TIMS with synchronized mobility filtering and analysis

Abstract

A method of data independent combined ion mobility and mass spectroscopy analysis, comprising the following steps: introducing precursor ions into two ion mobility separators (IMS), separating ions in the first ion mobility separator; transferring the ions of interest to the second ion mobility separator; and separating the ions in the second ion mobility separator. sequentially releasing ions from said second ion mobility separator according to their ion mobility, introducing the ions into a mass filter which selectively transmits ions having m / z values falling within a controllable mass window, fragmenting the ions transmitted through said mass filter to generate fragment ions, carrying out a mass spectroscopy measurement on said fragment ions, wherein each fragment ion is associated with a mass window and an ion mobility (IM) range, and associating detected fragments with its corresponding precursor ion, wherein said second ion mobility separator and said mass filter are controlled in a synchronized manner, and wherein a step of associating a detected fragment with its corresponding precursor ion is based on determining or utilizing the corresponding mass windows and IM ranges associated with various occurrences of said fragment in said mass spectrometry measurement.

Description

[0001] TITLE

[0002] TIMS WITH SYNCHRONIZED MOBILITY FILTERING AND ANALYSIS

[0003] TECHNICAL FIELD

[0004] The present invention relates to devices as well as methods for operating such devices in particular for the analysis of complex mixtures, especially mixtures of compounds that vary widely in abundance. In particular it relates to trapped ion mobility spectrometry with optimized mobility window filtering, accumulation, and mobility-mass analysis.

[0005] PRIOR ART

[0006] The motion of molecular ions in gases as compared to their motion in a condensed phase has established ion mobility as a powerful separation tool, especially in conjunction with mass spectrometry. Ion mobility spectrometry (IMS), such as drift tube ion mobility spectrometry (DTIMS), travelling wave ion mobility spectrometry (TWIMS), and differential mobility spectrometry (DMS), also known as field asymmetric ion mobility spectrometry (FAIMS), act as a post-ionization method for gas-phase filtering of ions in a controlled atmosphere, fractionating complex samples, suppressing chemical noise, separating isobaric mixtures, and characterizing molecular ions based on their mobility coefficients. These are methods that separate gas phase ions based on their interaction with a collision gas and their masses. In the first step, the ions are separated according to their mobility through a buffer gas on a millisecond timescale using an ion mobility spectrometer. The separated ions are then introduced into a mass analyzer in a second step where their mass- to-charge ratios can be determined on a microsecond timescale. The effective separation of analytes achieved with this method makes it widely applicable in the analysis of complex samples such as in proteomics and metabolomics.

[0007] US 7,838,826 B1 (M. A. Park, 2008) and the corresponding patent family members presents a small ion mobility analyzer / spectrometer which has become known under the acronym “TIMS” analyzer / spectrometer (TIMS = trapped ion mobility spectrometry). The terms ion mobility analyzer and ion mobility spectrometer are used interchangeably here. A TIMS analyzer comprises a gas flow that drives ions against a counter-acting electric field barrier such that the ions are at first trapped along the axis of the TIMS analyzer. The ions are confined in the radial direction by an electric RF field. After transferring ions from an ion source to the electric field barrier, the height of the electric field barrier or the gas velocity is adjusted such that ion species are released from the electric field barrier in the sequence of their mobility.

[0008] Commonly, the length of the ion mobility separation unit of a TIMS analyzer amounts to about five centimeters only. In a small tube with an inner diameter of about eight millimeters, a radial RF quadrupole field is generated to hold ions near to the axis. A gas flow inside a tube drives ions entrained in the gas flow against a ramped counter-acting electric DC field barrier where the ions are trapped and separated according to their mobilities at locations on the field ramp at which the friction force of the moving gas equals the counter-acting force of the electric DC field on the ramp. After loading the TIMS with ions, the height of the electric DC field barrier is decreased; this scan releases the ion species in the sequence of their mobility. Unlike many other trials to build small ion mobility spectrometers, the small device by M. A. Park has already achieved, with reduced scan speeds, ion mobility resolutions up to RmOb = 400, which is extraordinarily high.

[0009] Regarding the theoretical basis of TIMS, see the research article “Fundamentals of Trapped Ion Mobility Spectrometry”, K. Michelmann, J. A. Silveira, M. E. Ridgeway and M. A. Park, J. Am. Soc. Mass Spectrom., (2015) 26: 14-24.

[0010] US 8,766,176 B2 proposes improvements of the scan modes for TIMS analyzers by application of non-linear scans to achieve a linear mobility scale, a constant resolution along the mobility scale, or a temporal zoom. Furthermore, US 9,984,864 B2 describes a spatial zoom.

[0011] A TIMS analyzer with parallel ion accumulation is described in US 9,683,964; it improves the utilization of the ions from the ion source to nearly 100%. Importantly, many ions sources, such as electrospray ion sources produce ions continuously. TIMS with parallel accumulation can also be operated to collect and analyze ions continuously - that is, TIMS can operate at 100% duty cycle. Barring pseudopotential or space charge effects, substantially all ions of the ion source are collected and analyzed without loss. TIMS with parallel ion accumulation further provides the unique possibility to prolong the ion accumulation duration to find more detectable ion species, thereby even increasing the ion mobility resolution by a corresponding prolongation of the scan time. The ions are collected in an accumulator unit, preferably almost identical to the scanning unit, at a ramp of an electric DC field barrier such that they get spatially separated by their ion mobility along the ramp. Therefore, the accumulated ions are less influenced by space charge than in other types of accumulator units. Of greatest importance, however, is the unique feature of a TIMS analyzer that a longer accumulation period permits to increase the mobility resolution by choosing correspondingly longer mobility scan durations, e.g. 100 milliseconds scan duration with an ion mobility resolution of RmOb = 75 instead of 20 milliseconds scan duration with Rmob = 30. As a consequence of the higher number of ions collected and the better ion mobility resolution, more ion species can be detected and measured. Once an ion mobility scan is completed (optionally after twenty to some hundred milliseconds), the accumulated ions are transferred (in about a millisecond) from the accumulation unit to the scanning unit, and the next ion mobility scan can be started. In total, a skilled practitioner will appreciate that it will be possible to achieve a measurement rate of 300 to 450 ion species per second. If TIMS with parallel ion accumulation is installed in tandem mass spectrometer (MS / MS instrument) an MS-MS instrument, 300 to 450 characteristic fragment ion spectra per second may be measured quantitatively.

[0012] Some improvements for higher amounts of stored ions in selected regions of ion mobility, particularly for ions of low ion mobility, are given in US 9,304,106 B1. The higher loading capacity is based on non-linear electric DC field ramps, with flatter field ramps for ion species of interest, in order to diminish the effect of space charge for these ion species. But for precise ion mobility analyses of low abundant ion species in complex mixtures the influence of the space charge is still high.

[0013] Fig. 1 depicts a prior art, mobility-based filtering method according to Park et al. in US 2012 / 0273673. According to this document two mobility devices are provided. Ions to be filtered, entrained in a gas, are introduced at an entrance of the first mobility device, “... gas drives entrained ions against a first field barrier 50, keeping back all ions with mobilities p[> / =]p1. This case is schematically illustrated in Fig. 2 showing the disposition of ions 52 to 59 with different mobilities, indicated by different sizes of the dots representing the ions with their cross sections. The gas drives the ions 55 to 59, that have passed the first barrier 50 against a second field barrier 51 , keeping back the ions 55 and 56 with mobilities pt> / =pt2and thus keeping back and collecting all ions of a mobility in the range Apr between and p2with The ions 57 to 59 with p<p2pass the second barrier and disappear. To collect the ions 55 and 56, there has to be an ion storage device between the filters, e.g. by the provision of radial forces to keep the ions within the collection volume between the two barriers, such like a multipole field with its centripetally acting pseudopotential. The simplest way to generate the storage volume may be an enclosure of both filters in an RF multipole device, for instance, in an RF quadrupole system.”

[0014] US-A-2018340910 proposes a mass spectrometer comprising two ion mobility analyzers in tandem arrangement, of which at least one is a trapped ion mobility spectrometer (TIMS), and an ion gate which is located between the two ion mobility analyzers, and use thereof wherein ions are selectively transferred between the two ion mobility analyzers by adjusting the transmission of the ion gate while ions are separated in time according to ion mobility in the first ion mobility analyzer.

[0015] WO-A-2019096852 relates to use of an isobaric label in mass spectrometry (MS) analysis using data-independent acquisition (DIA), wherein said isobaric label comprises or consists of a group which fragments in the mass spectrometer (i) at an energy below the energy required for fragmenting analyte-derived precursor ions and / or a higher conversion rate than said precursor ions; and (ii) at said energy according to (i) and when coupled to a precursor ion, at a single site within said group, to yield a first moiety and a second moiety, said second moiety being coupled to said precursor ion. It proposes the use of a trapped ion mobility spectrometry- time of flight (timsTOF) instrument, equipped with parallel / serial fragmentation (PASEF); see, e.g. Meier et al. 2015, doi: 10.1021 / acs.jproteome.5b00932. US-A-2019371585 relates to selection of precursors from a measured mobility-mass map for tandem mass spectrometry and is based on processing a peak list from measured signals and clustering these peaks in the mobility-mass space.

[0016] US-A-2022034840 discloses an apparatus and a method of data independent combined ion mobility and mass spectroscopy analysis which includes introducing precursor ions into an ion mobility spectrometer (IMS), sequentially releasing precursor ions from said IMS according to their ion mobility, introducing said released precursor ions into a mass filter, fragmenting the precursor ions transmitted through said mass filter to generate fragment ions, and carrying out a mass spectroscopy measurement on said fragment ions. The IMS and mass filter are controlled in a synchronized manner to carry out a plurality of IM scans, wherein adjacent mass windows in said IM scan that are associated with consecutive mass spectroscopy measurements of fragment ions overlap, such that precursor ions transmitted through said mass filter during said IM scan are located in at least one continuous scan region in an m / z-IM plane which extends in a generally diagonal direction in said m / z-IM plane.

[0017] EP-A-4089714 discloses a method of and an apparatus for combined ion mobility and mass spectrometry analysis, the method comprising the following steps: introducing precursor ions into a trap configured for trapping ions and for selectively ejecting trapped ions according to their m / z ratio, selectively ejecting precursor ions having m / z values falling within at least one controllable ejection window from said trap, sequentially releasing precursor ions from said IMS according to their ion mobility, introducing said released precursor ions into a mass filter having a controllable mass window, fragmenting the precursor ions transmitted through said mass filter to generate fragment ions, and carrying out a mass spectrometry measurement on said fragment ions, wherein each fragment ion is associated with a mass window and an ion mobility (IM) range.

[0018] US-A-2022277949 provides hybrid mass spectrometric systems which comprise an ion source, a first trapped ion mobility spectrometry (TIMS) analyzer and a mass analyzer, wherein the TIMS analyzer is located and operated in a first vacuum chamber at an elevated pressure above 500 Pa, and methods for operating the hybrid mass spectrometric systems. Meier et al in Nature methods, vol 17 (2020), p 1229-1236 report, that data-independent acquisition modes isolate and concurrently fragment populations of different precursors by cycling through segments of a predefined precursor m / z range. Although these selection windows collectively cover the entire m / z range, overall, only a few per cent of all incoming ions are isolated for mass analysis. Here, we make use of the correlation of molecular weight and ion mobility in a trapped ion mobility device (timsTOF Pro) to devise a scan mode that samples up to 100% of the peptide precursor ion current in m / z and mobility windows. We extend an established targeted data extraction workflow by inclusion of the ion mobility dimension for both signal extraction and scoring and thereby increase the specificity for precursor identification. Data acquired from whole proteome digests and mixed organism samples demonstrate deep proteome coverage and a high degree of reproducibility as well as quantitative accuracy, even from 10 ng sample amounts.

[0019] Targeted selection of peptides from complex protein digests subjected to fragmentation is a performance-limiting factor in tandem mass spectrometry (MS). The problem is mainly attributed to the large number of co-eluting high abundance peptides and limitations in the duty cycle of the mass spectrometer arranged to operate in data-dependent acquisition (DDA) mode whereby precursor ions are selected sequentially for fragmentation in order of decreasing intensity. Furthermore, the depth of proteomic analyses is also limited by the overwhelming proportion of high abundance background ions that compromise the identification and quantification of low abundance peptides. Whether low-intensity precursors are selected for fragmentation is primarily dictated by the speed at which the mass analyzer can perform tandem MS analysis. The dynamic range of the analysis in DDA mode can be extended by excluding previously analyzed precursor ions, but this strategy is no longer efficient for highly complex samples with an extended dynamic range in concentration. The duty cycle of the MS remains a problem even in data-independent acquisition (DIA) mode where all ions within a selected mass-to-charge range are selected for fragmentation. The issue of sample complexity is routinely addressed by fractionating the sample.

[0020] Liquid chromatography coupled to Mass Spectrometry (LC-MS) has now been used for many years in the proteomic community for the identification and quantification of peptides (and thus proteins) from complex sample mixtures. In proteomics, the analytes are typically peptides generated by tryptic digestion of protein samples. The commonly most used approaches are variants of the so-called LC-MS / MS or “shotgun” MS approach that is based on the generation of fragment ions from precursor ions that are automatically selected based on the precursor ion profiles (data dependent analysis, DDA). A main shortcoming of these methods is poor reproducibility which results in only partially overlapping protein sets in repeated analysis of substantially similar samples. Several new approaches have recently been developed that address these limitations and which can conceptually be described as targeted proteomics approaches.

[0021] The most mature technology is called Selected Reaction Monitoring (SRM), frequently also referred to as Multiple Reaction Monitoring (MRM). The targets for MRM experiments are defined on a rational basis and depend on the hypothesis to be tested in the experiment. Selected combinations of precursor ions and fragment ions (so called transitions, the set of transitions for one target precursor is called MRM assays) for these targets are programmed into a mass spectrometer, which then generates measurement data only for the defined targets.

[0022] Another variant of targeted proteomics is data independent acquisition (DIA). Here, the targeted aspect is introduced only on the data analysis level. Contrary to MRM, this approach does not require any preliminary method design prior to the sample injection. Since the LC-MS acquisition covers the complete analyte contents of a sample through the entire mass and retention time (RT) ranges the data can be mined a posteriori for any peptide / precursor of interest. Data is acquired in a data independent manner, on the complete mass range (e.g. 200-2000 Thomson) and through the entire chromatography, disregarding of the content of the sample. This is commonly achieved by stepping the selection window of the mass analyzer step by step through the complete mass range. In effect, this data acquisition method generates a complete fragment ion map for all the analytes present in the sample and relates the fragment ion spectra back to the precursor ion selection window in which the fragment ion spectra were acquired. This is achieved by widening the precursor isolation windows on the mass analyzer and thus accounting a priori for multiple precursors co-eluting and concomitantly participating to the fragmentation pattern recorded during the analysis. Such a precursor window is called a precursor selection window. The result is complex fragment ion spectra from multiple precursor fragmentations, that require a more challenging data analysis.

[0023] SUMMARY OF THE INVENTION

[0024] As discussed above, when analyzing complex samples, for example samples of biological origin, it is necessary to separate sample components from each other that the various component species may be identified and quantified. In many cases, the sample components of interest have a relatively low abundance compared to uninformative, or less informative, species. Also, analytical instruments in general have a limited dynamic range - that is, the ability to simultaneously analyze very low abundance species in the presence of very high abundance species. It therefore occurs, for example in the analysis of blood plasma, that highly abundant components interfere with the analysis of low abundance components.

[0025] The solution to such a problem is to 1) improve the instrumentation by increasing its effective dynamic range or; 2) to eliminate, or reduce, the relative abundance of the high abundance components in the sample.

[0026] In the case of blood plasma, there are a number of approaches to reduce the relative abundance of the high abundance components in the liquid phase.

[0027] Two examples are “depletion”, wherein the high abundance proteins are largely removed from the sample; and “enrichment”, wherein the abundance of selected low abundance peptides is enhanced.

[0028] Although these approaches can achieve an improvement in effective dynamic range, such methods can be costly and time consuming and there is a concern that such methods may lack reproducibility and can lead to a distortion of relative abundance of the low abundance species as well - having a negative influence on protein quantitation.

[0029] Therefore, it is a first aim of the present invention to provide an additional, instrumentbased, more cost and time effective, approach to eliminate or substantially reduce the abundance of high abundance species from complex samples. This approach works by ionizing sample components and, in the gas phase, eliminating and / or reducing the high abundance, uninteresting ions based on their ion mobility. This approach can be used independently, or alongside prior art approaches, similar to those mentioned above for improving effective dynamic range.

[0030] It is a second aim of the present invention to provide an improved approach of fractionating ions, based on their gas phase ion mobilities, in order to reduce the complexity of resultant data sets, reduce the presence of high abundance species in at least some fractions, and improve the overall dynamic range of the sample analysis.

[0031] What is thus proposed are the following elements:

[0032] A method of filtering ions based on gas phase ion mobility - producing ion fractions. One aim is to provide an improved approach of fractionating ions, based on their gas phase ion mobilities, in order to reduce the complexity of resultant data sets, reduce the presence of high abundance species in at least some fractions, and improve the overall dynamic range of the sample analysis.

[0033] A method of eliminating undesired, uninformative, and / or high abundance species. One aim of the present invention is to provide an additional, instrument-based, more cost and time effective, approach to eliminate or substantially reduce the abundance of high abundance species from complex sample analysis.

[0034] The present invention accordingly relates to corresponding devices as well as methods for operating such devices in particular for the analysis of complex mixtures, especially mixtures of compounds that vary widely in abundance. In particular it relates to trapped ion mobility spectrometry with optimized mobility window filtering, accumulation, scan and detection window selection mobility-mass analysis.

[0035] A new protocol and system for LC-IMS-MS analyses is disclosed, including methods of operation thereof, in particular for data independent acquisition (DIA), to address the limitations in the art of analyzing complex protein samples summarized above. The new protocol features an IMS apparatus operated in the low-pressure regime enabling more tailored and selective as well as sensitive separations, including enhancing the duty cycle and the dynamic range of the instrument, thus increasing the depth of the analysis.

[0036] According to a first aspect of the present invention, it relates to a method of data independent combined ion mobility and mass spectroscopy analysis (selective transfer), comprising the following steps: introducing precursor ions into two ion mobility separators (IMS), of which at least one, preferably the second, is a trapped ion mobility spectrometry (TIMS) separator, an optional ion gate located between the two ion mobility separators, separating ions in time according to mobility in the first ion mobility separator; selecting ions of interest, preferably by adjusting the transmission of the ion gate during or after the separation in the first ion mobility separator; transferring the selected ions of interest to the second ion mobility separator; and separating the transferred ions according to mobility in the second ion mobility separator, sequentially releasing precursor ions from said second ion mobility separator according to their ion mobility, introducing said released precursor ions into a mass filter which selectively transmits precursor ions having m / z values falling within a controllable mass window, fragmenting the precursor ions transmitted through said mass filter to generate fragment ions, carrying out a mass spectroscopy measurement on said fragment ions, wherein each fragment ion is associated with a mass window and an ion mobility (IM) range, and associating detected fragments with its corresponding precursor ion.

[0037] According to the proposed protocol, said second ion mobility separator and said mass filter are controlled in a synchronized manner such as to carry out a plurality of IM scans, during which precursor ions of increasing or decreasing IM are successively released from said second IMS, and during which the mass window of said mass filter is shifted continuously or stepwisely towards lower or higher m / z values, respectively. So essentially a parallel accumulation serial fragmentation approach is used.

[0038] Furthermore, according to the proposed protocol, said step of associating a detected fragment with its corresponding precursor ion is preferably based on determining or utilizing the corresponding mass windows and IM ranges associated with various occurrences of said fragment in said mass spectrometry measurement.

[0039] Generally, such an ion mobility separator (IMS) can be a TIMS, an ion funnel, or an ion guide, in each case preferably having the capability to support radial RF ion confinement, a gas flow, and a DC electric field barrier capable of counteracting the drag force on the ions due to said gas flow.

[0040] A first preferred method of this approach is characterized as follows: In “bottom-up proteomics” LC-IMS-MS sample analysis - especially in cases in which the sample includes high abundance species which interfere with the analysis of species of interest. Such samples include ,for example, plasma and urine samples.

[0041] In this first preferred method,

[0042] (a) use one of the ion mobility separators only, preferably the second ion mobility separator (TIMS2), to obtain a low duty cycle survey scan (only a few % of the ions produced in the ion source are collected and analyzed);

[0043] (b) from the survey scan, identify the high abundance, undesirable species and determine the mobility fractions to be analyzed (normally mobility fraction windows excluding the high abundance, undesirable species);

[0044] (c) for a given mobility fraction window, set the barrier in the first ion mobility separator (TIMS1) to pass only ions within or below the mobility range of the given mobility fraction window (for selecting the desired fraction without high abundance, undesirable species);

[0045] (d) set the accumulation barrier in the second ion mobility separator (TIMS2) to pass only ions below the mobility range of the given mobility fraction window (again for selecting the desired fraction without high abundance, undesirable species) and accumulate ions of the desired mobility range in the second ion mobility separator (TIMS2) accumulation region for a predetermined time;

[0046] (e) transfer the accumulated ions to the analyzer section of the second ion mobility separator (TIMS2);

[0047] (f) perform a parallel accumulation serial fragmentation (PASEF), involving rapid switching of the quadrupole mass position to select multiple precursors at different m / z on the very same time scale, such that all targeted ions are fully used for fragmentation) analysis of the accumulated ion; and repeat steps (c) - (f) for all given mobility fraction windows (desired mobility fractions). According to the invention there is further provided an instrument I apparatus, including an ion source, (an ion optic device having the structure and features of) a first ion mobility separator (e.g. TIMS analyzer), a second ion mobility separator (e.g. TIMS analyzer) downstream from the first ion mobility separator, a (preferably quadrupole, e.g. time-of- flight) mass spectrometer downstream from said second ion mobility separator analyzer, and electronics, firmware, and software sufficient to operate and control the instrument to perform the above method.

[0048] According to a preferred embodiment, further an LC or other chromatographic separation upstream is provided of the above instrument.

[0049] The first ion mobility separator (e.g. TIMS1) may also be a simple funnel with a reverse DC at or near the funnel exit (as e.g. described in the prior art, Baykut et al, J. Am. Soc. Mass Spectrom. 2009, 20, 2070-2081 , which included by reference for this aspect of the present invention).

[0050] The above parallel accumulation serial fragmentation (PASEF, the expression including diaPASEF, MIDIA PASEF, slicePASEF, and synchroPASEF) may be a PASEF method including data dependent PASEF, data independent PASEF.

[0051] The proposed method may be used with any sample, any type of sample, any type of complex sample, and especially any type of sample comprised of components having a high dynamic range in abundance including proteomics, metabolomics, lipidomics, genomics, or any other “-omics” sample.

[0052] A “filter” method may be used to produce fractions without a survey scan, including using a table of predetermined species to be eliminated / fractions to be analyzed, using information about undesired species with a known LC and / or TIMS elution time / KO (scheduled species), using information about fractions of known LC and / or mobility range (scheduled fractions) to be analyzed.

[0053] According to a second aspect of the present invention, it relates to a method for fractionating and mass-mobility analyzing ions according to their mobility including the steps of (mobility filtering): a) providing an instrument including a source of ions, three ion mobility separators, and a mass spectrometer (mass spectroscopy measurement device); b) establishing a first mobility cutoff in said first ion mobility separator; c) establishing a second mobility cutoff in said second ion mobility separator; d) accumulating ions of interest in said second ion mobility separator wherein said ions of interest have a mobility between said first and second mobility cutoffs and the accumulated ions of interest constitute a mobility fraction; e) transferring said fraction to the third ion mobility separator; and f) using said third ion mobility separator in conjunction with said mass spectrometer to mobility mass analyze said fraction.

[0054] Said instrument may further include a first gate element between said first and second ion mobility separator.

[0055] In the analysis of complex samples, for example bottom-up plasma proteomics, it is generally advantageous to remove high abundance species (in the present case this means removing gas phase sample ions) which would otherwise mask low abundance species. In the examples shown further below it is estimated that removing one high abundance species, on average, in any given spectrum during the course of an LC-TI MS-MS analysis, will effectively improve the instrument’s dynamic range (especially the ability to see low abundant species) by a factor of about 6. Removing the 8 most abundant species results in a 100x improvement in effective dynamic range.

[0056] It is correspondingly desirable to remove a multitude of high abundance gas phase ions from a TIMS analysis.

[0057] According to of further element of the second aspect of the present invention, it is therefore proposed to have the first two of the above two ion mobility separators either extended to more than two such ion mobility separators, for example in the range of 3-8 such ion mobility separators or 4-6 such ion mobility separators, followed by a final ion mobility separator; and using said final ion mobility separator (preferably a TIMS analyzer with parallel accumulation and separation) in conjunction with said mass spectrometer to mobility mass analyze said fraction. A corresponding instrument may further include a gate element between the ion mobility separators.

[0058] These ion mobility separators upstream of the final ion mobility separator can be separate ion mobility separators, however the above two or more upstream ion mobility separators can also be realized as one trapped ion mobility separator device that is built to establish at least a first as well as a second mobility cutoff in the same ion mobility separator device. According to a preferred embodiment of the second aspect of the present invention it therefore also relates to using one single trapped ion mobility separator device which can establish at least two ion mobility cutoffs, preferably in the range of 2-8 or 3-6 ion mobility cutoffs.

[0059] So according this aspect of the invention, preferably a method for eliminating one or more high abundance species from a mobility analysis is proposed with the following elements / steps: a) provide an analyzer having a multitude of DC barriers, a radially confining RF pseudopotential, a gas flow from an entrance end to an exit end (alternatively ion gates between the barriers), b) set the barriers, c) introduce and accumulate ions, d) eliminate ions of high abundance by lowering, or substantially reducing the strength of the confining field in the accumulation region of the selected barriers (alternatively, the confining pseudopotential may be periodically, momentarily reduced or eliminated, for example for 1 ms every 30 ms during a 100 ms accumulation period, so as to eliminate or only reduce the number of high abundance ions in these regions. Alternatively, the high abundance ions may be eliminated by lowering the DC barrier and allowing the ions to be eliminated on a gate following the barrier), e) transfer the remaining ions to a downstream ion mobility (TIMS) analyzer (shown schematically in Fig. 18 d), so the ions are transferred first to the accumulation region of the downstream ion mobility (TIMS) analyzer (“TIMS 2”). After accumulation in the downstream ion mobility (TIMS) analyzer (“TIMS 2") accumulation region, the ions are transferred to the analyzer region of the downstream ion mobility (TIMS) analyzer ("TIMS 2") so they can be analyzed while the next group of ions is being accumulated and filtered).

[0060] Like that, high abundance species can be separated out in one or a series of ion mobility separator devices even if they are not located in the same ion mobility window, which allows for eliminating for example the above-mentioned five, six or even eight most abundant species by adjusting the corresponding cutoffs so as only to pass through to the final ion mobility separator the ions except these high abundant species, leading to the above- mentioned enormous improvement in effective dynamic range. According to the second aspect the invention relates not only to such a device but also to a corresponding method of analysis or to the use of such a device for eliminating several high abundance species from a complex sample which high abundance species are located in at least 2, preferably at least 3, at least 4, or in the range of 5-8 different ion mobility windows.

[0061] The means and method of the second aspect of the present invention can be used in conjunction with one or more of, liquid chromatography, electrospray ionization, in a data dependent manner in conjunction with a reduced duty cycle survey scan as described in this application, in conjunction with a schedule table, or wherein the selected / deselected mobility regions are predetermined and periodic during the course of the sample analysis (for example, and LC-IMS-MS analysis).

[0062] The selected fraction(s) are then analyzed via the downstream TIMS (or generally the above mentioned third ion mobility separator) and QTOF via any known prior art TIMS-TOF method including PASEF, dia PASEF, midia PASEF, synchroPASEF, etc.

[0063] The barriers defined by these ion mobility cutoffs are preferably set such that barriers retaining ions of low abundance are interleaved with barriers retaining ions of high abundance. A mobility region which includes one or more closely spaced high abundance species will normally be bounded by mobility regions that low abundance ions. This being the case, it is preferable also that a first RF generator produces the potential (pseudopotential) applied to the electrodes of the low abundance barriers and a second RF generator produces the RF potential applied to the electrodes of the high abundance barriers. The first generator according to the preferred method, can be operated to continuously confine ions - i.e. always producing a strong pseudopotential. The second generator can be operated at a high RF potential (e.g. 400 Vpp) most of the time so as to guide ions of lower abundance from the entrance end to the barrier region at which they will get trapped. However, periodically, or alternatively at the end of accumulation step c), the second generator can be set to a low RF potential, for example 150 Vpp or lower, for a short time, for example 1 or 2 ms, so as to allow ions in all the high abundance regions simultaneously to expand / diffuse radially and collide with the electrodes of the device - and thereby be destroyed. The advantage in this embodiment is the pseudopotential in all the high abundance regions can be controlled by a single RF generator - i.e. simplified electronics.

[0064] The “duty cycle” and thereby the relative abundance of the high abundance species can be controlled by the time between the last quench step - i.e. momentarily turning the second generator off or down - and end of accumulation step c). If, for example, the accumulation step lasts 100 ms, one can achieve a 10% duty cycle for the high abundance regions by momentarily reducing the potential on the second generator at 90 ms and then returning it to a confining potential for the last 10 ms of the accumulation cycle. One would then not quench any of the mobility regions at the end of the accumulation cycle, but rather, retain 100% of the low abundance species, and the 10% of the high abundance ions that accumulate in the final 10 ms of the accumulation cycle. That is, 90% of the high abundance species will be eliminated by quenching 90% of the way into the accumulation cycle. The remaining 10% of the high abundance species and 100% of the low abundance species would be transmitted on to “TIMS2”, or more generally the above mentioned third ion mobility separator.

[0065] It is noted that the method according to this second aspect of the present invention can be combined with the above-mentioned step of controlling the most downstream ion mobility separator and a mass filter in a synchronized manner such as to carry out a plurality of IM scans, during which precursor ions of increasing or decreasing IM are successively released from said IMS, and during which the mass window of said mass filter is shifted continuously or stepwisely towards lower or higher m / z values, respectively. So essentially a parallel accumulation serial fragmentation approach can be used combined with the method according to this second aspect of the present invention. This parallel accumulation serial fragmentation approach is an essential part of the method according to the first aspect of the present invention, but it this method according to this second aspect of the present invention can also be implemented without that parallel accumulation serial fragmentation approach step.

[0066] So, the above sequence of steps of the method according to this second aspect of the present invention can be regarded as a different approach to solving the same or a similar problem, and can also be considered an independent invention in as far as it does not include that parallel accumulation serial fragmentation step.

[0067] The method (this applies to the first and the second aspect of the invention equally) may further include a step of periodically quenching said first ion mobility separator by lowering said barrier such that substantially all ions exit the device and preferably simultaneously using a blocking potential on the gate element between the first and second ion mobility separator such that the ions exiting the first ion mobility separator are eliminated.

[0068] Preferably (this applies to the first and the second aspect of the invention equally) upstream of the first ion mobility separator there is no additional ion trap for selectively ejecting trapped ions according to their m / z ratio, in particular no high-capacity specific ejection (HCSE) trap, and in particular none configured for trapping ions within an m / z range of at least 400, preferably at least 600, and most preferably at least 800 (or even 1200 or more) for a time period of at least 0.3 s, preferably at least 1 .5 s, and for selectively ejecting trapped ions according to their m / z ratio.

[0069] The method (this applies to the first and the second aspect of the invention equally) may further include the step of periodically quenching said first ion mobility separator by momentarily reducing the confining RF potential such that all ions in the first ion mobility separator are lost radially.

[0070] Preferably, said second and third mobility devices are TIMS devices.

[0071] Said instrument may include a second gate element between the second and third ion mobility separator.

[0072] A blocking potential can be applied to said second gate element except during step e) such that when the blocking potential is applied to the second gate element, ions exiting the second ion mobility separator are eliminated.

[0073] Said mass spectrometer (this applies to the first and the second aspect of the invention equally) may further comprise or consist of any of an analytical quadrupole, a collision cell, a mass analyzer, and an ion detector. Said mass analyzer can be a time-of-flight, Paul trap, Penning trap, linear ion trap, or orbitrap mass analyzer.

[0074] The proposed method may further include the step of using any of the first, second, and third ion mobility separator and an ion detector to produce a mobility survey spectrum.

[0075] The method may further include the step of using any of the first, second, and third ion mobility separator, the mass analyzer, and an ion detector to produce a mass-mobility survey spectrum.

[0076] Any of the ion mobility separators (this applies to the first and the second aspect of the invention equally) can further be used with a duty cycle of between one and ten percent.

[0077] As mentioned above, and this applies to the first and the second aspect of the invention equally, the mobility range of the fractions can be determined from such a survey spectrum. The fractions are preferably selected to avoid mobility ranges containing high abundance ions and / or to avoid species in an exclusion list.

[0078] Such a method (this applies to the first and the second aspect of the invention equally) may further include the steps of: providing liquid chromatography up front of the ion source; loading a sample on the LC column; chromatographically separating sample components; and providing the separated sample components to the ion source for ionization.

[0079] Ion mobility fractions can be produced periodically during the course of the LC separation.

[0080] The mobility range selected for the fractions can vary with retention time during the course of the LC separation.

[0081] Also, the mobility range selected for the fractions can vary in a periodically repeating manner as a function of LC retention time.

[0082] The mobility range of the fractions produced can also be set as a function of LC retention time according to a predetermined schedule table.

[0083] Furthermore, the mobility range of the fractions can be determined from the survey spectrum, in particular to avoid mobility ranges containing high abundance ions and / or to avoid species in an exclusion list.

[0084] As mentioned, the method according to the second aspect may further include the further step of using said third ion mobility separator in combination with said mass spectrometer to mobility-mass analyze the fractions according to a method including PASEF, including diaPASEF, MIDIA PASEF, slicePASEF, and synchroPASEF.

[0085] Generally (so this applies to the first and the second aspect of the invention equally, as all of the following embodiments if not specified otherwise), according to a preferred embodiment, only two ion mobility devices are used - one primarily for accumulating ions of a selected mobility range and a second for analyzing the accumulated ions.

[0086] The method according to this embodiment preferably includes the steps of: a) Providing an instrument with at least two ion mobility separators and a mass spectrometer; b) Producing a survey scan, preferably at a low duty cycle; c) Using the survey scan to determine the presence and mobility of any high abundance, undesirable species; d) Determining a (or a plurality of) mobility range of interest which has a mobility higher than that of said high abundance species; e) Establishing a mobility cutoff in a first ion mobility separator; f) Accumulating ions of interest in said first ion mobility separator wherein the accumulated said ions of interest constitute a mobility fraction; g) transferring said fraction to the second ion mobility separator; and h) using said second mobility device in conjunction with said mass spectrometer to mobility mass analyze said fraction.

[0087] In relation with the above-mentioned first aspect of the invention (selective transfer principle) and with the above-mentioned second aspect of the invention (mobility filter principle), as concerns the use of a survey scan, the following is noted:

[0088] According to the present invention, several combinations of these concepts are possible, namely as follows:

[0089] • survey scan + selective transfer

[0090] • no survey scan + selective transfer

[0091] • survey scan + mobility filter

[0092] • no survey scan + mobility filter

[0093] As for the survey scan, this is preferably carried out with a low duty cycle (1 - 10%).

[0094] Further preferably the survey scan is preferably carried out in the second mobility separator (e.g. TIMS2).

[0095] If no survey scan is used, then preferably the method is carried out with foreknowledge of the sample, preferably recurring to database information in addition.

[0096] If the method is applied without foreknowledge, one may cycle repetitively through predetermined fractions.

[0097] As for the selective transfer, generally speaking the following is preferred:

[0098] The first ion mobility separator (timsl + optional gate) are used to produce and transfer mobility fraction to the second ion mobility separator (tims2); The first, ion mobility separator (timsl) may operate at a significantly (4x, 10x) higher repetition rate (number of mobility scans per second) than the second ion mobility separator (TIMS2). This allows the first ion mobility separator (TIMS1) to process significantly more ions per unit time than the second ion mobility separator (TIMS2) - effectively increasing overall dynamic range.

[0099] The first ion mobility separator can be operated using a high repetition rate in part, via a “non-linear” TIMS scan.

[0100] Preferably, the first ion mobility separator is operated with non-linear (TIMS1) scans so that one can achieve a high repetition rate.

[0101] As for the mobility filter, generally speaking the following is preferred:

[0102] A first and second ion mobility separator (TIMS-like device) produces mobility fractions (substantially without having to trap the high abundance species)

[0103] Preferably the method is generally combined with (liquid)chromatography + electrospray ionization upstream of the mobility analyzers.

[0104] Preferably in the method the second ion mobility separator (tims2) and the mass spectrometer perform PASEF on a selected fraction.

[0105] According to a particularly preferred embodiment, the second ion mobility separator is a TIMS analyzer, preferably a TIMS analyzer with parallel accumulation and separation, in particular operating using a method comprising the steps:

[0106] (a) accumulating ions from the first ion mobility separator in an RF ion trap;

[0107] (b) transferring at least a subset of the accumulated ions into a trapping ion mobility separator, in which the transferred ions are radially confined by an RF field and pushed by a gas flow against a rising edge of an axial electric DC field barrier such that the transferred ions are spatially separated along the rising edge according to ion mobility;

[0108] (c) successively releasing the transferred ions according to their ion mobility by decreasing the height of the electric DC field barrier while ions from the ion source are further accumulated in the RF ion trap; and

[0109] (d) restoring the height of the electric DC field barrier which triggers a consecutive transfer of the accumulated ions from the RF ion trap into the trapping ion mobility separator.

[0110] The first ion mobility separator can be operated such as to retain only ions having an ion mobility higher than an upper threshold value of an IM window for the selected ions of interest wherein either the second ion mobility separator is operated such as to allow ions having an ion mobility lower than a lower threshold value to pass through, or the second ion mobility separator is operated such that ions having an ion mobility lower than a lower threshold value are filtered out, or wherein the ion gate between the first ion mobility separator and the second ion mobility separator is operated such that ions having an ion mobility lower than a lower threshold value are not allowed to enter the second ion mobility separator.

[0111] Importantly, this particularly preferred embodiment of operating the first and the second ion mobility separator is a third aspect of the present invention which is to be regarded as a separate aspect which can also be implemented without the above-mentioned control of the second ion mobility separator and mass filter in a synchronized manner according to the first aspect of the present invention or according to the second aspect of the present invention.

[0112] According to a first preferred embodiment of this aspect of the present invention, but also in the context of the first and second aspect of the invention, the first ion mobility separator is a TIMS analyzer, in which the ions are radially confined by an RF field and pushed by a gas flow against a rising edge of an axial electric DC field barrier such that the transferred ions are spatially separated along the rising edge according to ion mobility, and wherein the height of the electric DC field barrier is chosen such as to retain only ions having an ion mobility higher than said upper threshold value, and the second ion mobility separator is a TIMS analyzer with parallel accumulation and separation, in which the transferred ions are accumulated in an accumulation section from the first ion mobility separator in an RF ion trap, in which the ions are radially confined by an RF field and pushed by a gas flow against a rising edge of an axial electric DC field barrier such that the transferred ions are spatially separated along the rising edge according to ion mobility, and wherein the height of the electric DC field barrier is chosen such as to retain only ions having an ion mobility higher than said lower threshold value followed by transferring at least a subset of the accumulated ions into a trapping ion mobility separator in a separation section, radially confined by an RF field and pushed by a gas flow against a rising edge of an axial electric DC field barrier such that the transferred ions are spatially separated along the rising edge according to ion mobility, and wherein the height of the electric DC field barrier at the beginning of the release is preferably chosen such as to retain only ions having an ion mobility higher than said lower threshold value.

[0113] For the collection of ions of a predetermined ion mobility range p1 >p>p2, a method can be used in any of the TIMS separators or a combination thereof, comprising: (a) passing ions from an ion source to two consecutive ion mobility low pass filters, a first filter followed by a second filter downstream from the first filter, each filter formed by entraining ions in a gas flowing away from the ion source and driving the entrained ions against a DC electric field barrier of predetermined, the first filter comprising a first DC electric field barrier blocking the passage of ions with a mobility of p^p1 and the second filter comprising a second DC electric field barrier blocking the passage of ions with a mobility of p^p2, where p2<p1 , wherein 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 the flow of the gas; and (b) collecting ions from the ion source in said predetermined ion mobility range in a space between the two filters.

[0114] Between the accumulation section and the separation section a second ion gate can be provided preventing ions having an ion mobility higher than said lower threshold value to enter the separation section.

[0115] According to yet another preferred embodiment of the any aspect of the present invention, the first ion mobility separator, at least once during a cycle of the second ion mobility separator, preferably at least twice or at least four times during a cycle of the second ion mobility separator, is operated such as to allow all ions to pass through while during this phase the gate between the first ion mobility separator and the second ion mobility separator is controlled not to allow any ions to enter the second ion mobility separator, or the second ion mobility separator is idle.

[0116] The first separation in the first ion mobility separator and the selective transfer can be 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 prior to separating them according to mobility.

[0117] Preferably the transmission of the ion gate is adjusted while the ions are separated in the first ion mobility separator such that the transmission for a highly abundant ion species of interest is lower than the transmission of less abundant ion species of interest.

[0118] 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 separating ions in time according to mobility in the first ion mobility separator involves driving the ions transferred to the first TIMS analyzer by a first gas flow against a first counter-acting electric DC field barrier such that the ions are trapped and spatially separated according to their mobilities at different positions along a ramp of the first electric DC field barrier at which a friction force of the first gas flow equals the counter-acting force of the first electric DC field barrier, and temporally separating ions according to mobility in the first TIMS analyzer by adjusting a height of the first electric DC field barrier or the velocity of the first gas flow.

[0119] According to another preferred embodiment, in the context of the any aspect of the invention, the second and / or third ion mobility separator is a TIMS analyzer, and wherein separating ions in time according to mobility in the second ion mobility separator involves driving the ions transferred to the second TIMS analyzer by a second gas flow against a second counter-acting electric DC field barrier such that the ions are trapped and spatially separated according to their mobilities at different positions along a ramp of the second electric DC field barrier at which a friction force of the first gas flow equals the counter-acting force of the second electric DC field barrier, and temporally separating ions according to mobility in the second TIMS analyzer by adjusting a height of the second electric DC field barrier or the velocity of the gas flow.

[0120] According to another preferred embodiment, in relation with the above mentioned survey scan approach for selecting the measurement IM windows in particular for excluding high abundance species, in the context of the first and / or the second aspect of the invention, at the beginning of one LC observation retention time window, preferably in the range of 1-15 seconds, particularly preferably in the range of 3-10 seconds, a survey scan is taken, wherein preferably for taking that survey scan only one of the ion mobility analyzes is operated in separating mode, and wherein in that survey scan a full ion mobility width of interest and a full m / z width of interest is scanned, and wherein as a function of that survey scan for the remainder of said LC observation window said second ion mobility separator and said mass filter are controlled in a synchronized manner such as to carry out a plurality of IM scans, during which precursor ions of increasing or decreasing IM are successively released from said IMS, and during which the mass window of said mass filter is shifted continuously or stepwisely towards lower or higher m / z values, respectively, to avoid peptides not of interest identified in the survey scan, and wherein said step of associating a detected fragment with its corresponding precursor ion is based on determining or utilizing the corresponding mass windows and IM ranges associated with various occurrences of said fragment in said mass spectrometry measurement.

[0121] Importantly, this preferred embodiment of using a survey scan is a fourth aspect of the invention which is to be regarded as a separate aspect which can also be implemented without the above-mentioned control of the second ion mobility separator and mass filter in a synchronized manner and without the specific arrangement of the second aspect and independent also of the third aspect.

[0122] For analyzing the survey scan information about LC retention time, ion mobility and m / z for peptides not of interest and / or for peptides of interest, a database containing respective information can be consulted and / or a list, which is preferably supplemented successively during one or a series of LC experiments, comprising peptides to be excluded, can be consulted. In said IM scans, adjacent mass windows that are associated with consecutive mass spectroscopy measurements of fragment ions may overlap, such that the precursor ions transmitted through said mass filter during one IM scan are located in at least one continuous scan region in an m / z-IM plane which extends in a generally diagonal direction in said m / z-IM plane, wherein adjacent scan regions associated with different IM scans overlap in the m / z-direction.

[0123] Preferably in said IM scans, adjacent mass windows that are associated with consecutive mass spectroscopy measurements of fragment ions overlap by at least 30% of their width, and / or wherein adjacent scan regions associated with different IM scans overlap in the m / z- direction by at least 33% of their width in m / z direction.

[0124] Further preferably the occurrence said fragment corresponds to a relative or absolute intensity of the fragment in the mass spectrometry measurement.

[0125] According to a fifth aspect of the present invention, it relates to an apparatus for data independent combined ion mobility and mass spectroscopy analysis.

[0126] This in particular for carrying out any of the methods as defined above.

[0127] Said apparatus preferably comprises: an ion mobility separator (IMS) for receiving and sequentially releasing precursor ions from said IMS according to their ion mobility, a mass filter arranged to receive said released precursor ions and to selectively transmit precursor ions having m / z values falling within a controllable mass window, a fragmentation device for fragmenting the precursor ions transmitted through said mass filter to generate fragment ions, an apparatus for carrying out a mass spectroscopy measurement on said fragment ions, wherein each fragment ion is associated with a mass window and an ion mobility (IM) range, and a control system, wherein preferably said control system is configured to control said IMS and said mass filter in a synchronized manner such as to carry out a plurality of TM scans, during which precursor ions of increasing or decreasing IM are successively released from said IMS, and during which the mass window of said mass filter is shifted continuously or stepwisely towards lower or higher m / z values, respectively, wherein said control system is preferably configured to control said IMS and said mass filter in a synchronized manner such that, in said IM scans, adjacent mass windows that are associated with consecutive mass spectroscopy measurements of fragment ions overlap, such that the precursor ions transmitted through said mass filter during said IM scan are located in at least one continuous scan region in an m / z-IM plane which extends in a generally diagonal direction in said m / z-IM plane, wherein further preferably said control system is configured to control said IMS and said mass filter in a synchronized manner such that adjacent scan regions associated with different IM scans overlap in the m / z-direction.

[0128] The second ion mobility separator can be a TIMS analyzer, preferably a TIMS analyzer with parallel accumulation and separation, in particular operating using a method comprising the steps:

[0129] (a) accumulating ions from the first ion mobility separator in an RF ion trap;

[0130] (b) transferring at least a subset of the accumulated ions into a trapping ion mobility separator, in which the transferred ions are radially confined by an RF field and pushed by a gas flow against a rising edge of an axial electric DC field barrier such that the transferred ions are spatially separated along the rising edge according to ion mobility;

[0131] (c) successively releasing the transferred ions according to their ion mobility by decreasing the height of the electric DC field barrier while ions from the ion source are further accumulated in the RF ion trap; and

[0132] (d) restoring the height of the electric DC field barrier which triggers a consecutive transfer of the accumulated ions from the RF ion trap into the trapping ion mobility separator.

[0133] The first ion mobility separator can be built and operated such as to retain only ions having an ion mobility higher than an upper threshold value of a IM window for the selected ions of interest and wherein either the second ion mobility separator is operated such as to allow ions having an ion mobility lower than a lower threshold value to pass through, or the second ion mobility separator is operated such that ions having an ion mobility lower than a lower threshold value are filtered out, or wherein the ion gate between the first ion mobility separator and the second ion mobility separator is operated such that ions having an ion mobility lower than a lower threshold value are not allowed to enter the second ion mobility separator.

[0134] The first ion mobility separator can be a TIMS analyzer, in which the ions are radially confined by an RF field and pushed by a gas flow against a rising edge of an axial electric DC field barrier such that the transferred ions are spatially separated along the rising edge according to ion mobility, and wherein the height of the electric DC field barrier is chosen such as to retain only ions having an ion mobility higher than said upper threshold value, and wherein the second ion mobility separator is a TIMS analyzer with parallel accumulation and separation, in which the transferred ions are accumulated in an accumulation section from the first ion mobility separator in an RF ion trap, in which the ions are radially confined by an RF field and pushed by a gas flow against a rising edge of an axial electric DC field barrier such that the transferred ions are spatially separated along the rising edge according to ion mobility, and wherein the height of the electric DC field barrier is chosen such as to retain only ions having an ion mobility higher than said lower threshold value followed by transferring at least a subset of the accumulated ions into a trapping ion mobility separator in a separation section, radially confined by an RF field and pushed by a gas flow against a rising edge of an axial electric DC field barrier such that the transferred ions are spatially separated along the rising edge according to ion mobility, and wherein the height of the electric DC field barrier at the beginning of the release is preferably chosen such as to retain only ions having an ion mobility higher than said lower threshold value, wherein preferably between the accumulation section and the separation section of the second ion mobility separator a second ion gate is provided preventing ions having an ion mobility higher than said lower threshold value to enter the separation section.

[0135] Further embodiments of the invention are laid down in the dependent claims.

[0136] BRIEF DESCRIPTION OF THE DRAWINGS

[0137] Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,

[0138] Fig. 1 schematically shows the principle as proposed in the prior art document US 2012 / 0273673 for sequential field barrier filtering in TIMS-like devices;

[0139] Fig. 2 shows the device and operation of a trapped ion mobility spectrometry (TIMS) analyzer. Top: Scheme of the TIMS device. Center: The profile of the electric field strength along the z axis. The size of the dots on the electric field ramp reflects their space charge. The scan releases the ion bunches in sequence of their mobilities. Bottom: The ion current of ion pulses separated in time according to mobility, representing an ion mobility spectrum.

[0140] Fig. 3 shows the device and operation of a trapped ion mobility spectrometry (TIMS) analyzer with parallel accumulation and separation. Top: Scheme of the TIMS device depicting “accumulation” (11a) and “analyzer” (11b) regions. Bottom: Plots representing the DC electric field gradient and ion positions during the accumulation and analysis steps. In the upper row a first group of ions are eluted from the analyzer region 11b by lowering the electric field gradient between points 32 and 34 while a second group of ions is simultaneously accumulated in the accumulation region on the electric field gradient between points 30 and 31 . Note the size of the dots represents the collision cross section of the ions. In the lower row, the field in the analyzer region 11 b is returned to its full strength after having eluted the first group of ions and the second group of ions is transferred from the accumulation region 11a to the analyzer region 11b by dropping the DC field strength in the accumulation region 11a to zero.

[0141] Fig. 4 shows a schematic overview of a mass spectrometer with two TIMS analyzers and an ion gate in between.

[0142] Fig. 5 shows schematically a device and operation of the tandem TIMS device of Fig. 4, with a first TIMS analyzer (TIMS 1), an ion gate, and a second TIMS analyzer (TIMS 2). The diagram below shows ions collected on the field ramp of TIMS 1 , with a marked range of ion mobility representing the ions of interest. The size of the dots represents the amount of ions of each type, thereby indicating the space charge of these ions. During the TIMS 1 scan the ions of interest are selected by the gate and collected on the electric field ramp of TIMS 2. The selected ions can be analyzed via TIMS 2 without further space charge disturbance.

[0143] Fig. 6 shows schematically a tandem TIMS device as in Fig. 5 wherein each TIMS device includes parallel accumulation as illustrated in Fig. 3. Figs. 6b) - 6e) depict schemes for operating such a device. The size of the dots represents the abundance of each type of ion. In each case the ions of interest are bounded by vertical dashed lines. In Fig. 6b) each field gradient is initially set to its maximum so as to analyze the maximum possible range of mobilities. In Fig. 6c) the field gradients in TIMS 2 are reduced so as to provide more space for ions in the range of interest. In Fig. 6d), the field gradients in TIMS 1 are lowered so as to never accumulate ions of lower mobility than the range of interest. In Fig. 6e) TIMS 1 is operated as a single region and the DC electric field gradient is set so as to accumulate only ions of higher mobility than the range of interest while allowing ions within the range of interest to pass to the accumulation region of TIMS 2.

[0144] Fig. 7 shows in a) a tandem TIMS device as in Fig. 6a), further including a gate between the accumulation and analyzer regions of TIMS 2 as well as a scheme for operating such a device. Of note, ions of lower mobility than the range of interest are eliminated at the second gate. Fig. 7b) is a timing diagram of one quench cycle.

[0145] Fig. 8 shows in a) plasma peptides as a function of their ion mobility and LC elution time during an LC-TIMS-MS analysis. As shown, high abundant and albumin peptides are interspersed amongst the lower abundance plasma peptides. Fig. 8b) shows mobility spectra of a single type of high abundance plasma peptide ions at various accumulation times. The bottom-most spectrum is a mobility spectrum obtained with a 100 ms accumulation time - corresponding to a 100 % duty cycle - whereas the top-most plot is a spectrum obtained with a 2 ms accumulation time (2% duty cycle). Together, the spectra of Fig. 8b) demonstrate that when a TIMS analyzer is overtimed with ions, the apparent mobility of the trapped ions can shift and the measured peak width can increase. Fig. 8c) shows how the filtering in TIMS 1 and TIMS 2 reflects in a corresponding spectrum.

[0146] Fig. 9 shows how a synchronous window selection and mass analysis can be used in a PASEF approach for further selectivity.

[0147] Fig. 10 shows in a) the unique peptide identification results as a function of the ion mobility window for 200 ng K562 at full and fractionated mobility ranges, and in b) the unique peptide IDs for 10 ng K562 at full and fractionated mobility ranges; Fig. 11 shows a comparison of LC peak shapes at different sample amounts for 2% duty cycle;

[0148] Fig. 12 shows in a) an IMS-MS survey spectrum obtained during an LC-IMS-MS analysis of a 300 ng neat plasma digest; in b) an IMS spectrum extracted from a survey scan, in c) an IMS spectrum extracted from a survey scan, in d) an IMS spectrum extracted from a survey scan.

[0149] Fig. 13 shows in a) BSA digest alone, in b) BSA digest plus bradykinin. Without filtering, BK2+ ions dominate - crowd out other ions, in c) mobility spectrum at 2% duty cycle. The BK2+ now appears at lower 1 / Ko and with multiple peaks representing multiple conformations, in d) low mobility fraction according to the present method, e) high mobility fraction according to the present method Fig. 14 shows a histogram of the data from the experiments of Fig. 13;

[0150] Fig. 15 shows in a) a mobilogram from a survey spectrum produced at a specific point in time during the course of an LC-IMS-MS analysis of plasma, in b) TIMS spectra of two fractions produced to avoid the high abundance species observed in the survey spectrum of Fig. a), and in c) the histogram of the results of the diaPASEF analysis of the unfractionated sample ions (left-most), and sample ions which have been mobility fractionated as depicted in Fig. b);

[0151] Fig. 16 shows in a) the Iog10 peptide quantity as a function of the peptide rank and in b) the counts as a function of the RT value, wherein the upper graph illustrates the situation in case of a sixfold reduction in dynamic range and the lower graph the situation in a 100 fold reduction in dynamic range; Fig. 17 shows schematically the principle as proposed with several for sequential field barrier filters in a TIMS or TIMS-like device;

[0152] Fig. 18 shows an elongated upstream ion separator followed by a gate and a second ion separator in a), and in b) - d) possible phases of operating the device with five filter potential barriers in the upstream ion separator device.

[0153] DESCRIPTION OF PREFERRED EMBODIMENTS

[0154] Fig. 2 outlines schematically a TIMS analyzer which can be used in the present context as referred to in US 7,838,826 B1 or in US 10,241 ,079 B1 and its operation. Entrained by a gas 7, ions 6 from an electrospray ion source (not shown) are introduced via capillary 8 into a first chamber of a vacuum system. A repeller plate 9 drives the ions 6 into an entrance funnel 10 of the mobility analyzer. Ion funnels 10, 12 usually are built as a stack of apertured diaphragms the openings of which taper to smaller diameters thus forming an inner volume in the shape of a funnel. Two phases of an RF voltage are applied alternately to the diaphragms to build up a pseudopotential which keeps the ions away from the funnel walls. The ions are driven to and through the narrow end of the first funnel 10 into the TIMS tube 11 by an axial gas flow 14 and optionally by an additional DC potential gradient along the diaphragms.

[0155] The axial gas flow 14 through the TIMS tube 11 is laminar and shows, in radial direction, a substantially parabolic velocity distribution. Nitrogen may serve as a preferred gas. The vacuum conditions around the TIMS tube 11 are chosen such that the maximum gas velocity amounts to about 100 to 150 meters per second, at a pressure of a few hectopascals. This velocity is only achieved near the axis. Further off axis, the velocity is considerably smaller, as indicated by the arrows 14 in Fig. 2.

[0156] The first funnel 10 guides the ions into the TIMS tube 11 forming a tunnel with internal RF quadrupole field in radial direction. The TIMS tunnel 11 comprises a stack of thin electrodes with central holes which form a circular tube arranged around the z-axis of the device. The thin electrodes are separated by insulating material closing the gaps between the electrodes around the tube. The electrodes of the TIMS tube 11 are segmented into quadrants 1 , 2, 3, 4, to allow for the generation of a radially confining quadrupolar electric RF field inside. The quadrants 1 , 2, 3, 4 of the tube electrodes are shown at the top of Fig. 2 with equipotential lines of the quadrupolar RF field inside 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 a lot of different possibilities including stacked PCB boards or even a rolled PCB board with printed electrodes.

[0157] Inside the TIMS tunnel 11 , the ions are blown by the gas flow 14 against an axial electric DC field barrier. In the center part of Fig. 2, the profile of the axial electric DC field barrier is shown for three phases of a scan. Between z locations 20 and 23, the electric DC field increases linearly, generated by a quadratically increasing electric potential. Between z locations 23 and 24, the electric DC field remains constant, forming a plateau of the electric DC field barrier, generated by a linear increase of the electrical potential. In a simple device, for instance, the complete field profile can be generated by a single voltage, applied to the diaphragm electrode at location 24, and divided by precision resistors along the diaphragm electrodes of the TIMS tube 11. The resistors between location 20) and 23 increase linearly, the resistors between 23 and 24 have equal resistance. In more complex devices, nonlinear field electric field profiles may be generated, even adjustable DC field profiles, e.g. by digital-to-analog converters (DAC).

[0158] The conventional operation of the TIMS analyzer starts with an “ion accumulation phase”, accumulating ions on the uppermost electric DC field ramp of the diagram. A voltage difference on the order of 300 volt produces the electric DC field barrier. The ions are blown by the gas flow, symbolically indicated by the arrows 16, against the electric DC field barrier and are stopped there because they cannot surmount the electric DC field barrier. It should be noted that the arrows 16 represent the maximum gas velocity of the parabolic gas velocity distribution 14 within the tube. The ions are accumulated on the rising edge of the electric DC field between locations 20 and 23, where ions of low mobility (mainly heavy ions with large collision cross section) gather in the high field near the upper end of the field ramp, whereas ions of high mobility gather in the low field near the foot of the ramp. The size of the dots represents the abundance of the ions of distinct ion mobility, indicating the strength of the space charge. In the subsequent “scan phase”, the supply voltage for the electric DC field barrier is steadily decreased, and ions of increasing mobility can escape towards an ion detector, particularly to a mass analyzer operating as ion detector. In the bottom of the figure, the resulting ion current of the released ion species is shown. The measured total ion current curve i= f(t) presents directly an ion mobility spectrum from low ion mobilities to high ion mobilities.

[0159] The ion mobility resolution RmOb depends on the scan speed. The lower the scan speed, the higher the resolution. As already mentioned, ion mobilities of RmOb = 400 have been achieved with the comparably small devices, using slow scans. Since the ions generated in the ion source are lost during the scan phases, the duty cycle (or the utilization rate of the ions) depends on the ratio of the accumulation time ta to the scan time ts.

[0160] Fig. 3 shows an embodiment of a mobility spectrometer which can be used in the present context and which combines accumulation and subsequent separation as e.g. described in US 9,683,964 B2. The TIMS comprises an elongated tunnel 11 , divided into an accumulation unit 11a and a scan unit 11b, and two voltage supply units (not shown) for the two tunnel units 11a, 11b, contacting the diaphragms at locations 31 and 34. Chains of resistors between the diaphragms in both tunnel units produce two axial electric DC field profiles, shown in the bottom part of the figure.

[0161] The operation of the TIMS device according to Fig. 3 comprises two phases: In the accumulation and scan phase D, ions from an ion source (not shown) are accumulated on the rising edge of the electric field profile in the accumulation unit 11a while, at the same time, ions in the scan unit 11b are scanned by decreasing the voltage supplied to location 34 of the scan unit 11 b, thereby releasing ions with higher and higher mobilities through the exit funnel 13 towards the ion detector.

[0162] In the transfer phase E, first the voltage of the scan unit 11 b is restored, and then the voltage of the accumulation unit 11a is switched off to let the ions be driven by the gas flow onto the rising edge of the electric field profile of the scan unit 11 b. The transfer is completed after only one millisecond, and the accumulation and scan phase may start again by switching on the voltage at location 31 .

[0163] Fig. 4 shows a time-of-flight mass spectrometer with two TIMS analyzers in a tandem arrangement as e.g. described in US-A-2018340910. In the present context, this is made use of in that the first ion mobility analyzer (TIMS 1 , which can be of the type as described in Fig. 2 or in Fig. 3) may scan a bunch of collected ions, thereby releasing ions in sequence of their ion mobilities. During the scan, the ion gate is alternately opened and closed, closed to reflect (or neutralize) unwanted ions and opened to pass ions in distinct ranges of mobility. In this way, highly abundant ions with their space charge can be reflected in full or at least partially. The passing ions enter the second ion mobility analyzer (TIMS 2, which can be of the type as described in Fig. 2 or in Fig. 3, but is preferably of the type of Fig. 3 in the present context) where they can be analyzed according to their ion mobility with high ion mobility resolution, undisturbed by space charge. The mass spectrometer then can measure their precise masses; a tandem mass spectrometer with quadrupole mass filter and time-of-flight analyzer, as presented in Fig. 3, even can measure fragment ion spectra for a better identification of the ion species.

[0164] The ions transferred to TIMS 2 may stem from a single range of mobility, or from several ranges, selected by switching the gate accordingly. In one embodiment, ions from a single range of mobility are collected on a flat ramp of TIMS 2, to spread the ions as far as possible along the z axis of TIMS 2. In this mode, ions may be accumulated and scanned in TIMS 1 several times to accumulate as many ions in TIMS 2 as required for an analysis of high quality. If there is a kind of ion with extremely high abundance within the range of interest, only a small portion of these ions may be transferred by reflecting the largest part of these ions. The ion gate preferably switches faster than the temporal width of ion pulses leaving the first ion mobility analyzer. The length of an ion pulse released by the scan is in the order of a millisecond, whereas the switching time for the gate can be below a microsecond; the ion pulse therefore can easily be cut into portions.

[0165] Whereas the radially confining RF field of TIMS 2 is preferably quadrupolar in order to achieve a high ion mobility resolution, TIMS 1 may show a tube with higher inner diameter, and / or with radial RF fields of higher multitude, like hexapole, octopole, or dodecapole, or with an RF tunnel. An ion trap can additionally be located upstream of TIMS 1 to accumulate ions from the ion source during the scan of TIMS 1. If the trap can be mass selectively unloaded, even TIMS 1 may be relieved from space charge.

[0166] Fig. 5 illustrates, in the top part, two tandem TIMS devices (TIMS 1 , TIMS 2) separated by an ion gate. The ion gate is formed as an ion-optical einzel lens. The gate can be switched on and off in less than a microsecond. During the scan of TIMS 1 , the gate may be opened and closed in a suitable manner to pick out ions of interest. The ions of interest transferred to TIMS 2 may stem from a single range of mobility, as shown in the bottom part of Fig. 5, which show the scheme for the situation where for TIMS1 and for TIMS 2 the type as described in Fig. 2 is used.

[0167] In this case, ions are eluted from the TIMS devices by raising the TIMS entrance potential at a constant rate. Low mobility ions elute first, medium mobility ions elute second, and high mobility last. However, the ion gate is open only when the desired mobility ions (illustrated schematically by vertical lines in TIMS 1) are eluting. Duty cycle (accumulation time I total time) in this case is rather limited.

[0168] The first separation in the first ion mobility analyzer TIMS 1 and the selective transfer of the ions of interest (illustrated schematically by vertical lines in TIMS 1) via the gate can be repeated and the second ion mobility analyzer TIMS 2 can be supplied with ions in the desired window to scan the repeatedly transferred ions of interest while separating them according to mobility.

[0169] The TIMS entrance potential in TIMS 1 and TIMS 2 can also be scanned in a non-linear manner so that the slow part of the scan (high resolution) occurs only at the time the ions of interest are eluting. Because the scans take less time, the TIMS fill and elute cycle can occur several times per mass analyzer (e.g. ICR or ToF) transient. Ions of the selected mobility are accumulated in the collision / fragmentation cell and then transferred to the mass analyzer (e.g. ICR or ToF) cell.

[0170] Also, the initial part of the TIMS scan can be cut off. Low mobility ions are then not accumulated and the elution scan starts right near the ion of interest. This means more of the ions of interest can be accumulated (i.e. they have more space available) and the scan time can be shorter so it can be repeated more times per mass analyzer (e.g. ICR or ToF) transient.

[0171] Fig. 6 illustrates, in the top part a), again two tandem TIMS devices (TIMS 1 , TIMS 2) separated by an ion gate, wherein however in this case for TIMS1 and for TIMS 2 the type as described in Fig. 3 is used. The ion gate is formed as an ion-optical einzel lens. The gate can be switched on and off in less than a microsecond. During the scan of TIMS 1 , the gate may be opened and closed in a suitable manner to pick out ions of interest. This setup is allowing for parallel accumulation and separation in each of the TIMS devices. So in this case both TIMS devices have an accumulation unit 11a and scan unit 11b.

[0172] The resulting scheme, if both TIMS 1 and TIMS 2 devices are operated in the same way, is illustrated in Fig. 6b. In the first TIMS1 device in the accumulation unit 11a, the full ion mobility range is accumulated and also the full ion mobility range is then entering the separation unit 11 b. The gate between the two TIMS is controlled such that only a desired ion mobility window (illustrated schematically by dashed vertical lines in TIMS 1) is allowed to enter the TIMS 2 device. In the latter again there is accumulation in the accumulation unit 11a and subsequent separation in the separation unit 11 b.

[0173] To achieve an optimum duty cycle and an optimum sensitivity of the ions of interest, not being hampered by strong signals of abundant ions which are not of interest, the operational scheme of such a tandem TIMS arrangement, if operated for DDA and in combination with a scheme, where TIMS 2 and the mass filter are controlled in a synchronized manner such as to carry out a plurality of IM scans, during which precursor ions of increasing or decreasing IM are successively released from the TIMS, and during which the mass window of said mass filter is shifted continuously or stepwisely towards lower or higher m / z values, respectively, can be optimized significantly, as shall be explained by the following schemes illustrated in Fig. 6c-e.

[0174] As illustrated in the scheme according to Fig. 6c, in order to achieve an as high as possible resolution focusing of the scan in TIMS 2, it is possible to focus just on the window of the ions of interest in the accumulation and scan of TIMS 2. The initial part of the scan can be cut off or, as illustrated, at least the slope of the scan can be adapted to the window of interest, such that essentially in TIMS 2 the ramp is reduced to scanning just the window of interest. If needed in combination with the velocity of the airflow, which can be selectively adapted in the two TIMS devices. This can be implemented in that the maximum potential which is reached in both the accumulation as well as the scan phase of TIMS 2 is rather low and somewhat below the low mobility ion threshold of the desired window. The TIMS 1 device is still operated the same way as in the scheme of Fig. 6b, so TIMS 1 accumulates and scans the desired window of interest at a higher frequency than TIMS 2 so that there is an optimum duty cycle. It is possible to operate the TIMS 1 device at a ten times higher cycle than TIMS 2. Each time in such a scan of TIMS 1 the ions of the desired window are eluted, the gate is opened, and the desired ions are entering the accumulation unit 11a of the TIMS 2 device.

[0175] Since the low mobility portion of the ions which are not of interest, if they are not gated off between the two TIMS devices, will pass through the TIMS 2 device before a corresponding scan is initiated, it is in fact possible to further improve that scheme as illustrated in Fig. 6c. It is possible to also limit the accumulation as well as scan voltage ramp in TIMS 1 so that it basically cuts off somewhat below the low mobility ion threshold of the desired window. This leads to a situation that ions that have a mobility below that low mobility ion threshold of the desired window will pass through TIMS 1 as well as TIMS 2. On the other hand, ions that have a mobility above the high mobility ion threshold of the desired window will remain and accumulate in TIMS 1. TIMS 1 correspondingly acts like a cut-off filter, cutting off any ions having an ion mobility above the high mobility ion threshold of the desired window. Eliminating the ions that have a mobility below the low mobility ion threshold is taken care of by TIMS 2, allowing those ions to pass through unimpeded.

[0176] This can further be optimized in the way as illustrated in the scheme of Fig. 6e. It is not necessary and in fact it is even leading to a steeper cutoff effect, if TIMS 1 is not operated involving an accumulation and a scan phase, but is operated in a way just making sure that any ions having an ion mobility above the high mobility ion threshold of the desired window are held back, and this with an as flat as possible voltage ramp so as to allow for optimum selectivity. The vertical dashed line illustrated in that scheme of TIMS 1 in Fig. 6e therefore represents the high mobility ion threshold 17 of the desired window in TIMS 1 . The vertical long dashed line illustrated in that scheme of TIMS 1 on the other hand represents the low mobility ion threshold 18 of the desired window in TIMS 2.

[0177] This means, that TIMS1 does not make a TIMS analysis but is operated only as a mobility cutoff.

[0178] To achieve this in a useful way, TIMS 1 is periodically quenched (for example every 100 ms) by, for example, lowering the TIMS potential to 0 V (or sufficiently low potential to allow all ions to escape out the exit). Ions released this way can be prevented from entering TIMS 2 by destroying the ions at the gate.

[0179] Ions passing the TIMS 1 cut-off 17 are accumulated in the TIMS 2 accumulation section. Ions of lower mobility than the TIMS 2 accumulation cut-off 18 can be eliminated at a further gate between TIMS 2 accumulation 11a and TIMS 2 analyzer 11 b regions. So in this case TIMS 1 is not operated at a higher frequency than TIMS 2, since accumulation is taken care off in the accumulation section of TIMS 2. Higher operation frequency of TIMS 1 however can still be an advantage if quenching of the higher mobility ions should take place periodically and as often as possible to reduce interference with the desired ion mobility window.

[0180] This is schematically illustrated in Fig. 7a), where in the upper part the TIMS arrangement including the additional gate (interstage optics) at the interface between the accumulation section 11a and the scan section 11 b is illustrated, and where in the lower part of the scheme according to Fig. 6e is given including the gating between the accumulation section 11a and the scan section 11 b. At this gate between the two sections 11a and 11 b ions of lower mobility than the cut-off 18 are eliminated so as not to enter the scan section 11 b in a way interfering with the scan and successive elution of the ions of interest as a function of their ion mobility from TIMS 2. However, it is to be noted that it is also possible to allow ions to pass through the downstream TIMS 2, as this might only lead to a constant low background however normally not interfering because it is in a region of the mass / mobility domain other than the selected range.

[0181] There is a huge and unexpected advantage associated with operating the two TIMS devices basically each as a cut-off device for the ion mobility window of interest, and this is the following: high abundant peaks show peak widths in the ion mobility scan which are significantly larger and which cover-up low abundance peaks which are present close to or in the tails of these high abundant peaks. This leads to a masking of the desired low abundance peaks and to a reduction of sensitivity if a cut-off value is selected insufficiently far away from a high abundance peak. These effects are due to the co-presence of the high abundance ions and the desired ions in the accumulation as well as in the scan phase of the TIMS devices. Using basically the cut-off tandem TIMS technology as presented here, in particular for DDA and combined with a scheme, where TIMS 2 and the mass filter are controlled in a synchronized manner such as to carry out a plurality of IM scans, during which precursor ions of increasing or decreasing IM are successively released from the TIMS, and during which the mass window of said mass filter is shifted continuously or stepwisely towards lower or higher m / z values, respectively, eliminates or at least significantly reduces these problems due to overlap in the mobility dimension. The proposed technology therefore allows for higher sensitivity and a better focus on a desired ion mobility window and a better elimination or keeping out of high abundance signals.

[0182] This is important because also in the LC dimension high abundance signals are broadened, and the proposed technology allows a more efficient blanking out of the high abundance signals also for broader LC peaks which may have peak with of up to 1.5 minutes, typically 20-30 seconds, leading to a peak width in the ion mobility dimension of 0.07 1 / K0.When TIMS1 is used to make a high mobility cutoff, ions of lower mobility than the cutoff pass through TIMS1 without being trapped. However, ions of higher mobility than the cutoff are accumulated in TIMS1. The trapped ion population and associated electrical charge will build up over time. To avoid being overloaded with trapped ions, TIMS1 must be periodically “quenched” - emptied of all ions. Fig. 7b) is an example timing diagram of one quench cycle. In practice this cycle of passing and quenching ions is repeated continuously and at regular intervals - in this example, every 30 ms - during mobility cutoff operation.

[0183] In Fig. 7b) the timing of the relevant potentials is depicted. In this example, the “T3” potential is applied to the entrance of the TIMS1 analyzer and sets the DC electrical barrier which determines the mobility cutoff. The “Deflector” and “Gate” potentials are applied to the deflector - at the entrance of TIMS1 - and gate - at the exit of TIMS1 - elements respectively. Initially, the Deflector potential is set to deflect ions from the ion source into TIMS1 , T3 is set to allow ions below a 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 to TIMS2. This state of passing ions continues, in this example, for 27 ms. After this time, all ions in TIMS1 are quenched.

[0184] During the quench, 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 pass out of the exit of TIMS1. During the quench, the Gate is set to a “closed” potential such that substantially all ions exiting the TIMS1 are eliminated. At the same time the Deflector potential is set to block ions from entering TIMS1 from the ion source. In this example, the “quench” takes about 3 ms.

[0185] In alternate methods, the ions may be quenched by temporarily reducing the TIMS1 RF ion confining potential.

[0186] In alternate methods, the Deflector potential need not be switched so as to block ions from entering TIMS1 during the quench step. Rather the Deflector potential may be set to continuously allow ions to pass into T I MS1. Before analysis, the blood plasma sample is digested - typically using trypsin - to produce peptides. The peptides and their abundances are indicative of the proteins from which they were generated and the proteins’ abundance in the original sample.

[0187] Importantly, in human blood plasma the «Top 14» most abundant proteins (the peptides of which are shown here in dark grey) constitute xx% of the sample - these are proteins and their corresponding peptides are easily observed and not of interest because they’re observed in every sample of blood plasma and are not particularly indicative of the state of system from which they were derived.

[0188] Albumin is one of the top proteins - the peptides of which are here shown in bright grey.

[0189] In Fig. 8a) the high abundant top 14 and albumin peptides are shown together with all other detected lower abundance plasma peptides as a function of their ion mobility and time of elution during an LC separation.

[0190] The point is, peptides from high abundance proteins appear frequently throughout the LC- IMS (and m / z) range and therefore often interfere with the measurement of other peptides (lower abundance peptides of interest, medium grey) that occupy near positions in the LC- IMS space. It’s therefore important to eliminate or substantially reduce these high abundance species.

[0191] If, for whatever reason, these high abundance species are not eliminated, or reduced, in relative abundance before the LC IMS analysis begins, then it is advantageous to do so within the instrument.

[0192] It’s important to use a low duty cycle when making a survey scan. The electrospray ion source produces ions continuously. “Duty cycle” corresponds to the fraction of time one accumulates ions from such a continuous source. This means, for example, that whereas the TIMS analysis may require 100 ms, the accumulation of ions for only 2 ms corresponds to a 2% duty cycle.

[0193] The measured total spectral information is a superposition of the lower abundance proteins, and of the high abundance (undesirable) peptides which usually appear near to the low abundance (desirable) peptides. The analytical challenge is how to avoid the high abundance peptides signals inasmuch as possible in the spectral analysis, so as to maintain high selectivity and high-sensitivity with respect to the lower abundance proteins at a high duty cycle.

[0194] The approach uses the above-mentioned combination of two sequential TIMS devices, preferably operated the way as described above. Upstream of the first TIMS 1 device a FAIMS filter can be located to filter away ions of a selected charge and to fractionate peptides based on differential mobility. The TIMS 1 with the downstream gate (or filter) provides for selective transfer and fractionates ions based on mobility. The second TIMS step with TIMS 2 accumulates ions and analyzes them for increased capacity. Said second ion mobility analyzer TIMS 2 and the downstream mass filter are controlled in a synchronized PASEF manner such as to carry out a plurality of IM scans, during which precursor ions of increasing or decreasing IM are successively released from said IMS, and during which the mass window of said mass filter is shifted continuously or stepwisely towards lower or higher m / z values, respectively, and wherein said step of associating a detected fragment with its corresponding precursor ion is based on determining or utilizing the corresponding mass windows and IM ranges associated with various occurrences of said fragment in said mass spectrometry measurement.

[0195] As depicted in the data of Fig. 8b), when a TIMS analyzer is overfilled with ions, the apparent mobility of the trapped ions can shift and the measured peak width can increase. Thus, the result of the survey scan, and the determination of the mobility range that should be excluded, can be distorted. Using a 2% duty cycle, for example, reduces the risk that the TIMS analyzer will be overloaded by high abundance ions during the survey measurement. Importantly, according to the present invention, by using a low duty cycle when making a survey scan, (1) only (or substantially only) high abundance species are visible; (2) by reducing the duty cycle, the TIMS is not overloaded; and (3) because the TIMS isn’t overloaded, a low duty cycle survey scan provides an undistorted measure of the ions’ mobility and mobility peak width.

[0196] It’s only by such a low duty cycle survey that one can determine the appropriate mobility windows for subsequent ion filtering - that is, a high duty cycle measure would produce distorted peaks.

[0197] The important point is that at low duty cycle (2 ms) the mobility peak is narrow and at high duty cycle (100 ms) the peak is broad and shifted because the TIMS is overloaded. The high duty cycle peak is not a true measure of the high abundance species’ mobility, rather the low duty cycle is.

[0198] An example how the scheme can be applied and was applied to a realistic situation is illustrated in Fig. 8c). The intensity of signals is given as a function of the barrier voltage in this illustration for different PEG fragments, and the high mobility threshold 17 is illustrated, on the left of which those systems are given, which are retained in TIMS 1 , then the window of interest is illustrated right of the dashed line 17, and left of the dashed line 18, illustrating the low mobility threshold 18. The area between these dashed lines is retained in the accumulation section 11a of TIMS 2. Anything right of the low mobility threshold 18, which is provided by TIMS 2, is discarded, either by way of the gate between the accumulation section 11a and the scan section 11 b of TIMS 2, or is lost downstream of TIMS 2. A very selective and high (steep) cutoff at 17 as well as 18 is possible using the proposed scheme. The effect of the synchronized control of the mass window filter as a function of the TIMS 2 ion mobility analyzer is partially illustrated in Fig. 9, showing heat maps for 200 ng K562 at full and fractionated mobility ranges. The results shown in Fig. 9 were produced using a conventional TIMS, not a tandem TIMS, instrument. These results were produced using 200 ng of K562 injected on a 25 cm LC column, an 85 minute gradient, and a flow rate of 300 nl / min. In these experiments, the mobility range over which ions were accumulated and analyzed was simply reduced to the ranges shown. In these experiments each fraction corresponds to a separate LC - “data dependent” PASEF run; 4 fractions = 4 complete LC runs. Also, because these experiments don’t include TIMS 1 selection, the scan range here doesn’t actually correspond to the mobility range of 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 the present invention, can exclude ions of both higher and lower mobility than the selected range leading to somewhat higher capacity for the selected range.

[0199] Fig. 10 shows, in histogram form, the results of the experiments from Fig. 9. In Fig. 10a) the unique peptide identification results as a function of the ion mobility window for 200 ng K562 at full and fractionated mobility ranges. Importantly, summing two, non-overlapping fractions - 1 / Ko=0.7-0.9 & 0.9-1.1 - leads to a significant increase in the number of peptides identified 30.4 vs 20.7k without selective transfer. Shown in Fig. 10b), the improvement in unique peptide identifications is somewhat reduced for lower sample amounts - in this case, 10 ng K562. Here, summing the results of two fractions leads to 37k peptides identified as compared to 31.8k peptides without fractionation.

[0200] According to the present invention, the selectivity and the synchronization of the TIMS 2 device and of the subsequent mass analysis preferably relies on a survey scan technique. One possibility to implement that is to, within a typical 3-10 second LC peak, first start with a survey scan, then followed by the selective and synchronized operation of the TIMS 2 device and of the subsequent mass analysis as a function of the survey scan. The survey scan can be recorded by operating only TIMS 1 or TIMS 2 over the full ion mobility width and recording the full survey scan in the sense of M / z as a function of 1 / K0, and this just for that small window in the LC dimension. Typically, that survey scan step takes 100 ms.

[0201] A computer algorithm can then be used to identify the high abundance peptides in that survey scan. This can be done by consulting a database if the high abundance peptides are known, and getting the corresponding LC and IM information of these high abundance peptides from the database. The corresponding regions in the survey scan can then be automatically blanked out and excluded from the selective and synchronized operation of the TIMS 2 device and of the subsequent mass analysis. In addition, or alternatively, it is possible to maintain a (preferably running) exclusion list, i.e. to exclude those peaks which from previous scans either have already been identified as high abundance signals not of interest or having been identified as low abundance signals already identified. The approach with a (running) exclusion list can also be implemented without survey scan.

[0202] So the steps can be implemented as follows: a) take a survey scan (1 % duty cycle) and look for what mobility ranges have high abundance peaks; b) carry out at least 2 selective TIMS 1 scans each of which deselects substantially all high abundance species as determined (by computer algorithm) via the survey scan; c) transmit results of selection to TIMS 2 for further analysis; d) TIMS 2 + rest of the instrument (analytical quadrupolar mass filter, collision cell, time of flight analyzer) is operated in to analyze the selected ion, but preferably according to PASEF method including DIA PASEF (see in particular Meier, F. et al. Parallel Accumulation-Serial Fragmentation (PASEF): multiplying sequencing speed and sensitivity by synchronized scans in a trapped ion mobility device. J. Proteome Res. 14, 5378-5387 (2015), which is included in this disclosure), sychro-PASEF (see in particular 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 in particular Meier et al., midiaPASEF maximizes information content in data-independent acquisition proteomics, bioRxiv, which is included in this disclosure).

[0203] The survey scan in a) may operate at a 1 - 10% duty cycle; ...at a 1 - 100% duty cycle.

[0204] The survey scan can be performed in TIMS2 (TIMS1 would be operated only to transmit ions).

[0205] The survey scan preferably analyzes ions over a 100 ms period; or the period over which TIMS 2 is operated in step d).

[0206] The selected range is preferably 10% of the survey scan range.

[0207] Any number of TIMS 1 selective PASEF scans may be used to accumulate selected ions for a single TIMS 2 scan.

[0208] In step b), TIMS 1 is preferably operated using a “non-linear” scan; any known scan + gating scheme.

[0209] Both TIMS 1 and TIMS 2 can be operated in “parallel accumulation” mode, preferably however only TIMS 2 is operated in that mode and TIMS 1 is operated only as a low IM pass filter or gate, as described above.

[0210] Steps a) - c) may be repeated 5 - 8 times per selected mobility range in order to achieve methods such as “synchro-PASEF”. Each such repetition would use a different quadrupole mass range in accordance with the synchro-PASEF method.

[0211] Note that an ion detector (for example a Faraday cup or a simple lens with electrometer) located between TIMS 1 and TIMS 2 (e.g. electrometer) may be used for the survey scan, making the survey scan independent of the rest of the instrument. This frees TIMS 2 to analyze a previously selected group of ions while TIMS 1 makes the survey scan.

[0212] Also, the survey scan can be implemented via TIMS 1 and a separate detector. The concept is to use TIMS 1 plus the separate ion detector to make a survey scan while TIMS 2 is performing the analysis of a previously selected group of ions. The ion detector may be any detector known in the prior art. In the preferred embodiment, the detector is fast enough to produce a mobility spectrum from the ions eluting from TIMS 1 (milliseconds) and sensitive enough to produce a spectrum of the most intense species when TIMS 1 is operated at a few percent duty cycle. Microchannel plate-based ion detectors or channeltron ion detectors are suitable, but may require a reduced pressure and therefore a separate chamber.

[0213] If the sample is generally known (i.e. it’s e.g. plasma) then the times at which the highest abundance and / or uninteresting species elute from the LC are substantially known. The actual elution times may differ from one LC run to the next, therefore methods have been developed to compensate for such variations (see e.g. US-A-20210033575). The mobilities of the highest abundance I uninteresting species can also be known ahead of time.

[0214] Therefore, according to this alternate embodiment, one may proceed as follows: determine the LC elution time and mobility of substantially all high abundance, uninteresting species present in a given sample type; produce a scheduled exclusion list containing the above elution times and mobilities which the instrument control computer can use; perform an LC-tandem TIMS-MS sample analysis using TIMS1 + gate, via the instrument control computer and the exclusion list, to selectively transfer mobility windows at selected LC elution times and mobilities which are not in the exclusion list to TIMS2 for further analysis.

[0215] An advantage of loading lower sample amounts, is decreased chromatographic peak width, as illustrated by Fig. 11 : High abundance species do not appear at one discrete point in the LC-IMS domain - rather, any particular high abundance species will appear over a range of elution times and mobilities. If the TIMS is overloaded, the abundant species will appear at a shifted and broader range of mobilities. Moreover (not shown) the high abundance species can affect other species in the TIMS analyzer - especially those of near mobility. In particular, the high abundance species can cause ions of other species to be lost from the TIMS analyzer and / or cause the mobility peak of other species to shift.

[0216] Likewise, if an LC column is overloaded with too much sample material, the retention time of the high abundance species can broaden and shift and cause other species in the LC column to shift in retention time. As an example, the figure shows the LC peak associated with a human albumin peptide from samples of neat plasma. These chromatograms were extracted from a 50 minute separation conducted with a Bruker nanoElute, an Ion Opticks 25 cm column, running at a flow rate of 300 nL / min detected with a Bruker timsTOF running at a 2% duty cycle. The amount of sample injected on column was 50, 100, 200, 300, and 600 ng to produce the chromatograms of Figs. 12 a), b), c), d), and e), respectively. Clearly, as the sample amount is increased the LC peak associated with this albumin peptide broadens and shifts to shorter elution times.

[0217] Using a more sensitive instrument (like a timsTOF SCP or ultra) and thereby reducing the amount of sample material loaded on the column can reduce the LC peak width and avoid shifting retention times. Importantly, reducing peak widths in any dimension (LC, IMS, or MS) reduces the overlap of near species in LC-IMS-MS space and thus the potential interference such overlap may cause. The disadvantage of loading less sample on the LC column is that chemical noise and cluster ions are more important.

[0218] In a single survey spectrum (2% duty, TIMS-MS spectrum taken at one given moment in the LC separation), as can be seen from Fig. 12, there are only a few high abundance species present.

[0219] The criteria for deciding which peaks are to be excluded from further analysis:

[0220] • abundance (an intensity threshold)

[0221] • species is known from prior experience to be uninteresting (user must upload a table)

[0222] • will its inclusion together with the rest of a selected mobility range lead to an overload of the TIMS 2 cartridge

[0223] Criteria for deciding on fraction ranges and what fractions are to be further analyzed:

[0224] • Excluded peaks

[0225] • Time available for analyzing all selected fractions

[0226] • Number of species likely to be in a fraction (the mobility and mobility width of the fraction

[0227] - the potential for the analysis of the fraction to return useful information)

[0228] In a) an IMS-MS survey spectrum (scan #11953) obtained during an LC-IMS-MS analysis of a 300 ng neat plasma digest is given.

[0229] In b) the IMS spectrum extracted from the survey scan of a). There are high abundance peaks only in the interval 0.78 < 1 / K0 < 0.84. Two fractions: 1) from 1 / K0 = 0.7 to 0.78; and 2) from 1 / K0 = 0.84 to 1.2 may be analyzed without substantial interference.

[0230] In c) the IMS spectrum extracted from survey scan #8623 obtained during an LC-IMS-MS analysis of a 50 ng neat plasma digest. There are high abundance peaks only in the interval 0.77 < 1 / K0 < 0.82. Two fractions: 1) from 1 / K0 = 0.7 to 0.77; and 2) from 1 / K0 = 0.82 to 1.2 may be analyzed without significant interference.

[0231] In d) the IMS spectrum extracted from survey scan #13177 obtained during an LC-IMS-MS analysis of a 50 ng neat plasma digest. There are high abundance peaks in two intervals 0.74 < 1 / K0 < 0.77 and 0.89 < 1 / K0 < 0.93. Three fractions: 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 may be analyzed without significant interference. However, fraction 1) might be discarded considering the time required for analyzing this fraction and the few species it’s likely to contain.

[0232] Further proof of concept is given in Fig. 13 (BSA digest + BK peptide).

[0233] To further demonstrate and provide an example of the method according to the present invention, a mixture of a protein digest and an unrelated, high abundant peptide was analyzed. Two samples were produced and analyzed - a 25 fmol / pL solution of a tryptic digest of bovine serum albumin (BSA) and a mixture of 25 fmol / pL of BSA with 5 pmol / pL of the peptide bradykinin (BK). Figure (a) shows a mobilogram from the TIMS analysis of a 25 fmol / pL BSA. PASEF analysis was performed on the relevant detected peptide ions. From the resultant data, 29 unique peptides were identified by a MASCOT search. These peptides represent the peptides of interest. Figure (b) is a TIMS mobility spectrum of the BSA plus BK mix performed with a 100% TIMS duty cycle. Here, the bradykinin 2+ ions (BK2+) are detected with an excessively high intensity - i.e. the TIMS analyzer is overloaded with BK2+ ions. Due to overloading, the BK2+ peak is broad and shifted in mobility. The PASEF analysis of the BSA plus BK mix resulted in the detection of just one BSA peptide. Figure (c) is a TIMS mobility spectrum of the BSA plus BK mix performed with a 2% TIMS duty cycle. Here, a moderate intensity BK2+ peak is detected. Of note, the BK2+ peak is seen to cover the range from about 0.8 to 0.9 1 / Ko. As a result, subsequent fractions (figures d and e) avoid this mobility range. Figure (d) shows the TIMS spectrum of the low mobility fraction produced according to the method of the present invention. Here the TIMS1 barrier is set to trap ions of higher mobility than 0.9 1 / Ko and allow ions of lower mobility to pass. The TIMS2 accumulation barrier is set to trap all ions of a higher mobility than a 1 / Ko of 1 .4. After accumulating ions for 100 ms, the ions were transferred to the TIMS2 analyzer and then mobility analyzed in a 100 ms scan. Performing a PASEF analysis followed by a MASCOT search of the data identified 12 unique peptides. Figure (e) shows the TIMS spectrum of the high mobility fraction produced according to the method of the present invention. Here the TIMS1 barrier is set to pass ions of all mobilities whereas the TIMS2 accumulation barrier is set to trap all ions of higher mobility than 0.78 1 / Ko. After accumulating ions for 100 ms, the ions were transferred to the TIMS2 analyzer and then mobility analyzed in a 100 ms scan. Performing a PASEF analysis followed by a MASCOT search of the data identified 15 unique peptides. The combined results from the low and high mobility fractions showed 23 unique peptides identified. By largely eliminating the high abundance BK2+ ion via the method of the present invention, 23 unique peptides were identified compared to the single unique BSA peptide identified by the prior art PASEF method.

[0234] Full MS for 25 fmol / uL BSA Digest in 5 uM Bradykinin; a simple sample was chosen to mimic plasma proteomics in which high intensity peptides from highly abundant proteins essentially flush out low intensity peptides; 5 pM bradykinin was spiked into; low and high mobility fractions were performed to exclude transmitting and accumulating bradykinin 2 - in TIMS-2; for both fractions, ddaPASEF was performed in TIMS-2 and the peptides identified were compared to those identified from running ddaPASEF of 25 fmol / pL BSA digest alone; the goal of the ddfPASEF method is to “recover” peptides lost when high intensity peptides are present.

[0235] Further proof of concept is given in Fig. 14. A histogram of the data from the experiments of Fig. 14. From the analysis of a 25 fmol / pL solution of a tryptic digest of BSA, 38 BSA peptides were identified, 29 of which were unique. From the analysis of a mixture of 25 fmol / pL of BSA with 5 pmol / pL of the peptide bradykinin (BK), only one BSA peptide was identified by a prior art PASEF method. Using the method according to the present invention to analyze the mixture of BSA and BK, 31 BSA peptides were identified, 23 of which are unique. At least three of BSA peptides undetected in the analysis of the mixture according to the present method were from a region in “mass-mobility space” near that of the BK2+ ions.

[0236] In the figure: performed PASEF on BSA alone (left bars); on fractions from sample with BSA+BK in accordance with steps (f) - (h) (middle bars); and on the sample with BSA+BK (right). Without filtering and fractionation, only one peptide was identified from the BSA digest. With filtering and fractionation according to the present method, many but not all (23 of 29) of the peptides were identified.

[0237] Further proof of concept is given in Fig. 15. A sample of 300 ng neat plasma was analyzed by LC-TIMS-TOFMS according to the ddfPASEF method of the current invention. A first sample was analyzed without fractionation according to the present invention and a second identical sample was analyzed with fractionation. Only the spectra associated with the fractionation results are shown in Fig. H. Figure (a) is the mobilogram from a survey spectrum - i.e. a 2% duty cycle TIMS mobility spectrum of the neat plasma - obtained 29.2 minutes into the LC run (frame #16444). Here it is seen that the most intense peak - an albumin peptide (564.85 m / z) - covers a mobility range from 0.85 to 0.91 1 / Ko. As a result, subsequent fractions - Fig. (b) - avoid this mobility range. Figure (b) shows the TIMS spectra of the two fractions - one from 0.60 to 0.85 1 / Ko and the other from 0.90 to 1.40 1 / Ko - combined into a single mobilogram. Using the TIMS2 analyzer, and the rest of the downstream instrument (quadrupole, collision cell, and orthogonal time-of-flight mass spectrometer), diaPASEF was performed on each fraction independent of the other. As shown in the histogram of Fig. (c), diaPASEF analysis of the high mobility fraction followed by a Spectromine search of the data identified 27 unique peptides and 18 unique protein groups. The same process identified 24 unique peptides and 18 unique protein groups from the low mobility fraction. For comparison, analyzing the neat plasma without filtering and fractionation according to the present invention revealed only 19 peptides and 5 protein groups in the corresponding period in the LC separation.

[0238] By largely eliminating the high abundance albumin ion via the method of the present invention, 50 unique peptides and 31 protein groups could be identified compared to 19 unique plasma peptides and 5 protein groups identified by the equivalent prior art PASEF method without the benefit of the present invention.

[0239] For 300 ng neat plasma, the albumin peptide peak elutes from 28.8-31 min (tailing out to 32.2 min but relatively low intensity). The peptides / proteins here have apex retention times ranging from 28.5-31.5 minutes.

[0240] In the analysis of complex samples, for example bottom-up plasma proteomics, it is advantageous to remove high abundance species (in present case this means removing gas phase sample ions) which would otherwise mask low abundance species. In the example shown in Fig. 16 it is estimated that removing one high abundance species, on average, in any given spectrum during the course of an LC-TIMS-MS analysis, will effectively improve the instrument’s dynamic range (esp. ability to see low abundant species) by a factor of about 6. Removing the 8 most abundant species results in a 100x improvement in effective dynamic range.

[0241] So it is desirable to remove a multitude of high abundance gas phase ions from our TIMS analysis. Reducing the peptide dynamic range in the measurement by 2 orders of magnitude requires removing the top - 1200 peptides (-1650 precursors) distributed throughout the LCMS run. This is not feasible in practice. However, as one can see in Fig. 16, a 6x reduction can be achieved if one excludes just one high abundance precursor in a 10 s retention time window, and removing the 8 most abundant species results in a 100x improvement in effective dynamic range.

[0242] This can be implemented by using a scheme as illustrated in Fig. 17, where a multitude of barriers such that a specific, predetermined mobility range is accumulated at each barrier, is established. The barriers are adjustable via the applied potentials such that the mobility ranges can be fixed (predetermined) throughout an LC-IMS-MS experiment or adjusted during the experiment according to a schedule or in a data dependent manner. In this example in Fig. 17, 8 barriers are available, but the last 3 are effectively not used. In this example, the width of the mobility range accumulated by the fifth barrier is larger than that of, for example, the second barrier.

[0243] The gas flow, esp. drag force, represented by the arrows, is substantially constant throughout the device. The height, Ez, of the DC electric field barriers is inversely proportional to the mobility, Ko, that can be retained against the gas flow.

[0244] Fig. 18 shows an example hardware and method:

[0245] As illustrated in a), the “TIMS1” hardware is elongated compared to “TIMS2” so as to make space for a multitude, in this case 5, barriers.

[0246] As illustrated in b), the electric field strength is structured as a function of position for the five barriers. The barriers in this case are of substantially the same length, but may differ in length in alternate embodiments. The electric field strength at the plateau of each barrier together with the previous barrier sets the mobility range to be accumulated on any barrier. The size of the dots represents the abundance of the ion species.

[0247] As illustrated in c), ions may be quenched at regular time intervals and / or at the end of the accumulation step. Here the first and third mobility range are quenched whereas the second and forth are retained.

[0248] As illustrated in d), retained ions are transferred to the accumulation region of “TIMS 2”. Notice all barriers in TIMS 1 are reduced to zero or alternatively a field whereas all ions of interest are released to TIMS 2. Notice also that as ions are being accumulated in TIMS1 , a previous group of ions is being analyzed in TIMS 2 (b) and c)). In essence, this provides a method for eliminating one or more high abundance species from a mobility analysis is proposed with the following elements / steps: a) provide an analyzer having a multitude of DC barriers, a radially confining RF pseudopotential, a gas flow from an entrance end to an exit end (alternatively ion gates between the barriers) (shown schematically in Fig. 18 a)), b) set the 5 barriers, c) introduce and accumulate ions (shown schematically in Fig. 18 b)), d) eliminate ions of high abundance by lowering, or substantially reducing the strength of the confining field in the accumulation region of the selected barriers (shown schematically in Fig. 18 d)). Alternatively, the confining pseudopotential may be periodically, momentarily reduced or eliminated, for example for 1 ms every 30 ms during a 100 ms accumulation period, so as to eliminate or only reduce the number of high abundance ions in these regions. Alternatively, the high abundance ions may be eliminated by lowering the DC barrier and allowing the ions to be eliminated on a gate following the barrier, e) transfer the remaining ions to a downstream ion mobility (TIMS) analyzer (shown schematically in Fig. 18 d)), so the ions are transferred first to the accumulation region of the downstream “TIMS 2”. After accumulation in the downstream “TIMS 2" accumulation region, the ions are transferred to the analyzer region of the downstream "TIMS 2" so they can be analyzed while the next group of ions is being accumulated and filtered).

[0249] LIST OF REFERENCE SIGNS

[0250] 1-4 quadrants of the TIMS tube 7 gas

[0251] 6 ions 8 capillary repeller plate 23 z location, end of rising edge entrance funnel 24 z location

[0252] TIMS tube 31 diaphragm locationa accumulation unit of 11 34 diaphragm locationb scan unit of 11 50 first field barrier exit funnel 51 second field barrier axial gas flow 52-59 ions with decreasing mobility maximum gas flow velocity

[0253] TIMS 1 cutoff D accumulation and scan

[0254] TIMS 2 cutoff phase z location, beginning of rising E transfer phase edge

Claims

CLAIMS1. A method of data independent combined ion mobility and mass spectroscopy analysis, comprising the following steps: introducing precursor ions into two ion mobility separators (IMS), of which at least one, preferably the second, is a trapped ion mobility spectrometry (TIMS) separator, an optional ion gate located between the two ion mobility separators, separating ions in time according to mobility in the first ion mobility separator; selecting ions of interest, preferably by adjusting the transmission of the ion gate during or after the separation in the first ion mobility separator; transferring the selected ions of interest to the second ion mobility separator; and separating the transferred ions according to mobility in the second ion mobility separator, sequentially releasing precursor ions from said second ion mobility separator according to their ion mobility, introducing said released precursor ions into a mass filter which selectively transmits precursor ions having m / z values falling within a controllable mass window, fragmenting the precursor ions transmitted through said mass filter to generate fragment ions, carrying out a mass spectroscopy measurement on said fragment ions, wherein each fragment ion is associated with a mass window and an ion mobility (IM) range, and associating detected fragments with its corresponding precursor ion, wherein said second ion mobility separator and said mass filter are controlled in a synchronized manner such as to carry out a plurality of ion mobility (IM) scans, during which precursor ions of increasing or decreasing IM are successively released from said second ion mobility separator (IMS), and during which the mass window of said mass filter is shifted continuously or stepwisely towards lower or higher m / z values, respectively, and wherein said step of associating a detected fragment with its corresponding precursor ion is based on determining or utilizing the corresponding mass windows and ion mobility (IM) ranges associated with various occurrences of said fragment in said mass spectrometry measurement.

2. Method according to claim 1 , wherein the second ion mobility separator is a TIMS analyzer,preferably a TIMS analyzer with parallel accumulation and separation, in particular operating using a method comprising the steps:(a) accumulating ions from the first ion mobility separator in an RF ion trap;(b) transferring at least a subset of the accumulated ions into a trapping ion mobility separator, in which the transferred ions are radially confined by an RF field and pushed by a gas flow against a rising edge of an axial electric DC field barrier such that the transferred ions are spatially separated along the rising edge according to ion mobility;(c) successively releasing the transferred ions according to their ion mobility by decreasing the height of the electric DC field barrier while ions from the ion source are further accumulated in the RF ion trap; and(d) restoring the height of the electric DC field barrier which triggers a consecutive transfer of the accumulated ions from the RF ion trap into the trapping ion mobility separator.

3. Method according to any of the preceding claims, wherein the first ion mobility separator is operated such as to retain only ions having an ion mobility higher than an upper threshold value (17) of a IM window for the selected ions of interest and wherein either the second ion mobility separator is operated such as to allow ions having an ion mobility lower than a lower threshold value (18) to pass through, or the second ion mobility separator is operated such that ions having an ion mobility lower than a lower threshold value (18) are filtered out, or wherein the ion gate between the first ion mobility separator and the second ion mobility separator is operated such that ions having an ion mobility lower than a lower threshold value (18) are not allowed to enter the second ion mobility separator and / or wherein the first ion mobility separator, preferably a TIMS analyzer with parallel accumulation and separation, is provided as at least one ion mobility separator which is(a) filtering the ions by passing the ions through one of(i) at least two consecutive ion mobility high pass filters and(ii) at least two consecutive low pass filters, each filter being formed by entraining the ions in a gas flowing in a direction and applying to the ions an electric field that causes movement of the ions in a direction opposite the gas flow direction; and(b) collecting ions of the predetermined ion mobility range in a space between the filters operated, wherein preferably the first ion mobility separator is provided as a series of ionmobility separators each providing such at least two consecutive ion mobility high-pass filters or at least two consecutive low-pass filters, and wherein in that series at least 2, or 3- 6 ion mobility separators are provided, or is provided as one ion mobility separator which is providing one of(i) at least three, preferably at least four, or 4-8 consecutive ion mobility high-pass filters and(ii) at least three, preferably at least four, or 4-8 consecutive ion mobility low-pass filters, collecting ions in predetermined ion mobility ranges in the space between the filters operated.

4. Method according to the claim 3, wherein the first ion mobility separator is a TIMS analyzer, in which the ions are radially confined by an RF field and pushed by a gas flow against a rising edge of an axial electric DC field barrier such that the transferred ions are spatially separated along the rising edge according to ion mobility, and wherein the height of the electric DC field barrier is chosen such as to retain only ions having an ion mobility higher than said upper threshold value (17), and wherein the second ion mobility separator is a TIMS analyzer with parallel accumulation and separation, in which the transferred ions are accumulated in an accumulation section (11a) from the first ion mobility separator in an RF ion trap, in which the ions are radially confined by an RF field and pushed by a gas flow against a rising edge of an axial electric DC field barrier such that the transferred ions are spatially separated along the rising edge according to ion mobility, and wherein the height of the electric DC field barrier is chosen such as to retain only ions having an ion mobility higher than said lower threshold value (18) followed by transferring at least a subset of the accumulated ions into a trapping ion mobility separator in a separation section (11b), radially confined by an RF field and pushed by a gas flow against a rising edge of an axial electric DC field barrier such that the transferred ions are spatially separated along the rising edge according to ion mobility, and wherein the height of the electric DC field barrier at the beginning of the release is preferably chosen such as to retain only ions having an ion mobility higher than said lower threshold value (18).

5. Method according to claim 4, wherein between the accumulation section (11a) and the separation section (11 b) a second ion gate is provided preventing ions having an ion mobility higher than said lower threshold value (18) to enter the separation section(11 b).

6. Method according to any of the preceding claims 4 and 5, wherein the first ion mobility separator, at least once during a cycle of the second ion mobility separator, preferably at least twice or at least four times during a cycle of the second ion mobility separator, is operated such as to allow all ions to pass through while during this phase the gate between the first ion mobility separator and the second ion mobility separator is controlled not to allow any ions to enter the second ion mobility separator, or the second ion mobility separator is idle.

7. Method according to any of the preceding claims, wherein the first separation in the first ion mobility separator and the selective transfer 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 prior to separating them according to mobility, and wherein preferably the transmission of the ion gate is adjusted while the ions are separated in the first ion mobility separator such that the transmission for a highly abundant ion species of interest is lower than the transmission of less abundant ion species of interest.

8. Method according to any of the preceding claims, wherein the first ion mobility separator is a TIMS analyzer, and wherein separating ions in time according to mobility in the first ion mobility separator involves driving the ions transferred to the first TIMS analyzer by a first gas flow against a first counter-acting electric DC field barrier such that the ions are trapped and spatially separated according to their mobilities at different positions along a ramp of the first electric DC field barrier at which a friction force of the first gas flow equals the counter-acting force of the first electric DC field barrier, and temporally separating ions according to mobility in the first TIMS analyzer by adjusting a height of the first electric DC field barrier or the velocity of the first gas flow; and / or wherein the second ion mobility separator is a TIMS analyzer, and wherein separating ions in time according to mobility in the second ion mobility separator involves driving the ions transferred to the second TIMS analyzer by a second gas flow against a second counter-acting electric DC field barrier such that the ions are trapped and spatially separated according to their mobilities at different positions along a ramp of the second electric DC field barrier at which a friction force of the first gas flow equals the counter-acting force of the second electric DC field barrier, and temporally separating ions according tomobility in the second TIMS analyzer by adjusting a height of the second electric DC field barrier or the velocity of the gas flow.

9. Method according to any of the preceding claims, wherein either at the beginning of one LC observation retention time window, preferably in the range of 1-15 seconds, particularly preferably in the range of 3-10 seconds, a survey scan is taken, wherein preferably for taking that survey scan only one of the ion mobility separators is operated in separating mode, and wherein in that survey scan a full ion mobility width of interest and a full m / z width of interest is scanned, and wherein as a function of that survey scan for the remainder of said LC observation window said second ion mobility separator and said mass filter are controlled in a synchronized manner such as to carry out a plurality of IM scans, during which precursor ions of increasing or decreasing IM are successively released from said IMS, and during which the mass window of said mass filter is shifted continuously or stepwisely towards lower or higher m / z values, respectively, to avoid peptides not of interest identified in the survey scan, and wherein said step of associating a detected fragment with its corresponding precursor ion is based on determining or utilizing the corresponding mass windows and IM ranges associated with various occurrences of said fragment in said mass spectrometry measurement or wherein a) a survey scan is produced, wherein preferably for taking that survey scan only one of the ion mobility separators is operated in separating mode, preferably at a low duty cycle; b) the survey scan is used to determine the presence and mobility of any high abundance, undesirable species; c) at least one, preferably a plurality of mobility ranges of interest, which has a mobility higher than that of said high abundance species, is determined; d) a mobility cutoff is established in a first ion mobility separator; e) ions of interest are accumulated in said first ion mobility separator, wherein the accumulated said ions of interest constitute a mobility fraction; f) said fraction is transferred to the second ion mobility separator; and g) said second ion mobility separator is used in conjunction with said mass spectrometer to mobility mass analyze said fraction.

10. Method according to claim 9, wherein for analyzing the survey scan information about LC retention time, ion mobility and m / z for peptides not of interest and / or for peptides of interest, a database containing respective information is consulted and / or a list, which is preferably supplemented successively during one or a series of LC experiments, comprising peptides to be excluded, is consulted.

11. Method according to any of the preceding claims, wherein in said IM scans, adjacent mass windows that are associated with consecutive mass spectroscopy measurements of fragment ions overlap, such that the precursor ions transmitted through said mass filter during one IM scan are located in at least one continuous scan region in an m / z-IM plane which extends in a generally diagonal direction in said m / z-IM plane, wherein adjacent scan regions associated with different IM scans overlap in the m / z-direction, wherein preferably in said IM scans, adjacent mass windows that are associated with consecutive mass spectroscopy measurements of fragment ions overlap by at least 30% of their width, and / or wherein adjacent scan regions associated with different IM scans overlap in the m / z-direction by at least 33% of their width in m / z direction and / or wherein preferably the occurrence said fragment corresponds to a relative or absolute intensity of the fragment in the mass spectrometry measurement12. An apparatus for data independent combined ion mobility and mass spectroscopy analysis, in particular for carrying out the method according to any of the preceding claims, said apparatus preferably comprising: an ion mobility separator (IMS) for receiving and sequentially releasing precursor ions from said IMS according to their ion mobility, a mass filter arranged to receive said released precursor ions and to selectively transmit precursor ions having m / z values falling within a controllable mass window, a fragmentation device for fragmenting the precursor ions transmitted through said mass filter to generate fragment ions, an apparatus for carrying out a mass spectroscopy measurement on said fragment ions, wherein each fragment ion is associated with a mass window and an ion mobility (IM) range, and a control system, wherein said control system is configured to control said IMS and said mass filter in a synchronized manner such as to carry out a plurality of TM scans, during whichprecursor ions of increasing or decreasing IM are successively released from said IMS, and during which the mass window of said mass filter is shifted continuously or stepwisely towards lower or higher m / z values, respectively, wherein said control system is preferably configured to control said IMS and said mass filter in a synchronized manner such that, in said IM scans, adjacent mass windows that are associated with consecutive mass spectroscopy measurements of fragment ions overlap, such that the precursor ions transmitted through said mass filter during said IM scan are located in at least one continuous scan region in an m / z-IM plane which extends in a generally diagonal direction in said m / z-IM plane, wherein further preferably said control system is configured to control said IMS and said mass filter in a synchronized manner such that adjacent scan regions associated with different IM scans overlap in the m / z-direction.

13. An apparatus according to claim 12, wherein the second ion mobility separator is a TIMS analyzer, preferably a TIMS analyzer with parallel accumulation and separation, in particular operating using a method comprising the steps:(a) accumulating ions from the first ion mobility separator in an RF ion trap;(b) transferring at least a subset of the accumulated ions into a trapping ion mobility separator, in which the transferred ions are radially confined by an RF field and pushed by a gas flow against a rising edge of an axial electric DC field barrier such that the transferred ions are spatially separated along the rising edge according to ion mobility;(c) successively releasing the transferred ions according to their ion mobility by decreasing the height of the electric DC field barrier while ions from the ion source are further accumulated in the RF ion trap; and(d) restoring the height of the electric DC field barrier which triggers a consecutive transfer of the accumulated ions from the RF ion trap into the trapping ion mobility separator.

14. An apparatus according to claim 12 or 13, wherein the first ion mobility separator is built and operated such as to retain only ions having an ion mobility higher than an upper threshold value (17) of a IM window for the selected ions of interest and wherein either the second ion mobility separator is operated such as to allow ions having an ion mobility lower than a lower threshold value (18) to pass through, or the second ion mobility separator is operated such that ions having an ion mobility lower than a lower threshold value (18) are filtered out,or wherein the ion gate between the first ion mobility separator and the second ion mobility separator is operated such that ions having an ion mobility lower than a lower threshold value (18) are not allowed to enter the second ion mobility separator.

15. An apparatus according to any of claim 12 -14, wherein the first ion mobility separator is a TIMS analyzer, in which the ions are radially confined by an RF field and pushed by a gas flow against a rising edge of an axial electric DC field barrier such that the transferred ions are spatially separated along the rising edge according to ion mobility, and wherein the height of the electric DC field barrier is chosen such as to retain only ions having an ion mobility higher than said upper threshold value (17), and wherein the second ion mobility separator is a TIMS analyzer with parallel accumulation and separation, in which the transferred ions are accumulated in an accumulation section (11a) from the first ion mobility separator in an RF ion trap, in which the ions are radially confined by an RF field and pushed by a gas flow against a rising edge of an axial electric DC field barrier such that the transferred ions are spatially separated along the rising edge according to ion mobility, and wherein the height of the electric DC field barrier is chosen such as to retain only ions having an ion mobility higher than said lower threshold value (18) followed by transferring at least a subset of the accumulated ions into a trapping ion mobility separator in a separation section (11b), radially confined by an RF field and pushed by a gas flow against a rising edge of an axial electric DC field barrier such that the transferred ions are spatially separated along the rising edge according to ion mobility, and wherein the height of the electric DC field barrier at the beginning of the release is preferably chosen such as to retain only ions having an ion mobility higher than said lower threshold value (18), wherein preferably between the accumulation section (11a) and the separation section (11 b) of the second ion mobility separator a second ion gate is provided preventing ions having an ion mobility higher than said lower threshold value (18) to enter the separation section (11b).

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