Ion recognition using ion mobility spectrometry

Abstract

A method of analyzing ions comprising: (i) subjecting ions of an analyte molecule to different levels of activation at different times so as to cause the ions to have different mobilities at the different times, wherein the level of activation varies over a plurality of cycles, and wherein during each of the cycles the level of activation varies between the different levels. The method uses an ion mobility separator or a scanning ion mobility filter to determine the mobilities of the ions at the different levels of activation; and correlates the determined mobilities with their respective levels of activation, thereby obtaining a fingerprint of the analyte molecule.

Description

[0001] Cross-references to related applications

[0002] This application claims priority and benefit to UK Patent Application No. 2012094.5, filed on 4 August 2020, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates generally to ion mobility spectrometry, and more specifically, to the analysis of how the mobility of an analyte changes after being subjected to different activation levels. Background Technology

[0004] Collision-induced unfolding (CIU) is a technique that can be used to distinguish ions with very similar structures, such as protein ions. This technique involves colliding the ion with gas molecules to activate its structure, causing it to unfold, and then determining the ionic mobility of the unfolded ion. This process is repeated, but with different collision energies, resulting in varying degrees of unfolding and thus different corresponding ionic mobilities for that ion. A fingerprint of the ion's mobility as a function of collision energy can then be obtained. Different ions with very similar structures may have the same or similar ionic mobilities at specific collision energies, especially when the resolution of the ion mobility separator is relatively low. Therefore, it can be difficult to distinguish two similar ions based solely on ionic mobility, such as conformational changes in the same protein. However, two such ions will have different mobility-collision energy fingerprints, making the CIU technique described above useful for distinguishing them. Summary of the Invention

[0005] In a first aspect, the present invention provides a method for analyzing ions, comprising: (i) subjecting ions of an analyte molecule to different activation levels at different times so that the ions have different mobilities at the different times, wherein the activation levels vary in a plurality of cycles, and wherein, during each period of the cycle, the activation levels vary between the different levels; (ii) determining the mobilities of the ions at the different activation levels using an ion mobility separator or a scanning ion mobility filter; and (iii) correlating the determined mobilities with their corresponding activation levels to obtain a fingerprint of the analyte molecule.

[0006] Because the activation levels cycle repeatedly between different activation levels, any given activation level is executed multiple times while analyzing the analyte. Therefore, even if the intensity of the analyte ions varies over time, this method can obtain spectral data for each activation level at each intensity. Consequently, the statistical precision of the spectral data obtained for different activation levels can be substantially the same.

[0007] It is possible to perform steps on ions in the gas phase to subject the ions of analyte molecules to different activation levels at different times.

[0008] Embodiments of the present invention relate to collision-induced unfolding (CIU) technology, although other activation techniques for unfolding analyte ions have also been considered.

[0009] An ion mobility separator can be an ion mobility separator in which ions are forced through a gas in a drift tube, thereby separating the ions according to their mobility through the gas. Ions can be forced through the device by a DC gradient and / or by repeatedly moving a DC barrier along the device. Alternatively, a scanning ion mobility filter, such as a differential mobility separator (DMS), such as a FAIMS device, can be used. Such devices are scanned to transmit ions with different mobilities at different times. This scanning can be performed by applying different voltages to the device at different times. The scanning ion mobility filter is scanned to transmit different mobilities during each occurrence of the activation level.

[0010] Different activation levels can cause analyte ions to expand and change their conformation to varying degrees, thus altering the collision cross-section of the analyte and consequently its ion mobility and drift time through the ion mobility separator or time to exit the ion mobility filter.

[0011] The analyte can be a biomolecule, such as a protein.

[0012] The same sequence of activation levels can be executed in each loop, meaning the loops can be copies of each other.

[0013] A single cycle of ion mobility separation can be performed after the ions have undergone each activation level, or alternatively, multiple ion mobility separation cycles can be performed after the ions have undergone each activation level.

[0014] The method may include: separating different analyte molecules in a sample using a first separator, then ionizing the separated analyte molecules eluted from the first separator, and then performing the steps of subjecting the analyte ions to the different activation levels at different times in the plurality of cycles; and / or separating analyte ions according to physicochemical properties using a second separator or filter, and then performing the steps of subjecting the analyte ions to the different activation levels at different times in the plurality of cycles.

[0015] The step of ionizing the separated analyte molecules eluted from the first separator can provide each analyte with ions whose intensity peaks over time, and / or ions of each analyte can exit the second separator with ions whose intensity peaks over time; and steps (i) to (iii) can be performed on the ions during at least a portion of the peak to determine the correlation between the ion mobility of the analyte and the corresponding activation level it has experienced.

[0016] The first separator can be a liquid chromatography (LC) separator, a gas chromatography (GC) separator, a size exclusion chromatography (SEC) separator, an ion exchange chromatography (IEX) separator, or a capillary electrophoresis (CE) separator.

[0017] The second separator or filter can be a mass-to-charge ratio separator, a scanned mass-to-charge ratio filter, an ion mobility separator, or a scanned ion mobility filter.

[0018] For example, a scanning ion mobility filter can be a differential mobility separator (DMS), such as a FAIMS device, or any scanning mobility filter. The device can be scanned so that it (only) transports ions with different mobilities at different times. Similarly, if a scanning mass-to-charge ratio filter is used, the device can be scanned so that it (only) transports ions with different mass-to-charge ratios at different times. In these scanning devices, scanning can be performed by varying the voltage applied to the device in a known manner.

[0019] The activation level can vary during each scan of the mass-to-charge ratio filter or the ion mobility filter.

[0020] According to the method described herein, a mass filter or ion mobility filter can be provided upstream or downstream of the region where ions are activated to selectively transport only ions with a selected range of mass-to-charge ratio or mobility. The mass filter can be configured, for example, to transport only ions with a fixed mass-to-charge ratio (or mass-to-charge ratio range) during the plurality of cycles. Similarly, if an ion mobility filter is used, it can be configured, for example, to transport only ions with a fixed mobility (or mobility range) during the plurality of cycles.

[0021] The method may include: using the ion mobility separator or scanning ion mobility filter described in step (ii) of claim 1, separating ions based on the ion mobility after experiencing each of the activation levels in each cycle, to obtain ion mobility spectral data for each of the activation levels; summing the spectral data obtained for the same activation level in multiple cycles to obtain summed spectral data for each of the activation levels; using the summed spectral data to determine the ion mobility of the analyte at different activation levels; and correlating these ion mobilities with their respective activation levels to generate a fingerprint of ion mobility as a function of the activation level of the analyte.

[0022] Ions of each analyte can be eluted from a first or second separator as elution peaks whose intensity varies over time, and the method may include summing spectral data obtained from only a portion of the ions in the elution peaks; or summing spectral data only for a portion of each separation cycle of the first or second separator.

[0023] The method may include normalizing the intensity of the spectral data for each activation level by using the number of times the activation level is used when obtaining the summation spectral data.

[0024] The method may include performing the plurality of cycles x times on each analyte eluted from a first separator or a second separator, wherein x is selected from the group consisting of: ≥2; ≥3; ≥4; ≥5; ≥6; ≥7; ≥8; ≥9; ≥10; ≥15; ≥20; ≥25; ≥30; ≥35; ≥40; or ≥45.

[0025] The method may include performing each cycle on a timescale selected from the following: ≤15s; ≤14s; ≤13s; ≤12s; ≤11s; ≤10s; ≤9s; ≤8s; ≤7s; ≤6s; or 5s.

[0026] In each cycle, there can be y different activation levels, where y is selected from the following groups: ≥2; ≥3; ≥4; ≥5; ≥6; ≥7; ≥8; ≥9; or ≥10.

[0027] Ions can be activated by colliding them with gas molecules, wherein the ions are subjected to different activation levels by colliding them with gas molecules at different energies and / or for different durations.

[0028] Less preferably, ions can be activated by other means (such as photoactivation), and different levels of activation can be performed using light of different frequencies and / or intensities. Alternatively, ions can be activated by reacting with reagent ions or molecules of different concentrations or types to perform different levels of activation.

[0029] The method may further include mass analysis of ions downstream of the ion mobility separator or scanning ion mobility filter described in step (ii) of the first aspect of the invention to determine their mass-to-charge ratio.

[0030] This method may include selecting a mass-to-charge ratio and filtering the spectral data obtained by the method to obtain correlation values ​​of the ion mobility and corresponding activation level of ions with that mass-to-charge ratio.

[0031] The method may include fragmenting or reacting ions downstream of the ion mobility separator or scanning ion mobility filter as described in step (ii) of claim 1 to form fragment or product ions; determining the mass-to-charge ratio of those fragment or product ions; and correlating the mass-to-charge ratio of the fragment or product ions with the activation level used on their respective precursor ions.

[0032] This provides a fingerprint of a given type of analyte molecule, containing correlation values ​​for ion mobility and activation level, as well as the type of fragment / product ions produced for each activation level.

[0033] The method may include determining the relative intensities of different fragment or product ions generated for each activation level and correlating these relative intensities with the corresponding activation levels.

[0034] This fragmentation can be performed through collision-induced dissociation.

[0035] However, other forms of fragmentation can be performed alternatively. For example, ions can be split through electron-trapping dissociation, electron-transfer dissociation, or surface-induced dissociation. Alternatively, ions can react with reagent ions or molecules according to any known technique to produce product ions.

[0036] This method may include using fingerprinting to identify the analyte.

[0037] The present invention also provides a method for screening analytes of interest or specific conformational isomers of an analyte of interest in a sample, comprising: providing a first fingerprint spectrum of how the ion mobility of the analyte of interest or conformational isomer changes after activation with different activation levels; performing the method described above on the analyte to obtain a second fingerprint spectrum of the analyte; comparing the first fingerprint spectrum and the second fingerprint spectrum to determine whether they match; and wherein if the fingerprint spectrum is determined to match, the presence of the analyte of interest or conformational isomer is determined.

[0038] The steps of providing a first fingerprint spectrum may include performing the methods described above on the analyte or conformational isomer of interest in order to obtain a first fingerprint spectrum.

[0039] Embodiments of the present invention include a method for identifying an analyte or analyte conformation, the method comprising: providing a database comprising known compounds or known conformational isomers of the same compound, wherein each compound or conformational isomer in the database is associated with a fingerprint of how its ion mobility changes after activation at different activation levels; performing the method described herein; comparing the fingerprint of the identified analyte molecule with fingerprints in the database to determine whether they match; and wherein if it is determined that the identified fingerprint matches fingerprints in the database, the analyte molecule is identified as a compound or conformational isomer in the database having a matching fingerprint.

[0040] For example, this method can be used to determine whether an analyzed sample contains two different analytes / components (e.g., within a specific mass-to-charge ratio range). If the analytes / components are separated as described above (e.g., via LC), a clean fingerprint corresponding to the first analyte / component will be detected during the first portion of the separation cycle, a clean fingerprint corresponding to the second different analyte / component will be detected during a later second portion of the separation cycle, and a fingerprint corresponding to a mixture of the first and second analytes / components will be detected during the time interval between the first and second portions of the separation cycle. Therefore, this method can determine the changes in the fingerprint spectrum during the separation cycle and use these changes to deconvolve the fingerprint spectra of the different analytes / components.

[0041] In the method described above, steps can be performed on gaseous ions to subject the analyte molecules to different activation levels at different times. However, it is also possible to perform activation on the analyte while it is still in the liquid phase.

[0042] Therefore, in a second aspect, the present invention also provides a method for analyzing ions, comprising: (i) activating an analyte and then ionizing the analyte to form analyte ions, wherein the analyte is subjected to different activation levels at different times, thereby ionizing to produce analyte ions with different mobilities at different times, wherein the activation level varies in multiple cycles, and wherein the activation level varies between the different levels during each period of the cycle; (ii) determining the mobilities of the ions at the different activation levels using an ion mobility separator or a scanning ion mobility filter; and (iii) correlating the determined mobilities with their corresponding activation levels to obtain a fingerprint of the analyte molecules.

[0043] This method may include any of the features described above related to the first aspect, except that activation is performed prior to ionization.

[0044] The method may include using a first separator to separate different analytes from a liquid sample, wherein: (a) the step of activating the analytes is performed before the analytes are separated in the first separator; or (b) steps (i) through (iii) are performed on each analyte eluted from the first separator.

[0045] The first separator can be a liquid chromatography (LC) separator, a size exclusion chromatography (SEC) separator, an ion exchange chromatography (IEX) separator, or a capillary electrophoresis (CE) separator.

[0046] For example, ions can be activated by varying the temperature of the sample solution containing the analyte, such as between the first separator and the ionization device. The sample solution can be heated to different temperatures to perform different levels of activation, for example, using a resistance heater or an electromagnetic heater, such as an infrared lamp or a microwave heater. The sample solution can then be cooled after heating. Alternatively, activation can be performed by changing the pH level of the sample solution. For example, the pH of the sample solution can be changed to perform different levels of activation by supplying acid or base to the sample solution.

[0047] Alternatively, activation can be induced by applying shear stress or mechanical vibration to the sample solution, and the activation level can be altered by changing the shear stress level of the mechanical vibration. These techniques can be particularly useful when the analyte is a protein.

[0048] A first aspect of the invention also provides an instrument for analyzing ions, comprising: a separator for providing ions of an analyte molecule with varying intensity over time; an activation region located downstream of the separator for activating the ions to alter their mobility; an ion mobility separator or scanning ion mobility filter located downstream of the activation region; and control circuitry arranged and configured to: (i) control the instrument to subject the ions of the analyte molecule to different activation levels at different times, thereby causing the ions to have different mobilities at the different times, wherein the activation level varies in a plurality of cycles, and wherein, during each period of the cycle, the activation level varies between the different levels; (ii) control the ion mobility separator or scanning ion mobility filter to determine the mobility of the ions at the different activation levels; and (iii) correlate the determined mobility with their respective activation levels to obtain a fingerprint of the analyte molecule.

[0049] The instrument can be configured to perform any of the methods described herein in relation to the first aspect of the invention.

[0050] A second aspect of the invention also provides an instrument for analyzing ions, comprising: an activation device for activating an analyte; an ion source located downstream of the activation device for ionizing the analyte; an ion mobility separator or scanning ion mobility filter located downstream of the ion source; and a control circuit arranged and configured to: (i) control the activation device to subject the analyte to activation, and control the ion source to ionize the analyte to form analyte ions, wherein the activation device subjects the analyte to different activation levels at different times, such that the ion source produces analyte ions with different mobilities at different times, wherein the activation level varies in multiple cycles, and wherein the activation level varies between the different levels during each period of the cycle; (ii) control the ion mobility separator or scanning ion mobility filter to determine the mobilities of the ions at the different activation levels; and (iii) correlate the determined mobilities with their corresponding activation levels to obtain a fingerprint of the analyte molecules.

[0051] This embodiment has been described, in which the activation level cycles between different values ​​as the ion peak elutes from the separator to obtain a fingerprint spectrum with similar statistical accuracy across all activation levels. However, it is considered that other operating parameters of the mass spectrometer or mobility spectrometer can cycle between different values ​​as the ion peak elutes from the separator to achieve similar statistical accuracy of the spectral data obtained at all operating parameter values.

[0052] Therefore, from a third aspect, the present invention also provides a method for analyzing ions, comprising: providing ions of an analyte molecule to a mass spectrometer or mobility spectrometer, the intensity of which varies as a peak over time; changing the operating parameters of the spectrometer over time such that the ions in the peak are subjected to different conditions at different times, wherein the operating parameters are varied in multiple cycles, and wherein the operating parameters vary between different values ​​during each period of the cycle; performing mass or mobility analysis on the ions using the spectrometer to obtain spectral data for each of the different values ​​of the operating parameters; and summing the spectral data obtained for the same value of the operating parameters in multiple cycles to obtain summed spectral data for each of the values ​​of the operating parameters.

[0053] Because the operating parameters cycle repeatedly between different values, any given value is executed multiple times during analyte analysis. Therefore, even if the intensity of the analyte ions varies over time, this method can obtain spectral data for each intensity at different operating parameter values. Consequently, the statistical precision of the spectral data obtained for different operating parameter values ​​can be substantially the same.

[0054] A separator can be used to provide ions as peaks. A separator can separate different analyte molecules in a sample, and these separated analyte molecules can then be ionized as they elute from the separator. For example, a separator can be a liquid chromatography (LC) separator, a gas chromatography (GC) separator, a size exclusion chromatography (SEC) separator, an ion exchange chromatography (IEX) separator, or a capillary electrophoresis (CE) separator.

[0055] Alternatively, a separator can be used to separate analyte ions based on physicochemical properties, and the ions of each analyte can be eluted from the separator as peaks. For example, the separator can be a mass-to-charge ratio separator, a mass filter, an ion mobility separator, or an ion mobility filter.

[0056] This method may include summing only the spectral data obtained from ions in a portion of the peak.

[0057] The method may include normalizing the intensity of the spectral data for each value of the operating parameter by the number of times the value is used when the summed spectral data is obtained.

[0058] The method described in this paper can be executed during a single experimental run.

[0059] The method may include performing the plurality of cycles x times on the peak, wherein x is selected from the group consisting of: ≥2; ≥3; ≥4; ≥5; ≥6; ≥7; ≥8; ≥9; ≥10; ≥15; ≥20; ≥25; ≥30; ≥35; ≥40; or ≥45.

[0060] The method may include performing each cycle on a timescale selected from the following: ≤15s; ≤14s; ≤13s; ≤12s; ≤11s; ≤10s; ≤9s; ≤8s; ≤7s; ≤6s; or 5s.

[0061] In each loop, there can be y different values ​​for the operation parameter, where y is selected from the following groups: ≥2; ≥3; ≥4; ≥5; ≥6; ≥7; ≥8; ≥9; or ≥10.

[0062] The mobility spectrometer may be a differential ion mobility spectrometer, wherein a time-varying electric field is applied to cause ions to move in a time-averaged manner in a first direction and an electrostatic field is applied to drive ions in a direction opposite to the first direction, wherein the operating parameter that varies with time is the magnitude of the electrostatic field.

[0063] A third aspect of the invention also provides an instrument for analyzing ions, comprising: a separator for providing ions of analyte molecules with intensities varying over time; a mass or mobility spectrometer for determining the mass-to-charge ratio or mobility of the ions; and control circuitry arranged and configured to: (i) change operating parameters of the spectrometer over time such that ions in the peaks are subjected to different conditions at different times, wherein the operating parameters are varied in a plurality of cycles, and wherein the operating parameters vary between different values ​​during each period of the cycle; (ii) perform mass or mobility analysis on the ions using the spectrometer to obtain spectral data for each of the different values ​​of the operating parameters; and (iii) sum the spectral data for the same values ​​of the operating parameters obtained in the plurality of cycles to obtain summed spectral data for each of the values ​​of the operating parameters.

[0064] The instrument can be configured to perform any of the methods described herein that relate to the third aspect of the invention. Attached Figure Description

[0065] Various embodiments will now be described by way of example only, and with reference to the accompanying drawings, in which:

[0066] Figure 1 A schematic diagram of an embodiment of the present invention is shown;

[0067] Figures 2A to 2B Known CIU technologies are shown;

[0068] Figure 3A CIU technology according to an embodiment of the present invention is illustrated, and Figure 3B It shows according to Figure 3A The analyte mobility-collision energy fingerprint was analyzed; and

[0069] Figure 4 A schematic diagram of another embodiment of the present invention is shown. Detailed Implementation

[0070] Figure 1 A schematic diagram of an embodiment of the present invention is shown, including an ion source 2, an activation unit 4 for activating ions, an ion mobility separator (IMS) 6, and a mass analyzer 8.

[0071] In operation, ions are generated by ion source 2 and delivered to activation unit 4. The ions are activated within activation unit 4, thereby altering their structure. For example, this may cause the ion structure to unfold or otherwise change shape. Ions can be activated by axially accelerating them into activation unit 4 or by causing them to collide with background gas molecules within the activation unit. Ions can be accelerated in this way by applying a voltage to the instrument to generate a potential difference. Alternatively or additionally, ions can be activated by causing them to radially oscillate within activation unit 4, resulting in collisions with the background gas. This can be achieved, for example, by applying an alternating current voltage to the electrodes of activation unit 4 to cause ion oscillation. Activation unit 4 may include an ion guide through which ions can pass and be activated without being axially trapped therein, or activation unit 4 may include an ion trapping device that axially traps ions when they are activated. The ion guide or ion trapping device may be formed by an ion tunnel or a multipole assembly.

[0072] Alternative or additional activation methods may be considered, such as supplying reagent molecules or ions to the analyte ions to activate them and / or photoactivating them by directing photons toward them.

[0073] Ions are transported from activation unit 4 to IMS device 6. IMS device 6 may include an ion accumulation region that accumulates ions and periodically pulses them into a separation region containing a background gas to separate the ions based on their ion mobility across the background gas. Alternatively, a separate accumulation device (not shown) may be provided upstream of IMS device 6 for accumulating ions and periodically pulsed into IMS device 6 containing a background gas. IMS device may be a drift tube IMS device, in which a static DC gradient causes ions to pass through the background gas present in the separation region, resulting in ion separation based on their mobility. Alternatively, IMS device 6 may be a traveling wave IMS device, in which one or more DC barriers repeatedly travel along the separation region of IMS device 6 to cause ions to pass through the background gas therein at different rates based on the ion mobility across the gas (e.g., as described in US6791078, which is incorporated herein).

[0074] It is considered that ion activation can be performed in the accumulation region or accumulation device associated with IMS device 6, rather than in the upstream region.

[0075] Ions eluted from IMS device 6 can be detected at an ion detector. The ion mobility of any given ion can then be determined by the duration it takes for the ion to travel through the separation region of IMS device 6. Therefore, the duration of ion travel through the separation region of IMS device 6 can be determined by the time elapsed between the ion being pulsed into the separation region of IMS device 6 and its subsequent detection at the detector. Ions can be eluted from IMS device 6 into a mass analyzer 8 (such as a time-of-flight (TOF) mass analyzer) and subjected to mass analysis. Therefore, the mass-to-charge ratio of the ions can be detected, and the ion mobility of the ions can be determined using the detector of the mass analyzer. Thus, the data recorded by the mass analyzer can be filtered according to the mass-to-charge ratio to obtain ion mobility spectra specific to any given mass-to-charge ratio. This allows the instrument to simultaneously analyze the ion mobility of multiple different ions.

[0076] In embodiments where mass analyzer 8 is a TOF mass analyzer, the extraction region of the TOF mass analyzer can intermittently push ions into the time-of-flight region at a certain rate, allowing it to perform multiple mass analyses for each separation cycle of the IMS device 6, i.e., multiple mass analyses between adjacent pulses when ions enter the separation region of the IMS device 6. This enables the detection of different elution times of ions from the IMS device 6. The time of flight of ions from the extraction region of the TOF mass analyzer to its detector can also be recorded and used to determine the mass-to-charge ratio of the ions in a normal manner.

[0077] Although mass analyzer 8 has been described as a TOF mass analyzer, it can also be another type of mass analyzer. For example, mass analyzer 8 could be a scanning quadrupole mass analyzer with a quadrupole mass filter followed by an ion detector.

[0078] To perform a CIU experiment, a first analysis of the ions is performed as described above, simultaneously using a first level of ion activation in activation unit 4. A second analysis of the ions is also performed as described above, but with a different level of ion activation in activation unit 4, thereby activating the given ions to a different degree than in the first analysis. For example, in the second analysis, the ions may expand or change shape to a greater extent compared to the first analysis. At least a third analysis can be performed as described above, but with at least a third different level of ion activation in activation unit 4. Any number of such analyses can be performed, each with a different level of ion activation in activation unit 4.

[0079] As described above, ions can be activated using any known technique. For example, in the case of ion activation by colliding ions with background gas molecules, different levels of ion activation in activation unit 4 can be performed by providing different acceleration rates through the background gas (e.g., by using different DC potential gradients, or AC voltages of different frequencies and / or amplitudes). Alternatively or additionally, different levels of ion activation in activation unit 4 can be performed by accelerating ions through the background gas for different durations. In the case of photoactivation, different levels of ion activation can be performed using light of different frequencies and / or intensities. Similarly, in the case of ions reacting with reagent ions or molecules, different concentrations or types of reagent ions or molecules can be used to perform different levels of ion activation.

[0080] In at least some of these different activation levels, different levels of ion activation can cause a given ion to unfold or change shape to varying degrees. This, in turn, affects the ion mobility of the ion and therefore its ion mobility through the IMS device 6. Therefore, the techniques disclosed herein are able to determine how the ion mobility of an ion varies as a function of the ion's activation level, thereby providing a fingerprint of the ion. Although it is conventionally difficult to distinguish different ions with very similar structures using a single activation level, especially when the resolution of the ion mobility separator is relatively low, the techniques described herein provide fingerprints of ion mobility at a range of different activation levels. Different ions will have different fingerprints, and therefore can be more easily distinguished according to the CIU techniques described herein.

[0081] For example, the techniques described herein can be used to identify or differentiate analytes such as biomolecules. These techniques are particularly suitable for the analysis of protein molecules, such as identifying or differentiating conformational changes within the same protein. The techniques described herein are also particularly suitable for drug discovery, biotherapeutic characterization, or screening compounds of interest.

[0082] For example, fingerprints of ion mobility as a function of activation level can be determined for various known reference ions (standards) to generate a classification database. Samples can then be analyzed based on the experiments described in this paper to determine such fingerprints for analyte ions. The resulting fingerprints can then be compared with fingerprints in the database to determine if there are any matches. If the fingerprint of the analyte ion matches a fingerprint in the database, the analyte ion is identified. Alternatively, experimental data for ions of interest can be screened by comparing known fingerprints of the ions of interest with those of the experimentally analyzed ions to determine if any of them match a known fingerprint. If a known fingerprint matches the fingerprint of the analyte ion, the presence of the analyte of interest in the sample is determined.

[0083] Embodiments of the present invention can also be used to determine information about any given analyte ion. For example, the CIU analysis described above can be performed on the analyte ion after it has been subjected to different conditions, and the fingerprints obtained under those corresponding conditions can then be compared to determine information about the ion, such as its structure. Examples of conditions that can be modified include changing the degree of binding of another molecule to the analyte or changing the heat applied to the analyte.

[0084] Other embodiments of the invention include performing the aforementioned CIU analysis on an analyte (e.g., a protein) multiple times after the analyte has been exposed to a variety of different conditions. For example, the multiple conditions of analyte exposure could be exposing the analyte to a variety of different temperatures or light intensities, or storing the analyte for a variety of different durations. The different CIU fingerprints obtained under these multiple conditions can then be used to detect the analyte in samples subsequently subjected to CIU analysis. For example, CIU fingerprints obtained under multiple different conditions can be used to screen samples for subsequent analysis to identify changes in the analyte that may have occurred under different conditions. This can be performed as part of a quality control process, such as checking whether the analyte has the same CIU fingerprint in each subsequently analyzed sample, or more generally determining whether the analyte has been degraded or altered.

[0085] CIU technology is known in which ion mobility spectra are obtained for ions that have undergone different collision energies. As ions are transported to an IMS device, the collision energy steps over time, resulting in IMS spectra recorded at different collision energies, thereby producing a two-dimensional mobility versus collision energy fingerprint spectrum that reflects the unfolding of proteins in the gas phase. Figure 2A and Figure 2B An example of this type of known technology is shown.

[0086] Figure 2A A known technique is illustrated in which the analyte is injected at a substantially constant intensity (as shown in the figure above), while the collision energy is gradually increased to different values ​​over time (as shown in the figure below). Based on the shown collision energies (CE1, CE2, CE3), the ion migrations of the ions are determined after the analyte is activated. More specifically, for each collision energy level, the ions are repeatedly pulsed into the IMS device, and thus for each collision energy, multiple ion mobility spectra are summed until sufficient statistical precision is achieved for robust classification of the analyte.

[0087] The aim is to combine chromatographic introduction techniques for biotherapeutic drugs (such as SEC, IEX, CE, or LC) with CIU to improve the quality of the obtained spectra (sample purification) and achieve automation. Figure 2B An example of this type of known technology is shown.

[0088] Figure 2B A known technique is shown in which an analyte is eluted from a chromatographic separator, and its intensity profile is displayed as a peak value changing over time (as shown in the figure above). With Figure 2A Similarly, over time, the collision energy increases to different values ​​(such as...). Figure 2B (As shown in the figure below), and the ion mobility of the ions was determined. More specifically, for each level of collision energy, the ions were repeatedly pulsed into the IMS device, so for each collision energy, multiple ion mobility spectra were summed. However, since the intensity of the analyte eluted from the separator varies over time, the statistical precision of the IMS spectral data obtained for different collision energy values ​​also varies.

[0089] Compared to such conventional techniques, embodiments of the present invention repeatedly change the activation level within a certain range as the analyte is eluted from the upstream separator equipment.

[0090] Figure 3A An example according to an embodiment of the invention is shown, wherein the ion source 2 includes a separator for separating the analytes before ionization, such as a SEC, IEX, CE, or LC separator. However, alternatively or additionally, it may be considered to ionize the sample and then separate the analytes according to their physicochemical properties. After separation, the analytes are activated as described above. Figure 3A As shown in the figure above, the intensity I of the analyte varies as a function of the retention time RT in the separator and has the form of a peak. Figure 3AThe lower graph represents the collision energy at which the analyte is activated as a function of time. In the example shown, the collision energy is cycled repeatedly between three different collision energies, CE1, CE2, and CE3, during the elution of the analyte from the separator to activate the ions. As described above, the activated ions are then passed to the IMS device 6 to determine their ion mobility. For each level of collision energy in each cycle, the ions may be pulsed into the separation region of the IMS device only once to perform only a single IMS separation cycle. Alternatively, for each level of collision energy in each cycle, the ions may be pulsed into the separation region of the IMS device multiple times to obtain multiple ion mobility spectra. In both cases, the ion mobility spectra obtained at the same collision energy in multiple different cycles occurring during peak elution can be summed. For the example shown, the ion mobility spectra obtained using collision energy CE1 in all five cycles can be summed. Similarly, the ion mobility spectra obtained using collision energy CE2 in all five cycles can be summed, and the ion mobility spectra obtained using collision energy CE3 in all five cycles can be summed. Alternatively, the ion mobility spectra obtained from the same collision energies in multiple different cycles occurring within a specific range (or multiple ranges) of peak elution time can be summed. For example, refer again... Figure 3A The ion mobility spectra obtained using the collision energy CE1 between retention times RT1 and RT2 can be summed. Similarly, the ion mobility spectra obtained using the collision energy CE2 between retention times RT1 and RT2 can be summed, and the ion mobility spectra obtained using the collision energy CE3 between retention times RT1 and RT2 can also be summed. The values ​​of RT1 and RT2 can be selected to include only the duration of significant changes in the analyte ion signal intensity (e.g., above a threshold). Alternatively, this range can be a predetermined range or can be automatically selected in a data-dependent manner, i.e., based on the detected ion signal.

[0091] By cyclically acquiring data using collision energies in this manner, the strength and statistical precision of the data are ensured to be similar for each collision energy value, independent of the elution curve of the chromatographic peak. For example, from Figure 2B It is evident that in conventional methods, the collision energy changes relatively little as the ion peak elutes from the separator. Therefore, the ion signal intensity at collision energy value CE2 is significantly different compared to values ​​CE1 and CE3. Consequently, the statistical precision of the data for these collision energy values ​​will differ significantly. In contrast, from... Figure 3AAs can be clearly seen in the embodiments of the present invention, the collision energy changes relatively rapidly as the ion peaks elute from the separator, thus exposing the ions to all collision energies within a relatively short retention time range in the separator. Therefore, the ion signal intensity (and statistical precision) is substantially the same for the spectral data obtained at each collision energy value in each cycle.

[0092] IMS data for each collision energy value obtained from multiple cycles can be summed either after data acquisition or in real time. The intensity of the summed data for each collision energy value can be normalized based on the number of times the collision energy was at that value at the time of data acquisition. For example, refer again... Figure 3A If we sum only the data for the duration between retention times RT1 and RT2, then we will sum the three sets of data for collision energy value CE1, the three sets of data for collision energy value CE2, but the four sets of data for collision energy value CE3. The data for collision energy CE3 can therefore be normalized to the data for each of collision energies CE1 and CE2 by multiplying its intensity by 3 / 4. This data can then be used to correlate how the ion mobility of the analyte varies as a function of collision energy.

[0093] Figure 3B It has been shown that it has been based on Figure 3A The graph shows the ion mobility of the analyzed analyte ions as a function of collision energy. The vertical dashed line on the left represents the collision energy value CE1, the middle vertical dashed line represents the collision energy value CE2, and the right vertical dashed line represents the collision energy value CE3. In this example, the ion mobility values ​​will be determined only for these three collision energies CE1, CE2, and CE3. However, as described below, each cycle can include more than three collision energy values, allowing for a more detailed fingerprint of ion mobility and collision energy.

[0094] Figure 3A Five collision energy cycles are shown as the analyte peaks elute from the separator, with the collision energy stepping between three distinct values ​​during each cycle. However, it is considered that any number of such cycles (greater than one) can be performed, although a relatively high number is desirable. Alternatively, it is considered that the collision energy can step between any number of values ​​(greater than one) during each cycle, although a relatively high number of cycles is desirable. For example, the collision energy could be stepped between 40 distinct values ​​in each cycle, with multiple ion mobility separations performed for each collision energy step, resulting in IMS spectral data acquired within 0.1 seconds for each step. This results in a cycle time of 4 seconds. However, in the limiting case, only one ion mobility separation can be performed in each collision energy step; for example, if the ion mobility separation takes 20 milliseconds, then the cycle time for 40 steps would only be 0.8 seconds.

[0095] Figure 4 Another embodiment identical to that described above is shown, except that the ions are fragmented or reacted by device 10 to generate fragment or product ions downstream of IMS device 6. The fragment or product ions, along with any remaining precursor ions thereof, are then reacted with the ions described above. Figure 1 The same method described above is used for downstream detection and / or quality analysis. Ions can split in any known manner (e.g., collision-induced dissociation, electron-capture dissociation, electron-transfer dissociation, or surface-induced dissociation). Similarly, ions can react with reagent ions or molecules according to any known technique to produce product ions.

[0096] According to such embodiments, the time of detection of fragment or product ions represents the time taken for their precursor ions to travel through IMS device 6. Therefore, the re-acquired spectral data allows for obtaining a fingerprint of ion mobility as a function of the activation level (occurring before IMS device 6) for each precursor ion. However, fragmentation or reaction of ions downstream of IMS device 6 enables the generation of fragment / product ions, which may be specific to the conformation of the ions eluted from IMS device 6, or may be specific to the intensity of the conformation of the ions eluted from IMS device 6. For example, precursor ions that have already undergone a first activation level upstream of IMS device 6 can generate a first set of fragment / product ions downstream of IMS device 6, while the same precursor ions that have undergone different second activation levels upstream of IMS device 6 can generate different second sets of fragment / product ions downstream of IMS device 6 (i.e., at least some fragment / product ions are of different types). Alternatively or additionally, precursor ions that have undergone a first activation level upstream of IMS device 6 can generate fragment / product ions with a first relative intensity downstream of IMS device 6, while the same precursor ions that have undergone a second different activation level upstream of IMS device 6 can generate fragment / product ions with a second different relative intensity downstream of IMS device 6.

[0097] Mass analyzer 8 detects the mass-to-charge ratio of fragment / product ions and correlates this data with the drift time of their corresponding precursor ions through IMS device 6. The fragment / product ion data can thus be added to the fingerprint spectrum of each analyte ion, making the fingerprint spectrum contain both ion mobility data and fragment / product ion data, which can vary as a function of activation level. Further details in the fingerprint spectrum can aid in ion characterization using CIU techniques.

[0098] like Figure 4As shown, an ion separator 12 can be provided between the ion source 2 and the activation unit 4 for separating ions based on physicochemical properties such as mass-to-charge ratio or ion mobility. The ion separator can be a filter that selectively transports only certain ions at any given time, filtering out and discarding other ions, and its transported ions change over time. For example, the ion separator 12 can be a mass-to-charge ratio filter (e.g., a quadrupole mass filter) or a differential mobility analyzer. Alternatively, the ion separator 12 can separate ions without discarding them, such as a mass-selective ion trap or ion mobility separator of the type described above. It should be understood that the ion mobility separator 12 can also be provided in embodiments without the fragmentation / reaction unit 10.

[0099] Although the invention has been described with reference to preferred embodiments, those skilled in the art will understand that various changes in form and detail may be made without departing from the scope of the invention as set forth in the appended claims.

[0100] An embodiment has been described in which the collision energy steps between multiple different, gradually increasing values ​​in each cycle, and the collision energy is held at each value before stepping to the next value. However, it is also conceivable that the collision energy can alternate between multiple different, gradually decreasing values ​​in each cycle. Alternatively, the collision energy can gradually increase and then decrease during each cycle, or gradually decrease and then increase during each cycle. It is also conceivable that the collision energy can be continuously and progressively scanned in each cycle, rather than being stepped and held in each step. The collision energy can vary in a linear, non-linear, sawtooth, or pseudo-random manner in each cycle. Each spectrum can have associated metadata to allow the collision energy value to be determined at any point.

[0101] Although specific embodiments of collision energy changes have been described, as described above, ions can be activated by means other than colliding with gas molecules, and these other activation methods can be used in the described embodiments.

[0102] It has also been considered that the analyte can be activated in the liquid phase rather than the gas phase. For example, the analyte can be in the sample solution and activated before ionization. This activation can be performed by changing the temperature of the sample solution, for example, between the LC separator and the ionization device. For example, the sample solution can be heated to different temperatures to perform different levels of activation, such as using a resistance heater or an electromagnetic heater, such as an infrared lamp or a microwave heater. The sample solution can be cooled after heating. Alternatively, activation can be performed by changing the pH of the sample solution, for example, between the LC separator and the ionization device. For example, the pH of the sample solution can be changed to perform different levels of activation by supplying acid or base to the sample solution. Alternatively, activation can be induced by applying shear stress or mechanical vibration to the sample solution, and the level of shear stress of the mechanical vibration can be changed, thereby changing the level of activation. These techniques may be particularly useful when the analyte is a protein.

[0103] Data can be summed using a moving RT window (e.g., based on knowledge of typical peak widths) to produce a file containing a series of CIU fingerprints against the separator RT. This can generate CIU fingerprints for different analytes eluted from the separator at different times. Ions in a single charge state can be selected, for example, by using a mass filter upstream of the IMS device (or by using reconstructed mass chromatography). CIU plots (ion mobility versus activation level) can be obtained at multiple retention times in the separator. Each CIU plot can be spaced apart by the time taken to perform a single cycle at a different activation level. (The above is about...) Figures 3A to 3B The described embodiments involve identifying peaks and summing the data between RT1 and RT2 for each activation level to generate a single CIU fingerprint. Figure 3B However, instead of identifying peaks before summing, the process can include automatically moving the RT window over which the data is summed. For example, assuming the expected RT range of the elution peak corresponds to the retention time for obtaining x CIU plots, the data from the nth to (n+x)th CIU plots (for each activation level) are summed to generate a first summed CIU plot. The summing window is then moved to sum the data from the (n+1)th to (n+1+x)th CIU plots (for each activation level), thus generating a second summed CIU plot. This process is repeated multiple times, but each time the upper and lower limits of the summing window are moved by one CIU plot, resulting in a new summed CIU plot. This process provides statistically good CIU plots within the expected RT range of the elution peak. These data can then be questioned in a targeted or non-targeted manner. For example, several different variants of the same protein can be discovered, or variations in the CIU plots at separating peaks indicating interference, such as shoulders on peaks with different patterns, can be identified.

[0104] Examples have been described in which the activation level cycles between different values ​​as the ion peak elutes from the separator to obtain a fingerprint spectrum with good statistical accuracy for all activation levels. However, it is also considered that other operating parameters of the mass spectrometer or mobility spectrometer can cycle between different values ​​as the ion peak elutes from the separator to achieve good statistical accuracy of the spectral data obtained at all values ​​of the operating parameters. For example, a differential ion mobility spectrometer (DIMS) can be positioned downstream of a separator (e.g., an LC device), and the parameters of the DIMS device, such as the magnitude of the compensation voltage, can be periodically varied as the peak elutes from the separator. The spectral data obtained for each value of the operating parameters (e.g., for each magnitude of the compensation voltage) can then be summed, enabling the acquisition of spectral data with similar accuracy for each value of the operating parameters.

[0105] Alternatively, an energy-resolved mass spectrometer (ERMS) can be provided downstream of the separator, and the activation energy of the ERMS device can be varied periodically as peaks elute from the separator. The spectral data obtained at each activation energy can then be summed, enabling the acquisition of spectral data with similar accuracy for each activation energy.

Claims

1. A method for analyzing ions, comprising: Different analyte molecules in a sample are separated using a first separator, and then the separated analyte molecules eluted from the first separator are ionized, wherein the ionization of the separated analyte molecules eluted from the first separator provides ions with a peak intensity over time for each analyte; and / or analyte ions are separated according to physicochemical properties using a second separator or filter, wherein ions of each analyte exit the second separator with a peak intensity over time; and then (i) Exposing the ions of the analyte molecules to different activation levels at different times so that the ions have different mobilities at the different times, wherein the activation level varies in multiple cycles and wherein the activation level varies between different levels during each period of the cycle. (ii) Use an ion mobility separator or a scanning ion mobility filter to determine the mobility of the ions at the different activation levels; and (iii) Correlating the determined mobilities with their corresponding activation levels to obtain the fingerprint spectrum of the analyte molecules, And wherein steps (i) to (iii) are performed on the ions during at least a portion of the peak in order to determine the correlation between the ion mobility of the analyte and the corresponding activation level it has experienced.

2. The method according to claim 1, wherein the first separator is a liquid chromatography (LC) separator, a gas chromatography separator, a size exclusion chromatography separator, an ion exchange chromatography separator, or a capillary electrophoresis separator.

3. The method according to claim 1, wherein the second separator or filter is a mass-to-charge ratio separator, a scanned mass-to-charge ratio filter, an ion mobility separator, or a scanned ion mobility filter.

4. The method according to claim 1, comprising: Using the ion mobility separator or scanning ion mobility filter described in step (ii) of claim 1, the ions are separated based on the ion mobility after each activation level in each cycle, so as to obtain ion mobility spectral data for each activation level. The spectral data obtained for the same activation level in the plurality of cycles are summed to obtain the summed spectral data for each of the activation levels; The summative spectral data were used to determine the ion mobility of the analytes at different activation levels; and These ion mobilities are correlated with their corresponding activation levels to generate a fingerprint of ion mobility as a function of the activation level of the analyte.

5. The method of claim 4, wherein ions of each analyte are eluted from the first separator or the second separator as elution peaks with intensity varying over time, and the method comprises summing the spectral data obtained only from ions in a portion of the elution peaks; or wherein the spectral data are summed only for a portion of each separation cycle of the first separator or the second separator.

6. The method of claim 4, further comprising normalizing the intensity of the spectral data for each activation level by using the number of activation levels used when obtaining the total spectral data.

7. The method of claim 1, comprising performing the plurality of cycles x times on each analyte eluted from the first separator or the second separator, wherein x is selected from the group consisting of: ≥2; ≥3; ≥4; ≥5; ≥6; ≥7; ≥8; ≥9; ≥10; ≥15; ≥20; ≥25; ≥30; ≥35; ≥40; or ≥45.

8. The method of claim 1, wherein there are y different activation levels in each cycle, and wherein y is selected from the group consisting of: ≥2; ≥3; ≥4; ≥5; ≥6; ≥7; ≥8; ≥9; or ≥10.

9. The method of claim 1, wherein the ions are activated by colliding with gas molecules, and the ions are subjected to the different activation levels by colliding with gas molecules at different energies and / or for different durations.

10. The method of claim 1, wherein the ions are activated by photoactivation, and different levels of activation are performed using light of different frequencies and / or intensities.

11. The method of claim 1, wherein the ions are activated by reacting with reagent ions or molecules of different concentrations or types to perform different levels of activation.

12. The method of claim 1, further comprising performing mass analysis on ions downstream of the ion mobility separator or scanning ion mobility filter as described in step (ii) of claim 1 to determine their mass-to-charge ratio.

13. The method of claim 9, further comprising selecting a mass-to-charge ratio and filtering the spectral data obtained by the method to obtain correlation values ​​of ion mobility and corresponding activation levels of ions having the mass-to-charge ratio.

14. The method of claim 1, further comprising fragmenting or reacting ions downstream of the ion mobility separator or scanning ion mobility filter as described in step (ii) of claim 1 to form fragment or product ions; determining the mass-to-charge ratio of those fragment or product ions; and correlating the mass-to-charge ratio of the fragment or product ions with the activation level used on their respective precursor ions.

15. The method of claim 14, further comprising determining the relative intensities of different fragment or product ions generated for each activation level and relating these relative intensities to the respective activation level.

16. The method of claim 14, wherein the fragmentation is performed by collision-induced dissociation.

17. A method for screening analytes of interest or specific analyte conformational isomers of interest in a sample, comprising: Provides a first fingerprint of how the ion mobility of the analyte or conformational isomer of interest changes after activation at different activation levels; Perform the method according to claim 1 on the analyte to obtain a second fingerprint spectrum of the analyte; The first fingerprint and the second fingerprint are compared to determine if they match; and If the fingerprint spectrum is determined to match, then the presence of the analyte of interest or conformational isomer is determined.

18. A method for analyzing ions, comprising: (i) Activating an analyte and then ionizing the analyte to form analyte ions with peak intensity over time, wherein the analyte is subjected to different activation levels at different times, such that the ionization produces analyte ions with different mobilities at different times, wherein the activation level varies in multiple cycles, and wherein the activation level varies between different levels during each period of the cycle. (ii) Use an ion mobility separator or a scanning ion mobility filter to determine the mobility of the ions at the different activation levels; and (iii) Correlate the determined mobility with their corresponding activation levels to obtain fingerprints of the analyte molecules.

19. The method of claim 18, further comprising using a first separator to separate different analytes in a liquid sample, wherein... (a) The step of activating the analyte is performed before the analyte is separated in the first separator; or (b) Perform steps (i) to (iii) on each analyte eluted from the first separator.

20. An instrument for analyzing ions, comprising: A separator is used to provide ions of analyte molecules whose intensity changes over time; An activation region located downstream of the separator is used to activate the ions to alter their ion mobility; An ion mobility separator or scanning ion mobility filter located downstream of the activation region; and The control circuit is arranged and configured as follows: (i) Controlling the instrument to subject the ions of the analyte molecules to different activation levels at different times, thereby causing the ions to have different mobilities at the different times, wherein the activation level varies in multiple cycles, and wherein during each of the cycles, the activation level varies between different levels; (ii) Control the ion mobility separator or scan ion mobility filter to determine the mobility of the ions at the different activation levels; and (iii) Correlate the determined mobility with their corresponding activation levels to obtain fingerprints of the analyte molecules.

21. An instrument for analyzing ions, comprising: Activation equipment for activating analytes; An ion source located downstream of the activation device is used to ionize the analyte; An ion mobility separator or scanning ion mobility filter located downstream of the ion source; and The control circuit is arranged and configured as follows: (i) Controlling the activation device to activate the analyte and controlling the ion source to ionize the analyte to form analyte ions with peak intensity over time, wherein the activation device subjectes the analyte to different activation levels at different times, thereby generating analyte ions with different mobilities at different times, wherein the activation level varies in multiple cycles, and wherein the activation level varies between different levels during each period of the cycle; (ii) Control the ion mobility separator or scan ion mobility filter to determine the mobility of the ions at the different activation levels; and (iii) Correlate the determined mobility with their corresponding activation levels to obtain fingerprints of the analyte molecules.

22. A method for analyzing ions, comprising: Provide the ions of the analyte molecules to a mass spectrometer or mobility spectrometer, and use the change in intensity over time as the peak value; The operating parameters of the mass spectrometer or mobility spectrometer are changed over time, such that the ions in the peak are subjected to different conditions at different times, wherein the operating parameters are varied in multiple cycles, and wherein the operating parameters vary between different values ​​during each period of the cycle; The ions are analyzed by mass spectrometry or mobility spectrometry to obtain spectral data for each of the different values ​​of the operating parameters. and The spectral data obtained for the same value of the operating parameter in the plurality of loops are summed to obtain the summed spectral data for each of the values ​​of the operating parameter.

23. The method of claim 22, wherein the mobility spectrometer is a differential ion mobility spectrometer, wherein a time-varying electric field is applied to cause the ions to move in a time-averaged manner in a first direction and an electrostatic field is applied to drive the ions in a direction opposite to the first direction, wherein the time-varying operating parameter is the magnitude of the electrostatic field.

24. An instrument for analyzing ions, comprising: A separator is used to provide ions of analyte molecules whose intensity changes over time; A mass spectrometer or mobility spectrometer is used to determine the mass-to-charge ratio or mobility of the ions; and The control circuit is arranged and configured as follows: (i) The operating parameters of the mass spectrometer or mobility spectrometer are changed over time such that the ions in the peak are subjected to different conditions at different times, wherein the operating parameters are varied in multiple cycles and wherein the operating parameters vary between different values ​​during each period of the cycle; (ii) Perform mass or mobility analysis on the ions using the mass spectrometer or mobility spectrometer to obtain spectral data for each of the different values ​​of the operating parameters; and (iii) Summing the spectral data obtained for the same value of the operating parameter in the plurality of loops to obtain summed spectral data for each of the values ​​of the operating parameter.

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