Multiple compensation voltage fraction collection

Abstract

To provide a technology that uses a field asymmetric-waveform ion-mobility spectrometry (FAIMS) device.SOLUTION: A system for collecting compensation voltage fractions includes a field asymmetric-waveform ion-mobility spectrometry (FAIMS) device. A mass spectrometer injection gate is configured to inject, when in an open state, ions to be transmitted by the FAIMS device into a mass analyzer of a mass spectrometer. A controller circuit is configured to open the injection gate and apply a dynamic compensation voltage (CV) to an electrode of the FAIMS device. The dynamic CV may have a waveform shaped into a ramp, triangle, wavelet, sinusoid, or a different shape. The dynamic CV includes the CV associated with a plurality of adjacent CV fractions, and the FAIMS device is configured to transmit the plurality of adjacent CV fractions into the mass analyzer via the open injection gate so as to be scanned together by the mass analyzer in a single experiment.SELECTED DRAWING: Figure 5B

Description

[Technical field]

[0001] The field is electric field asymmetric waveform ion mobility spectrometry. [Background technology]

[0002] Field asymmetric waveform ion mobility spectrometry Asymmetric-waveform ion-mobility spectrometry (FAIMS) devices can be used in conjunction with mass spectrometers to transmit different gas-phase ion populations to the mass spectrometer at each compensation voltage (CV) setting. The CV setting refers to the direct current (DC) voltage applied to the electrodes of the FAIMS device. This gas-phase fractionation occurs on the millisecond time scale within the ion source housing and is integrated into the scan matrix of the mass spectrometer. In a typical experiment, the CV is set within a range of voltages that transmits multiply charged ion currents, generally between -30V and -100V. This results in an enhanced signal-to-noise mass spectrum, after which the distribution of individual ion species can be examined in post-acquisition software. Unique identifications are performed at each CV within this range. However, ions that are not transmitted at the CV setting are lost at the FAIMS electrodes and cannot be identified. This generally results in fewer ion species being identified in a single CV experiment compared to when FAIMS is not employed. Thus, a technique is needed to increase the number of ion species identified during each scan for mass spectrometers that utilize FAIMS devices. Summary of the Invention

[0003] According to one aspect of the disclosure, a system includes a FAIMS device configured to receive ions from an ionization source configured to receive a sample and generate ions from the sample, and an injection gate configured to inject ions transmitted by the FAIMS device into a mass analyzer when in an open state. The system also includes a controller circuit configured to open the injection gate and apply a dynamic compensation voltage to an electrode of the FAIMS device while the injection gate is open. Other embodiments of this aspect include corresponding computer systems, apparatus, and computer programs recorded on one or more computer storage devices, each configured to perform the operations of the method.

[0004] The system may include an ionization source and a mass analyzer, the mass analyzer may include a mass analyzer and an injection gate. The time-varying voltage waveform may include a ramp pattern, a triangular pattern, a sawtooth pattern, and / or a wavelet pattern. The mass analyzer may be configured to collectively scan the ions of the multiple CV fractions in a single scan, and the controller circuit may be configured to generate a mass spectrum representing the ions of the multiple CV fractions based on the scan data received from the mass analyzer. The controller circuit may be configured to apply a dynamic CV with the time-varying voltage waveform during an opening period of the injection gate. The CV fraction associated with the multiple CVs may include adjacent CV fractions. The FAIMS device may be configured to receive the multiple CV fractions from the ionization source and transmit one of the CV fractions in response to application of the associated CV. The dynamic CV may include a CV associated with the CV fraction such that two or more of the CV fractions are transmitted while the injection gate is open. In some examples, the sample is a biological sample, and the controller circuit may be further configured to transmit the mass spectrum to software after acquisition to determine the peptide distribution of the biological sample.

[0005] According to another aspect of the disclosed technique, a method includes introducing ions from an ion source into a FAIMS device, opening an injection gate of a mass analyzer coupled to an outlet of the FAIMS device, and dynamically varying a CV applied to an electrode of the FAIMS device while the injection gate is open to transmit ions from the FAIMS device into a mass analyzer of the mass analyzer. The method may also include scanning the transmitted ions in a single scan with the mass analyzer. Scanning the transmitted ions may further include collectively scanning ions of the first and second groups of ions in a single scan with the mass analyzer, and the method may further include receiving data from the mass analyzer including results of the single scan with the controller, and generating a mass spectrum representative of the ions of the first and second groups of ions based on the received data. The CV applied to the electrodes while the injection gate is open may have a waveform that begins with an initial voltage, ends with a final voltage, and varies between the initial voltage and the final voltage. The initial voltage and the final voltage are the same value. The waveform is shaped to correspond to a single cycle of a triangular, sawtooth, or wavelet waveform. The initial and final voltages have different values. The waveform has a ramp pattern. The ions introduced into the FAIMS device may include at least a first group of ions and a second group of ions, the first group of ions may include ions that are transmitted by the FAIMS device when a CV within a predetermined range of the first CV is applied, and the second group of ions may include ions that are transmitted by the FAIMS device when a CV within a predetermined range of the second CV is applied, the first and second CVs having different values ​​from each other, and varying the CV applied to the electrodes while the injection gate is open may include continuously varying the CV over a range of values ​​including the first CV and the second CV to cause transmission of ions in the first and second groups of ions into the mass analyzer while the injection gate is open.The ions introduced into the FAIMS device may further include one or more additional groups of ions, ions of each additional group of ions being transmitted by the FAIMS device upon application of a respective associated CV, and the range of values ​​over which the CV is varied while the injection gate is open may further include CVs associated with the additional groups of ions, thereby resulting in transmission of the additional groups of ions into the mass spectrometer while the injection gate is open.

[0006] According to another aspect of the disclosed technology, a method for identifying peptides in a biological sample includes receiving ions generated by an ionization source from the biological sample in a FAIMS device. The ions may include a plurality of groups of ions, each group of ions having an associated CV that, when applied to an electrode of the FAIMS device, causes the group of ions to be transmitted by the FAIMS device. The method also includes dynamically varying a CV applied to an electrode of the FAIMS device over a range of DC voltages, the range including a CV for at least two of the groups of ions. The method also includes opening an injection gate of a mass spectrometer while applying the varying CV to the electrode, resulting in transmission of the at least two groups of ions into a mass analyzer of the mass spectrometer. The method may also include collectively scanning the at least two groups of ions in a single scan with the mass analyzer. In some example methods, the controller generates a mass spectrum based on the results of the single scan, the generated mass spectrum representing the at least two groups of ions. In some example methods, the mass spectrum is interrogated using post-acquisition software to determine the peptide distribution of the biological sample. In some example methods, the DC voltage in the range is greater than or equal to -100V and less than or equal to 0V. Opening the injection gate of the mass analyzer while applying the varying CV to the electrodes may include maintaining the injection gate of the mass analyzer open while dynamically varying the CV applied to the electrodes over a range of DC voltages. In some example methods, the range of DC voltages is a first range, and the method may include performing a single scan of the at least two ion groups, then synchronously opening the injection gate of the mass analyzer and continuously varying the CV applied to the electrodes over a second range of DC voltages, the second range including CVs associated with at least two other ion groups of the plurality of ion groups, thereby resulting in transmission of the at least two other ion groups into the mass analyzer. In some example methods, the single scan is a first single scan, the mass spectrum is a first mass spectrum, and the method includes collectively scanning the at least two other ion groups in a second single scan with the mass analyzer.The controller can generate a second mass spectrum representative of the at least two other groups of ions based on results of the second single scan, and combine the first mass spectrum and the second mass spectrum into a third mass spectrum representative of the at least two groups of ions and the at least two other groups of ions. The method can further include examining the third mass spectrum with post-acquisition software to determine a peptide distribution within the at least two groups of ions and the at least two other groups of ions.

[0007] According to another aspect of the disclosed technology, one or more non-transitory computer readable media include computer executable instructions for causing an apparatus to perform operations including: introducing ions from an ion source into a FAIMS device, opening an injection gate of a mass spectrometer coupled to an outlet of the FAIMS device, and applying a dynamic CV to an electrode of the FAIMS device while the injection gate is open to transmit at least two adjacent CV fractions of ions from the FAIMS device into a mass analyzer of the mass spectrometer.

[0008] According to another aspect of the disclosed technology, a method for operating a FAIMS device includes operating the FAIMS device in a robustness mode in which a CV applied to an electrode of the FAIMS device is dynamically varied within a first voltage range associated with the transmission of only one CV fraction during a single opening period of an injection gate of a mass spectrometer. The method also includes operating the FAIMS device in a coverage mode in which the coverage mode includes dynamically varying the CV applied to the electrode within a second voltage range associated with the transmission of two or more CV fractions during a single opening period of an injection gate.

[0009] The foregoing and other objects, features, and advantages of the disclosed technology will become more apparent from the following detailed description which proceeds with reference to the accompanying drawings. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram of a mass spectrometer including a FAIMS device in which dynamic CV is applied to the FAIMS electrodes to increase ion identification in each scan. [Figure 2A] FIG. 1 is a graph of ionic strength versus applied CV showing the range of CVs that result in the identification of multiply charged peptides. [Figure 2B] FIG. 1 is a graph of ionic strength versus applied CV showing the loss of ions to the FAIMS electrode, making it impossible to identify specific ions during a static CV experiment. [Figure 2C] 1 is a graph of ionic strength vs. applied CV, showing the results of an approach in which multiple discrete CVs are applied to the FAIMS electrode. [Figure 2D] 1 is a graph of ionic strength vs. applied CV, showing the results of an approach in which multiple discrete CVs are applied to the FAIMS electrode. [Diagram 3] 1 shows HeLa chromatograms generated from scans performed by a mass spectrometer incorporating a FAIMS device at different CV values. [Figure 4] A graphical representation of the proteins, peptides, and peptide spectral matches (PSMs) identified within each CV fraction of the scan results shown in FIG. 3 is shown. [Figure 5A] A cross-sectional view of each of the FAIMS devices during application of three different static CVs is shown with associated graphs of transmitted ion intensity. [Figure 5B] 1 shows a graph comparing the transmitted ion intensities of three static CV scans to the transmitted ion intensities of three dynamic CV scans. [Figure 6] 1 is a flowchart illustrating various methods according to various disclosed examples. [Figure 7] FIG. 1 shows a cross-sectional view of a FAIMS device during application of a dynamic CV with a ramp waveform. [Figure 8] FIG. 1 shows a cross-sectional view of a FAIMS device during application of a dynamic CV with a triangular waveform. [Figure 9]FIG. 1 shows a cross-sectional view of a FAIMS device during application of a dynamic CV with a wavelet waveform. [Figure 10] 1 is a flowchart illustrating various methods according to various disclosed examples. [Figure 11] 1 is a flowchart illustrating various methods according to various disclosed examples. [Figure 12] 1 illustrates a generalized example of a suitable computing environment in which the described innovations may be implemented. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] FAIMS Overview FAIMS devices can be used with mass spectrometers, such as hybrid and tribrid mass spectrometers, to improve the identification rate of ions in a sample (e.g., the identification rate of peptides in a biological sample). Typically, FAIMS devices are located between an ionization source and a mass spectrometer and perform gas-phase ion separation based on ion mobility. In particular, FAIMS devices implement differential ion mobility, where the electric field strength changes many times during the ion's residence in the device. FAIMS devices include two electrodes with a gap between them. In some examples, the electrodes are cylindrical electrodes, one electrode disposed or positioned within the other electrode. Alternatively, FAIMS devices can have a "flat" shape, where the two electrodes form parallel plates, or some other shape / relative electrode arrangement.

[0012] As an overview of FAIMS device operation, an RF DC field is applied between two electrodes, and gas phase ion separation occurs in the gap between the two electrodes. RF offsets the ions entering the cell, discharging them toward one of the electrodes. A DC component of the field, referred to as CV, is applied to compensate for that phenomenon. As explained further below, different CV values ​​are applied to the FAIMS electrodes to compensate for different ion species. When a given CV is applied to the FAIMS electrodes, the ions that are transmitted by the FAIMS (rather than ejected toward the electrodes) may be referred to as the "CV fraction" associated with that CV. In some cases, ions in the CV fraction for a given CV may also be transmitted by the FAIMS, albeit in lower amounts, at values ​​within a certain range of that CV (e.g., 20V).

[0013] An exemplary system 100 for analyzing multiple CV fractions in a single scan is shown diagrammatically in FIG. 1. A sample 102, such as a biological sample containing one or more peptides, is introduced into an ion source 104. The ion source 104 ionizes the material under analysis (e.g., sample 102) by removing or adding charge-carrying entities (e.g., hydrogen nuclei or electrons) from the material to provide the material with a positive or negative charge. This results in the formation of ions from the ionization of the sample 102. The ion source 104 may be of the electrospray ionization (ESI) type, or may alternatively utilize another suitable ionization technique, including atmospheric-pressure chemical ionization (APCI) or atmospheric pressure photoionization (APPI). Ionization of the sample typically produces both singly and multiply charged ions.

[0014] The ions generated by the ion source then enter the FAIMS device 108. In the illustrated example, the FAIMS device 108 includes a FAIMS cell with cylindrical inner and outer electrodes having radially opposed surfaces that define an annular separation region ("analysis gap") between which ions are transported. This type of FAIMS cell geometry, shown in cross section in Figures 5A and 7-9, may be generally referred to as a "side-to-side FAIMS cell," with the longitudinal axes of the inner and outer electrodes (axes of the cylindrical surfaces facing out of the page) oriented transversely to the general direction of ion flow. However, the FAIMS cell may also have the flat plate geometry discussed above, or another geometry / relative electrode arrangement, without departing from the scope of this disclosure. The design and principles of operation of FAIMS cells and other ion mobility spectrometry devices have been extensively described elsewhere in the art and will therefore not be described in detail herein. See, for example, U.S. Patent No. 6,639,212 to Guevremont et al., U.S. Patent No. 10,325,767 to Prasad et al., and U.S. Patent No. 11,293,898 to Prasad et al., which are incorporated herein by reference. Briefly, a carrier gas flows with ions through a separation region from an entrance orifice of the FAIMS device to an exit orifice of the FAIMS device. Ion separation is performed in the separation region (analytical gap) of the FAIMS device by applying an asymmetric waveform having a peak voltage (DV) and a CV to one of the inner or outer electrodes. The values ​​of CV and DV are set to allow selected ion species to transmit through the separation region. Other ion species with different relative values ​​of high and low field mobilities migrate to the surface of one of the electrodes and are neutralized.

[0015] The selected ions exit the FAIMS device through an exit orifice and pass through a small gap that separates the FAIMS device from the mass spectrometer 116. While most of the carrier gas is exhausted through the gap at atmospheric pressure, the ions are electrostatically guided through an orifice in the mass spectrometer or through a capillary inlet or ion transport tube into at least one reduced pressure chamber 118 of the mass spectrometer 116. The at least one reduced pressure chamber 118 may be evacuated by a vacuum port. From the at least one reduced pressure chamber 118, the ions are transferred (e.g., through an orifice in a skimmer) into a high vacuum chamber 120 that is maintained at a lower pressure (typically about 100 mTorr) than the reduced pressure chamber 118. The high vacuum chamber 120 may be evacuated by a turbo pump or similar high vacuum pump via a vacuum port.

[0016] Ions transferred into the high vacuum chamber may be focused or guided through an electrical injection gate 122 into a mass analyzer 124 for mass analysis. In some examples, the injection gate 122 is part of an ion optics assembly that includes various electrodes forming an ion lens, an ion guide, an ion gate, a quadrupole or octapole rod set, etc. When a closing voltage is applied to the injection gate, ion transmission into the mass analyzer is blocked. The closing voltage may be pulsed down to a lower opening voltage for a certain amount of time (referred to herein as the "opening period" of the injection gate) to allow ions to enter the mass analyzer. The mass analyzer 124 may be implemented as any one or combination of conventional mass analyzers, including (but not limited to) a quadrupole mass analyzer, an ion trap, or a time-of-flight analyzer. Although the mass analyzer 124 is shown in the high vacuum chamber 120, in other examples it may be located elsewhere. After the ions transmitted through the open injection gate to the mass analyzer are analyzed, a controller 126 in communication with the mass analyzer generates data based on the analysis that can be used to generate a mass spectrum.

[0017] Limitations of a typical FAIMS experiment In a typical FAIMS experiment, only one CV value is applied during the time that the mass spectrometer injects ions into the mass analyzer (e.g., during a single opening period of the injection gate). The ions associated with this single CV fraction fill the mass analyzer and produce a mass spectrum with reduced chemical interferences. However, ions that do not transmit at that CV setting are lost to the FAIMS electrodes and cannot be identified. As a result, a single CV experiment may result in the same number of identified ion species, or perhaps even fewer identified ion species, compared to the same experiment performed with a mass spectrometer without FAIMS.

[0018] In the example shown in FIG. 2A, the complete cohort of multiply charged peptides is transmitted over a CV range of approximately 50 V (-30 V to -80 V). During proteomics experiments, the CV can be set within the range of -30 V to -80 V to generate enhanced signal-to-noise spectra for subsequent interrogation by post-acquisition software to determine individual PSM, peptide, and protein distributions. However, in other examples, multiply charged peptides may be transmitted over a different CV range (e.g., -30 to -100 V). In contrast, singly charged peptides are typically transmitted in the range of 0 V to -30 V, and any multiply charged peptides transmitting in this range tend not to be analytically valuable.

[0019] Using standard FAIMS parameters, a single CV setting transmits ion current over approximately 20V of CV. The resulting CV fractions transmitted by FAIMS exclude undesired singly charged ions from different CV regions. However, the CV fractions consisting of the remaining 75% of peptide ion current are also excluded. For example, as shown in FIG. 2B, a single CV setting of -55V transmits ion current over a range of approximately -45V to -65V, while excluding CV fractions associated with compensation voltages below approximately -65V and above approximately -45V. Specifically, ion detection peak 202 represents ions transmitting at a CV of -55V. The majority (e.g., 100%) of these ions may be transmitted by the FAIMS device during an experiment. In contrast, ion detection peaks 204 and 206, which are more attenuated than peak 202, represent ions transmitting at -60V and -50V, respectively. Only a portion (e.g., 50%) of the ions in ion detection peaks 204 and 206 are transmitted during an experiment using a static CV setting of -55V because many are lost due to having different differential mobilities, inappropriate initial energies or velocities, etc. As shown, a static tolerance curve 208 for the experiment can correspond to an envelope encompassing peaks 202, 204, and 206. In this case, static tolerance curve 208 has a tolerance width of approximately 20V, as shown at 210.

[0020] In an attempt to increase the amount of ion species (e.g., peptides) identified, multiple scans (also referred to as "runs" or "experiments") can be performed by the mass analyzer, each scan utilizing a different static CV applied to the FAIMS electrodes. Such an operation can also be described as "scanning through multiple CV values" or "sweeping the CV". The results of scans of ions transmitted at different static CVs can then be processed together, as shown in Figures 3 and 4. In particular, Figure 3 is a graph of a HeLa chromatogram digestion run at different static CV values. The CV values ​​for the experiment were selected in the range of -30V to -100V. During a typical proteomics experiment, the static CV is set within the range of voltages that transmit multiply charged peptide ion currents, generally -30V to -100V. Because only one CV fraction can be analyzed per single scan, the graph shown in Figure 3 includes mass spectra from multiple scans plotted on the same plot. These mass spectra can then be interrogated by post-acquisition software to determine individual PSM, peptide, and protein distribution among the analyzed ions, as shown in Figure 4.

[0021] Similarly, FIG. 5A shows the results of three separate scans, with reference to three ion groups with different CV optima. Although the term "ion group" is used in this example, each ion group can alternatively be referred to as an ion type, ion class, ion family, or CV fraction. Each ion group can include a large number of ion species, for example hundreds or thousands of ion species. An example of an ion species is a peptide. However, other ion species may be analyzed without departing from the scope of the present disclosure.

[0022] As shown in Figure 5A, ions from ion group 1 transmit through the FAIMS electrode at a CV of -65V, while ions from ion groups 2 and 3 are undercompensated at this CV and collide with the outer electrode before reaching the outlet. Thus, the resulting mass analysis shown consists of ions only from ion group 1. When the CV is increased to -60V, as shown in Figure 5A, ions from ion group 1 are overcompensated and discharge onto the inner electrode. Ions from ion group 2 are properly compensated and therefore transmitted, while ions from ion group 3 remain undercompensated and collide with the outer electrode. When the CV is increased to -55V, as shown in Figure 5A, ions from ion group 3 transmit, while the rest of the ions are overcompensated and lost. In each of the above cases, the associated graphs of transmitted ion intensity show the transmission of only the properly compensated ion type, without any chemical background contributed by the other ion types. As shown in Figure 5A, the graph of relative ion intensity resulting from the application of a static CV has a shape similar to a Gaussian curve.

[0023] In another example, the FAIMS device can internally switch (e.g., apply consecutively) multiple static CVs within the same scan so that ions can be filtered through the FAIMS device according to their relative mobility. In such an approach, if the static CVs applied do not include a CV associated with a given ion type, the FAIMS device effectively acts as a filter for that ion type. When switching CVs internally, it is important to select voltages that are well spaced apart so that overlap of the transmitted gas phase portions is limited. For example, a 3CV approach using CV fractions of -40V, -55V, and -70V introduced to the mass analyzer in a loop during a single scan is shown in FIG. 2C. Introducing more CV fractions, such as the CV fractions associated with the five CVs shown in FIG. 2D, provides even more coverage of this region. However, there is a timing delay associated with setting the CVs, making it prohibitive to set more than three additional static CVs in a single scan.

[0024] To overcome some of these shortcomings, techniques are disclosed herein for alternatively applying dynamic CVs during a single experiment. In an example of the disclosed technique discussed further below, the CV applied to the FAIMS electrodes can be dynamically varied through a single opening period of the injection gate of the mass spectrometer. Ions of adjacent CV fractions can thereby be transmitted to the mass analyzer of the mass spectrometer and analyzed collectively in a single scan. The disclosed method can be applied to systems for mass analysis utilizing FAIMS devices or other types of IMS devices. When used with a FAIMS device, the exemplary technique described below can enable a user to obtain additional ion species identifications from CV fractions directly adjacent to the original CV setting via a single scan. Furthermore, multiple such scans can be performed to more efficiently collect ion currents across the entire CV range.

[0025] Exemplary Techniques for Combining Adjacent CV Fractions in a Single Scan Described herein is a technique for varying the CV applied to the FAIMS electrodes, combining ions of adjacent CV fractions, and transmitting these ions to be analyzed in a single scan of a mass analyzer. As mentioned above, FAIMS has traditionally been used to exclude ions from a given ion pool and probe deeper within that pool. The technique disclosed herein may therefore appear counterintuitive as it intentionally reduces the resolution of the separation technique by putting more than one CV fraction into the mass analyzer per scan. However, the large storage capacity of state-of-the-art ion trap devices used in mass spectrometers (e.g., those described in U.S. Pat. No. 11,293,898 to Prasad et al.) in combination with the decaying ion current associated with fraction techniques allows adjacent CV fractions (e.g., in data-dependent proteomics experiments) to be combined to increase the number of targets. Employing the proposed technique allows the user to continue to enjoy the benefits of FAIMS electrodes that keep the mass spectrometer clean, with the added benefit of additional ion species identification from CV fractions directly adjacent to the original setup. In addition, users who wish to increase the total ion species coverage of their sample can use multi-fraction loading to more efficiently collect ion current across the entire CV range (e.g., as discussed below with reference to FIG. 10).

[0026] FIG. 5B shows a graph 500 comparing the results of three mass analyzer scans using dynamic CV (features 502a-c) with the results of three mass analyzer scans using static CV (features 504a-c). The results of the three static CV scans, denoted 504a-c, are identical to those shown in FIG. 2C. Similar to the graph of relative ion intensity shown in FIG. 5A, the features 504a-c have a shape similar to a Gaussian curve. In contrast, the features 502a-c have broader, rounded peaks that are somewhat flattened relative to the peaks of the features 504a-c. The broadening of the peaks reflects an increase in the number of individual ion species transmitted in each of the dynamic CV scans compared to the static CV scans. In various disclosed examples, dynamic variation of the CV can be applied to a single mass analyzer scan to increase the full-width half maximum (FWHM) of the mass analyzer scan measurement of ion intensity relative to the compensation voltage. In some examples, an increase of 5%, 10%, 25%, 50%, 100%, 200% or more may be obtained over a similar scan measurement using a fixed compensation voltage. The increase may be used to expand and / or adjust the range of ion species collected.

[0027] Figure 6 shows an example flow chart of a method 600 for operating a system including a FAIMS device and a mass spectrometer, such as system 100 of Figure 1. The operations of method 600 may be performed by a controller, such as controller 126 of Figure 1, in conjunction with other components of the system, such as the FAIMS device and a mass spectrometer.

[0028] At 602, a sample is received by a mass spectrometer. In some examples, the sample is a biological sample containing one or more peptides. For example, the sample may be analyzed by liquid chromatography (LC), gas chromatography (GC), or other methods. The separate components of the mixture may be peptides that have been separated from other peptides (and other components) in the mixture using capillary chromatography (GC), capillary electrophoresis (CE), or another type of system used to separate components of a mixture. In the example of a protein that has undergone digestion, the separate components of the mixture are peptides (e.g., fragments of the protein). Although biological samples are described herein for illustrative purposes, the disclosed techniques may be used with other types of samples without departing from the scope of the present disclosure.

[0029] The sample is then ionized to form ions, at 604. Ionization may be performed by an ionization source, such as ion source 104 of FIG. 1. Ionization of the sample produces both singly and multiply charged ions. The ions produced by the ion source are then introduced, at 606, into a FAIMS device, such as FAIMS device 108 of FIG. 1. In particular, the ions are introduced into an analytical gap between electrodes of the FAIMS device.

[0030] At 608, the injection gate of the mass spectrometer is opened and a dynamic CV is applied to the FAIMS device electrode throughout the injection gate opening period. These operations are performed synchronously, e.g., the opening of the injection gate occurs simultaneously with or in a predetermined relationship to the initial application of the dynamic CV, and the dynamic CV continues to be applied (e.g., the CV continues to be dynamically varied) until the injection gate is closed or in a predetermined relationship to the injection gate closing. In some examples, the dynamic variation throughout the opening period can include one or more periods of constant voltage. The injection gate can be an electric gate disposed in the high vacuum chamber of the mass spectrometer, such as injection gate 122 of FIG. 1. Opening the injection gate can include lowering the voltage applied to the injection gate from a first higher potential to a second lower potential for a duration, and then returning the voltage applied to the injection gate to the first higher potential once the duration has elapsed. Alternatively, the CV can be continuously varied over a range of voltages that includes CVs associated with two or more CV fractions. An exemplary representation of the synchronous application of dynamic CV and opening of injection is shown graphically at 128 in Figure 1. While the illustrated example shows a ramp waveform, the dynamic CV applied during the injection gate opening duration can have a waveform having any shape (e.g., triangular, sawtooth, wavelet, sinusoidal, stepped, another shape, or a combination) that reflects the dynamic distribution of the CV. In some examples, the waveform is selected such that its initial value is equal to its final value, for example, a triangular, sawtooth, or wavelet waveform, to advantageously reduce the time required to reset the FAIMS device between repeated experiments.

[0031] At 610, ions of adjacent CV fractions are transmitted through the open injection gate to the mass analyzer. The CV fractions for a given CV are alternatively referred to herein as the ion group / type / species / class / family associated with that CV, and represent the ions transmitted by the FAIMS when the associated CV is applied to the FAIMS electrodes. In some examples, the CV fraction associated with a CV is transmitted when the CV applied to the FAIMS electrodes is within about 20V of the CV associated with that CV fraction. However, in other examples, the "width" of the CV fraction associated with a given CV may be greater or less than 20V. As further described below with reference to Figures 7-9, the dynamic CV applied to the FAIMS electrodes varies over time over a range of values ​​that includes CVs associated with multiple CV fractions, some of which are associated with adjacent CVs. Ions of the CV fraction associated with a CV within the range over which the dynamic CV varies are transmitted through the open injection gate to the mass analyzer.

[0032] At 612, the mass analyzer collectively analyzes the transmitted ions in a single scan. In particular, multiple CV fractions transmitted into the mass analyzer while the injection gate is in an open state are analyzed together by the mass analyzer in a single experiment. At 614, a mass spectrum is generated based on the analysis performed by the mass analyzer. A controller (e.g., controller 126 of FIG. 1) can receive a signal from the mass analyzer with the results of the analysis and then generate a mass spectrum based on this data. An exemplary representation of a mass spectrum generated based on a single mass analyzer scan and including multiple CV fractions is shown diagrammatically at 130 of FIG. 1. In some examples, the characteristics of the generated mass spectrum can be related to the applied dynamic CV waveform, for example, as a convolution of a static CV mass spectrum (often having a Gaussian shape) with the dynamic CV waveform.

[0033] At 616, the mass spectrum is transmitted to post-acquisition software for further processing. For example, the controller can transmit the generated mass spectrum (or multiple mass spectra generated via the illustrated methods) to the post-acquisition software. The post-acquisition software may be hosted locally or remotely, and the generated mass spectrum may be transmitted via a wired or wireless connection to a computing device hosting the post-acquisition software. In some examples, the post-acquisition software includes code executable by the computing device to interrogate the mass spectrum to determine the distribution of individual PSMs, peptides, and proteins within the mass spectrum.

[0034] 7-9 show mass spectra generated by a system (e.g., system 100 of FIG. 1) for the same three ion groups shown in FIG. 5A, but now with the dynamic CV settings synchronized with respect to the opening of the injection gate. In each of FIGS. 7-9, ions of ion group 1 are transmitted from the FAIMS device into the mass analyzer through the open injection gate when the applied CV is within about 5 V of −65 V. Ions of ion group 2 are transmitted from the FAIMS device into the mass analyzer through the open injection gate when the applied CV is within about 5 V of −60 V, and ions of ion group 3 are transmitted from the FAIMS device into the mass analyzer through the open injection gate when the applied CV is within about 5 V of −55 V. However, in other examples, ions may be transmitted by the FAIMS device when CV values ​​within different ranges of the ion's associated CV value are applied (e.g., greater or less than 5 V). The range of CVs that result in the transmission of a given ion species may alternatively be referred to as the "static tolerance envelope" for that ion species. In some cases, peptides may have a relatively large static tolerance envelope for an applied static CV (e.g., about 20V FWHM), while smaller molecules may have a relatively small tolerance envelope (e.g., about 5V FWHM).

[0035] Additionally, while the dynamic CVs shown in Figures 7-9 vary within a range of 10 V, this value was chosen for illustrative purposes and is not intended to be limiting. The range of dynamic CVs applied to the FAIMS electrodes may be greater or less than 10 V in other examples.

[0036] 7 shows an example 700 in which a dynamic CV with a ramp waveform is applied to the FAIMS electrodes during the injection gate opening period. The CV values ​​over time are shown in graph 702 and the injection gate states are shown in graph 704. As shown in graph 702, the applied CV varies dynamically (e.g., continuously) from an initial voltage of -65V to a final voltage of -55V during the injection gate opening period, thereby resulting in the transmission of ion current to the mass analyzer from ion groups (e.g., CV fractions) associated with the CV within the range of -65V to -55V, or ±5V of that range. This then causes the mass analyzer (and thus the mass analyzer scan matrix) to be filled with ions from all three ion groups.

[0037] Graph 712 illustrates the relative ion intensity of the CV fraction transmitted into the mass analyzer in response to application of the ramp-shaped dynamic CV waveform shown in graph 702. Specifically, graph 712 illustrates the respective ion identification peaks for each of the three ion groups. These peaks form a dynamic tolerance curve 714. The width of the dynamic tolerance curve 714 defines a tolerance range 716 for the experiment of approximately 30V. CV fractions transmitting at voltages outside the boundaries of the tolerance range 716 are not identified in the present scan because the dynamic waveform applied during the injection period for the scan does not include their associated CVs. As shown, the end of the tolerance range 716 corresponds to a relative ion intensity of substantially zero, although other measures such as FWHM may be used. As shown in cross-sectional view 706 and graph 712, ions in ion group 1 are transmitted from the FAIMS device through the open injection gate into the mass analyzer as the CV dynamically ramps up from -65V to approximately -62.5V. As shown in cross section 708 and graph 712, as the CV is dynamically ramped from -62.5V to approximately -57.5V, ions in ion group 2 are transmitted from the FAIMS device through the open injection gate into the mass analyzer. Ions from ion group 1 are no longer transmitted. As shown in cross section 710 and graph 712, as the CV is dynamically ramped from approximately -57.5V to -55V, ions from ion group 3 are transmitted from the FAIMS device through the open injection gate into the mass analyzer. Ions from ion groups 1 and 2 are no longer transmitted. Considering the adjacency of the CVs that cause the transmission of those ions, ion groups 1 and 2 may be referred to as "adjacent" CV fractions, as well as ion groups 2 and 3. In contrast, ion groups 1 and 3 may not be considered adjacent CV fractions. Although three ion detection peaks are shown in graph 712 for example, it should be understood that any additional ion species injected into the FAIMS device during the experiment that transmit at a CV within the tolerance range 716 will also be transmitted. Additionally, although a rising ramp waveform is shown in FIG. 7, a falling ramp waveform could alternatively be used without departing from the scope of the present disclosure.

[0038] In the example 800 shown in FIG. 8, the dynamic CV applied to the FAIMS electrodes during the injection gate opening period has a triangular waveform (specifically, a single period of a triangular waveform). The CV values ​​over time are shown in graph 802, and the injection gate state is shown in graph 804. As shown in graph 802, a single period of a triangular waveform can be characterized as having a linear rise and fall similar to a ramp, but reversing direction at the peak amplitude instead of abruptly resetting, thus forming a triangular shape. The implementation of dynamic CV in a triangular pattern allows for the collection of ion fractions on both sides of a focused CV target, but may possibly require additional cooling at the PCB level. The same may be true for a dynamic CV with a waveform of a single period of a sawtooth waveform. Similar to a triangular waveform, a single period of a sawtooth waveform can be characterized as having a linear ramp up followed by an abrupt reset to its starting point (initial value) so that it can rise or fall, similar to the teeth of a saw.

[0039] Graph 812 shows the relative ion intensities of the CV fractions transmitted into the mass analyzer in response to application of the triangular shaped dynamic CV waveform shown in graph 802. Specifically, graph 812 shows the respective ion identification peaks for each of the three ion groups. These peaks form a dynamic tolerance curve 814. The width of the dynamic tolerance curve 814 defines a tolerance range 816 for the experiment of about 30V. CV fractions transmitting at voltages outside the boundaries of the tolerance range 816 are not identified in the scan because the dynamic waveform applied during the injection period for the scan does not include their associated CVs. In the example shown in cross section 806 and graph 812, ions in ion group 1 are transmitted from the FAIMS device through the open injection gate into the mass analyzer when the CV dynamically swings from about -65V to about -62.5V (before the peak of the waveform is reached) and when the CV decreases from about -62.5 to -65V. In the example shown in cross section 808 and graph 812, ions in ion group 2 are transmitted from the FAIMS device through the open injection gate into the mass analyzer when the applied CV dynamically ramps up from about -62.5V to about -57.5V (before the peak of the waveform is reached) and when the applied CV ramps down from about -57.5V to -62.5V. As shown in cross section 810 and graph 812, ions only from ion group 3 are transmitted from the FAIMS device through the open injection gate into the mass analyzer while the applied CV is dynamically varied from about -57.5V to -55V and then back down to -57.5V after the peak of the waveform is reached.

[0040] Three ion detection peaks are shown in graph 812 for example, but it should be understood that any additional ion species injected into the FAIMS device during the experiment that transmit in CV within the tolerance range 816 will also be transmitted.

[0041] In the example 900 shown in FIG. 9, the dynamic CV applied to the FAIMS electrodes during the injection gate opening period has a waveform that can be described as a wavelet. The CV values ​​over time are shown in graph 902, and the injection gate states are shown in graph 904. As shown in graph 902, a single period of the illustrated wavelet waveform has equal initial and final values. It rises from the initial value to a peak, then drops to a negative value with the same amplitude as the peak, and then rises again to the final value. In other words, in the illustrated wavelet implementation, the mass spectrometer injection window starts and ends at a focused CV target, but briefly scans over a wider CV range in the middle of the window. Other types of wavelet waveforms can be used without departing from the scope of this disclosure.

[0042] Graph 912 shows the relative ion intensities of the CV fraction transmitted into the mass analyzer in response to application of the wavelet shaped dynamic CV waveform shown in graph 902. Specifically, graph 912 shows the respective ion identification peaks for each of the three ion groups. These peaks form a dynamic tolerance curve 914. The width of the dynamic tolerance curve 914 defines a tolerance range 916 for the experiment of approximately 30V. CV fractions transmitting at voltages outside the boundaries of the tolerance range 916 are not identified in the scan because the dynamic waveform applied during the injection period for the scan does not include their associated CVs. In the example shown in cross-sectional views 906-910 and graph 912, when the applied CV ramps up from its initial value of -60V to approximately -57.5V, ramps down from -60V (after reaching its peak value) back down to approximately -62.5V, and then ramps up from approximately -62.5V back up to -60V (after reaching the trough of the waveform), only ions in ion group 2 are transmitted from the FAIMS device through the open injection gate into the mass analyzer. In contrast, the applied CV is dynamically varied from approximately -57.5V to the peak (-55V) and then back down to approximately -57.5V, only ions in ion group 3 are transmitted from the FAIMS device into the mass analyzer. Finally, when the applied CV is continuously decreased from approximately -62.5V to the trough of the waveform (-65V), and then increased again to -62.5V, only ions in ion group 1 are transmitted from the FAIMS device into the mass analyzer.

[0043] For illustrative purposes, three ion detection peaks are shown in graph 912, however, it will be understood that any additional ion species injected into the FAIMS device during an experiment and transmitting at a CV within the tolerance range 916 may also be transmitted.

[0044] In each of the examples shown in Figures 7-9, the dynamic application of CV can cause ions in three ion groups to be scanned continuously across the outlet of the FAIMS electrode. In contrast, in FAIMS experiments where static or discrete CVs are applied, a minimum switching time is used each time the CV value changes to clear undesired ions from the electrode before injection into the mass spectrometer. When using a dynamic CV setting, this minimum switching time can be significantly reduced or even negligible, since adjacent CV fractions can be scanned directly into the mass spectrometer inlet. Furthermore, as shown, using dynamic CV rather than static CV when performing one or more scans can advantageously result in a wider tolerance curve. For example, the tolerance curve for a static CV experiment can be about 20V, as shown in Figure 2B, while a dynamic CV experiment can achieve a tolerance curve of about 30V, as shown in Figures 7-9. The specific tolerance curve width values ​​discussed herein are selected for illustrative purposes only and are not meant to be limiting.

[0045] In addition to the exemplary waveforms shown in Figures 7-9, other dynamic CV settings and shapes are contemplated. For example, the applied dynamic CV can have a sinusoidal waveform, a waveform having multiple cycles (e.g., a triangle, sawtooth, ramp, wavelet, or two or more cycles of a sine wave).

[0046] Exemplary Techniques for Producing Mass Spectra Based on Multiple Scans, Each of Which Combines Multiple CV Fractions The amount of time that an ion spends in the presence of a particular CV value, also known as the ion's CV residence time, may be attenuated when a dynamic CV setting is used compared to a static CV setting. In some cases, this reduced residence time may be mitigated by performing multiple consecutive scans, each using a dynamic CV setting (compared to cycling through multiple static CV loops). Increasing the number of FAIMS scans across an LC peak should improve quantification as well as increase qualitative analysis (e.g., PSM / peptide / protein identification).

[0047] In some examples, a user of the FAIMS device may provide, via a user interface, an input indicating a desired number of experiments to run for a given injection of ions into the FAIMS device, or an input specifying a number of CV fractions at which to run experiments. While a conventional approach to running multiple such experiments has involved setting multiple static CVs ("set points"), one for each desired experiment or CV fraction, the inventors herein have recognized that it may instead be advantageous to utilize dynamic CV setting on an experiment-by-experiment basis. For example, instead of setting a set point for each of two consecutive experiments, a range may be set for each of the experiments, with each range including only those CVs that carry a desired CV fraction. In some examples, the ranges may be entered by a user of the FAIMS device, for example, via a user interface. In other examples, the ranges to be set may be determined by a controller based on user input or other data.

[0048] Figure 10 shows an exemplary flowchart 1000 for operating a system including a FAIMS device and a mass spectrometer, such as system 100 of Figure 1. The operations of flowchart 1000 may be performed by a controller, such as controller 126 of Figure 1, in conjunction with other components of the system, such as the FAIMS device and a mass spectrometer.

[0049] At 1002, steps 602-614 of the method 600 of Figure 6 are performed to generate a mass spectrum representative of ions of a sample analyzed in a single scan of a mass spectrometer. As discussed above with reference to Figure 6, these ions include a number of CV fractions, at least two of which are adjacent CV fractions.

[0050] At 1004, steps 606-614 of method 600 are repeated one or more times to perform multiple successive scans of ions from the sample, each scan using a dynamic CV setting. As described above, such operation can advantageously increase ion residence time within the FAIMS device. As shown at 1006, the range and / or waveform of the dynamic CV applied to the FAIMS electrodes at step 608 for each iteration is varied (relative to the dynamic CV applied for the first scan at 1002 and any other dynamic CVs applied during the iteration of method 600) such that additional mass spectra generated by the iterations represent different CV fractions of the sample. In other words, the dynamic CV applied in each iteration of steps 606-614 is selected (e.g., by a controller of the system) to deliver a different CV fraction of the sample than the CV fraction delivered in any previous iteration. Varying the range of the applied dynamic CV relative to the dynamic CV applied in any previous iteration may include applying a dynamic CV that varies within a range that includes at least some CV values ​​not included in the range of any dynamic CV applied in the previous iteration. Similarly, varying the waveform of the applied dynamic CV relative to the dynamic CV applied in any previous iteration may include applying a dynamic CV having a waveform that differs from the waveform of the dynamic voltage applied in any previous iteration.

[0051] At 1008, the mass spectrum generated at 1002 is combined with one or more mass spectra generated at 1004. For example, a controller (e.g., controller 126 of FIG. 1) can combine the generated mass spectra into a single combined mass spectrum.

[0052] At 1010, the combined mass spectrum is transmitted to post-acquisition software for further processing. For example, the controller can transmit the combined mass spectrum to locally or remotely hosted post-acquisition software. As described above with reference to Figure 6, the post-acquisition software can include code executable by a computing device to examine the combined mass spectrum to determine individual ion species distributions (e.g., PSM, peptide, and protein distributions) within the combined mass spectrum.

[0053] Exemplary Techniques for Selecting Between Robustness and Coverage Modes for Operating a Mass Spectrometer Having a FAIMS Device In some cases, it may be advantageous for the user of the mass spectrometer used with the FAIMS device to choose between a robustness mode of operation and a coverage mode. As mentioned above, utilizing a FAIMS with a single CV setting in conjunction with a mass spectrometer to scan a sample may result in approximately the same number, or even fewer, identifications of multiply charged ions (e.g., peptides) compared to scanning a sample with a mass spectrometer without using FAIMS. Thus, utilizing a FAIMS with a single CV setting may seem counterintuitive. However, the inventors herein have recognized certain advantages of using FAIMS in this context that are unrelated to ion identification. In particular, certain structural features of the FAIMS device can advantageously prevent debris from entering the mass spectrometer, thereby keeping the mass spectrometer "clean" for a longer period of time and therefore more robust (e.g., can continue to operate longer without service). For example, a large electrode in a FAIMS device (e.g., a large inner electrode in a FAIMS device with a radial electrode arrangement) can physically block debris (e.g., neutral particles, solvent clusters, etc.) from entering the relatively clean vacuum system in the mass spectrometer. In certain circumstances, a user of a mass spectrometer may wish to obtain detailed information about a single ion type in a sample, as opposed to obtaining less detailed information about multiple ion types. In such cases, according to the techniques disclosed herein, a user can adjust the settings of the FAIMS device (e.g., via an associated user interface) and select a robustness mode, in which the CV applied to the FAIMS is varied within a range of values ​​that transmits a single CV fraction while the mass spectrometer injection gate is open. In some examples, the range of values ​​can span approximately 20V and can be centered around the CV associated with the CV fraction the user desires to analyze. In other examples, a different range smaller or larger than 20V can be used.Operating in robustness mode can advantageously reduce debris entering the mass spectrometer while also providing the user with the desired detailed investigation of the CV fraction associated with the selected CV setting. Operating in coverage mode, which may alternatively be referred to as enhanced identification mode, can increase ion identification per CV (e.g., resulting in more peptide targets) compared to methods using multiple static CV settings. Coverage mode can replace typical operating modes that utilize multiple static CV settings (one CV per MS scan for each CV run in the loop) within the same LC / MS run. Experiments using dynamic CV settings are set up similarly, but should result in more ion species targets per CV. Examples are envisioned in which dynamic CV settings can be optimized to explore the entire multiply charged ion range with only a single input from the user.

[0054] 11 shows an exemplary flow chart of a method 1100 for selectively operating a system including a FAIMS device and a mass spectrometer in a robustness mode or a coverage mode. The operations of the method 1100 may be performed by a controller, such as the controller 126 of FIG. 1, in conjunction with other components of the system, such as the FAIMS device and the mass spectrometer. In some examples, the controller communicates with an input device, such as a keyboard, to receive user input indicating a desired mode (e.g., via a user interface that also allows the user to enter values ​​for other parameters of interest). The user may enter additional information, such as a desired CV fraction to be transmitted, a CV associated with the desired CV fraction, a desired range to vary the CV during the injection period, a desired CV waveform, etc.

[0055] At 1102, an operational mode of the FAIMS device is selected. In some examples, a user selects the operational mode, for example, via a user interface. In other examples, the controller defines which operational mode to execute based on parameters entered by the user.

[0056] At 1103, a coverage mode is selected. As described above, a coverage mode may be defined to prioritize the amount of identified ions. At 1104, operating the FAIMS device in a coverage mode includes performing either method 600 of FIG. 6 or method 1100 of FIG. 11. In instances where a relatively complete investigation of a small number of CV fractions is desired, method 600 may be preferred. In contrast, in instances where a less complete investigation of a large number of CV fractions is desired, method 1100 may be preferred. The decision to perform either method 600 or method 1100 may be performed by the controller based on various parameters (e.g., parameters entered by the user), or the user may provide an input explicitly indicating which method is preferred.

[0057] At 1105, a robust mode is selected. As discussed above, a robust mode may be defined to reduce debris entering the mass analyzer by transmitting only a single CV fraction to the mass analyzer for analysis. However, as discussed below, the voltage applied to the FAIMS electrodes is dynamically varied over a range associated with the desired single CV fraction. Such operation advantageously provides a more detailed analysis of a single CV fraction as compared to data obtained via a static CV setting.

[0058] At 1106, operating in robustness mode includes performing steps 602-606 of method 600 of FIG. 6. At 1108, the CV applied to the FAIMS device electrodes is dynamically varied over a voltage range associated with a single CV fraction during the open period of the injection gate of the mass spectrometer. In other words, rather than simply applying a static voltage pulse with the value of the CV, the voltage applied to the FAIMS electrodes is continuously varied throughout the time the injection gate is open within a voltage range that transmits only the desired CV fraction. The dynamic CV can have a waveform with a ramp, triangular, sawtooth, or wavelet shape, or another waveform shape. In some examples, the voltage range over which the CV varies during operation in robustness mode is smaller than the voltage range over which the CV varies during operation in coverage mode. However, in other examples, the voltage range over which the CV is varied during operation in robustness mode has the same width or is larger than the voltage range over which the CV is varied during operation in coverage mode, even though it only results in the transmission of a single CV fraction. For example, while operating in coverage mode, a relatively narrow voltage range can be used as long as it includes CVs associated with at least two different CV fractions.

[0059] At 1110, ions of the specified single CV fraction are transmitted through an open injection gate to a mass analyzer. At 1112, the mass analyzer analyzes the ions of the single CV fraction in a single scan. At 1114, a mass spectrum is generated based on the analysis performed by the mass analyzer (e.g., via a controller in communication with the mass analyzer). At 1116, the mass spectrum is transmitted to post-acquisition software for further processing (e.g., in the manner described above for step 616 of method 600).

[0060] Exemplary Techniques for Trapping Ions The techniques described herein for dynamically varying the CV applied to the FAIMS electrodes may also be used in conjunction with ion trapping techniques. In particular, ions may be collected in one or more ion traps between the ionization source and the mass analyzer. The release of collected ions can be controlled such that only a fraction of the ions collected during a given ionization event are released at one time for mass analysis.

[0061] For example, referring to the system 100 of FIG. 1, the mass analyzer 116 may further include one or more ion traps disposed upstream of the high vacuum chamber 120 (e.g., between the reduced pressure chamber 118 and the high vacuum chamber 120, or within the reduced pressure chamber 118). The ion traps may include, for example, a quadrupole ion trap or a linear ion trap. Alternatively, the mass analyzer may be an orbitrap incorporating an ion trap. In either case, the controller 126 or another controller may be configured to control the opening and closing of the ion trap to collect and eject ions from the ion trap on a desired schedule. In particular, ions output by the FAIMS device 108 during application of a dynamic CV to the FAIMS electrodes may be collected in an ion trap in the mass analyzer 116, and the controller 126 may operate to coordinate the opening times of the ion trap and the injection gate 122 such that a desired group of ions are analyzed together by the mass analyzer 124.

[0062] Exemplary Computing Environment 12 illustrates a generalized example of a suitable computing environment 1200 in which the described innovations relating to dynamically varying the CV applied to the FAIMS electrodes may be implemented. The computing environment 1200 is not intended to suggest any limitation as to scope of use or functionality, and the innovations may be implemented in a variety of general-purpose or special-purpose computing systems. For example, the computing environment 1200 may be any of a variety of computing devices (e.g., a desktop computer, a laptop computer, a server computer, a tablet computer, etc.).

[0063] Referring to Figure 12, a computing environment 1200 includes one or more processing units 1210, 1215, and memory 1220, 1225. In Figure 12, this basic configuration 1230 is included within the dashed line. The processing units 1210, 1215 execute computer-executable instructions. A processing unit may be a general-purpose central processing unit (CPU), an application-specific integrated circuit (IC), or a combination of both. The tangible memory 1220, 1225 may be a processor in a general-purpose integrated circuit (ASIC), or any other type of processor. In some examples, the controller / controller circuitry described herein is implemented by one or more such processing units. In a multiprocessing system, multiple processing units execute computer-executable instructions to increase processing power. For example, FIG. 12 shows a central processing unit 1210 and a graphics processing unit or co-processing unit 1215. The tangible memory 1220, 1225 may be volatile memory (e.g., registers, cache, RAM), non-volatile memory (e.g., ROM, EEPROM, flash memory, etc.), or some combination of the two, accessible by the processing units. The memory 1220, 1225 stores software 1280 in the form of computer-executable instructions suitable for execution by the processing units, which implements one or more innovations described herein, such as dynamically varying CV, ion gating, generating mass spectra, interrogating mass spectra using post-acquisition software, etc.

[0064] A computing system may have additional features. For example, computing environment 1200 includes storage 1240, one or more input devices 1250, one or more output devices 1260, and one or more communication connections 1270. An interconnection mechanism (not shown), such as a bus, controller, or network, interconnects the components of computing environment 1200. Typically, operating system software (not shown) provides an operating environment for other software executing within computing environment 1200 (e.g., the FAIMS user interface and post-acquisition software described herein) and coordinates the activities of the components of computing environment 1200.

[0065] Tangible storage 1240 may be removable or non-removable and includes magnetic disks, magnetic tapes or cassettes, CD-ROMs, DVDs, or any other medium that can be used to store information in a non-transitory manner and that can be accessed within computing environment 1200. Storage 1240 stores instructions for software 1280 that implements one or more innovations described herein, such as instructions that cause the FAIMS device to apply a dynamically varying CV to the electrodes.

[0066] The input device 1250 may be a touch input device such as a keyboard, mouse, pen, or trackball, a voice input device, a scanning device, or another device that provides input to the computing environment 1200. A user of the FAIMS device may use an input device such as the input device 1250 to input desired parameter values ​​for operation of the FAIMS. The output device 1260 may be a display, a printer, a speaker, a CD-writer, or another device that provides output from the computing environment 1200. In some examples, the output device 1260 includes a monitor or other screen that displays a user interface, and a user of the FAIMS device may input desired parameter values ​​for operation of the FAIMS device into fields of the user interface.

[0067] The communication connection 1270 enables communication over a communication medium to another computing entity. The communication medium carries information such as computer executable instructions, audio or video input or output, or other data in a modulated data signal. A modulated data signal is a signal having one or more characteristics set or changed in such a manner as to encode information in the signal. By way of example and not limitation, the communication medium may use electrical, optical, RF, or other carrier waves. For example, in the context of the system 100 of FIG. 1, a mass spectrum generated by the controller circuitry may be communicated over the communication connection 1270 to another computing entity hosting post-acquisition software for examining the mass spectrum to determine individual ion species distributions (e.g., PSM, peptide, and protein distributions) in a sample associated with the mass spectrum.

[0068] Exemplary Implementations You can implement one of the following:

[0069] Clause 1. A system comprising: a field asymmetric waveform ion mobility spectrometry (FAIMS) device configured to receive ions from an ionization source, the ionization source configured to receive a sample and generate ions from the sample; an injection gate configured, when in an open state, to inject ions transmitted by the FAIMS device into a mass analyzer; and a controller circuit configured to open the injection gate and apply a dynamic compensation voltage (CV) to an electrode of the FAIMS device while the injection gate is open.

[0070] Clause 2. The system of clause 1, further comprising an ionization source and a mass spectrometer comprising a mass analyzer and an injection gate.

[0071] Clause 3. The system of clause 1 or 2, wherein the controller circuit is configured to apply a dynamic CV during the opening period of the injection gate using a time-varying voltage waveform.

[0072] Clause 4. The system of clause 3, wherein the time-varying voltage waveform comprises a ramp pattern, a triangular pattern, a sawtooth pattern, and / or a wavelet pattern.

[0073] Clause 5. A system as described in any one of clauses 1 to 4, wherein the FAIMS device is configured to receive a plurality of CV fractions from the ionization source, the FAIMS device is configured to transmit one of the CV fractions in response to application of an associated CV, and the dynamic CV includes respective CVs associated with two or more of the CV fractions such that two or more of the CV fractions are transmitted while the injection gate is open.

[0074] Clause 6. The system of clause 5, wherein the CV fraction associated with the plurality of CVs includes adjacent CV fractions.

[0075] Clause 7. The system of clause 5 or 6, further comprising a mass analyzer, the mass analyzer configured to collectively scan ions of the multiple CV fractions in a single scan, and the controller circuitry configured to generate a mass spectrum representing ions of the multiple CV fractions based on scan data received from the mass analyzer.

[0076] Clause 8. The system of clause 7, wherein the sample is a biological sample and the controller circuitry is further configured to acquire and transmit the mass spectrum to the software to determine the peptide distribution of the biological sample.

[0077] Clause 9. A method comprising: introducing ions from an ion source into a field asymmetric waveform ion mobility spectrometry (FAIMS) device; opening an injection gate of a mass spectrometer coupled to an outlet of the FAIMS device; and, while the injection gate is open, dynamically varying a compensation voltage (CV) applied to an electrode of the FAIMS device to transmit ions from the FAIMS device into a mass analyzer of the mass spectrometer.

[0078] Clause 10. The method of clause 9, further comprising scanning the transmitted ions in a single scan with a mass analyzer.

[0079] Clause 11. The method of clause 9 or 10, wherein the CV applied to the electrodes while the injection gate is open has a waveform that begins at an initial voltage, ends at an end voltage, and varies between the initial voltage and the end voltage.

[0080] Clause 12. The method of clause 11, wherein the initial voltage and the final voltage have the same value.

[0081] Clause 13. The method of clause 12, wherein the waveform has a shape corresponding to a single cycle of a triangular waveform, a sawtooth waveform, or a wavelet waveform.

[0082] Clause 14. The method of any one of clauses 11 to 13, wherein the initial voltage and the final voltage have different values.

[0083] Clause 15. The method of clause 14, wherein the waveform has a ramp pattern.

[0084] Clause 16. A method according to any one of clauses 9 to 15, wherein the ions introduced into the FAIMS device include at least a first group of ions and a second group of ions, the first group of ions including ions that are transmitted by the FAIMS device when a CV within a predetermined range of the first CV is applied, and the second group of ions including ions that are transmitted by the FAIMS device when a CV within a predetermined range of the second CV is applied, the first and second CVs having different values, and varying the CV applied to the electrodes while the injection gate is open includes continuously varying the CV through a range of values ​​including the first CV and the second CV to result in transmission of ions in the first and second groups of ions into the mass analyzer while the injection gate is open.

[0085] Clause 17. The method of clause 16, wherein scanning the transmitted ions further includes collectively scanning the first and second groups of ions in a single scan using a mass analyzer, and the method further includes receiving, using a controller, data from the mass analyzer including results of the single scan, and generating a mass spectrum representative of the first and second groups of ions based on the received data.

[0086] Clause 18. A method as described in clause 16 or 17, wherein the ions introduced into the FAIMS device further include one or more additional groups of ions, ions of each additional group of ions being transmitted by the FAIMS device upon application of a respective associated CV, and the range of values ​​over which the CV is varied while the injection gate is open further includes the CV associated with the additional group of ions, thereby resulting in transmission of the additional group of ions into the mass spectrometer while the injection gate is open.

[0087] Clause 19. A method for identifying peptides in a biological sample, comprising: receiving ions generated by an ionization source from the biological sample in a field asymmetric waveform ion mobility spectrometry (FAIMS) device, the ions comprising a plurality of groups of ions, each group of ions having an associated compensation voltage (CV) that, when applied to an electrode of the FAIMS device, causes the group of ions to be transmitted by the FAIMS device; dynamically varying a CV applied to an electrode of the FAIMS device over a range of direct current (DC) voltages including a CV associated with at least two of the groups of ions; opening an injection gate of a mass analyzer while applying the varying CV to the electrode, resulting in the transmission of the at least two groups of ions into a mass analyzer of the mass analyzer; collectively scanning the at least two groups of ions in a single scan with the mass analyzer; and generating a mass spectrum based on results of the single scan with a controller, the generated mass spectrum being representative of the at least two groups of ions.

[0088] Clause 20. The method of clause 19, further comprising examining the mass spectrum using post-acquisition software to determine the peptide distribution of the biological sample.

[0089] Clause 21. The method of clause 19 or 20, wherein the range of DC voltage is greater than or equal to -100V and less than or equal to 0V.

[0090] Clause 22. A method according to any one of clauses 19 to 21, wherein opening the injection gate of the mass spectrometer whilst applying a varying CV to the electrodes comprises maintaining the injection gate of the mass spectrometer open whilst dynamically varying the CV applied to the electrodes over a range of DC voltages.

[0091] Clause 23. A method according to any one of clauses 19 to 22, wherein the range of DC voltages is a first range, and the method further comprises, after performing a single scan of the at least two groups of ions, synchronously opening an injection gate of the mass spectrometer and continuously varying the CV applied to the electrodes over a second range of DC voltages, the second range including CVs associated with at least two other groups of ions of the plurality of groups of ions, thereby resulting in transmission of the at least two other groups of ions into the mass analyzer.

[0092] Clause 24. The method of clause 23, wherein the single scan is a first single scan and the mass spectrum is a first mass spectrum, and the method further comprises: collectively scanning at least two other ion groups in a second single scan with a mass analyzer; and generating, with a controller, a second mass spectrum based on results of the second single scan, the second mass spectrum representing the at least two other ion groups; combining the first mass spectrum and the second mass spectrum into a third mass spectrum representing the at least two ion groups and the at least two other ion groups; and examining the third mass spectrum with post-acquisition software to determine a peptide distribution within the at least two ion groups and the at least two other ion groups.

[0093] Clause 25. One or more non-transitory computer-readable media comprising computer-executable instructions for causing an apparatus to perform operations, the operations including: introducing ions from an ion source into a field asymmetric waveform ion mobility spectrometry (FAIMS) device; opening an injection gate of a mass spectrometer coupled to an outlet of the FAIMS device; and, while the injection gate is open, applying a dynamic compensation voltage (CV) to an electrode of the FAIMS device to transmit at least two adjacent CV fractions of ions from the FAIMS device into a mass analyzer of the mass spectrometer.

[0094] Clause 26. A method for operating a field asymmetric waveform ion mobility spectrometry (FAIMS) device, comprising: operating the FAIMS device in a robustness mode in which a compensation voltage (CV) applied to an electrode of the FAIMS device is dynamically varied within a first voltage range associated with the transmission of only one CV fraction during a single opening period of an injection gate of a mass spectrometer; and operating the FAIMS device in a coverage mode in which the CV applied to the electrode is dynamically varied within a second voltage range associated with the transmission of two or more CV fractions during a single opening period of an injection gate of a mass spectrometer.

[0095] Clause 27. The method of clause 26, wherein the CV fraction associated with the second voltage range includes at least two adjacent CV fractions.

[0096] General Considerations As used in this application and the claims, the singular forms "a," "an," and "the" include the plural forms unless the content clearly dictates otherwise. Furthermore, the term "includes" means "comprises." Furthermore, the term "coupled" does not exclude the presence of intermediate elements between the coupled items.

[0097] The systems, devices, and methods described herein should not be construed as limiting in any way. Instead, the present disclosure is directed to all novel and non-obvious features and aspects of the various disclosed embodiments, alone and in various combinations and subcombinations with each other. The disclosed systems, methods, and devices are not limited to any particular aspect or feature or combination thereof, and the disclosed systems, methods, and devices do not require that any one or more particular advantages exist or problems be solved. Any theory of operation is intended for ease of explanation, but the disclosed systems, methods, and devices are not limited to such theories of operation.

[0098] Although some operations of the disclosed methods are described in a particular sequential order for convenient presentation, it should be understood that this style of description encompasses reordering unless a particular ordering is required by specific language described below. For example, operations described in sequence may in some cases be reordered or performed simultaneously. Furthermore, for the sake of simplicity, the accompanying drawings may not show the various ways in which the disclosed systems, methods, and apparatuses may be used in conjunction with other systems, methods, and apparatuses. Furthermore, the description may use terms such as "produce" and "provide" to describe the disclosed methods. These terms are high-level abstractions of actual operations that are performed. The actual operations that correspond to these terms vary depending on the particular implementation and are readily discernible by those of ordinary skill in the art.

[0099] In some examples, values, procedures, or devices are referred to as "lowest," "best," "smallest," etc. It will be understood that such descriptions are intended to indicate that selections can be made from among many functional alternatives used, and that such selections are not necessarily better, lesser, or otherwise preferred than other selections.

[0100] The disclosed techniques for applying dynamic CV to FAIMS electrodes can be embodied as software or firmware instructions executed, for example, by a digital computer. For example, any of the disclosed techniques can be executed by a computer or other computing hardware (e.g., ASIC or FPGA) that is part of an apparatus (e.g., a mass spectrometer) that includes or is used in conjunction with a FAIMS device. The computer can be connected to or otherwise in communication with an ion analysis device (e.g., a mass analyzer) and can be programmed or configured to receive data from the ion analysis device and use the data to generate a mass spectrum. The computer may be a computer system that includes one or more processors (processing devices) and tangible, non-transitory computer readable media, such as one or more optical media disks, volatile memory devices (such as DRAM or SRAM), or non-volatile memory or storage devices (such as hard drives, NVRAM, and solid-state drives (e.g., flash drives)). The one or more processors may execute computer-executable instructions stored on one or more of the tangible, non-transitory computer readable media, thereby performing any of the disclosed techniques. For example, software for performing any of the disclosed embodiments may be stored on one or more volatile, non-transitory computer readable media as computer-executable instructions that, when executed by one or more processors, cause the one or more processors to perform any of the disclosed techniques for applying dynamic CV to FAIMS electrodes. Results of the calculations may be stored in one or more tangible, non-transitory computer readable storage media (e.g., in suitable data structures or lookup tables) and / or output to a user, for example, by displaying the super-resolution image on a display device using a graphical user interface.

[0101] In view of the many possible embodiments to which the principles of the disclosed technology may be applied, it should be recognized that the illustrated embodiments are merely representative examples and should not be construed as limiting the scope of the present disclosure. The alternatives specifically addressed in these sections are merely exemplary and do not constitute all possible alternatives to the embodiments described herein. For example, various components of the systems described herein may be combined in function and use. We therefore claim all that comes within the scope of the following claims.

Claims

1. 1. A system comprising: a Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) device configured to receive ions from an ionization source, the ionization source configured to receive a sample and generate the ions from the sample; an injection gate configured, when in an open state, to inject ions transmitted by said FAIMS device into a mass analyzer; a controller circuit configured to open the injection gate and apply a dynamic compensation voltage (CV) to an electrode of the FAIMS device while the injection gate is open.

2. the ionization source; The system of claim 1 further comprising a mass spectrometer comprising said mass analyzer and said injection gate.

3. 10. The system of claim 1, wherein the controller circuit is configured to apply the dynamic CV during an opening period of the injection gate with a time-varying voltage waveform.

4. The system of claim 3 , wherein the time-varying voltage waveform comprises a ramp pattern, a triangular pattern, a sawtooth pattern, and / or a wavelet pattern.

5. 2. The system of claim 1, wherein the FAIMS device is configured to receive a plurality of CV fractions from the ionization source, the FAIMS device is configured to transmit one of the CV fractions in response to application of an associated CV, and the dynamic CV includes respective CVs associated with two or more of the CV fractions such that two or more of the CV fractions are transmitted while the injection gate is open.

6. The system of claim 5 , wherein the CV fractions associated with a plurality of CVs include adjacent CV fractions.

7. 6. The system of claim 5, further comprising the mass analyzer, the mass analyzer configured to collectively scan the ions of the multiple CV fractions in a single scan, and the controller circuit configured to generate a mass spectrum representative of the ions of the multiple CV fractions based on scan data received from the mass analyzer.

8. 8. The system of claim 7, wherein the sample is a biological sample, and the controller circuit is further configured to transmit the mass spectrum after acquisition to software for determining a peptide distribution of the biological sample.

9. 1. A method comprising: introducing ions from an ion source into a field asymmetric waveform ion mobility spectrometry (FAIMS) device; opening an injection gate of a mass spectrometer coupled to an outlet of the FAIMS device; while the injection gate is open, dynamically varying a compensation voltage (CV) applied to an electrode of the FAIMS device to transmit ions from the FAIMS device into a mass analyzer of the mass spectrometer.

10. 10. The method of claim 9, further comprising scanning the transmitted ions with the mass analyzer in a single scan.

11. 10. The method of claim 9, wherein the CV applied to the electrode while the injection gate is open has a waveform that begins with an initial voltage, ends with a final voltage, and varies between the initial voltage and the final voltage.

12. The method of claim 11 , wherein the initial voltage and the final voltage have the same value.

13. The method of claim 12 , wherein the waveform has a shape corresponding to a single cycle of a triangular waveform, a sawtooth waveform, or a wavelet waveform.

14. The method of claim 11 , wherein the initial voltage and the final voltage have different values.

15. The method of claim 14 , wherein the waveform has a ramp pattern.

16. the ions introduced into the FAIMS device include at least a first group of ions and a second group of ions; the first set of ions includes ions that are transmitted by the FAIMS device when a CV within a first predetermined range of CV is applied; the second set of ions includes ions that are transmitted by the FAIMS device when a CV within a second predetermined range of CV is applied; the first and second CVs have different values; 10. The method of claim 9, wherein varying the CV applied to the electrodes while the injection gate is open comprises continuously varying the CV over a range of values ​​including the first CV and the second CV to effect transmission of the ions in the first and second groups of ions into the mass analyzer while the injection gate is open.

17. 17. The method of claim 16, wherein scanning the transmitted ions further comprises collectively scanning the ions of the first and second groups of ions in a single scan with the mass analyzer, the method further comprising: receiving, with a controller, data from the mass analyzer including results of the single scan; and generating a mass spectrum representative of the ions of the first and second groups of ions based on the received data.

18. 17. The method of claim 16, wherein the ions introduced into the FAIMS device further comprise one or more additional groups of ions, the ions of each additional group of ions being transmitted by the FAIMS device upon application of a respective associated CV, and the range of values ​​over which the CV is varied while the injection gate is open further comprises the CV associated with the additional group of ions, thereby resulting in transmission of the additional group of ions into the mass spectrometer while the injection gate is open.

19. 17. The method of claim 16, wherein the range of values ​​over which the CV is continuously varied is a range of direct current (DC) voltages, the DC voltages within the range being greater than or equal to -100V and less than or equal to 0V.

20. One or more non-transitory computer-readable media comprising computer-executable instructions for causing an apparatus to perform operations, the operations including: introducing ions from an ion source into a field asymmetric waveform ion mobility spectrometry (FAIMS) device; opening an injection gate of a mass spectrometer coupled to an outlet of the FAIMS device; and applying a dynamic compensation voltage (CV) to an electrode of the FAIMS device while the injection gate is open to transmit at least two adjacent CV fractions of ions from the FAIMS device into a mass analyzer of the mass spectrometer.

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