22. The method according to claim 20 or claim 21, wherein, (v)(a) includes: Collect the charge amount of all OFR and CM pairs in the filtered file whose oscillation frequencies in the ELIT or orbital trap are within the frequency window of the OFR and CM pairs in the recovered file, which are located within the oscillation frequencies in the ELIT or orbital trap. Randomly select one of the collected charge quantities, and The charge amount of the OFR and CM pair in the recovery file is modified depending on the randomly selected charge amount.

23. The method according to claim 22, wherein, Modifying the charge amount includes: The charge quantity is modified by adding a noise value to one of the randomly selected charge quantities from the collected charge quantities, and The modified charge amounts are used to replace the charge amounts of the corresponding OFR and CM pairs.

24. The method according to any one of claims 20 to 23, wherein, (iii) Further includes: excluding from the set of OFR and CM pairs all OFR and CM value pairs of ions that were not captured in the ELIT or orbital trap during the entire duration of the ion capture event.

25. The method according to any one of claims 20 to 24, wherein, (iii) Further includes: The calculation sequentially steps through a series of short-time overlapped Fourier transforms (STFTs) of the collected charge detection data, wherein the STFTs include STFT charge values ​​and STFT frequency values, and The standard deviation of the oscillation frequency of each of the OFR and CM pairs is determined from the STFT charge and frequency values.

26. The method according to claim 25, wherein, (iv) Further includes: excluding from the filter file and from the recovery file all OFR and CM pairs whose oscillation frequency standard deviation is greater than a second threshold.

27. The method according to claim 25 or claim 26, wherein, (iii) Further includes: The calculation sequentially steps through a series of short-time overlapped Fourier transforms (STFTs) of the collected charge detection data, wherein the STFTs include STFT charge values ​​and STFT frequency values, and The standard deviation of the charge amount for each of the OFR and CM pairs is determined from the STFT charge and frequency values. Furthermore, (iv) further includes: requiring the OFR and CM pairs in the filter file to have a charge standard deviation less than a first threshold, and requiring the OFR and CM pairs in the recovery file to have a charge standard deviation greater than the first threshold.

28. The method according to any one of claims 20 to 27, further comprising: Execute (vi) for each of the OFR and CM pairs in the recovery file.

29. The method according to any one of claims 25 to 28, wherein, (iii) includes: determining the set of OFR and CM pairs from the STFT charge and frequency values, And wherein, (v)(a) includes: determining the first and second OFR values ​​of the OFR and CM pair based on the STFT charge value and the STFT frequency value.

30. The method according to any one of claims 25 to 29, wherein, (v)(a) includes determining the first and second OFR values ​​by depending on the STFT charge and frequency values ​​of the selected OFR and CM pair: (1) Determine the center frequency from the mode of the STFT frequency values ​​of the selected OFR and CM pairs. (2) Fit a sine function to the STFT charge values ​​of the selected OFR and CM pairs to determine the frequency difference. (3) Add half the frequency difference to the center frequency to determine the first OFR value, and (4) Subtract half the frequency difference from the center frequency to determine the second OFR value.

31. The method according to claim 29 or claim 30, wherein, Creating the recovery file further includes processing the STFT, and the recovery file contains only the following OFR and CM pairs: The change in the corresponding STFT charge value across the STFT exceeds a first percentage. The change in the corresponding STFT charge value across the STFT is non-linear in shape, and Less than a second percentage of the corresponding STFT frequencies across the STFT are within the frequency threshold of the oscillation frequency of the corresponding OFR and CM pair.

32. The method according to any one of claims 20 to 31, wherein, (vii) includes: determining at least one of the mass-to-charge ratio and mass of the corresponding ion for each OFR and CM pair in the updated filter file, and including one or any combination of the corresponding mass, mass-to-charge ratio and charge (CM) for each OFR and CM pair in the updated filter file in the spectral distribution.

33. A method of operating a charge detection mass spectrometer, the charge detection mass spectrometer comprising an electrostatic linear ion trap (ELIT) or an orbital trap, the method comprising: (i) A plurality of ions generated from the sample are captured in the ELIT or orbital trap, such that during an ion capture event, the plurality of captured ions oscillate back and forth through or around a charge detector of the ELIT or orbital trap. (ii) During the ion trapping event, charge detection data is collected from the detection of charges induced on the charge detector by the plurality of ions. (iii) Determine a set of oscillation frequency (OFR) and charge quantity (CM) pairs from the collected charge detection data, each pair corresponding to a different ion among the plurality of captured ions. (iv) From the set of OFR and CM pairs, a filter file and first and second recovery files are formed, the filter file comprising OFR and CM pairs from which the spectral distribution of the sample is generated, and the first and second recovery files each comprising OFR and CM pairs having oscillation frequencies that overlap with the oscillation frequencies of other OFR and CM pairs, wherein the overlapping oscillation frequencies in the first recovery file are distinguishable from each other, and the overlapping oscillation frequencies in the second recovery file are indistinguishable from each other. (v) For at least one OFR and CM pair in the first recovery file, (a) the charge of the OFR and CM pair is modified based on the charge of one of the OFR and CM pairs in the filter file, wherein the oscillation frequency of the OFR and CM pair in the ELIT or orbital trap is within a frequency window of the oscillation frequency of the OFR and CM pair in the ELIT or orbital trap, and (b) the filter file is updated by adding the OFR and the modified CM pair to the filter file. (vi) For at least one OFR and CM pair in the second recovery file, (a) first and second OFR values ​​of the OFR and CM pair are determined from the collected charge detection data, and (b) the second recovery file is modified by replacing the OFR and CM pair in the second recovery file with the first and second OFR and CM pairs, the first OFR and CM pair having the determined first OFR value and the charge amount of the OFR and CM pair, and the second OFR and CM pair having the determined second OFR value and the charge amount of the OFR and CM pair. (vii) For each OFR and CM pair in the modified second recovery file, (a) the charge of the OFR and CM pair is modified based on the charge of one of the OFR and CM pairs in the filter file, wherein the oscillation frequency of the OFR and CM pair in the ELIT or orbital trap is within a frequency window of the oscillation frequency of the OFR and CM pair in the ELIT or orbital trap, and (b) the filter file is updated by adding the OFR and CM pair with the modified charge to the filter file, and (viii) Generate the spectral distribution of the sample from the updated filtered file.

34. The method of claim 33, further comprising: Execute (i)-(vii) multiple times, and then execute (viii) using the updated filter file containing both OFR and CM pairs from the multiple executions of (i)-(vii).

35. The method according to claim 33 or claim 34, further comprising: Execute (v) for each of the OFR and CM pairs in the first recovery file.

36. The method according to any one of claims 33 to 35, further comprising: Execute (vi) for each of the OFR and CM pairs in the second recovery file.

37. The method according to any one of claims 33 to 36, wherein, (iii) Further includes: The calculation sequentially steps through a series of short-time overlapped Fourier transforms (STFTs) of the collected charge detection data, wherein the STFTs include STFT charge values ​​and STFT frequency values, and The standard deviation of the charge amount for each of the OFR and CM pairs is determined from the STFT charge and frequency values. Furthermore, (iv) further includes: requiring the OFR and CM pairs in the filtered file to have a charge standard deviation less than a first threshold, and requiring the OFR and CM pairs in each of the first and second recovery files to have a charge standard deviation greater than the first threshold.

38. The method according to any one of claims 33 to 37, wherein, (iv) Further includes: excluding from the set of OFR and CM pairs all OFR and CM value pairs of ions that were not captured in the ELIT or orbital trap during the entire duration of the ion capture event.

39. The method according to any one of claims 33 to 38, wherein, (iii) Further includes: The calculation sequentially steps through a series of short-time overlapped Fourier transforms (STFTs) of the collected charge detection data, wherein the STFTs include STFT charge values ​​and STFT frequency values, and The standard deviation of the oscillation frequency for each of the OFR and CM pairs is determined from the STFT charge and frequency values. Furthermore, (iv) further includes: excluding from the filter file and from each of the first and second recovery files all OFR and CM pairs whose oscillation frequency standard deviation is greater than a second threshold.

40. The method according to any one of claims 33 to 39, wherein, (v)(a) includes: Collect the charge amount of all OFR and CM pairs in the filtered file whose oscillation frequency in the ELIT or orbital trap is within the frequency window of the OFR and CM pairs in the first recovery file in the ELIT or orbital trap. Randomly select one of the collected charge quantities, and The charge amount of the OFR and CM pair in the first recovery file is modified depending on the randomly selected charge amount.

41. The method according to claim 40, wherein, Modifying the charge amount includes: The charge quantity is modified by adding a noise value to one of the randomly selected charge quantities from the collected charge quantities, and The modified charge values ​​are used to replace the charge values ​​of the OFR and CM pairs in the first recovery file.

42. The method according to any one of claims 33 to 41, wherein, (iii) Includes: The calculation sequentially steps through a series of short-time overlapped Fourier transforms (STFTs) of the collected charge detection data, wherein the STFTs include STFT charge values ​​and STFT frequency values, and The set of OFR and CM pairs is determined from the STFT charge and frequency values.

43. The method according to claim 42, wherein, (vi)(a) includes determining the first and second OFR values ​​by depending on the STFT charge and frequency values ​​of the selected OFR and CM pair: (1) Determine the center frequency from the mode of the STFT frequency values ​​of the selected OFR and CM pairs. (2) Fit a sine function to the STFT charge values ​​of the selected OFR and CM pairs to determine the frequency difference. (3) Add half the frequency difference to the center frequency to determine the first OFR value, and (4) Subtract half the frequency difference from the center frequency to determine the second OFR value.

44. The method according to claim 42 or claim 43, wherein, Forming the second recovery file further includes processing the STFT, and the second recovery file contains only the following OFR and CM pairs: The change in the corresponding STFT charge value across the STFT exceeds a first percentage. The change in the corresponding STFT charge value across the STFT is non-linear in shape, and Less than a second percentage of the corresponding STFT frequencies across the STFT are within the frequency threshold of the oscillation frequency of the corresponding OFR and CM pair.

45. The method according to any one of claims 33 to 44, wherein, (vii)(a) includes: Collect the charge amount of all OFR and CM pairs in the filtered file whose oscillation frequencies in the ELIT or orbital trap are within the frequency window of the OFR and CM pairs in the modified second recovery file in the ELIT or orbital trap. Randomly select one of the collected charge quantities, and The charge amount of the OFR and CM pair in the modified second recovery file is modified depending on the randomly selected charge amount.

46. ​​The method according to claim 45, wherein, Modifying the charge amount includes: The charge quantity is modified by adding a noise value to one of the randomly selected charge quantities from the collected charge quantities, and The charge amounts of the OFR and CM pairs in the modified second recovery file are replaced with the modified charge amounts.

47. The method according to any one of claims 33 to 46, wherein, (viii) includes determining at least one of the mass-to-charge ratio and mass of the corresponding ion for each OFR and CM pair in the updated filter file, and including one or any combination of the corresponding mass, mass-to-charge ratio and charge (CM) for each OFR and CM pair in the updated filter file in the spectral distribution.

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