Tims with simultaneous mobility filtering and analysis

Abstract

A data-independent method for combined ion mobility and mass spectrometry analysis includes the following steps: introducing precursor ions into two ion mobility separators (IMS); separating the ions in a first ion mobility separator; transferring the ion of interest to a second ion mobility separator; and separating the ions in the second ion mobility separator. Ions are sequentially released from the second ion mobility separator according to their ion mobility; the ions are introduced into a mass filter that selectively transmits ions with m / z values ​​falling within a controllable mass window; the ions transmitted through the mass filter are fragmented to generate fragment ions; mass spectrometry measurements are performed on the fragment ions; each fragment ion is associated with a mass window and an ion mobility (IM) range; and detected fragments are associated with their corresponding precursor ions. The second ion mobility separator and the mass filter are controlled synchronously, and the step of associating detected fragments with their corresponding precursor ions is based on determining or utilizing corresponding mass windows and IM ranges associated with each occurrence of the fragments in the mass spectrometry measurements.

Description

Technical Field

[0001] This invention relates to apparatus and methods for operating such apparatus, particularly for analyzing complex mixtures, especially mixtures of compounds with wide variations in abundance. In particular, this invention relates to captured ion mobility spectrometry with optimized mobility window filtering, accumulation, and mobility mass analysis. Existing technology

[0002] Compared to the movement of molecular ions in condensed phases, the movement of molecular ions in gases has established ion mobility as a powerful separation tool, especially when combined with mass spectrometry. Ion mobility spectrometry (IMS) (such as drift tube ion mobility spectrometry (DTIMS), traveling wave ion mobility spectrometry (TWIMS), and differential mobility spectrometry (DMS), also known as field asymmetric ion mobility spectrometry (FAIMS)) serves as a post-ionization method for gas-phase filtration of ions in controlled atmospheres, fractionation of complex samples, suppression of chemical noise, separation of isotopic mixtures, and characterization of molecular ions based on their mobility coefficients. These are methods for separating gas-phase ions based on the interaction between gas-phase ions and colliding gases and the mass of the gas-phase ions. In the first step, ions are separated on a millisecond timescale using an ion mobility spectrometer based on their mobility through a buffer gas. Then, in the second step, the separated ions are introduced into a mass analyzer, where the mass-to-charge ratio of the separated ions can be determined on a microsecond timescale. The efficient separation of analytes achieved using this method makes it widely applicable to the analysis of complex samples in fields such as proteomics and metabolomics.

[0003] US 7,838,826 B1 (MAPark, 2008) and its corresponding patent family members disclose a small ion mobility analyzer / spectrometer, which has become known by the acronym "TIMS" analyzer / spectrometer (TIMS = Trapped Ion Mobility Spectrometer). The terms ion mobility analyzer and ion mobility spectrometer are used interchangeably herein. A TIMS analyzer comprises a gas flow that drives ions against a counteracting electric field barrier, such that ions are initially trapped along the axis of the TIMS analyzer. The ions are confined radially by an RF electric field. After the ions have been transferred from the ion source to the electric field barrier, the height of the electric field barrier or the gas velocity is adjusted so that the ion species are released from the electric field barrier in order of their mobility.

[0004] Typically, the ion mobility separation unit of a TIMS analyzer is only about 5 cm long. A radial RF quadrupole field is generated within a small tube with an inner diameter of approximately 8 mm to hold ions near the axis. A gas flow within the tube drives the ions entrained in the gas flow against a tilted, counteracting DC electric field barrier, where ions are trapped and separated according to their mobility at various locations on the field ramp, where the friction of the moving gas equals the reaction force of the DC electric field on the ramp. After ion loading into the TIMS, the height of the DC electric field barrier decreases; the scan releases ion species in order of mobility. Unlike many other experiments constructing small ion mobility spectrometers, MAPark's small device has achieved high R0 values ​​while reducing scan speed. mob An ion mobility resolution of 400 is very high.

[0005] For the theoretical basis of TIMS, see the research article “Fundamentals of Trapped Ion Mobility Spectrometry” by K. Michelmann, J.A. Silvira, M.M. D. Geway, and M.A. Mapark in J. Am. Soc. Mass Spectrom, (2015) 26: 14-24.

[0006] US 8,766,176 B2 proposes improvements to the scanning modes of TIMS analyzers by applying nonlinear scanning to achieve linear mobility scaling, constant resolution along the mobility scale, or time scaling. Furthermore, US 9,984,864 B2 describes spatial scaling.

[0007] US 9,683,964 describes a TIMS analyzer with parallel ion accumulation; it increases the utilization of ions from the ion source to nearly 100%. Importantly, many ion sources (such as electrospray ion sources) continuously generate ions. A TIMS with parallel accumulation can also be operated to continuously collect and analyze ions—that is, the TIMS can operate at 100% duty cycle. Essentially all ions from the ion source are collected and analyzed without loss, except for pseudopotential or space charge effects. A TIMS with parallel ion accumulation also offers the unique possibility of extending the ion accumulation duration to discover more detectable ion species, thereby improving ion mobility resolution even through a corresponding extension of the scan time. Ions are collected at the ramp of the DC electric field barrier in an accumulator unit preferably almost identical to the scan unit, such that ions are spatially separated by their ion mobility along the ramp. Therefore, the accumulated ions are less affected by space charge compared to other types of accumulator units. However, the most important feature is the unique characteristic of the TIMS analyzer: the longer accumulation period allows for improved mobility resolution by selecting a correspondingly longer mobility scan duration, for example, an ion mobility resolution of R. mob With =75, the scan duration is 100 milliseconds, instead of R. mob With a TIMS concentration of 30, the scan duration is 20 milliseconds. Due to the higher number of ions collected and better ion mobility resolution, more ion species can be detected and measured. Once the ion mobility scan is complete (optionally after twenty to several hundred milliseconds), the accumulated ions are transferred from the accumulation unit to the scan unit (within approximately one millisecond), and the next ion mobility scan can begin. Overall, skilled practitioners will understand that measurement rates of 300 to 450 ion species per second can be achieved. If a TIMS with parallel ion accumulation is installed in a tandem mass spectrometer (MS / MS instrument) or MS-MS instrument, quantitative measurements of 300 to 450 characteristic fragment ion spectra per second are possible.

[0008] US 9,304,106 B1 presents some improvements for storing a greater number of ions in selected ion mobility regions, particularly for ions with low ion mobility. The higher loading capacity is based on a nonlinear DC electric field ramp with a flatter field ramp for the ion species of interest, reducing the influence of space charge on these ion species. However, for accurate ion mobility analysis of low-abundance ion species in complex mixtures, the influence of space charge remains significant.

[0009] Figure 1The prior art, based on a mobility-based filtration method according to Park et al. in US 2012 / 0273673, is described. According to that document, two mobility devices are provided. Ions to be filtered, entrained in a gas, are introduced at the inlet of the first mobility device. "...the gas drives the entrained ions against a first field barrier 50, thereby blocking the ions with mobility..." All ions. This situation exists in Figure 2 The diagram illustrates that... Figure 2 An arrangement of ions 52 to 59 with different mobilities is shown, indicated by dots representing the ions and different sizes of ion cross-sections. Gas-driven ions 55 to 59 that have passed through the first barrier 50 resist the second field barrier 51, thereby blocking ions with different mobilities. Ions 55 and 56, and therefore block and collect range The mobility of all ions within, exist and Between, among them, .have Ions 57 to 59 pass through the second potential barrier and disappear. To collect ions 55 and 56, an ion storage device must exist between the filters, such as a multipole field with a centripetal pseudopotential that holds the ions within a collection volume between the two barriers by providing a radial force. The simplest way to generate the storage volume is to encapsulate the two filters within an RF multipole device, such as an RF quadrupole system.

[0010] US-A-2018340910 discloses a mass spectrometer and its uses, comprising two ion mobility analyzers arranged in series and an ion gate located between the two ion mobility analyzers, at least one of which is a captured ion mobility spectrometer (TIMS), wherein ions are selectively transferred between the two ion mobility analyzers by adjusting the transmission of the ion gate when ions are separated in time according to ion mobility in the first ion mobility analyzer.

[0011] WO-A-2019096852 relates to the use of isotopic tags in mass spectrometry (MS) analysis using data-independent acquisition (DIA), wherein the isotopic tag comprises or is composed of a group that fragments in the mass spectrometer (i) at an energy lower than that required to fragment the precursor ion from which the analyte originates and / or at a higher conversion rate than the precursor ion; and (ii) at the energy according to (i), and when coupled with the precursor ion, fragments at a single site of the group to produce a first portion and a second portion, the second portion being coupled with the precursor ion. WO-A-2019096852 proposes the use of a captured ion mobility spectroscopy time-of-flight (timsTOF) instrument equipped with parallel / serial fragmentation (PASEF); see, for example, Meier et al., 2015, doi:10.1021 / acs.jproteome.5b00932.

[0012] US-A-2019371585 relates to selecting precursors for tandem mass spectrometry from a measured mobility-mass map, and based on processing a list of peaks according to the measured signal and clustering these peaks in the mobility-mass space.

[0013] US-A-2022034840 discloses an apparatus and method for data-independent combined ion mobility and mass spectrometry analysis. The method includes: introducing precursor ions into an ion mobility spectrometer (IMS); sequentially releasing the precursor ions from the IMS according to their ion mobility; introducing the released precursor ions into a mass filter; fragmenting the precursor ions transmitted through the mass filter to generate fragment ions; and performing mass spectrometry measurements on the fragment ions. The IMS and mass filter are controlled synchronously to perform multiple IM scans, wherein adjacent mass windows associated with consecutive mass spectrometry measurements of fragment ions overlap during the IM scans, such that the precursor ions transmitted through the mass filter during the IM scans are located in at least one consecutive scan region in the m / z-IM plane, the at least one consecutive scan region extending in a generally diagonal direction in the m / z-IM plane.

[0014] EP-A-4089714 discloses a method and apparatus for combined ion mobility and mass spectrometry analysis. The method includes the following steps: introducing precursor ions into a trap configured to capture ions and selectively eject the captured ions according to the m / z ratio of the ions; selectively ejecting precursor ions from the trap whose m / z values ​​fall within at least one controllable ejection window; sequentially releasing the precursor ions from the IMS according to the ion mobility of the precursor ions; introducing the released precursor ions into a mass filter having a controllable mass window; fragmenting the precursor ions delivered through the mass filter to generate fragment ions; and performing mass spectrometry measurements on the fragment ions, wherein each fragment ion is associated with a mass window and an ion mobility (IM) range.

[0015] US-A-2022277949 discloses a mixed mass spectrometry system and a method for operating the mixed mass spectrometry system, which includes an ion source, a first captured ion mobility spectrometer (TIMS) analyzer, and a mass analyzer, wherein the TIMS analyzer is located in a first vacuum chamber and operates in the first vacuum chamber at an increased pressure of more than 500 Pa.

[0016] Meier et al., in *Nature Methods*, Vol. 17 (2020), pp. 1229–1236, reported a data-independent acquisition mode that isolates and simultaneously fragments populations of different precursors by iteratively traversing segments of a predefined precursor m / z range. Although these selection windows collectively cover the entire m / z range, only a few percent of all incoming ions are isolated for mass analysis. Here, we utilize the correlation between molecular weight and ion mobility in a captured ion mobility device (timsTOF Pro) to design a scanning mode that samples up to 100% of peptide precursor ion currents within m / z and mobility windows. The established targeted data extraction workflow is extended by including the ion mobility dimension for both signal extraction and scoring, thereby improving the specificity of precursor identification. Data acquired from whole proteomic digests and mixed biological samples demonstrate deep proteomic coverage and high reproducibility and quantitative accuracy, even with sample amounts as low as 10 ng.

[0017] Targeted selection of peptides from fragmented complex protein digests is a performance limiting factor in tandem mass spectrometry (MS). This problem is primarily attributed to the large amounts of high-abundance peptides co-eluted and the limited duty cycle of mass spectrometers configured for data-dependent acquisition (DDA) mode, which sequentially selects precursor ions for fragmentation in descending order of intensity. Furthermore, the depth of proteomics analysis is also limited by the vast majority of high-abundance background ions that impair the identification and quantification of low-abundance peptides. Whether to select low-intensity precursors for fragmentation is primarily determined by the speed at which the mass analyzer can perform tandem MS analyses. The analytical dynamic range in DDA mode can be extended by excluding previously analyzed precursor ions, but this strategy is no longer effective for highly complex samples with extended concentration dynamic ranges. Even in data-independent acquisition (DIA) mode, the MS duty cycle remains an issue, where all ions within the selected mass charge range are selected for fragmentation. Sample complexity issues are typically addressed by fractionating the sample.

[0018] Liquid chromatography-mass spectrometry (LC-MS) has been used in the field of proteomics for many years for the identification and quantification of peptides (and therefore proteins) from complex mixtures of samples. In proteomics, analytes are typically peptides generated from the trypsin breakdown of protein samples. The most common methods are variants of the so-called LC-MS / MS or "shotgun" MS methods, which are based on the generation of fragment ions from precursor ions, automatically selected based on the distribution of precursor ions (data-dependent analysis, DDA). The main drawback of these methods is poor reproducibility, which results in only partially overlapping proteomes in repeated analyses of substantially similar samples. Recently, several new methods have been developed that address these limitations, and these new methods can be conceptually described as targeted proteomics approaches.

[0019] The most mature technique is called Selected Reaction Monitoring (SRM), often also referred to as Multiple Reaction Monitoring (MRM). The targets for MRM experiments are defined on a reasonable basis and depend on the hypotheses to be tested in the experiment. Selected combinations of precursor and fragment ions for these targets (so-called transitions; a set of transitions for a target precursor is called an MRM determination) are programmed into the mass spectrometer, which then generates measurement data only for the defined targets.

[0020] Another variant of targeted proteomics is data-independent acquisition (DIA). Here, the targeting aspect is introduced only at the data analysis level. Unlike MRM, this approach requires no prior method design before sample injection. Because LC-MS acquisition traverses the entire mass and retention time (RT) range, covering the full analyte content of the sample, data can be a posteriori mined for any peptide / precursor of interest. Data is acquired in a data-independent manner across the entire mass range (e.g., 200 Thomson to 2000 Thomson) and traversing the entire chromatogram, regardless of sample content. This is typically achieved by progressively stepping through the mass analyzer's selection window across the entire mass range. In effect, this data acquisition method generates a complete fragment ion map for all analytes present in the sample and then correlates the fragment ion spectra again with the precursor ion selection window from which the fragment ion spectra are acquired. This is achieved by widening the precursor isolation window on the mass analyzer, thus a priori considering multiple precursors co-eluting and contributing to the fragmentation patterns recorded during analysis. Such a precursor window is called the precursor selection window. The result is a complex fragment ion spectrum from multiple precursor fragments, which requires more challenging data analysis. Summary of the Invention

[0021] As discussed above, when analyzing complex samples, such as those of biological origin, it is essential to separate the sample components to allow for the identification and quantification of various component species. In many cases, the sample component of interest has a relatively low abundance compared to species that are uninformative or have limited information. Furthermore, analytical instruments typically have a limited dynamic range—that is, the ability to analyze very low abundance species simultaneously while very high abundance species are present.

[0022] Therefore, for example, the presence of high abundance components in plasma analysis can interfere with the analysis of low abundance components.

[0023] The solutions to such problems are: 1) improve the instrument by increasing its effective dynamic range; 2) eliminate or reduce the relative abundance of high-abundance components in the sample.

[0024] In the case of plasma, there are many methods to reduce the relative abundance of high-abundance components in the liquid phase.

[0025] Two examples are: “depletion,” in which high-abundance proteins are largely removed from the sample; and “enrichment,” in which the abundance of selected low-abundance peptides is enhanced.

[0026] While these methods can improve the effective dynamic range, they can be costly and time-consuming, and have the following problems: they may lack reproducibility and can lead to distortion of the relative abundance of low-abundance species, which has a negative impact on protein quantification.

[0027] Therefore, a primary objective of this invention is to provide an additional, instrument-based, more cost-effective and time-efficient method for eliminating or significantly reducing the abundance of high-abundance species from complex samples. This method operates by ionizing sample components and eliminating and / or reducing high-abundance, uninteresting ions in the gas phase based on the ion mobility of the sample components. This method can be used independently or in conjunction with prior art methods, similar to those mentioned above for improving effective dynamic range.

[0028] A second objective of this invention is to provide an improved method for fractional separation of ions based on their gas-phase ion mobility, thereby reducing the complexity of the resulting dataset, decreasing the presence of high-abundance species in at least some fractions, and improving the overall dynamic range of sample analysis.

[0029] Therefore, the following elements are proposed:

[0030] Methods for filtering ions based on gas-phase ion mobility to generate ionic components. One objective is to provide an improved method for fractional separation of ions based on their gas-phase ion mobility, thereby reducing the complexity of the resulting dataset, decreasing the presence of high-abundance species in at least some components, and improving the overall dynamic range of sample analysis.

[0031] Methods for eliminating unwanted, uninformative, and / or high-abundance species. One object of the present invention is to provide additional, instrument-based, more cost-effective and time-efficient methods for eliminating or significantly reducing the abundance of high-abundance species from complex sample analysis.

[0032] Therefore, the present invention relates to corresponding apparatus and methods for operating such apparatus, particularly for analyzing complex mixtures, especially mixtures of compounds with wide variations in abundance. Specifically, the present invention relates to captured ion mobility spectrometry with optimized mobility window filtering, accumulation, scanning, and detection window selection for mobility quality analysis.

[0033] New protocols and systems for LC-IMS-MS analysis, including their operating methods, are disclosed, particularly for data-independent acquisition (DIA), to address the limitations summarized above in the analysis of complex protein samples. The new protocols are characterized by the operation of the IMS instrument under low-pressure conditions to achieve more customized, selective, and sensitive separations, including increased instrument duty cycle and dynamic range, thus increasing the depth of analysis.

[0034] according to First aspect of the invention This invention relates to a method for data-independent combined ion mobility and mass spectrometry analysis (selective transfer), comprising the following steps:

[0035] Precursor ions are introduced into two ion mobility separators (IMS), at least one of which, preferably the second, is a captured ion mobility spectrometry (TIMS) separator, wherein an optional ion gate is located between the two ion mobility separators.

[0036] In the first ion mobility separator, ions are separated over time according to their mobility.

[0037] Preferably, the ions of interest are selected by adjusting the transport of the ion gate during or after separation in the first ion mobility separator;

[0038] The selected ions of interest are transferred to the second ion mobility separator; and

[0039] In the second ion mobility separator, the transferred ions are separated according to their mobility.

[0040] Precursor ions are sequentially released from the second ion mobility separator according to the ion mobility of the precursor ions;

[0041] The released precursor ions are introduced into a mass filter that selectively delivers precursor ions with m / z values ​​falling within a controllable mass window.

[0042] The precursor ions delivered through the mass filter are fragmented to generate fragment ions.

[0043] Mass spectrometry measurements are performed on the fragment ions, wherein each fragment ion is associated with a mass window and an ion mobility (IM) range, and the detected fragments are associated with their corresponding precursor ions.

[0044] According to the proposed protocol, the second ion mobility separator and the mass filter are controlled synchronously to perform multiple IM scans, during which precursor ions with increasing or decreasing IM are sequentially released from the second IMS, and during the multiple IM scans, the mass window of the mass filter is continuously or gradually shifted toward lower or higher m / z values, respectively. Therefore, a parallel cumulative serial fragmentation method is essentially used.

[0045] Furthermore, according to the proposed protocol, the step of associating the detected fragments with their corresponding precursor ions is preferably based on determining or utilizing the corresponding mass window and IM range associated with each occurrence of the fragments in the mass spectrometry measurement.

[0046] Typically, such ion mobility separators (IMS) can be TIMS, ion funnels, or ion guides. In each case, they preferably have the ability to support radial RF ion confinement, gas flow, and a DC electric field barrier that can counteract the drag force on ions caused by the gas flow.

[0047] The first preferred method is characterized as follows: in bottom-up proteomics LC-IMS-MS sample analysis—especially when the sample includes high-abundance species that interfere with the analysis of the species of interest. Such samples include, for example, plasma and urine samples.

[0048] In this first preferred method,

[0049] (a) Using only one of the ion mobility separators, preferably the second ion mobility separator (TIMS2), to obtain a low duty cycle first-stage full scan (collecting and analyzing only a few percent of the ions generated in the ion source);

[0050] (b) Based on the first-level full scan, identify high-abundance, undesirable species and determine the mobility components to be analyzed (usually the mobility component window that excludes high-abundance, undesirable species).

[0051] (c) For a given mobility component window, a barrier is set in the first ion mobility separator (TIMS1) to allow only ions within or below the mobility range of the given mobility component window to pass through (for selecting desired components that are not highly abundant or of undesirable species).

[0052] (d) An accumulation barrier is set in the second ion mobility separator (TIMS2) to allow only ions below the mobility range of a given mobility component window to pass through (again used to select desired components without high abundance or desired species), and to accumulate ions of the desired mobility range in the accumulation region of the second ion mobility separator (TIMS2) for a predetermined time.

[0053] (e) The accumulated ions are transferred to the analyzer section of the second ion mobility separator (TIMS2);

[0054] (f) Performing Parallel Accumulation Serial Fragmentation (PASEF) involves rapidly switching quadrupole mass positions to select multiple precursors at different m / z values ​​on very similar timescales, ensuring that all target ions are fully utilized for fragmentation analysis of the accumulated ions; and

[0055] Repeat steps (c) through (f) for all given mobility component windows (desired mobility components).

[0056] According to the invention, an instrument / device is also provided, comprising an ion source, an ion optics having the structure and features of a first ion mobility separator (e.g., a TIMS analyzer), a second ion mobility separator (e.g., a TIMS analyzer) downstream of the first ion mobility separator, a mass spectrometer (preferably a quadrupole, e.g., time-of-flight) downstream of the second ion mobility separator analyzer, and electronic equipment, firmware, and software sufficient to operate and control the instrument to perform the methods described above.

[0057] According to a preferred embodiment, LC or other chromatographic separation is also provided upstream of the instrument.

[0058] The first ion mobility separator (e.g., TIMS1) may also be a simple funnel with reverse DC at or near the funnel outlet (as described, for example, in the prior art by Baykut et al., J. Am. Soc. Mass Spectrom. 2009, 20, 2070-2081, which is incorporated herein by reference for this aspect of the invention).

[0059] The above parallel cumulative serial fragmentation (PASEF, including expressions such as diaPASEF, MIDIA PASEF, slicePASEF, and synchronous PASEF) can be PASEF methods, including data-dependent PASEF and data-independent PASEF.

[0060] The proposed method can be used for any sample, any type of sample, any type of complex sample, and especially any type of sample including components with a high dynamic abundance range, including proteomics, metabolomics, lipidomics, genomics or any other "-omics" sample.

[0061] The “filtering” method can be used to generate components without performing a first-level full scan, including using a table of predetermined species to be eliminated / components to be analyzed, using information about undesirable species (planned species) with known LC and / or TIMS elution times / K0, and using information about components with known LC and / or mobility ranges to be analyzed (planned components).

[0062] according to Second aspect of the invention This invention relates to a method for fractionating and separating ions based on their mobility and for mass mobility analysis, comprising the following steps (mobility filtering):

[0063] a) Provide an instrument including an ion source, three ion mobility separators, and a mass spectrometer (mass spectrometry measurement device);

[0064] b) Establish a first mobility cutoff in the first ion mobility separator;

[0065] c) Establish a second mobility cutoff in the second ion mobility separator;

[0066] d) Accumulate ions of interest in the second ion mobility separator, wherein the ions of interest have a mobility between the first mobility cutoff and the second mobility cutoff, and the accumulated ions of interest constitute a mobility component;

[0067] e) Transferring the component to a third ion mobility separator; and

[0068] f) The third ion mobility separator is used in conjunction with the mass spectrometer to perform mobility mass analysis on the components.

[0069] The instrument may also include a first gate element between the first ion mobility separator and the second ion mobility separator.

[0070] In the analysis of complex samples, such as bottom-up plasma proteomics, it is often advantageous to remove high-abundance species (in this case, removing ions from the gas phase sample), which would otherwise mask low-abundance species. In the example further illustrated below, it is estimated that removing one high-abundance species on average in any given spectrum during an LC-TIMS-MS analysis would effectively improve the instrument's dynamic range (especially its ability to visualize low-abundance species) by approximately 6-fold. Removing the eight most abundant species resulted in a 100-fold improvement in effective dynamic range.

[0071] Correspondingly, it is desired to remove multiple high-abundance gaseous ions from TIMS analysis.

[0072] According to another element of the second aspect of the invention, it is therefore proposed to extend the first two of the two ion mobility separators above into more than two such ion mobility separators, for example, in the range of 3 to 8 or 4 to 6 such ion mobility separators, followed by a final ion mobility separator; and the final ion mobility separator (preferably having a TIMS analyzer with parallel accumulation and separation) is used in conjunction with the mass spectrometer to perform mobility mass analysis of the components. The corresponding instrument may also include gate elements between the ion mobility separators.

[0073] The ion mobility separators upstream of the final ion mobility separator can be individual ion mobility separators; however, two or more upstream ion mobility separators can also be implemented as a single captured ion mobility separator device, configured to establish at least a first mobility cutoff and a second mobility cutoff within the same ion mobility separator device. Therefore, according to a preferred embodiment of the second aspect of the invention, the invention also relates to the use of a single captured ion mobility separator device capable of establishing at least two ion mobility cutoffs, preferably in the range of 2 to 8 or 3 to 6 ion mobility cutoffs.

[0074] Therefore, according to this aspect of the invention, preferably, a method for eliminating one or more highly abundant species based on mobility analysis is provided, the method comprising the following elements / steps:

[0075] a) Provide an analyzer with multiple DC barriers, radially confined RF pseudopotentials, and a gas flow from the inlet to the outlet (optionally, an ion gate between the barriers).

[0076] b) Set up barriers

[0077] c) Introducing and accumulating ions,

[0078] d) Eliminating high-abundance ions by reducing or significantly decreasing the strength of the confinement field in the accumulation region of the selected barrier (alternatively, the confinement pseudopotential may be reduced or eliminated periodically or instantaneously, for example, by reducing or eliminating it by 1 ms every 30 ms during a 100 ms accumulation period, to eliminate or only reduce the number of high-abundance ions in these regions. Alternatively, high-abundance ions may be eliminated by lowering the DC barrier and allowing elimination of ions at the gates after the barrier).

[0079] e) Transfer the remaining ions to a downstream ion mobility analyzer (TIMS) (e.g.) Figure 18 As schematically shown in d), ions are first transferred to the accumulation area of ​​the downstream ion mobility (TIMS) analyzer (“TIMS 2”). After accumulation in the accumulation area of ​​the downstream ion mobility (TIMS) analyzer (“TIMS 2”), ions are transferred to the analyzer area of ​​the downstream ion mobility (TIMS) analyzer (“TIMS 2”) so that they can be analyzed when the next set of ions is accumulated and filtered.

[0080] In this way, even if high-abundance species are not located in the same ion mobility window, they can be separated in one or a series of ion mobility separator devices. This allows for the elimination of, for example, the five, six, or even eight most abundant species mentioned above by adjusting the corresponding cutoff, so that only ions other than these high-abundance species pass through to the final ion mobility separator, thereby causing the aforementioned significant improvement in the effective dynamic range. According to a second aspect, the invention relates not only to such devices but also to corresponding analytical methods or uses of such devices for eliminating several high-abundance species from complex samples, which are located within a range of at least two ion mobility windows, preferably at least three ion mobility windows, at least four ion mobility windows, or five to eight different ion mobility windows.

[0081] The apparatus and method of the second aspect of the invention can be used in combination with one or more of liquid chromatography, electrospray ionization, in a data-dependent manner with reduced duty cycle first-level full scan as described in this application, in combination with a schedule, or wherein selected / deselected mobility regions are predetermined and periodic during the sample analysis (e.g., and LC-IMS-MS analysis).

[0082] The selected components are then analyzed via downstream TIMS (or the third ion mobility separator mentioned above) and QTOF, using any known existing TIMS-TOF method (including PASEF, dia PASEF, midia PASEF, synchro PASEF, etc.).

[0083] The barriers defined by these ion mobility cutoffs are preferably configured such that barriers retaining low-abundance ions are alternated with barriers retaining high-abundance ions. Mobility regions comprising one or more closely spaced high-abundance species are typically defined by mobility regions of low-abundance ions. In this case, it is also preferred that a first RF generator generates a potential (pseudopotential) applied to the electrode of the low-abundance barrier, and a second RF generator generates an RF potential applied to the electrode of the high-abundance barrier. The first generator according to the preferred method can be operated to continuously confine ions—that is, always generating a strong pseudopotential. The second generator can operate at a high RF potential (e.g., 400 Vpp) for most of the time to guide lower-abundance ions from the inlet to the barrier region where they will be trapped. However, periodically or alternatively, at the end of accumulation step c), the second generator can be set to a low RF potential, such as 150 Vpp or lower, for a short time (e.g., 1 ms or 2 ms) to allow ions in all high-abundance regions to simultaneously expand / diffuse radially and collide with the electrodes of the device—and thus be destroyed. An advantage of this implementation is that the pseudopotentials in all high-abundance regions can be controlled by a single RF generator—i.e., simplified electronics.

[0084] The duty cycle, and thus the relative abundance of high-abundance species, can be controlled by the time between the final quenching step—i.e., momentarily shutting off or lowering the second generator—and the end of accumulation step c). For example, if the accumulation step lasts 100 ms, a 10% duty cycle in the high-abundance region can be achieved by momentarily lowering the potential on the second generator at 90 ms and then restoring it to the limiting potential in the last 10 ms of the accumulation cycle. Then, at the end of the accumulation cycle, no one in the mobility region is quenched; instead, 100% of the low-abundance species and 10% of the high-abundance ions are retained, which accumulate in the last 10 ms of the accumulation cycle. That is, 90% of the high-abundance species will be eliminated by quenching 90% of those entering the channel in the accumulation cycle. The remaining 10% of the high-abundance species and 100% of the low-abundance species will be transferred to "TIMS2," or more generally to the third ion mobility separator mentioned above.

[0085] Note that the method according to the second aspect of the invention can be combined with the steps mentioned above: synchronously controlling the downstream ion mobility separator and mass filter to perform multiple IM scans, during which precursor ions with increased or decreased IM are sequentially released from the IMS, and during the multiple IM scans, the mass window of the mass filter is continuously or gradually shifted toward lower or higher m / z values, respectively. Therefore, the parallel cumulative serial fragmentation method can essentially be combined with the method according to the second aspect of the invention. This parallel cumulative serial fragmentation method is an important part of the method according to the first aspect of the invention, but the method according to the second aspect of the invention can also be implemented without this parallel cumulative serial fragmentation method step.

[0086] Therefore, the above sequence of steps of the method according to the second aspect of the invention can be regarded as a different method for solving the same or similar problems, and can also be regarded as an independent invention as long as it does not include parallel cumulative serial fragmentation steps.

[0087] The method (which also applies to the first and second aspects of the invention) may further include the step of periodically quenching the first ion mobility separator by lowering the potential barrier so that substantially all ions leave the device, and preferably simultaneously using a blocking potential on a gate element between the first and second ion mobility separators so that ions leaving the first ion mobility separator are eliminated.

[0088] Preferably (this also applies to the first and second aspects of the invention), upstream of the first ion mobility separator, there is no additional ion trap for selectively ejecting captured ions according to the m / z ratio of the captured ions, in particular no high-capacity specific ejection (HCSE) trap, and in particular no trap configured to capture ions in the m / z range of at least 400, preferably at least 600, and most preferably at least 800 (or even 1200 or greater) for a time period of at least 0.3 s, preferably at least 1.5 s, and to selectively eject the captured ions according to the m / z ratio of the captured ions.

[0089] The method (which also applies to the first and second aspects of the invention) may further include the step of periodically quenching the first ion mobility separator by instantaneously reducing the limiting RF potential, causing all ions in the first ion mobility separator to be radially lost.

[0090] Preferably, the second mobility device and the third mobility device are TIMS devices.

[0091] The instrument may include a second gate element between the second ion mobility separator and the third ion mobility separator.

[0092] Except during step e), a blocking potential may be applied to the second gate element such that when a blocking potential is applied to the second gate element, ions leaving the second ion mobility separator are eliminated.

[0093] The mass spectrometer (this also applies to the first and second aspects of the invention) may further include any one of an analytical quadrupole, a collision chamber, a mass analyzer, and an ion detector, or be composed of any one of a quadrupole, a collision chamber, a mass analyzer, and an ion detector. The mass analyzer may be a time-of-flight, Paul trap, Penning trap, linear ion trap, or orbital trap mass analyzer.

[0094] The proposed method may further include the following steps: using any one of a first ion mobility separator, a second ion mobility separator, and a third ion mobility separator, as well as an ion detector, to generate a first-order full-spectrum mobility spectrum.

[0095] The method may also include the following steps: using any one of a first ion mobility separator, a second ion mobility separator, and a third ion mobility separator, a mass analyzer, and an ion detector to generate a first-order full spectrum of mass mobility.

[0096] Any of the ion mobility separators (this also applies to the first and second aspects of the invention) can also be used with a duty cycle between one percent and ten percent.

[0097] As mentioned above, and this also applies to the first and second aspects of the invention, the migration range of the components can be determined based on such a first-order full spectrum.

[0098] Preferably, the components are selected to avoid including the migration range of high-abundance ions and / or to avoid species in the exclusion list.

[0099] Such a method (which also applies to the first and second aspects of the invention) may further include the following steps:

[0100] A liquid chromatography phase is provided in front of the ion source;

[0101] Load the sample onto the LC column;

[0102] Chromatographic separation of sample components; and

[0103] The separated sample components are provided to an ion source for ionization.

[0104] Ion mobility components can be generated periodically during the LC separation process.

[0105] During the LC separation process, the range of migration rates for selected components can vary with retention time.

[0106] Furthermore, the migration range for selected components can be varied in a periodic repeating manner based on LC retention time.

[0107] Alternatively, the migration range of the generated components can be set as a function of the LC retention time according to a predetermined schedule.

[0108] In addition, the migration range of components can be determined based on the first-order full spectrum, in particular to avoid migration ranges containing high-abundance ions and / or to avoid species in the exclusion list.

[0109] As mentioned, the method according to the second aspect may further include the following additional step: the third ion mobility separator is used in conjunction with the mass spectrometer to perform mobility mass analysis of the components according to a method including PASEF containing diaPASEF, MIDIA PASEF, slicePASEF and synchroPASEF.

[0110] Typically (and thus equally applicable to the first and second aspects of the invention, and in all embodiments as described below, unless otherwise specified), according to a preferred embodiment, only two ion mobility devices are used—one primarily for accumulating ions within a selected mobility range, and a second for analyzing the accumulated ions.

[0111] The method according to this embodiment preferably includes the following steps:

[0112] a) Provide an instrument with at least two ion mobility separators and a mass spectrometer;

[0113] b) Preferably, a first-level full scan is generated with a low duty cycle;

[0114] c) Use a first-level full scan to determine the presence and migration rate of any high-abundance, unwanted species;

[0115] d) Determine one (or more) ranges of interest in mobility, which have a mobility higher than that of the high-abundance species;

[0116] e) Establish a mobility cutoff in the first ion mobility separator;

[0117] f) Accumulate the ions of interest in the first ion mobility separator, wherein the accumulated ions of interest constitute a mobility component;

[0118] g) Transferring the component to a second ion mobility separator; and

[0119] h) The second mobility device is used in conjunction with the mass spectrometer to perform mobility mass analysis on the components.

[0120] Regarding the first aspect (selective transfer principle) and the second aspect (mobility filter principle) of the present invention mentioned above, the following should be noted regarding the use of a first-level full scan:

[0121] According to the present invention, several combinations of these concepts are possible, namely:

[0122] Level 1 full scan + selective transfer

[0123] No Level 1 Full Scan + Selective Transfer

[0124] Level 1 full scan + mobility filter

[0125] No first-level full scan + mobility filter

[0126] Regarding Level 1 Full scan Preferably, a first-level full scan is performed with a low duty cycle (1% to 10%).

[0127] More preferably, a first-level full scan is performed in a second mobility separator (e.g., TIMS2).

[0128] If using none Level 1 Full scan Preferably, the method is executed using prior knowledge of the sample, and more preferably, it also utilizes database information.

[0129] If this method is applied without prior knowledge, it can be repeatedly cycled using predetermined components.

[0130] about Selective transfer Generally speaking, the following are preferred:

[0131] The first ion mobility separator (tims1+ optional gate) is used to generate the mobility component and transfer it to the second ion mobility separator (tims2).

[0132] The first ion mobility separator (TIMS1) can operate at a significantly higher repetition rate (mobility scans per second) than the second ion mobility separator (TIMS2) (4x, 10x). This allows the first ion mobility separator (TIMS1) to process significantly more ions per unit time than the second ion mobility separator (TIMS2) – thus effectively increasing the overall dynamic range.

[0133] The first ion mobility separator can operate at a high repetition rate in part via “non-linear” TIMS scanning.

[0134] Preferably, the first ion mobility separator operates in a nonlinear (TIMS1) scanning manner, which enables a high repetition rate.

[0135] about Mobility Filter Generally speaking, the following are preferred:

[0136] The first and second ion mobility separators (similar to TIMS devices) generate mobility components (essentially eliminating the need to capture high-abundance species).

[0137] Preferably, this method is typically combined with (liquid) chromatography + electrospray ionization upstream of a mobility analyzer.

[0138] Preferably, in this method, a second ion mobility separator (tims2) and a mass spectrometer perform PASEF on the selected component.

[0139] According to a particularly preferred embodiment, the second ion mobility separator is a TIMS analyzer, preferably a TIMS analyzer with parallel accumulation and separation, and specifically, it is operated using a method comprising the following steps:

[0140] (a) Ions from the first ion mobility separator are accumulated in the RF ion trap;

[0141] (b) At least a subset of the accumulated ions are transferred to a captured ion mobility separator, in which the transferred ions are radially confined by an RF field and pushed by a gas flow against the rising edge of an axial DC electric field barrier, such that the transferred ions are spatially separated along the rising edge according to the ion mobility.

[0142] (c) As ions from the ion source further accumulate in the RF ion trap, the transferred ions are released sequentially according to their ion mobility by lowering the height of the DC electric field barrier; and

[0143] (d) Restore the height of the DC electric field barrier, which triggers the continuous transfer of accumulated ions from the RF ion trap to the captured ion mobility separator.

[0144] The first ion mobility separator can be operated to retain only ions whose ion mobility is higher than the upper threshold of the IM window of the selected ion of interest.

[0145] in,

[0146] The second ion mobility separator is operated to allow ions with ion mobility below a lower threshold to pass through.

[0147] Alternatively, the second ion mobility separator can be operated such that ions with ion mobility below a lower threshold are filtered out.

[0148] Alternatively, the ion gate between the first ion mobility separator and the second ion mobility separator is operated such that ions with ion mobility below a lower threshold are not allowed to enter the second ion mobility separator.

[0149] Importantly, this particularly preferred embodiment of operating the first ion mobility separator and the second ion mobility separator is Third aspect of the invention This will be considered a separate aspect, which can also be implemented without the simultaneous control of the second ion mobility separator and the mass filter according to the first aspect of the invention or the second aspect of the invention mentioned above.

[0150] According to a first preferred embodiment of this aspect of the invention, but still within the context of the first and second aspects of the invention, the first ion mobility separator is a TIMS analyzer, wherein ions are radially confined by an RF field and propelled by a gas flow against the rising edge of an axial DC electric field barrier, such that the transferred ions are spatially separated along the rising edge according to their ion mobility, and wherein the height of the DC electric field barrier is selected so as to retain only ions with ion mobility higher than the upper threshold.

[0151] The second ion mobility separator is a TIMS analyzer with parallel accumulation and separation, wherein the transferred ions are accumulated in the accumulation section of the RF ion trap from the first ion mobility separator, wherein the ions are radially confined by the RF field and propelled by the gas flow against the rising edge of the axial DC electric field barrier, such that the transferred ions are spatially separated according to the ion mobility along the rising edge, and wherein the height of the DC electric field barrier is selected so as to retain only ions with ion mobility higher than the lower threshold.

[0152] The accumulated ions are then transferred to a capture ion mobility separator in the separation section, which is radially confined by an RF field and pushed by a gas flow against the rising edge of an axial DC electric field barrier, such that the transferred ions are spatially separated along the rising edge according to their ion mobility, and wherein the height of the DC electric field barrier at the start of release is preferably selected so as to retain only ions with ion mobility higher than the lower threshold.

[0153] For a predetermined ion mobility range The collection of ions can be performed using a method in any of a TIMS separator or a combination thereof, the method comprising: (a) passing ions from an ion source to two consecutive low-pass ion mobility filters, the first filter being followed by a second filter downstream of the first filter, each filter being formed by entraining ions in a gas exiting from the ion source and driving the entrained ions against a predetermined DC electric field barrier. The first filter includes a barrier having The first DC electric field barrier allows ions with high mobility to pass through, and the second filter includes a barrier having... The mobility of ions passing through the second DC electric field barrier, where, (a) The DC electric field barrier of the first filter and the DC electric field barrier of the second filter are each stationary along an axis parallel to the direction of gas flow; and (b) Ions from the ion source within the predetermined ion mobility range are collected in the space between the two filters.

[0154] A second ion gate may be provided between the accumulation section and the separation section to prevent ions with ion mobility higher than the lower threshold from entering the separation section.

[0155] According to another preferred embodiment of any aspect of the invention, the first ion mobility separator is operated at least once during the cycle of the second ion mobility separator, preferably at least twice or at least four times during the cycle of the second ion mobility separator, in order to allow all ions to pass through, while during this phase, the gate between the first and second ion mobility separators is controlled to prevent any ions from entering the second ion mobility separator, or the second ion mobility separator is left idle.

[0156] The first separation and selective transfer in the first ion mobility separator can be repeated, and the second ion mobility separator or an additional ion trap between the second ion mobility separator and the ion gate can be operated to accumulate the repeatedly transferred ions of interest before separating the repeatedly transferred ions of interest according to their mobility.

[0157] Preferably, when separating ions in the first ion mobility separator, the transmission of the ion gate is adjusted so that the transmission of ion species with high abundance of interest is lower than that of ion species with low abundance of interest.

[0158] According to yet another preferred embodiment, in the context of any aspect of the invention, the first ion mobility separator is a TIMS analyzer, and wherein, in the first ion mobility separator, the separation of ions in time according to mobility involves: driving ions transferred to the first TIMS analyzer by a first gas flow against a first reaction DC electric field barrier, such that the ions are captured and spatially separated according to their mobility at different locations along the slope of the first DC electric field barrier, at which the frictional force of the first gas flow is equal to the reaction force of the first DC electric field barrier; and separating ions in time according to mobility in the first TIMS analyzer by adjusting the height of the first DC electric field barrier or the velocity of the first gas flow.

[0159] According to another preferred embodiment, in the context of any aspect of the invention, the second ion mobility separator and / or the third ion mobility separator is a TIMS analyzer, and wherein, in the second ion mobility separator, the separation of ions in time according to mobility involves: driving ions transferred to the second TIMS analyzer by a second gas flow against a second reaction DC electric field barrier, such that the ions are captured and spatially separated according to their mobility at different locations along the slope of the second DC electric field barrier, at which the frictional force of the first gas flow is equal to the reaction force of the second DC electric field barrier; and separating ions in time according to mobility in the second TIMS analyzer by adjusting the height of the second DC electric field barrier or the velocity of the gas flow.

[0160] According to another preferred embodiment, regarding the aforementioned method for selecting the measurement IM window, particularly for excluding high-abundance species, in the context of the first and / or second aspects of the invention, at the start of an LC observation retention time window, preferably within the range of 1 to 15 seconds, particularly preferably within the range of 3 to 10 seconds, wherein preferably only one of the ion mobility analyzers is operated in separation mode for performing this first-level full scan, and wherein the complete ion mobility width of interest and the complete m / z width of interest are scanned in this first-level full scan, and wherein, based on the... The first-stage full scan of the remaining portion of the LC observation window is synchronously controlled with the second ion mobility separator and the mass filter to perform multiple IM scans, during which precursor ions with increasing or decreasing IM are sequentially released from the IMS, and during this time, the mass window of the mass filter is continuously or gradually shifted toward lower or higher m / z values ​​to avoid identifying uninteresting peptides in the first-stage full scan, and wherein the step of associating detected fragments with their corresponding precursor ions is based on determining or utilizing the corresponding mass window and IM range associated with the various occurrences of the fragments in the mass spectrometry measurement.

[0161] Importantly, this preferred embodiment using a first-level full scan is the fourth aspect of the invention, which is considered an independent aspect, and can also be implemented without the aforementioned synchronous control of the second ion mobility separator and mass filter, without the specific arrangement of the second aspect, and also independently of the third aspect.

[0162] To analyze first-level full-scan information on LC retention time, ion mobility, and m / z for peptides of uninterested interest and / or of interest, a database containing the relevant information can be queried and / or a list of peptides to be excluded can be queried, preferably being continuously supplemented during one or a series of LC experiments.

[0163] In the IM scan, adjacent mass windows associated with continuous mass spectrometry measurements of fragment ions can overlap such that the precursor ions delivered through the mass filter during an IM scan are located in at least one continuous scan region in the m / z-IM plane, which extends in a generally diagonal direction in the m / z-IM plane, wherein adjacent scan regions associated with different IM scans overlap in the m / z direction.

[0164] Preferably, in the IM scan, adjacent mass windows associated with continuous mass spectrometry measurements of fragment ions overlap by at least 30% of their width, and / or wherein adjacent scan regions associated with different IM scans overlap by at least 33% of their width in the m / z direction.

[0165] Preferably, the presence of the fragment corresponds to the relative or absolute intensity of the fragment in the mass spectrometry measurement.

[0166] according to Fifth aspect of the invention It involves equipment for data-independent combined ion mobility and mass spectrometry analysis.

[0167] This is particularly suitable for executing any of the methods defined above.

[0168] The device preferably includes:

[0169] An ion mobility separator (IMS) is used to receive precursor ions and sequentially release them from the IMS according to their ion mobility.

[0170] A mass filter is arranged to receive the released precursor ions and selectively deliver precursor ions whose m / z values ​​fall within a controllable mass window.

[0171] A fragmentation device for fragmenting precursor ions conveyed through the mass filter to generate fragment ions.

[0172] A device for mass spectrometry measurement of the fragment ions.

[0173] Each fragment ion is associated with a mass window and an ion mobility (IM) range.

[0174] and control systems,

[0175] Preferably, the control system is configured to synchronously control the IMS and the mass filter for multiple TM scans, during which precursor ions with increasing or decreasing IM values ​​are sequentially released from the IMS, and during this time, the mass window of the mass filter is continuously or gradually shifted toward lower or higher m / z values, respectively.

[0176] The control system is preferably configured to synchronously control the IMS and the mass filter such that, during the IM scan, adjacent mass windows associated with continuous mass spectrometry measurements of fragment ions overlap, and the precursor ions delivered through the mass filter during the IM scan are located in at least one continuous scan region in the m / z-IM plane, which extends approximately diagonally in the m / z-IM plane.

[0177] In another preferred embodiment, the control system is configured to control the IMS and the quality filter in a synchronous manner, such that adjacent scan regions associated with different IM scans overlap in the m / z direction.

[0178] The second ion mobility separator can be a TIMS analyzer.

[0179] Preferably, it is a TIMS analyzer with parallel accumulation and separation, and is specifically operated using a method including the following steps:

[0180] (a) Ions from the first ion mobility separator are accumulated in the RF ion trap;

[0181] (b) At least a subset of the accumulated ions are transferred to a trapped ion mobility separator, in which the transferred ions are radially confined by an RF field and propelled by a gas flow against the rising edge of an axial DC electric field barrier, such that the transferred ions are spatially separated according to the ion mobility along the rising edge.

[0182] (c) As ions from the ion source further accumulate in the RF ion trap, the transferred ions are released sequentially according to their ion mobility by lowering the height of the DC electric field barrier; and

[0183] (d) The height of the DC electric field barrier is restored, which triggers the continuous transfer of accumulated ions from the RF ion trap to the captured ion mobility separator.

[0184] The first ion mobility separator can be constructed and operated to retain only ions with ion mobilities higher than the upper threshold of the IM window for the selected ions of interest.

[0185] And among them,

[0186] The second ion mobility separator is operated to allow ions with ion mobility below a lower threshold to pass through.

[0187] Alternatively, the second ion mobility separator can be operated such that ions with ion mobility below a lower threshold are filtered out.

[0188] Alternatively, the ion gate between the first ion mobility separator and the second ion mobility separator is operated such that ions with ion mobility below a lower threshold are not allowed to enter the second ion mobility separator.

[0189] The first ion mobility separator can be a TIMS analyzer, wherein ions are radially confined by an RF field and propelled by a gas flow against the rising edge of an axial DC electric field barrier, such that the transferred ions are spatially separated along the rising edge according to their ion mobility, and wherein the height of the DC electric field barrier is selected so as to retain only ions with ion mobility higher than the upper threshold.

[0190] Furthermore, the second ion mobility separator is a TIMS analyzer with parallel accumulation and separation capabilities.

[0191] The transferred ions accumulate in an accumulation section within the RF ion trap from the first ion mobility separator, wherein the ions are radially confined by the RF field and propelled by the gas flow against the rising edge of the axial DC electric field barrier, such that the transferred ions are spatially separated according to their ion mobility along the rising edge, and wherein the height of the DC electric field barrier is selected so as to retain only ions with ion mobility higher than the lower threshold.

[0192] Subsequently, at least a subset of the accumulated ions are transferred to a trap ion mobility separator in the separation section, which is radially confined by an RF field and propelled by a gas flow against the rising edge of an axial DC electric field barrier, such that the transferred ions are spatially separated according to their ion mobility along the rising edge, and wherein the height of the DC electric field barrier at the start of release is preferably selected so as to retain only ions with ion mobility higher than the lower threshold.

[0193] Preferably, a second ion gate is provided between the accumulation section and the separation section of the second ion mobility separator to prevent ions with ion mobility higher than the lower threshold from entering the separation section.

[0194] Further embodiments of the invention are described in the dependent claims. Attached Figure Description

[0195] Preferred embodiments of the present invention will now be described with reference to the accompanying drawings, which are for illustrative purposes only and not for limiting the scope of the invention. In the drawings,

[0196] Figure 1 The principle of sequential field barrier filtering in a TIMS-like device, as proposed in prior art document US 2012 / 0273673, is illustrated schematically.

[0197] Figure 2The apparatus and operation of a captured ion mobility spectroscopy (TIMS) analyzer are illustrated. Top: Schematic diagram of the TIMS apparatus. Center: Distribution of electric field intensity along the z-axis. The size of the points on the electric field ramp reflects their space charge. The ion beams are released in order of mobility according to the ion beams. Bottom: Ion currents of ion pulses separated in time according to mobility, representing the ion mobility spectrum.

[0198] Figure 3 The apparatus and operation of a captured ion mobility spectroscopy (TIMS) analyzer with parallel accumulation and separation are shown. Top: Schematic diagram of the TIMS apparatus depicting the "accumulation" (11a) and "analyzer" (11b) regions. Bottom: Graph showing the DC electric field gradient and ion positions during the accumulation and analysis steps. In the top row, the first set of ions is eluted from analyzer region 11b by reducing the electric field gradient between points 32 and 34, while the second set of ions is simultaneously accumulated in the accumulation region on the electric field gradient between points 30 and 31. Note that the size of the points represents the collision cross-section of the ions. In the bottom row, after the first set of ions has been eluted, the field in analyzer region 11b returns to its full intensity, and the second set of ions is transferred from accumulation region 11a to analyzer region 11b by reducing the DC field intensity in accumulation region 11a to zero.

[0199] Figure 4 A schematic overview of a mass spectrometer with two TIMS analyzers and an ion gate between them is shown.

[0200] Figure 5 schematically shown Figure 4 The apparatus and operation of a tandem TIMS device are described, comprising a first TIMS analyzer (TIMS 1), an ion gate, and a second TIMS analyzer (TIMS 2). The figure below shows ions collected on the field ramp of TIMS 1, where the marked ranges of ion mobility represent the ions of interest. The size of the dots represents the amount of each type of ion, thus indicating the space charge of these ions. During a scan of TIMS 1, the ions of interest are gated and collected on the electric field ramp of TIMS 2. The selected ions can then be analyzed by TIMS 2 without additional space charge interference.

[0201] Figure 6 It schematically shows, as Figure 5 The cascaded TIMS devices shown herein, wherein each TIMS device includes, as Figure 3 The parallel accumulation shown. Figure 6 Schemes for operating this apparatus are depicted in sections b) through e). The size of the dots represents the abundance of each type of ion. In each case, the ion of interest is bounded by a vertical dashed line. Figure 6In b), each field gradient is initially set to its maximum value in order to analyze the maximum possible range of mobility. Figure 6 In c), the field gradient in TIMS 2 is reduced to provide more space for ions within the range of interest. Figure 6 In d), the field gradient in TIMS 1 is reduced so that ions with mobility below the range of interest never accumulate. Figure 6 In (e), TIMS 1 operates as a single region, and the DC electric field gradient is set so that only ions with mobility higher than the region of interest are accumulated, while allowing ions within the region of interest to pass through to the accumulation region of TIMS 2.

[0202] Figure 7 As shown in a), Figure 6 The tandem TIMS device in (a) also includes a gate between the accumulation region of TIMS 2 and the analyzer region, and a scheme for operating such a device. Note that ions with mobilities below the range of interest are eliminated at the second gate. Figure 7 b) is a timing diagram of a quenching cycle.

[0203] Figure 8 Plasma peptides as a function of ion mobility and LC elution time during LC-TIMS-MS analysis are shown in a). As indicated, high-abundance peptides and albumin peptides are distributed among lower-abundance plasma peptides. Figure 8 Mobility spectra of a single type of high-abundance plasma peptide ion at different accumulation times are shown in b). The bottom spectra are the mobility spectra obtained at an accumulation time of 100 ms—corresponding to 100% duty cycle—while the top spectra are the spectra obtained at an accumulation time of 2 ms (2% duty cycle). Figure 8 The spectra in b) together indicate that when the TIMS analyzer is overfilled with ions, the apparent mobility of the captured ions shifts, and the measured peak width increases. Figure 8 c) shows how the filtering in TIMS 1 and TIMS 2 is reflected in the corresponding spectra.

[0204] Figure 9 This demonstrates how synchronous window selection and quality analysis can be used in the PASEF method to obtain further selectivity.

[0205] Figure 10 In a), the unique peptide recognition results of 200 ng K562 as the ion mobility window varies across the full mobility range and the fractional mobility range are shown, and in b), the unique peptide ID of 10 ng K562 is shown across the full mobility range and the fractional mobility range.

[0206] Figure 11 The comparison of LC peak shapes at different sample amounts with a 2% duty cycle is shown.

[0207] Figure 12 IMS-MS primary full spectrum obtained during LC-IMS-MS analysis of 300 ng pure plasma hydrolysate is shown in a), IMS spectrum extracted from primary full scan is shown in b), IMS spectrum extracted from primary full scan is shown in c), and IMS spectrum extracted from primary full scan is shown in d).

[0208] Figure 13 BSA hydrolysate alone is shown in a), and BSA hydrolysate plus bradykinin is shown in b). Without filtration, BK2+ ions dominate—displacing other ions, and the mobility spectra at 2% duty cycle are shown in c). BK2+ now appears at a lower 1 / Ko and as multiple peaks representing multiple conformations; the low-mobility component according to this method is shown in d), and the high-mobility component according to this method is shown in e).

[0209] Figure 14 Showing from Figure 13 Histogram of experimental data;

[0210] Figure 15 Figure a) shows the migration spectra from the primary full spectrum generated at specific time points during the LC-IMS-MS analysis of plasma; Figure b) shows the TIMS spectra of the two components generated to avoid the high abundance species observed in the primary full spectrum of Figure a); and Figure c) shows the histograms of the results of diaPASEF analysis of unseparated sample ions (leftmost) and sample ions that have undergone migration fractionation as depicted in Figure b).

[0211] Figure 16 a) shows the number of log10 peptides as peptide ranking changes, and b) shows the count as RT value changes, where the upper graph shows the dynamic range shrinking by a factor of six, and the lower graph shows the dynamic range shrinking by a factor of 100.

[0212] Figure 17 The principle of using several sequential field barrier filters in a TIMS device or a similar device as proposed is illustrated schematically.

[0213] Figure 18 A) shows an elongated upstream ion separator followed by a gate and a second ion separator, and b) through d) show possible stages of operation of the device with five filter potential barriers in the upstream ion separator assembly. Detailed Implementation

[0214] Figure 2 A TIMS analyzer and its operation are schematically outlined, which can be used in this context as referenced in US 7,838,826B1 or US 10,241,079 B1. Ions 6 from an electrospray ion source (not shown) are entrained by gas 7 and introduced into the first chamber of a vacuum system through capillary 8. Repulsion plates 9 drive ions 6 into the inlet funnel 10 of the mobility analyzer. Ion funnels 10, 12 are typically constructed as a stack of perforated septa with openings gradually tapering to a smaller diameter, thus forming a funnel-shaped internal volume. Two phases of an RF voltage are alternately applied to the septa to establish a pseudopotential that keeps the ions away from the funnel walls. Ions are driven by an axial gas flow 14 and optionally by an additional DC potential gradient along the septa and through the narrow end of the first funnel 10 into the TIMS tube 11.

[0215] The axial gas flow 14 through the TIMS tube 11 is laminar and exhibits a generally parabolic velocity distribution in the radial direction. Nitrogen is preferred as the gas. The vacuum conditions around the TIMS tube 11 are selected such that the maximum gas velocity reaches approximately 100 to 150 meters per second at pressures of several hundred Pascals. This velocity is achieved only near the axis. The velocity decreases significantly further away from the axis, such as... Figure 2 As indicated by the middle arrow 14.

[0216] The first funnel 10 guides ions into the TIMS tube 11, forming a tunnel with an internal RF quadrupole field in the radial direction. The TIMS tunnel 11 comprises a stack of thin electrodes with a central aperture, forming a circular tube arranged around the z-axis of the device. The thin electrodes are separated by an insulating material that seals the gaps between the electrodes surrounding the tube. The electrodes of the TIMS tube 11 are divided into quadrants 1, 2, 3, and 4 to allow the generation of a radially confined quadrupole RF electric field internally. Figure 2 The top shows quadrants 1, 2, 3, and 4 of the tube electrodes, as well as the equipotential lines of the quadrupole RF field within the tube at a given time. It should be mentioned here that the design of a quadrupole tunnel does not necessarily consist of metal electrode sheets; there are many different possibilities, including stacked PCBs, or even rolled PCBs with printed electrodes.

[0217] Inside the TIMS tunnel 11, ions are propelled by the gas flow 14 against the axial DC electric field barrier. Figure 2The middle section shows the distribution of the axial DC electric field barrier for the three stages of the scan. Between z-positions 20 and 23, the DC electric field generated by the quadratic increase in potential increases linearly. Between z-positions 23 and 24, the DC electric field remains constant, thus forming a plateau of the DC electric field barrier generated by the linear increase in potential. For example, in a simple device, the complete field distribution can be generated by a single voltage applied to the separator electrode at position 24, and voltage division is achieved by precision resistors along the separator electrode of the TIMS tube 11. The resistors between positions 20 and 23 increase linearly, and the resistors between 23 and 24 have equal resistance. In more complex devices, nonlinear field distributions, or even adjustable DC field distributions, can be generated, for example, by a digital-to-analog converter (DAC).

[0218] The routine operation of the TIMS analyzer begins with the "ion accumulation phase," where ions accumulate on the DC electric field ramp at the top of the diagram. A voltage difference of approximately 300 volts creates a DC electric field barrier. A gas flow, symbolically indicated by arrow 16, propels the ions against the DC electric field barrier, where they stop because they cannot cross it. Note that arrow 16 represents the maximum gas velocity in the parabolic gas velocity distribution 14 within the tube. Ions accumulate on the rising edge of the DC electric field between positions 20 and 23, where low-mobility ions (primarily heavy ions with large collision cross-sections) accumulate in the high field near the upper end of the ramp, while high-mobility ions accumulate in the low field near the bottom of the ramp. The size of the dots represents the abundance of ions with different ionic mobilities, indicating the strength of the space charge. In the subsequent "scan phase," the supply voltage of the DC electric field barrier steadily decreases, and ions with increased mobility can escape toward the ion detector, specifically toward the mass analyzer, which operates as the ion detector. At the bottom of the figure, the ion currents for the released ion species are shown. The measured total ion current curve i = f(t) directly presents the ion mobility spectrum from low ion mobility to high ion mobility.

[0219] Ion mobility resolution R mob It depends on the scan speed. The lower the scan speed, the higher the resolution. As already mentioned, using slow scans, R² has been achieved with relatively small devices. mob = 400 ion mobility. Since ions generated in the ion source are lost during the scanning phase, the duty cycle (or ion utilization) depends on the cumulative time ta and the scan time t. s The ratio.

[0220] Figure 3An embodiment of a mobility spectrometer, which can be used in this context, is shown, and which combines accumulation and subsequent separation, as described, for example, in US 9,683,964 B2. The TIMS includes an elongated tunnel 11 divided into accumulation units 11a and scanning units 11b, and two voltage supply units (not shown) for the two tunnel units 11a, 11b, which contact partitions at positions 31 and 34. A resistor chain between the partitions in the two tunnel units generates two axial DC electric field distributions, as shown in the bottom portion of the figure.

[0221] according to Figure 3 The operation of the TIMS device includes two phases: in the accumulation phase and the scanning phase D, ions from the ion source (not shown) accumulate on the rising edge of the electric field distribution in the accumulation unit 11a, while at the same time, the ions in the scanning unit 11b are scanned by reducing the voltage supplied to position 34 of the scanning unit 11b, thereby releasing ions with increasingly higher mobility toward the ion detector through the outlet funnel 13.

[0222] In the transfer phase E, the voltage of the scanning unit 11b is first restored, and then the voltage of the accumulation unit 11a is turned off, so that the ions are driven by the gas flow to the rising edge of the electric field distribution of the scanning unit 11b. The transfer is completed in just one millisecond, and the accumulation and scanning phases can be restarted by turning on the voltage at position 31.

[0223] Figure 4 A time-of-flight mass spectrometer is shown having two TIMS analyzers arranged in tandem, as described, for example, in US-A-2018340910. In this context, this is utilized because the first ion mobility analyzer (TIMS 1, which can be as described in US-A-2018340910)... Figure 2 or Figure 3 The type described herein) can scan the collected ion beam, thereby releasing ions in order of their ion mobilities. During the scan, ion gates are alternately opened and closed, closed to reflect (or neutralize) unwanted ions, and opened to allow ions of different mobility ranges to pass. In this way, high-abundance ions with their space charge can be reflected in whole or at least partially. The passing ions enter a second ion mobility analyzer (TIMS 2, which can be as follows) Figure 2 or Figure 3 The type described herein, but preferably in this context is Figure 3 (types), which can be analyzed at high ion mobility resolution based on their ion mobility, unaffected by space charge. The mass spectrometer can then measure their precise mass; such as Figure 3The tandem mass spectrometer shown in the paper, equipped with a quadrupole mass filter and time-of-flight analyzer, can even measure fragment ion spectra for better identification of ion species.

[0224] Ions transferred to TIMS 2 may originate from a single mobility range or from several ranges, selected by switching the gate accordingly. In one embodiment, ions from a single mobility range are collected on a flat ramp of TIMS 2 to propagate ions as far as possible along the z-axis of TIMS 2. In this mode, ions can be accumulated and scanned several times in TIMS 1 to accumulate as many ions as required for high-quality analysis in TIMS 2. If an ion with a very high abundance exists in the range of interest, only a small fraction of these ions may be transferred by reflecting the largest portion of these ions. The ion gate is preferably switched faster than the time width of the ion pulse leaving the first ion mobility analyzer. The length of the ion pulse released by the scan is on the order of milliseconds, while the gate switching time can be less than microseconds; therefore, the ion pulse can be easily segmented.

[0225] While the radially confined RF field of TIMS 2 is preferably quadrupole for high ion mobility resolution, TIMS 1 can show tubes with larger inner diameters and / or radial RF fields with higher multiplicity, such as hexa, octupole, or dodecupole, or tubes with RF tunnels. An ion trap can also be located upstream of TIMS 1 to accumulate ions from the ion source during TIMS 1 scans. Even TIMS 1 can be freed from space charge effects if the trap can be mass-selectively unloaded.

[0226] Figure 5 The top section shows two tandem TIMS devices (TIMS 1, TIMS 2) separated by ion gates. The ion gates are formed as single-lenses for ion optics. These gates can be turned on and off in less than microseconds. During a scan of TIMS 1, the gates can be opened and closed in a suitable manner to select ions of interest. Ions of interest transferred to TIMS 2 can originate from a single mobility range, such as... Figure 5 The bottom section shows the usage for TIMS1 and TIMS2 as follows. Figure 2 The solution for the type of situation described.

[0227] In this configuration, ions are eluted from the TIMS device by increasing the TIMS inlet potential at a constant rate. Low-mobility ions are eluted first, followed by medium-mobility ions, and finally high-mobility ions. However, the ion gate only opens when ions with the desired mobility (schematically shown as vertical lines in TIMS 1) have been eluted. The duty cycle (cumulative time / total time) in this configuration is quite limited.

[0228] The first separation and selective transfer of ions of interest (schematically shown by vertical lines in TIMS 1) in the first ion mobility analyzer 1 can be repeated via a gate, and ions can be supplied to the second ion mobility analyzer 2 in the desired window to scan the repeatedly transferred ions of interest while separating ions according to mobility.

[0229] The TIMS inlet potentials in TIMS 1 and TIMS 2 can also be scanned nonlinearly, allowing the slow portion (high resolution) of the scan to occur only during the elution of the ions of interest. Because the scan takes less time, the TIMS filling and elution cycles can occur several times in each mass analyzer (e.g., ICR or ToF) transient. Ions with the selected mobility accumulate in the collision / fragmentation chamber and are then transferred to the mass analyzer (e.g., ICR or ToF) chamber.

[0230] Similarly, the initial portion of a TIMS scan can be truncated. Low-mobility ions then do not accumulate, and the elution scan begins immediately near the ions of interest. This means that more ions of interest can accumulate (i.e., they have more available space), and the scan time can be shorter, allowing for more repetitions in each mass analyzer (e.g., ICR or ToF) transient.

[0231] Figure 6 In the top section a), two tandem TIMS devices (TIMS 1, TIMS 2) separated by ion gates are shown again, where, however, in this case, for TIMS 1 and for TIMS 2, the following is used: Figure 3 The type described herein. The ion gate is formed as an ion optical single lens. The gate can be turned on and off in less than microseconds. During the scan of TIMS 1, the gate can be opened and closed in a suitable manner to select ions of interest. This setup allows for parallel accumulation and separation in each of the TIMS devices. Therefore, in this case, both TIMS devices have an accumulation unit 11a and a scanning unit 11b.

[0232] If TIMS 1 and TIMS 2 operate in the same manner, the resulting scheme is as follows: Figure 6As shown in b. In the accumulation unit 11a of the first TIMS 1 device, the entire ion mobility range is accumulated, and the entire ion mobility range then enters the separation unit 11b. The gate between the two TIMSs is controlled such that only the desired ion mobility window (schematically shown by the vertical dashed line in TIMS 1) is allowed to enter the TIMS 2 device. In the latter, accumulation is again performed in the accumulation unit 11a, and then separation is performed in the separation unit 11b.

[0233] To achieve optimal duty cycle and sensitivity for the ions of interest without being hindered by strong signals from uninteresting abundance ions, this tandem TIMS arrangement can be significantly optimized when operated for DDA in combination with the following scheme: in which TIMS 2 and the mass filter are controlled synchronously to perform multiple IM scans, during which precursor ions with increasing or decreasing IM are sequentially released from the TIMS, and during this time, the mass window of the mass filter is continuously or gradually shifted toward lower or higher m / z values, as if passing through Figure 6 The following schemes shown in c to e will be described.

[0234] According to Figure 6 As shown in scheme c, to achieve the highest possible resolution focusing in TIMS 2, focusing can be performed only on the window of ions of interest during both accumulation and scanning in TIMS 2. The initial portion of the scan can be truncated, or, as shown, at least the scan slope can be adapted to the window of interest, such that, substantially in TIMS 2, the slope is reduced to scan only the window of interest. This can be selectively adapted in both TIMS devices, if desired, in conjunction with the gas flow rate. This can be achieved such that the maximum potential reached in both the accumulation and scanning phases of TIMS 2 is relatively low and slightly below the low-mobility ion threshold of the desired window. The TIMS 1 device remains focused with... Figure 6 The same method is used in scheme b, therefore TIMS 1 accumulates and scans the desired window of interest at a higher frequency than TIMS 2, resulting in an optimal duty cycle. The TIMS 1 device can be cycled at a rate ten times higher than TIMS 2. In each such scan of TIMS 1, ions in the desired window are eluted, the gate is opened, and the desired ions enter the accumulation cell 11a of the TIMS 2 device.

[0235] Due to the low mobility of the ions of uninterested interest, if they are not gated between the two TIMS devices, they will pass through the TIMS 2 device before the corresponding scan is initiated, which can actually be further improved as... Figure 6The scheme shown in c can also limit the accumulation and scan voltage ramps in TIMS 1 so that it is essentially cut off at a low mobility ion threshold slightly below the desired window.

[0236] This results in ions with mobility below the desired window's low-mobility ion threshold passing through TIMS 1 and TIMS 2. Conversely, ions with mobility above the desired window's high-mobility ion threshold are retained and accumulate in TIMS 1. TIMS 1 thus acts as a cutoff filter, blocking any ions with mobility above the desired window's high-mobility ion threshold. TIMS 2 is responsible for eliminating ions with mobility below the low-mobility ion threshold, allowing these ions to pass through unimpeded.

[0237] This can be Figure 6 The approach shown in scheme e is further optimized. This is not necessary if the TIMS 1 operation does not involve accumulation and scanning phases, but instead operates in a manner that only ensures any ions with ion mobility above the high-mobility ion threshold of the desired window are blocked, and this has the flattest possible voltage ramp to allow for optimal selectivity; in fact, it may even lead to a steeper cutoff effect. Therefore, Figure 6 In the scheme of TIMS 1, the vertical dashed line represents the high mobility ion threshold 17 of the desired window in TIMS 1. On the other hand, the long vertical dashed line in the scheme of TIMS 1 represents the low mobility ion threshold 18 of the desired window in TIMS 2.

[0238] This means that TIMS1 does not perform TIMS analysis, but only operates as a mobility truncation.

[0239] To achieve this in a useful manner, TIMS 1 is periodically quenched (e.g., every 100 ms) by, for example, reducing the TIMS potential to 0 V (or a potential low enough to allow all ions to escape from the outlet). Ions released in this way can be prevented from entering TIMS 2 by destroying the ions at the gate.

[0240] Ions cut off by TIMS 1 at 17 accumulate in the accumulation section of TIMS 2. Ions with mobilities lower than the TIMS 2 accumulation cutoff at 18 can be eliminated at an additional gate between the TIMS 2 accumulation region 11a and the TIMS 2 analyzer region 11b. Therefore, in this case, TIMS 1 does not operate at a higher frequency than TIMS 2 because accumulation is handled in the accumulation section of TIMS 2. However, a higher operating frequency of TIMS 1 would still be advantageous if the quenching of higher-mobility ions should occur periodically and as frequently as possible to reduce interference with the desired ion mobility window.

[0241] This is Figure 7 Schematably shown in a), wherein the upper portion shows a TIMS arrangement including additional gates (interstage optics) at the interface between the accumulation portion 11a and the scanning portion 11b, and wherein, according to Figure 6 The scheme described below includes a gating mechanism between the accumulation section 11a and the scanning section 11b. At this gate between the two sections 11a and 11b, ions with mobilities below the cutoff 18 are eliminated, thus preventing them from entering the scanning section 11b in a way that interferes with the scan, and the ions of interest are eluted sequentially from TIMS 2 according to their ion mobilities. However, it should be noted that ions may also be allowed to pass through the downstream TIMS 2, as this may only result in a consistently low background, which generally does not interfere because it is in the region of the mass / mobility domain, rather than in the selected range.

[0242] There are significant and unexpected advantages associated with operating two TIMS devices essentially as cutoff devices for the ion mobility window of interest, one by one, as follows: high-abundance peaks exhibit significantly larger peak widths in ion mobility scans and mask low-abundance peaks present at or near the tails of these high-abundance peaks. If the cutoff value is not chosen sufficiently far from the high-abundance peaks, this results in the masking of desired low-abundance peaks and reduced sensitivity. These effects are due to the co-existence of high-abundance ions and desired ions during the accumulation and scanning phases of the TIMS devices. Essentially using the cutoff tandem TIMS technique proposed herein, particularly for DDA, and in combination with a scheme in which TIMS 2 and a mass filter are controlled synchronously to perform multiple IM scans, during which precursor ions of increasing or decreasing IM are sequentially released from the TIMS, and during this time, the mass window of the mass filter is shifted continuously or gradually toward lower or higher m / z values, respectively. Therefore, the proposed technique allows for higher sensitivity and better focusing on the desired ion mobility window, as well as better elimination or blocking of high abundance signals.

[0243] This is important because the high abundance signal also broadens in the LC dimension, and the proposed technique allows for more efficient elimination of the high abundance signal, as well as for broader LC peaks, which can have peaks up to 1.5 minutes long (typically 20 to 30 seconds), resulting in a peak width of 0.07 1 / K0 in the ion mobility dimension. When using TIMS1 for high mobility cutoff, ions with mobilities below the cutoff pass through TIMS1 without being trapped. However, ions with mobilities above the cutoff accumulate in TIMS1. The trapped ion population and associated charge will increase over time. To avoid overloading of trapped ions, TIMS1 must be periodically “quenched”—clearing all ions. Figure 7 b) is an example timing diagram of a quenching cycle. In practice, during the mobility cutoff operation, the cycle of ions passing through and being quenched is repeated continuously at regular intervals—every 30 ms in this example.

[0244] exist Figure 7 In section b), the timing of the relevant potentials is depicted. In this example, a potential “T3” is applied to the inlet of the TIMS1 analyzer, and a DC barrier is set to determine the mobility cutoff. “Deflector” and “gate” potentials are applied to the deflector at the inlet of the TIMS1 element and the gate at the outlet of the TIMS1 element, respectively. Initially, the deflector potential is set to deflect ions from the ion source into TIMS1, T3 is set to allow ions below the predetermined high mobility cutoff to pass through TIMS1, while trapping ions above the high mobility limit in TIMS1, and the gate is set to allow ions to pass through TIMS2. ​​In this example, this state of ion passage lasts for 27 ms. After this time, all ions in TIMS1 are quenched.

[0245] During quenching, all ions in TIMS1 are released by reducing the DC potential on T3 to 0 V. Lowering the T3 potential eliminates the DC barrier in TIMS1, allowing all ions to exit from the TIMS1 outlet. During quenching, the gate is set to a "closed" potential, ensuring that virtually all ions leaving TIMS1 are eliminated. Simultaneously, the deflector potential is set to prevent ions from entering TIMS1 from the ion source. In this example, "quenching" takes approximately 3 ms.

[0246] In alternative methods, ions can be quenched by temporarily reducing the TIMS1 RF ion confinement potential.

[0247] In alternative methods, it is not necessary to switch the deflector potential to prevent ions from entering TIMS1 during the quenching step. Instead, the deflector potential can be set to continuously allow ions to pass through into TIMS1. Prior to analysis, the plasma sample is digested—typically using trypsin—to produce peptides. The peptides and their abundance indicate the abundance of the proteins that produce them and the proteins in the original sample.

[0248] Importantly, in human plasma, the 14 most abundant proteins (whose peptides are shown here as dark gray) constitute xx% of the sample—these are proteins and their corresponding peptides are easily observed and not of interest, as they are observed in every plasma sample and do not specifically indicate the state of the system from which they originate.

[0249] Albumin is one of the proteins at the top—its peptides are shown here as light gray.

[0250] exist Figure 8 In a), the top 14 most abundant peptides and albumin peptides, along with all other detected less abundant plasma peptides, were shown to vary with their ion mobility and elution time during LC separation.

[0251] The key point is that peptides from high-abundance proteins frequently appear throughout the LC-IMS (and m / z) range, and therefore often interfere with the measurement of other peptides occupying nearby positions in the LC-IMS space (lower-abundance peptides of interest, medium gray). Therefore, eliminating or significantly reducing these high-abundance species is important.

[0252] For whatever reason, it is advantageous to do so within the instrument if these high-abundance species have not been eliminated or their relative abundance reduced before the start of LC IMS analysis.

[0253] Using a low duty cycle is important when performing a single-level full scan. Electrospray ionization sources continuously generate ions. The "duty cycle" corresponds to the fraction of time it takes for ions to accumulate from this continuous source. This means, for example, that although a TIMS analysis might require 100 ms, only 2 ms of ion accumulation corresponds to a 2% duty cycle.

[0254] The measured total spectral information is a superposition of low-abundance proteins and high-abundance (undesired) peptides, which typically appear near low-abundance (desired) peptides. The challenge of the analysis is to minimize the signal of high-abundance peptides in the spectral analysis in order to maintain high selectivity and sensitivity for low-abundance proteins at high duty cycles.

[0255] This method uses a combination of the two sequential TIMS devices mentioned above, preferably operated in the manner described. A FAIMS filter can be located upstream of the first TIMS 1 device to filter out ions with selected charges and to fractionate peptides based on different mobilities. TIMS 1, with a downstream gate (or filter), provides selective transfer and fractionates ions based on mobility. The second TIMS step is TIMS 2, which accumulates ions and analyzes them to increase capacity. The second ion mobility analyzer TIMS 2 and the downstream mass filter are controlled in a synchronized PASEF manner to perform multiple IM scans, during which precursor ions with increasing or decreasing IM are sequentially released from the IMS, and during this time, the mass window of the mass filter is continuously or gradually shifted toward lower or higher m / z values, respectively. The step of associating detected fragments with their corresponding precursor ions is based on determining or utilizing the corresponding mass window and IM range associated with the various occurrences of the fragments in the mass spectrometry measurement.

[0256] like Figure 8 As depicted in the data in b), when the TIMS analyzer is overfilled with ions, the apparent mobility of the captured ions shifts, and the measured peak width increases. Therefore, the results of the first-stage full scan and the determination of the mobility range to be excluded become distorted. For example, using a 2% duty cycle reduces the risk of the TIMS analyzer being overloaded by high-abundance ions during the first-stage full scan. Importantly, according to the invention, by using a low duty cycle during the first-stage full scan, (1) only (or essentially only) high-abundance species are visible; (2) by reducing the duty cycle, the TIMS is not overloaded; and (3) because the TIMS is not overloaded, the low duty cycle first-stage full scan provides a distortion-free measurement of ion mobility and mobility peak width.

[0257] The appropriate mobility window for subsequent ion filtration can be determined using only this low duty cycle full scan – that is, high duty cycle measurements will produce distorted peaks.

[0258] Importantly, at low duty cycles (2 ms), the mobility peak is narrow, while at high duty cycles (100 ms), the peak is broad and shifted due to TIMS overload. High duty cycle peaks are not a true measure of the mobility of high-abundance species; low duty cycle peaks are.

[0259] exist Figure 8Example of how this scheme can be applied and put into practice is shown in section c). In this illustration, the signal intensity is given as a function of the barrier voltage for different PEG fragments, and a high mobility threshold 17 is shown, with those systems retained in TIMS 1 to its left. The window of interest is then shown to the right of dashed line 17 and to the left of dashed line 18, which shows a low mobility threshold 18. The region between these dashed lines is retained in the accumulation section 11a of TIMS 2. Anything to the right of the low mobility threshold 18 provided by TIMS 2 is discarded either through the gate between the accumulation section 11a and the scan section 11b of TIMS 2 or by way of loss downstream of TIMS 2. High selectivity and high (steep) cutoff at 17 and 18 are possible using the proposed scheme.

[0260] exist Figure 9 The effect of synchronous control of the mass window filter, which functions as a feature of the TIMS 2 ion mobility analyzer, is partially shown in the middle section. Heatmaps of 200 ng K562 are shown in the full mobility range and the fractionated mobility range. Figure 9 The results shown were generated using conventional TIMS instruments instead of tandem TIMS instruments. These results were generated using 200 ng of K562 injected onto a 25 cm LC column, an 85-minute gradient, and a flow rate of 300 nl / min. In these experiments, the ion accumulation and analyzed mobility ranges were simply narrowed down to the ranges shown. In these experiments, each fraction corresponded to a separate LC – a “data-dependent” PASEF run; 4 fractions = 4 complete LC runs. Furthermore, because these experiments do not include TIMS 1 selection, the scan range here does not actually correspond to the mobility range of the accumulated ions. Low-mobility ions may be excluded from accumulation, but high-mobility ions will always be accumulated. In contrast, true selective transfer, as proposed in this invention, can exclude ions with mobilities higher and lower than the selected range, resulting in a slightly higher capacity for the selected range.

[0261] Figure 10 The histogram shows the data from... Figure 9 The experimental results. Figure 10 In (a), the unique peptide recognition results varied with the ion mobility windows of 200 ng K562 in both the full mobility range and the fractionated mobility range. Importantly, adding the two non-overlapping components—1 / Ko = 0.7–0.9 and 0.9–1.1—resulted in a significant increase in the number of peptides recognized without selective transfer, 30.4 kJ vs. 20.7 kJ. Figure 10As shown in b), for lower sample amounts—in this case 10 ng K562—the improvement in unique peptide recognition is slightly reduced. Here, the results of the two components are added together, as compared to the unfractionated 31.8 kJ peptide, resulting in the identification of a 37 kJ peptide.

[0262] According to the present invention, the selectivity and synchronicity of the TIMS 2 apparatus and subsequent mass analysis preferably rely on a first-level full scan technique. One possibility for achieving this is to begin with a first-level full scan within a typical 3-10 second LC peak, followed by selective and synchronized operation of the TIMS 2 apparatus and subsequent mass analysis based on the first-level full scan. The first-level full scan can be recorded by operating only TIMS 1 or TIMS 2 across the full ion mobility width and recording the complete first-level full scan in the sense that M / z varies with 1 / K0, and this is only for a small window in the LC dimension. Typically, the first-level full scan step takes 100 ms.

[0263] Computer algorithms can then be used to identify high-abundance peptides in the first-stage full scan. If the high-abundance peptides are known, this can be done by querying a database and obtaining the corresponding LC and IM information for these high-abundance peptides. The corresponding regions in the first-stage full scan can then be automatically cleared and excluded from the selective and synchronous operation of the TIMS 2 device and subsequent quality analyses. Alternatively, an exclusion list can be maintained (preferably running) to exclude peaks from previous scans that have been identified as high-abundance signals of uninterested interest or as low-abundance signals already identified. The method with an exclusion list can also be implemented without a first-stage full scan.

[0264] Therefore, the steps can be implemented as follows:

[0265] a) Perform a first-level full scan (1% duty cycle) and look for mobility ranges with high abundance peaks;

[0266] b) Perform at least two selective TIMS 1 scans, each of which substantially deselects all high-abundance species as determined (by computer algorithms) via a first-level full scan;

[0267] c) Transfer the selected results to TIMS 2 for further analysis;

[0268] d) Operate the remainder of the TIMS 2+ instrument (analytical quadrupole mass filter, collision chamber, time-of-flight analyzer) to analyze the selected ions, but preferably according to the PASEF method, which includes DIA PASEF (see Meier, F. et al., Parallel Accumulation-Serial Fragmentation (PASEF): Sequencing Speed ​​and Sensitivity by Simultaneous Scan Doubling in Capture Ion Mobility Devices, J. Proteome Res. 14, 5378–5387 (2015), which is included in this disclosure), sychro-PASEF (see Skowronek P. et al., Synchro-PASEF Allows Precursor-Specific Fragment Ion Extraction and Interference Removal in Data-Independent Acquisition, bioRxiv, https: / / doi.org / 10.1101 / 2022.11.01.514654, which is included in this disclosure), or MIDIA. PASEF (see Meier et al., midiaPASEF maximizes the information content in proteomics by data independence, bioRxiv, https: / / doi.org / 10.1101 / 2023.01.30.526204, which is included in this disclosure).

[0269] The first-level full scan in a) can operate with a duty cycle of 1% to 10%; ... with a duty cycle of 1% to 100%.

[0270] A full scan can be performed in TIMS2 (TIMS1 will only be operated to deliver ions).

[0271] The first-level full scan is preferably performed within a 100 ms time period; or within the time period of the TIMS 2 operation in step d).

[0272] The selected range is preferably 10% of the first-level full scan range.

[0273] Any number of TIMS 1 selective PASEF scans can be used to accumulate selected ions for a single TIMS 2 scan.

[0274] In step b), a “non-linear” scan is preferably used; any known scan + gating scheme can be used to operate TIMS1.

[0275] Both TIMS 1 and TIMS 2 can operate in “parallel accumulation” mode; however, as mentioned above, preferably only TIMS 2 operates in this mode, while TIMS 1 operates only as an IM low-pass filter or gate.

[0276] Steps a) through c) can be repeated 5 to 8 times for each selected mobility range to achieve, for example, the “synchro-PASEF” method. According to the synchro-PASEF method, each such repetition will use a different quadrupole quality range.

[0277] Note that an ion detector (e.g., a Faraday cup or a simple lens with an electrometer) located between TIMS 1 and TIMS 2 (e.g., an electrometer) can be used for a first-level full scan, thus making the first-level full scan independent of the rest of the instrument. This allows TIMS 2 to analyze a previously selected set of ions, while TIMS 1 performs the first-level full scan.

[0278] Furthermore, a first-level full scan can be achieved using TIMS 1 and a separate detector. The concept is to perform a first-level full scan using TIMS 1 with a separate ion detector, while TIMS 2 performs analysis on a previously selected set of ions. The ion detector can be any detector known in the art. In a preferred embodiment, the detector is fast enough to generate mobility spectra (in milliseconds) based on the ions eluted from TIMS 1, and sensitive enough to produce spectra of the strongest class when TIMS 1 is operating at a duty cycle of a few percent. Microchannel plate-based ion detectors or channel electron multiplier ion detectors are suitable, but may require depressurization and therefore a separate chamber.

[0279] If the sample is generally known (i.e., it is plasma, for example), then the elution time of the most abundant and / or least interesting species from the LC is essentially known. The actual elution time can vary from one LC run to the next, and therefore methods have been developed to compensate for this variation (see, for example, US-A-20210033575). The migration rates of the most abundant / least interesting species can also be known in advance.

[0280] Therefore, according to this alternative implementation method, it can be carried out as follows:

[0281] Determine the LC elution time and mobility of virtually all high-abundance, uninterested species present in a given sample type;

[0282] A planned exclusion list containing the above-mentioned elution times and migration rates is generated and can be used by the instrument control computer;

[0283] Using the instrument control computer and exclusion list, the TIMS1+ gate is used to selectively transfer the migration window at the selected LC elution time and migration rate that is not in the exclusion list to TIMS2 for further analysis, thereby performing LC tandem TIMS-MS sample analysis.

[0284] The advantage of loading a lower sample volume is a reduction in chromatographic peak width, such as... Figure 11 As shown: High abundance species do not appear at a discrete point in the LC-IMS domain—instead, any particular high abundance species will appear within a range of elution times and mobilities. If TIMS is overloaded, the abundance species will appear at a shifted and wider range of mobilities. Furthermore (not shown), high abundance species affect other species in the TIMS analyzer—especially those with similar mobilities. In particular, high abundance species can cause the loss of other species ions from the TIMS analyzer and / or cause a shift in the mobility peaks of other species.

[0285] Similarly, if the LC column is overloaded with too much sample material, the retention times of high-abundance species will broaden and shift, causing other species in the LC column to also shift in retention time. As an example, this figure shows the LC peaks associated with human albumin peptides from a pure plasma sample. These chromatograms were extracted from a 50-minute separation performed under the following conditions: Bruker nanoElute, Ion Opticks 25 cm column, run at 300 nL / min, and detection using Bruker timsTOF running at a 2% duty cycle. Sample amounts injected onto the column were 50 ng, 100 ng, 200 ng, 300 ng, and 600 ng, respectively, to produce… Figure 12 Chromatograms of a), b), c), d), and e). Clearly, with increasing sample amount, the LC peaks associated with this albumin peptide broaden and shift to shorter elution times.

[0286] Using more sensitive instruments (such as timsTOF SCP or Ultra), and thus reducing the amount of sample material loaded onto the column, can reduce LC peak widths and avoid retention time shifts. Importantly, reducing peak widths in any dimension (LC, IMS, or MS) reduces overlap of similar species in the LC-IMS-MS space, and therefore reduces potential interference from such overlap. A disadvantage of loading less sample onto an LC column is that chemical noise and cluster ions become more pronounced.

[0287] As from Figure 12 As can be seen, in a single first-order full spectrum (2% duty cycle, TIMS-MS spectrum obtained at a given time in LC separation), only a few species with high abundance exist.

[0288] The criteria used to determine which peaks will be excluded from further analysis:

[0289] Abundance (Intensity Threshold)

[0290] Based on past experience, we know which categories users are not interested in (users must upload a form).

[0291] Will including it along with the rest of the selected mobility range cause TIMS 2 box overload?

[0292] Criteria used to determine the range of components and which components should be further analyzed:

[0293] Excluded peaks

[0294] Time available for analyzing all selected components

[0295] The number of species that may exist in a component (the component's mobility and mobility width—its potential to be analyzed to return useful information).

[0296] In a), the first-order full spectrum of IMS-MS obtained during LC-IMS-MS analysis of 300 ng of pure plasma hydrolysate is given (scan #11953).

[0297] In b), the IMS spectrum extracted from the first-order full scan of a). High abundance peaks are only present in the interval 0.78 < 1 / K0 < 0.84. Two components: 1) from 1 / K0 = 0.7 to 0.78; and 2) from 1 / K0 = 0.84 to 1.2, can be analyzed without significant interference.

[0298] In c), the IMS spectrum extracted from a full scan #8623 was obtained during LC-IMS-MS analysis of 50 ng of pure plasma hydrolysate. High abundance peaks were present only in the 0.77 < 1 / K0 < 0.82 interval. Two components: 1) from 1 / K0 = 0.7 to 0.77; and 2) from 1 / K0 = 0.82 to 1.2, could be analyzed without significant interference.

[0299] In d), the IMS spectrum extracted from a first-level full scan #13177 was obtained during LC-IMS-MS analysis of 50 ng of pure plasma hydrolysate. High abundance peaks were observed in two intervals: 0.74 < 1 / K0 < 0.77 and 0.89 < 1 / K0 < 0.93. Three components: 1) from 1 / K0 = 0.7 to 0.74; 2) from 1 / K0 = 0.77 to 0.89; and 3) from 1 / K0 = 0.93 to 1.2, could be analyzed without significant interference. However, considering the time required to analyze component 1) and the limited number of species it may contain, this component might be discarded.

[0300] exist Figure 13 Further evidence of the concept is provided in the paper (BSA hydrolysate + BK peptide).

[0301] To further illustrate and provide an example of the method according to the invention, a mixture of protein hydrolysates and unrelated high-abundance peptides was analyzed. Two samples were prepared and analyzed—a 25 fmol / μL solution of bovine serum albumin (BSA) trypsin hydrolysate and a mixture of 25 fmol / μL BSA and 5 pmol / μL bradykinin (BK). Figure (a) shows the migration spectra from the TIMS analysis of 25 fmol / μL BSA. PASEF analysis was performed on the relevant detected peptide ions. From the obtained data, 29 unique peptides were identified by a MASCOT search. These peptides represent the peptides of interest. Figure (b) shows the TIMS mobility spectra of the BSA plus BK mixture performed at 100% TIMS duty cycle. Here, bradykinin 2+ ions (BK2+) were detected with excessively high intensity—i.e., the TIMS analyzer was overloaded with BK2+ ions. Due to the overload, the BK2+ peak broadened and shifted in terms of mobility. PASEF analysis of the BSA+BK mixture resulted in the detection of only one BSA peptide. Figure (c) shows the TIMS mobility spectra of the BSA+BK mixture performed with a 2% TIMS duty cycle. Here, a moderate intensity BK2+ peak was detected. Notably, the BK2+ peak was observed to cover the range from approximately 0.8 1 / Ko to 0.9 1 / Ko. Therefore, subsequent components (Figures d and e) avoided this mobility range. Figure (d) shows the TIMS spectrum of the low-mobility component produced by the method according to the invention. Here, the TIMS1 barrier was set to trap ions with mobilities higher than 0.9 1 / Ko and allow ions with lower mobilities to pass through. The TIMS2 accumulation barrier was set to trap all ions with mobilities higher than 1.4 1 / Ko. After 100 ms of accumulation, the ions were transferred to the TIMS2 analyzer and then mobility analysis was performed in a 100 ms scan. PASEF analysis was performed, and then the data were subjected to a MASCOT search, identifying 12 unique peptides. Figure (e) shows the TIMS spectra of the high-mobility fraction generated according to the method of the present invention. Here, the TIMS1 barrier was set to allow ions of all mobilities to pass through, while the TIMS2 accumulation barrier was set to capture all ions with mobilities higher than 0.78 1 / Ko. After 100 ms of accumulation, the ions were transferred to the TIMS2 analyzer, and mobility analysis was performed in a 100 ms scan. PASEF analysis was performed, followed by a MASCOT search of the data, identifying 15 unique peptides. The combined results of the low-mobility and high-mobility fractions show 23 unique peptides identified. Compared to a single unique BSA peptide identified by the prior art PASEF method, 23 unique peptides were identified by significantly eliminating high-abundance BK2+ ions via the method of the present invention.

[0302] For complete MS analysis of 25 fmol / uL BSA hydrolysate in 5 μM bradykinin; a simple sample was selected to mimic plasma proteomics, where high-intensity peptides from high-abundance proteins essentially washed away low-intensity peptides; 5 μM bradykinin was incorporated; low-mobility and high-mobility fractions were performed to exclude the transport and accumulation of bradykinin 2+ in TIMS-2; for both fractions, ddaPASEF was performed in TIMS-2, and the identified peptides were compared with those identified from ddaPASEF analysis of 25 fmol / uL BSA hydrolysate alone; the goal of the ddfPASEF method is to “recover” lost peptides in the presence of high-intensity peptides.

[0303] Figure 14 Further proof of the idea is provided. (From...) Figure 14 Histograms of experimental data. Analysis of a 25 fmol / μL BSA trypsin hydrolysate solution identified 38 BSA peptides, 29 of which were unique. Analysis of a mixture of 25 fmol / μL BSA and 5 pmol / μL bradykinin (BK) identified only one BSA peptide using the existing PASEF method. Analysis of a mixture of BSA and BK according to the method of the invention identified 31 BSA peptides, 23 of which were unique. At least three of the BSA peptides not detected in the mixture analysis according to the method of the invention originated from regions near BK2+ ions in the mass-mobility space.

[0304] In the figure: PASEF was performed on BSA alone (left bar); PASEF was performed on the components from the sample containing BSA+BK according to steps (f) to (h) (middle bar); and PASEF was performed on the sample containing BSA+BK (right bar). Without filtration and fractionation, only one peptide was identified from the BSA hydrolysate. Using filtration and fractionation according to the method of the invention, many, but not all (23 of 29) of the peptides were identified.

[0305] Figure 15Further proof of the concept is given. A 300 ng pure plasma sample was analyzed by LC-TIMS-TOFMS according to the ddfPASEF method of the present invention. A first sample was analyzed without fractionation according to the invention, and a second identical sample was analyzed with fractionation. Figure H shows only the spectra associated with the fractionation results. Figure (a) is the migration spectrum from the first full spectrum—i.e., the 2% duty cycle TIMS migration spectrum of pure plasma—obtained at LC run for 29.2 minutes (frame #16444). Here the strongest peak—albumin peptide (564.85 m / z)—can be seen, covering a migration range from 0.85 l / Ko to 0.91 l / Ko. Therefore, the subsequent fraction—Figure (b)—avoids this migration range. Figure (b) shows the TIMS spectra of the two fractions—one from 0.60 l / Ko to 0.85 l / Ko, and the other from 0.90 l / Ko to 1.40 l / Ko—combined into a single migration spectrum. Using the TIMS2 analyzer and the remainder of the downstream instruments (quadrupole, collision chamber, and orthogonal time-of-flight mass spectrometer), diaPASEF was performed on each fraction independently of the others. As shown in the histogram in Figure (c), diaPASEF analysis of the high-mobility fraction, followed by a Spectromine search of the data, identified 27 unique peptides and 18 unique proteomes. The same treatment identified 24 unique peptides and 18 unique proteomes from the low-mobility fraction. For comparison, analysis of pure plasma without the filtration and fractionation according to the invention revealed only 19 peptides and 5 proteomes at the corresponding time points of LC separation.

[0306] Compared to identifying 19 unique plasma peptides and 5 proteomes using the equivalent prior art PASEF method without the benefits of this invention, 50 unique peptides and 31 proteomes can be identified by significantly eliminating high-abundance albumin ions via the method of this invention.

[0307] For 300 ng of pure plasma, the albumin peptide peak eluted from 28.8 min to 31 min (tailing to 32.2 min, but at a relatively low intensity). Here, the peptide / protein had a peak retention time ranging from 28.5 min to 31.5 min.

[0308] In the analysis of complex samples, such as bottom-up plasma proteomics, it is advantageous to remove high-abundance species (in the current case, this means removing ions from the gas phase sample), otherwise high-abundance species will mask low-abundance species. Figure 16In the example shown, it is estimated that, during the LC-TIMS-MS analysis, removing one high-abundance species from any given spectrum on average would effectively improve the instrument's dynamic range (particularly the ability to see low-abundance species) by approximately 6-fold. Removing the eight most abundant species resulted in a 100-fold improvement in effective dynamic range.

[0309] Therefore, removing a large number of high-abundance gas-phase ions from our TIMS analysis is desirable. Reducing the dynamic range of peptides measured by two orders of magnitude would require removing approximately 1200 of the highest-abundance peptides (approximately 1650 precursors) distributed throughout the LCMS run. This is not feasible in practice. However, from Figure 16 As can be seen, if only one high-abundance precursor is excluded within the 10 s retention time window, a 6-fold reduction can be achieved, and removing the 8 most abundant species results in a 100-fold improvement in the effective dynamic range.

[0310] This can be achieved by using, for example Figure 17 The scheme shown achieves this by establishing a series of potential barriers, such that a specific, predetermined mobility range accumulates at each barrier. The barriers can be adjusted by the applied potential, allowing the mobility range to be fixed (predetermined) throughout the LC-IMS-MS experiment, or adjusted during the experiment either according to a plan or in a data-dependent manner. Figure 17 In this example, eight barriers are available, but the last three are not actually used. In this example, the width of the mobility range accumulated by the fifth barrier is greater than, for example, the width of the mobility range accumulated by the second barrier.

[0311] The gas flow, especially the drag force indicated by the arrow, is essentially constant throughout the device. The height E of the DC electric field barrier... z With the mobility K that can resist gas flow and be retained o Inversely proportional.

[0312] Figure 18 Example hardware and methods are shown:

[0313] As shown in a), the hardware of “TIMS1” is extended compared to “TIMS2” in order to make room for many (in this case, 5) barriers.

[0314] As shown in b), the electric field strength is constructed as a function of the five barrier locations. In this case, the barriers have substantially the same length, but in alternative embodiments the lengths can be different. The electric field strength at the plateau of each barrier, together with the previous barriers, sets the range of mobility to be accumulated on any barrier. The size of the point represents the abundance of the ion species.

[0315] As shown in c), ions can be quenched at regular time intervals and / or at the end of the accumulation step. Here, the first and third mobility ranges are quenched, while the second and fourth are retained.

[0316] As shown in d), the retained ions are transferred to the accumulation region of “TIMS 2”. Note that all barriers in TIMS 1 are reduced to zero, or alternatively, are fields, while all ions of interest are released into TIMS 2. Also note that while ions accumulate in TIMS 1, a previous set of ions is analyzed in TIMS 2 (b) and c). Essentially, this provides a method for eliminating one or more high-abundance species from mobility analysis, a method proposed with the following elements / steps:

[0317] a) An analyzer providing a gas flow with multiple DC barriers, radially confined RF pseudopotentials, and from the inlet to the outlet (alternatively, ion gates between barriers) Figure 18 (illustrated in a))

[0318] b) Set up 5 barriers.

[0319] c) Introduction and accumulation of ions (in) Figure 18 (shown schematically in b)

[0320] d) Eliminating high-abundance ions (in the accumulation region of the selected barrier) by reducing or significantly reducing the intensity of the confinement field. Figure 18 (Illustrated schematically in d). Alternatively, the limiting pseudopotential can be periodically or instantaneously reduced or eliminated, for example, by 1 ms every 30 ms during a 100 ms accumulation period, in order to eliminate or reduce only the number of high-abundance ions in these regions. Alternatively, high-abundance ions can be eliminated by lowering the DC barrier and allowing the ions to be eliminated at the gates behind the barrier.

[0321] e) Transfer the remaining ions to a downstream ion mobility (TIMS) analyzer (in... Figure 18 (Illustrated schematically in d), so the ions are first transferred to the downstream "TIMS 2" accumulation region. After accumulation in the downstream "TIMS 2" accumulation region, the ions are transferred to the downstream "TIMS 2" analyzer region so that they can be analyzed when the next set of ions is accumulated and filtered.

[0322] List of reference numerals

[0323] Quadrants of 1-4 TIMS tubes

[0324] 6 ions

[0325] 7 gases

[0326] 8 capillaries

[0327] 9 repulsion board

[0328] 10 Inlet Funnel

[0329] 11 TIMS tube

[0330] 11a11 cumulative unit

[0331] 11b11 scanning unit

[0332] 13 Outlet Funnel

[0333] 14-axis gas flow

[0334] 16 Maximum gas flow velocity

[0335] 17TIMS 1 truncation

[0336] 18TIMS 2-truncation

[0337] 20z position, the start of the rising edge

[0338] At position 23z, the end of the rising edge.

[0339] 24z position

[0340] 31. Partition position

[0341] 34 partition position

[0342] 50 First Base

[0343] 51 Second Base

[0344] Ions with decreased mobility (52-59)

[0345] D Accumulation and Scanning Phase

[0346] E-transfer phase

Claims

1. A data-independent method for combined ion mobility and mass spectrometry analysis, comprising the following steps: Precursor ions are introduced into two ion mobility separators (IMS), at least one of which, preferably the second, is a captured ion mobility spectrometry (TIMS) separator, wherein an optional ion gate is located between the two ion mobility separators. In the first ion mobility separator, ions are separated over time according to their mobility. Preferably, the ions of interest are selected by adjusting the transport of the ion gate during or after separation in the first ion mobility separator; The selected ions of interest are transferred to the second ion mobility separator; and In the second ion mobility separator, the transferred ions are separated according to their mobility. The precursor ions are sequentially released from the second ion mobility separator according to their ion mobility. The released precursor ions are introduced into a mass filter that selectively delivers precursor ions with m / z values ​​falling within a controllable mass window. The precursor ions delivered through the mass filter are fragmented to generate fragment ions. Mass spectrometry measurements are performed on the fragment ions, wherein each fragment ion is associated with a mass window and an ion mobility (IM) range, and the detected fragments are correlated with their corresponding precursor ions. The second ion mobility separator (IMS) and the mass filter are controlled synchronously to perform multiple ion mobility (IM) scans. During these multiple IM scans, precursor ions with increasing or decreasing IM values ​​are sequentially released from the second ion mobility separator (IMS). During these multiple IM scans, the mass window of the mass filter continuously or gradually shifts towards lower or higher m / z values, respectively. Furthermore, the step of associating the detected fragments with their corresponding precursor ions is based on determining or utilizing the corresponding mass window and ion mobility (IM) range associated with each occurrence of the fragments in the mass spectrometry measurement.

2. The method according to claim 1, wherein, The second ion mobility separator is a TIMS analyzer. Preferably, a TIMS analyzer with parallel accumulation and separation is used, particularly by a method comprising the following steps: (a) Ions from the first ion mobility separator are accumulated in the RF ion trap; (b) At least a subset of the accumulated ions are transferred to a captured ion mobility separator, in which the transferred ions are radially confined by an RF field and propelled by a gas flow against the rising edge of an axial DC electric field barrier, such that the transferred ions are spatially separated along the rising edge according to the ion mobility. (c) As ions from the ion source further accumulate in the RF ion trap, the transferred ions are released sequentially according to their ion mobility by lowering the height of the DC electric field barrier; and (d) Restore the height of the DC electric field barrier, which triggers the continuous transfer of accumulated ions from the RF ion trap to the captured ion mobility separator.

3. The method according to any one of the preceding claims, wherein, The first ion mobility separator is operated so as to retain only ions whose ion mobility is higher than the upper threshold (17) of the IM window of the selected ion of interest. And among them, The second ion mobility separator is operated to allow ions with ion mobility below the lower threshold (18) to pass through. Alternatively, the second ion mobility separator may be operated such that ions with ion mobility below a lower threshold (18) are filtered out. Alternatively, the ion gate between the first ion mobility separator and the second ion mobility separator is operated such that ions with ion mobility below a lower threshold (18) are not allowed to enter the second ion mobility separator. And / or wherein the first ion mobility separator, preferably having a TIMS analyzer with parallel accumulation and separation, is configured as at least one ion mobility separator, said at least one ion mobility separator (a) Filtering the ions by passing them through one of the following methods. (i) at least two consecutive high-pass ion mobility filters, and (ii) At least two consecutive low-pass filters, Each filter is formed by entraining the ions in a gas flowing in one direction and applying an electric field to the ions, the electric field causing the ions to move in the opposite direction to the gas flow; and (b) Collect ions within a predetermined ion mobility range in the space between the operating filters. Preferably, the first ion mobility separator is configured as a series of ion mobility separators, each ion mobility separator providing at least two consecutive high-pass ion mobility filters or at least two consecutive low-pass ion mobility filters, and wherein, in this series, at least two or three to six ion mobility separators are provided, or the first ion mobility separator is configured to provide one of the following: (i) at least three, preferably at least four, or four to eight consecutive high-pass ion mobility filters, and (ii) At least three, preferably at least four, or four to eight consecutive low-mobility ion-pass filters, Ions within a predetermined ion mobility range are collected in the space between the operating filters.

4. The method according to claim 3, wherein, The first ion mobility separator is a TIMS analyzer, wherein the ions are radially confined by an RF field and propelled by a gas flow against the rising edge of an axial DC electric field barrier, such that the transferred ions are spatially separated along the rising edge according to their ion mobility, and wherein the height of the DC electric field barrier is selected so as to retain only ions with ion mobility higher than the upper threshold (17). And wherein, the second ion mobility separator is a TIMS analyzer with parallel accumulation and separation, In this process, the transferred ions accumulate in the accumulation section (11a) of the RF ion trap from the first ion mobility separator, wherein the ions are radially confined by the RF field and propelled by the gas flow against the rising edge of the axial DC electric field barrier, such that the transferred ions are spatially separated along the rising edge according to their ion mobility, and wherein the height of the DC electric field barrier is selected so as to retain only ions with ion mobility higher than the lower threshold (18). Subsequently, at least a subset of the accumulated ions are transferred to a captured ion mobility separator in the separation section (11b), which is radially confined by an RF field and pushed by a gas flow against the rising edge of an axial DC electric field barrier, such that the transferred ions are spatially separated along the rising edge according to their ion mobility, and wherein the height of the DC electric field barrier at the start of release is preferably selected so as to retain only ions with ion mobility higher than the lower threshold (18).

5. The method according to claim 4, wherein, A second ion gate is provided between the accumulation portion (11a) and the separation portion (11b) to prevent ions with ion mobility higher than the lower threshold (18) from entering the separation portion (11b).

6. The method according to any one of claims 4 and 5, wherein, During the cycle of the second ion mobility separator, the first ion mobility separator is operated at least once, preferably at least two or at least four times, to allow all ions to pass through, while during this phase, the gate between the first and second ion mobility separators is controlled to prevent any ions from entering the second ion mobility separator, or the second ion mobility separator is left idle.

7. The method according to any one of the preceding claims, wherein, The first separation and selective transfer in the first ion mobility separator are repeated, and the second ion mobility separator or an additional ion trap between the ion gate and the second ion mobility separator is operated to accumulate the repeatedly transferred ions of interest before separating them according to their mobility. Furthermore, preferably, the transport of the ion gate is adjusted during the separation of the ions in the first ion mobility separator, such that the transport of the high-abundance ion species of interest is lower than the transport of the low-abundance ion species of interest.

8. The method according to any one of the preceding claims, wherein, The first ion mobility separator is a TIMS analyzer, and wherein, in the first ion mobility separator, the separation of ions in time according to mobility involves: driving the ions transferred to the first TIMS analyzer by a first gas flow against a first reaction DC electric field barrier, such that the ions are captured and spatially separated according to the mobility of the ions at different locations along the slope of the first DC electric field barrier, at which the frictional force of the first gas flow is equal to the reaction force of the first DC electric field barrier; and separating the ions in time according to mobility in the first TIMS analyzer by adjusting the height of the first DC electric field barrier or the velocity of the first gas flow. And / or wherein the second ion mobility separator is a TIMS analyzer, and wherein the temporal separation of ions in the second ion mobility separator based on mobility involves: driving the ions transferred to the second TIMS analyzer by a second gas flow against a second reaction DC electric field barrier, such that the ions are captured and spatially separated based on their mobility at different locations along the slope of the second DC electric field barrier, at which the frictional force of the first gas flow is equal to the reaction force of the second DC electric field barrier; and separating the ions in the second TIMS analyzer based on mobility by adjusting the height of the second DC electric field barrier or the velocity of the gas flow.

9. The method according to any one of the preceding claims, wherein At the beginning of an LC observation retention time window, preferably within the range of 1 to 15 seconds, and particularly preferably within the range of 3 to 10 seconds, a first-stage full scan is performed. Preferably, for this first-stage full scan, only one of the ion mobility separators operates in separation mode. And among them, In this first-level full scan, the full ion mobility width and the full m / z width of interest are scanned, as well as Specifically, based on this first-stage full scan targeting the remainder of the LC observation window, the second ion mobility separator and the mass filter are controlled synchronously to perform multiple IM scans. During these multiple IM scans, precursor ions with increasing or decreasing IM values ​​are sequentially released from the IMS, and during these multiple IM scans, the mass window of the mass filter is continuously or gradually shifted toward lower or higher m / z values ​​to avoid identifying uninteresting peptides in the first-stage full scan. The step of associating detected fragments with their corresponding precursor ions is based on determining or utilizing corresponding mass windows and IM ranges associated with each occurrence of the fragments in the mass spectrometry measurement. Or one of them a) To generate a first-level full scan, wherein, preferably, only one of the ion mobility separators operates in separation mode for the purpose of performing the first-level full scan, preferably with a low duty cycle; b) Use the first-level full scan to determine the presence and migration rate of any high-abundance, unwanted species; c) Determine at least one range of migration rates of interest, preferably multiple ranges of migration rates of interest, wherein the at least one range of migration rates of interest has a migration rate higher than that of the high-abundance species; d) Establish a mobility cutoff in the first ion mobility separator; e) Ions of interest accumulate in the first ion mobility separator, wherein the accumulated ions of interest constitute a mobility component; f) The component is transferred to the second ion mobility separator; and g) The second ion mobility separator is used in conjunction with the mass spectrometer to perform mobility mass analysis on the components.

10. The method according to claim 9, wherein, To analyze first-level full-scan information on LC retention time, ion mobility, and m / z for peptides of no interest and / or interest, a database containing the relevant information is queried and / or a list of peptides to be excluded is queried, the list preferably being continuously updated during one or a series of LC experiments.

11. The method according to any one of the preceding claims, wherein, In the IM scan, adjacent mass windows associated with continuous mass spectrometry measurements of fragment ions overlap, such that the precursor ions delivered through the mass filter during a single IM scan lie within at least one continuous scan region in the m / z-IM plane, the at least one continuous scan region extending in a generally diagonal direction in the m / z-IM plane. Among them, adjacent scan regions associated with different IM scans overlap in the m / z direction. Preferably, in the IM scan, adjacent mass windows associated with continuous mass spectrometry measurements of fragment ions overlap by at least 30% of the width of the adjacent mass windows, and / or wherein adjacent scan regions associated with different IM scans overlap by at least 33% of the width of the adjacent scan regions in the m / z direction. And / or preferably, the presence of the fragment corresponds to the relative or absolute intensity of the fragment in the mass spectrometry measurement.

12. An apparatus for data-independent combined ion mobility and mass spectrometry analysis, particularly for performing the method according to any one of the preceding claims, said apparatus preferably comprising: An ion mobility separator (IMS) is used to receive precursor ions and sequentially release the precursor ions from the IMS according to their ion mobility. A mass filter is arranged to receive the released precursor ions and selectively deliver precursor ions whose m / z values ​​fall within a controllable mass window. A fragmentation device for fragmenting the precursor ions conveyed through the mass filter to generate fragment ions. A device for performing mass spectrometry measurements on the fragment ions. Each fragment ion is associated with a mass window and an ion mobility (IM) range, and Control system The control system is configured to synchronously control the IMS and the mass filter to perform multiple TM scans, during which precursor ions with increased or decreased IM values ​​are sequentially released from the IMS, and during the multiple TM scans, the mass window of the mass filter is continuously or gradually shifted toward lower or higher m / z values, respectively. The control system is preferably configured to synchronously control the IMS and the mass filter such that adjacent mass windows associated with continuous mass spectrometry measurements of fragment ions overlap during the IM scan, and that the precursor ions delivered through the mass filter during the IM scan are located in at least one continuous scan region in the m / z-IM plane, the at least one continuous scan region extending in a generally diagonal direction in the m / z-IM plane. In a further preferred embodiment, the control system is configured to control the IMS and the quality filter in a synchronous manner, such that adjacent scan regions associated with different IM scans overlap in the m / z direction.

13. The device according to claim 12, wherein, The second ion mobility separator is a TIMS analyzer. Preferably, a TIMS analyzer with parallel accumulation and separation is used, particularly by a method comprising the following steps: (a) Ions from the first ion mobility separator are accumulated in the RF ion trap; (b) At least a subset of the accumulated ions are transferred to a captured ion mobility separator, in which the transferred ions are radially confined by an RF field and propelled by a gas flow against the rising edge of an axial DC electric field barrier, such that the transferred ions are spatially separated along the rising edge according to the ion mobility. (c) As ions from the ion source further accumulate in the RF ion trap, the transferred ions are released sequentially according to their ion mobility by lowering the height of the DC electric field barrier; and (d) Restore the height of the DC electric field barrier, which triggers the continuous transfer of accumulated ions from the RF ion trap to the captured ion mobility separator.

14. The device according to claim 12 or 13, wherein, The first ion mobility separator is constructed and operated so as to retain only ions whose ion mobility is higher than the upper threshold (17) of the IM window of the selected ion of interest. And among them, The second ion mobility separator is operated to allow ions with ion mobility below the lower threshold (18) to pass through. Alternatively, the second ion mobility separator may be operated such that ions with ion mobility below a lower threshold (18) are filtered out. Alternatively, the ion gate between the first ion mobility separator and the second ion mobility separator is operated such that ions with ion mobility below a lower threshold (18) are not allowed to enter the second ion mobility separator.

15. The device according to any one of claims 12 to 14, wherein, The first ion mobility separator is a TIMS analyzer, wherein the ions are radially confined by an RF field and propelled by a gas flow against the rising edge of an axial DC electric field barrier, such that the transferred ions are spatially separated along the rising edge according to their ion mobility, and wherein the height of the DC electric field barrier is selected so as to retain only ions with ion mobility higher than the upper threshold (17). Furthermore, the second ion mobility separator is a TIMS analyzer with parallel accumulation and separation capabilities. In this process, the transferred ions accumulate in the accumulation section (11a) of the RF ion trap from the first ion mobility separator, wherein the ions are radially confined by the RF field and propelled by the gas flow against the rising edge of the axial DC electric field barrier, such that the transferred ions are spatially separated along the rising edge according to their ion mobility, and wherein the height of the DC electric field barrier is selected so as to retain only ions with ion mobility higher than the lower threshold (18). Subsequently, at least a subset of the accumulated ions are transferred to a trap ion mobility separator in the separation section (11b), which is radially confined by an RF field and propelled by a gas flow against the rising edge of an axial DC electric field barrier, such that the transferred ions are spatially separated along the rising edge according to their ion mobility, and wherein the height of the DC electric field barrier at the start of release is preferably selected so as to retain only ions with ion mobility higher than the lower threshold (18). Preferably, a second ion gate is provided between the accumulation section (11a) and the separation section (11b) of the second ion mobility separator to prevent ions with ion mobility higher than the lower threshold (18) from entering the separation section (11b).

Images