TIMS with synchronized mobility and quadrupole filtering

Abstract

A method of combined ion mobility and mass spectrometry analysis, comprising the following steps: introducing precursor ions into an ion mobility separator, e.g. a TIMS separator, separating them in the TIMS separator, sequentially releasing precursor ions from said TIMS separator and introducing them into a mass filter, fragmenting the precursor ions transmitted to generate fragment ions, carrying out a mass spectrometry measurement on said fragment ions, wherein said TIMS separator and said mass filter in at least one spectrometry measurement frames are controlled in a synchronized manner and wherein in a least one mass spectrometry measurement frame said mass filter is operated in a mode in which, within an overall filter width thereof, it acts as a band stop filter having at least one band stop mass range, wherein precursor ions having m / z values falling within the band stop mass range are not transmitted.

Description

[0001] F07355 11.9.2025

[0002] 1

[0003] TITLE

[0004] TIMS WITH SYNCHRONIZED MOBILITY AND QUADRUPOLE FILTERING

[0005] TECHNICAL FIELD

[0006] 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 mobility accumulation, quadrupole filtering and respective mobility-mass analysis, in particular also corresponding encoding schemes for data analysis.

[0007] PRIOR ART

[0008] 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.

[0009] US 7,838,826 B1 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.

[0010] Commonly, the length of the ion mobility separation unit of a TIMS analyzer amounts to F07355 11.9.2025

[0011] 2 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 has already achieved, with reduced scan speeds, ion mobility resolutions up to RmOb = 400, which is extraordinarily high.

[0012] 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.

[0013] Acquisition methods which sample the ion cloud more efficiently have been proposed, F07355 11.9.2025

[0014] 3 namely synchro-PASEF (Skowronek P. et al. "Synchro-PASEF allows precursor-specific fragment ion extraction and interference removal in data-independent acquisition"; Mol. Cel. Prot. 2022; 22(2)) and midia-PASEF (Distler II. et al. "midiaPASEF maximizes information content in data-independent acquisition proteomics"; bioRxiv. 2023). The latter distinguishes from the former by using overlapping mass windows in the IM and m / z plane, and US-A-2022034840 discloses such an apparatus and a method of data independent combined ion mobility and mass spectrometry 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 spectrometry 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 spectrometry 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.

[0015] Skowronek et al in Mol Cell Proteomics, 22(2):100489 (doi: 10.1016 / j.mcpro.2022.100489) report that data-independent acquisition (DIA) methods have become increasingly popular in mass spectrometry-based proteomics because they enable continuous acquisition of fragment spectra for all precursors simultaneously. However, these advantages come with the challenge of correctly reconstructing the precursor-fragment relationships in these highly convoluted spectra for reliable identification and quantification. Here, they introduce a scan mode for the combination of trapped ion mobility spectrometry with parallel accumulation-serial fragmentation (PASEF) that seamlessly and continuously follows the natural shape of the ion cloud in ion mobility and peptide precursor mass dimensions. Termed synchro-PASEF, it increases the detected fragment ion current several-fold at subsecond cycle times. Consecutive quadrupole selection windows move synchronously through the mass and ion mobility range. In this process, the quadrupole slices through the peptide precursors, which separates fragment ion signals of each precursor into adjacent synchro-PASEF scans. This precisely defines precursor-fragment relationships in ion mobility and mass dimensions and effectively deconvolutes the DIA fragment space. Importantly, the partitioned parts of the fragment ion transitions provide a further dimension of specificity via a lock-and-key mechanism. This is also advantageous for quantification, where signals from interfering precursors in the DIA selection window do not affect all partitions of the fragment ion, allowing to retain only the specific parts for quantification. F07355 11.9.2025

[0016] 4

[0017] Overall, we establish the defining features of synchro-PASEF and explore its potential for proteomic analyses.

[0018] Distler et al in biorxiv (https: / / doi.Org / 10.1101 / 2023.01.30.526204) report that data- independent acquisition (DIA) approaches provide comprehensive records of all detectable pre-cursor and fragment ions. Here we introduce midiaPASEF, a novel DIA scan mode using mobility-specific micro-encoding of overlapping quadrupole windows to optimally cover the ion population in the ion mobility-mass to charge plane. Using overlapping ion mobility-encoded quadrupole windows, midiaPASEF maximizes information content in DIA acquisitions which enables the determination of the precursor m / z of each fragment ion with a precision of less than 2 Th. The Snakemake-based MIDIAID pipeline integrates algorithms for multidimensional peak detection and for machine-learning-based classification of precursor-fragment relationships. The MIDIAID pipeline enables fully automated processing and multidimensional deconvolution of midia-PASEF files and exports highly specific DDA- like MSMS spectra which are suitable for de novo sequencing and can be searched directly with established tools including PEAKS, FragPipe and Mascot. midiaPASEF acquisition identifies over 40 unique peptides per second and provides powerful library-free DIA analyses including phosphopeptidome and immunopeptidome samples.

[0019] 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.

[0020] US-A-2020266041 proposes a data acquisition method in a mass spectrometer which includes a. providing an ion source to generate precursor ions; b. feeding the precursor ions into a first mass analyzer that selects one mass window such that the precursor ions located outside the mass window pass through the first mass analyzer and the precursor ions located within the mass window cannot pass through the first mass analyzer; c. feeding the precursor ions passing through the first mass analyzer into a collision cell for collisional dissociation, to generate product ions; d. feeding the product ions into a second mass F07355 11.9.2025

[0021] 5 analyzer for mass analysis and recording a spectrum; and e. repeating Steps b-d. Each time when Step b is repeatedly performed, the selected mass window does not overlap with all the mass windows previously selected. After all the mass windows in a mass range are selected, the repetition is stopped.

[0022] US-A-2015279640 describes a tandem mass spectrometry system, in which a first mass analyzer filters parent ions using a wide mass passband with a narrow rejection notch defined according to a modulation format. A wide mass range of parent ions is transmitted to an ion fragmentation device. Daughter ions produced thereby are transmitted to a second mass analyzer to produce a daughter ion mass spectrum. The modulation of the measured daughter ion mass spectrum, when correlated with the passband modulation of the first mass analyzer (i.e., parent ion spectrum), allows definitive identification of each daughter mass peak with the appropriate parent ion. Due to the wide mass passband, the ion detector signal is in proportion to the increased ion flux passed by the first mass analyzer.

[0023] SUMMARY OF THE INVENTION

[0024] As mentioned, synchro PASEF is a technique for correlating fragment ions with their precursors in TIMS-MS2 analysis of complex samples. The TIMS device is ramped at a rate of ~10 Hz for the duration of a chromatographic separation. LC peaks at a specific RT value or window may be ~5 second wide. In this approach, several TIMS ramps are grouped together for each PASEF experiment in a RT window, with each ramp now comprising a PASEF Frame during which the quadrupole is used as a synchronized DC mass filter to transmit a unique but rather broad (10-100 Da) mass pass band to the collision cell for fragmentation. The ions observed in the mass spectrum in MS2 are the fragments of species transmitted in each Frame. For each ion mobility value, each precursor m / z is only transmitted during a single Frame which permits coarse fragment-precursor correlation. The PASEF Frames are looped until the chromatographic runs terminates.

[0025] The number of PASEF Frames in each loop is tuned to suit the upstream chromatographic peak width and the required sensitivity, but trade-offs exist:

[0026] Increasing the number of Frames per PASEF loop: Improves precision of fragmentprecursor correlation by narrowing the mass isolation; reduces ion utilization by reducing the fraction of Frames during which each precursor is transmitted; risks chromatographic distortion as a PASEF loop becomes long enough to sample different regions of a single LC peak

[0027] Midia PASEF is similar, but transmits each precursor in several consecutive frames to improve ion utilization without changing fragment-precursor correlation.

[0028] The approach proposed here, also termed Knockout PASEF, is the photo negative of F07355 11.9.2025

[0029] 6

[0030] Synchro PASEF. One (or several) narrow mass bands are ejected in each Frame instead of transmitting one narrow mass band. Each m / z is transmitted in most Frames so full mass range is analyzed repeatedly. Each m / z is ejected in at least one Frame to ensure full coverage of fragment-precursor correlation. Knockout PASEF applies the quadrupole selection e.g. via Resonant Ejection wherein a low amplitude supplemental waveform is applied to multipole rods to selectively eject ions.

[0031] This disclosure is covering both critical aspects of Knockout PASEF:

[0032] • Time-variant resonant ejection as a means of precursor ion selection following a mobility separation; o As described, the resonant ejection waveform is to be changed between each mobility ramp to generate Frames (additionally the main RF amplitude - or frequency - can be varied to change the position in mass for a given AC frequency); o the ejection waveform may be changed within a single mobility ramp or may be held constant for several ramps;

[0033] • A binary encoding scheme used to generate resonant ejection waveforms for tuneable sensitivity and precursor selectivity.

[0034] The present invention relates to a method as defined in claim 1 and as further specified in the respective dependent claims.

[0035] Definitions:

[0036] LC-MS / MS: Tandem mass spectrometry coupled to a liquid chromatography system, a technique in instrumental analysis where one or more mass analyzers are coupled together behind a liquid chromatography system using an additional reaction step to increase their abilities to analyse chemical samples.

[0037] MS1, MS2: The molecules of a given sample in an LC-MS / MS experiment are ionized and their mass-to-charge ratio (often given as m / z or m / Q) is measured / selected by the mass analyzer (designated MS1). Ions of a particular m / z-ratio coming from MS1 are selected and then made to split into smaller fragment ions, e.g. by collision-induced dissociation, ionmolecule reaction, or photo-dissociation. These fragments are then introduced into the mass analyzer (MS2), which in turn measures the fragments by their m / z-ratio. The fragmentation step makes it possible to identify and separate ionized molecules that have very similar m / z-ratios but produce different fragmentation patterns in MS2. The unfragmented peptide ion that dissociates to a smaller fragment ion, usually as a result of collision-induced dissociation in an MS / MS experiment, is typically referred to as precursor. F07355 11.9.2025

[0038] 7

[0039] Data dependent acquisition (DDA): LC-MS / MS or “shotgun” MS approach that is based on the generation of fragment ions from precursor ions that are automatically selected in the first (MS1) dimension based on the precursor ion profiles in that dimension. The window for the second (MS2) dimension is chosen as a function MS1 output (single precursor peak) automatically by the machine. This means that in this mode the MS2 dimension is not continuously sampled but only selectively as a function of the MS1 signal. In a typical shotgun acquisition method, top 10 precursor ions are selected for fragmentation per MS1 scan by the MS for measurement in MS2 with a relatively narrow isolation width of 1-2 Thomson. Precursor ions that have been selected for fragmentation are also typically ignored by the MS in the subsequent scans to allow fragmentation of new precursor ions.

[0040] Data independent acquisition (DIA): LC-MS / MS approach, in this mode, all ionized compounds of a given sample that fall within a specified mass range in the first MS1 dimension are fragmented in a systematic and unbiased fashion resulting in corresponding spectra in the MS2 dimension. In contrast to DDA, in this case the MS2 space is continuously sampled. This not only leads to a larger data volume, but also has the effect that the spectra measured in the MS2 space comprise fragments not just from one precursor in the MS1 dimension but potentially from several such precursors. The common feature of DIA methods is that instead of selecting and sequencing a single precursor peak, wider m / z windows are fragmented resulting in complex spectra containing fragment ions of several precursors. In DIA acquisition, windows size in MS2 dimension is often more than 30 Thomson. This means that a typical MS2 scan in DIA is more complex than in DDA because of significantly more precursor ions being co-fragmented.

[0041] Mobilogram: T race of an ion in the ion mobility dimension. It is usually generated by tracing an ion in a single scan. The x-axis is a metric for ion mobility (e.g. reversed ion mobility, collision cross section) and y-axis is intensity.

[0042] Micro scan: A MS1 or MS2 scan performed with a specific precursor isolation width at a specific ion mobility and retention time. The x-axis is m / z and y-axis is intensity. So it is a single TIMS-push event resulting in a list of m / z intensity pairs at one single point in ionmobility dimension.

[0043] TIMS scan: In a single TIMS scan, ions from the selected mass ranges are fragmented to record ion mobility-resolved MS2 spectra of all precursors. A TIMS scan typically consists of hundreds of micro scans where each micro scan is a MS2 spectrum of precursors fragmented with a selected mass range at a specific ion mobility.

[0044] Mass spectrometry measurement frame: A set of microscans carried out within one RT window (LC observation retention time window), in which the mass filter has the same filter characteristics while being shifted along the mass dimension in synchrony with the TIMS F07355 11.9.2025

[0045] 8 separator being shifted along the ion mobility dimension. Within one RT window, several mass spectrometry measurement frames with different filter characteristics of the mass filter are carried out. The set of mass spectrometry measurement frames carried out within one RT window is called PASEF loop.

[0046] The present invention according to a first aspect thereof relates to a method of combined ion mobility and mass spectrometry analysis, comprising the following steps:

[0047] • introducing precursor ions into an ion mobility separator, in particular a trapped ion mobility spectrometry (TIMS) separator,

[0048] • separating the ions according to mobility in the ion mobility separator, preferably the trapped ion mobility spectrometry (TIMS) separator,

[0049] • sequentially releasing precursor ions from said ion mobility separator (preferably said trapped ion mobility spectrometry (TIMS) separator) according to their ion mobility,

[0050] • introducing said released precursor ions into a mass filter which selectively transmits precursor ions having m / z values falling within one or several controllable mass windows,

[0051] • fragmenting the precursor ions transmitted through said mass filter to generate fragment ions,

[0052] • 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, and associating detected fragments with its corresponding precursor ion.

[0053] Said ion mobility separator is preferably a trapped ion mobility spectrometry (TIMS) separator. However, it can also be a traveling wave ion mobility spectrometry (TWIMS) or a drift tube ion mobility spectrometer (DTIMS).

[0054] Said ion mobility separator, preferably a trapped ion mobility spectrometry (TIMS), and said mass filter in at least one, preferably all mass spectrometry measurement frames 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 trapped ion mobility spectrometry (TIMS) separator, and during which the mass window of said mass filter is shifted continuously or stepwisely towards lower or higher m / z values, respectively.

[0055] Further, in a least one mass spectrometry measurement frame said mass filter is operated in a mode in which, within an overall filter width thereof, it acts as a band stop filter having at least one band stop mass range, and on both sides thereof band pass mass ranges, wherein precursor ions having m / z values falling within the band stop mass range are not F07355 11.9.2025

[0056] 9 transmitted, while precursor ions having m / z values falling within any the band pass mass ranges are transmitted.

[0057] As for the band stop mass range this means that ions having m / z values falling within the band stop mass range are not substantially transmitted, as the ejection efficiency may not be 100%. Typically, less than 5%, preferably less than 3% or less than 2 % or less than 1% of the ions having m / z values falling within the band stop mass range are transmitted by the filter.

[0058] Overall filter widths of the mass filter that are associated with consecutive mass spectrometry measurement frames of target ions can be adjacent (not overlapping, preferred) or may also overlap with each other.

[0059] Preferably in said IM scans, adjacent mass windows that are associated with consecutive mass spectrometry measurements of fragment ions are adjacent or 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 may or may not overlap in the m / z-direction.

[0060] The proposed method also works with the application of purely vertical mass bands i.e. constant throughout a given mobility scan. The scans can additionally be diagonal against the trend line of the mass mobility relationship to give different “slicing” behavior.

[0061] Preferably in said IM scans, adjacent mass windows that are associated with consecutive mass spectrometry measurements of fragment ions overlap by at least 30% of their width, and / or wherein adjacent scan regions associated with different IM scans overlap in the m / z- direction by at least 33% of their width in m / z direction.

[0062] The area covered by the overall filter widths of the mass spectrometry measurement frames in the m / z-IM plane in one IM scan is preferably arranged generally diagonally.

[0063] The term "generally diagonal" is to be interpreted in a broad manner, and in particular does not necessarily require that the frame extends along a straight line. Instead, the term "generally diagonal" mainly reflects the fact that in the continuous scan region, both the m / z range and the IM range concurrently varied.

[0064] Within the overall filter width there can be provided one single band stop mass range, which, in consecutive measurement frames, preferably shifts its position, preferably sequentially, along the m / z dimension, to preferably cover the full m / z range of the overall filter width of the mass filter in a full set of mass spectrometry measurement frames within one LC observation retention time window. Normally, the overall filter width of the mass filter only needs to cover the known m / z mobility correlation region which is limited to preferably -300 m / z. F07355 11.9.2025

[0065] 10

[0066] Alternatively and preferably, within the overall filter width there can be provided more than one individual band stop mass ranges (so multiple stop bands, e.g. 2, 3, 4 or even 5 individual band stop mass ranges within the overall filter width), which, in consecutive measurement frames, preferably shift their position, preferably sequentially, along the m / z dimension, to preferably, both or each alone, cover the full m / z range of the overall filter width of the mass filter in a full set of mass spectrometry measurement frames within one LC observation retention time window. Normally, the overall filter width of the mass filter only needs to cover the known m / z mobility correlation region which is limited to preferably -300 m / z.

[0067] So encoding is possible by applying multiple stop bands simultaneously and by turning off / on each MS1 stop band in a defined pattern over several scans. The scheme can differentiate between N precursor mass bands in as few as log2(N) sequential MS1 scans, which is a significant improvement over the linear method in the prior art and is unique to the proposed approach. These additional bands are illustrated in the figures described in detail further below. An encoding / decoding scheme to assign MS2 fragments to their precursor mass band is also described below. Some encoding / decoding can be required for pulling usable information from the chimeric spectra that result from applying multiple stop (or pass) bands.

[0068] In a set of mass spectrometry measurement frames within one LC observation retention time window frames with different mass filter patterns can be combined, including at least one mass spectrometry measurement frame with at least one band stop mass range or at least two band stop mass ranges, preferably in combination with at least one mass spectrometry measurement frame with the mass filter being structured as a high pass filter, and / or as a low-pass filter, and / or as a bandpass filter.

[0069] According to yet another preferred embodiment, the mass filter patterns of the mass filter are associated with a code, preferably a binary code, uniquely identifying the patterns used in each mass spectrometry measurement frame at least within the respective LC observation retention with time window, and wherein the corresponding code is used for the data analysis for associating detected fragments with its corresponding precursor ion.

[0070] The mass filter pattern can be selected to be different between different LC observation retention time windows, and / or between different frames within one LC observation retention time window and / or for different areas in the ion mobility-m / z plane.

[0071] According to a further preferred embodiment, 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, and in that survey scan a full ion mobility width of interest and a full m / z width of interest is scanned. As a function of that survey scan F07355 11.9.2025

[0072] 11 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, wherein 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. Preferably a mass filter (Knockout) pattern is selected as a function of the survey scan for the different frames within the LC observation retention time window.

[0073] Said step of associating a detected fragment with its corresponding precursor ion can be 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.

[0074] Yet another preferred embodiment is characterized in that said mass filter is a quadrupole mass filter, and wherein preferably the band stop is implemented by applying a respective excitation waveform in addition to the confining radiofrequency to lead to resonant ejection, wherein preferably for the implementation of several band stop ranges, a corresponding number of different excitation frequencies are applied, if need be in combination with DC isolation techniques.

[0075] A liquid phase separation device, preferably a capillary electrophoresis device or preferably a liquid chromatography (LC) device, is preferably provided, loading a sample on the liquid phase separation (LC) device, in particular a column, (chromatographically) separating sample components; and providing the separated sample components as ion source for ionization of said precursor ions.

[0076] The TIMS analyzer is preferably a TIMS analyzer with parallel accumulation and separation, in particular operating using a method comprising the steps:

[0077] (a) accumulating ions from the first ion mobility separator in an RF ion trap;

[0078] (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;

[0079] (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 F07355 11.9.2025

[0080] 12

[0081] (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.

[0082] According to another aspect of the present invention, it relates to an apparatus suitable and adapted for carrying out the above method.

[0083] According to this aspect, it relates to an apparatus for combined ion mobility and mass spectrometry analysis, in particular for carrying out the method according to any of the preceding claims, said apparatus preferably comprising: an ion mobility separator, preferably a trapped ion mobility spectrometry (TIMS) separator, for receiving and sequentially releasing precursor ions from said ion mobility separator (preferably from said trapped ion mobility spectrometry (TIMS) separator) 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 spectrometry 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 ion mobility separator (preferably said trapped ion mobility spectrometry (TIMS) separator) and said 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 ion mobility separator (preferably said trapped ion mobility spectrometry (TIMS) separator), and during which the mass window of said mass filter is shifted continuously or stepwise towards lower or higher m / z values, respectively, and wherein said mass filter can be operated to act as a bandstop filter, which, within an overall filter width, it acts as a band stop filter having at least one band stop mass range, and on both sides thereof band pass mass ranges, wherein precursor ions having m / z values falling within the band stop mass range are not transmitted, while precursor ions having m / z values falling within any the band pass mass ranges are transmitted.

[0084] Upstream of the trapped ion mobility spectrometry (TIMS) separator preferably a liquid chromatography (LC) device is provided and downstream thereof an ionization device, preferably an electrospray ionization device, for providing said precursor ions.

[0085] Further embodiments of the invention are laid down in the dependent claims. F07355 11.9.2025

[0086] 13

[0087] BRIEF DESCRIPTION OF THE DRAWINGS

[0088] 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,

[0089] Fig. 1 shows how in a set of four PASEF frames in the IM (y-axis) and m / z (x-axis) plane, using synchro PASEF, the mass filter (quadrupole) is operated as a selective bandpass with synchronized travelling mass filter window synchronized over the course of the ramp of the TIMS device;

[0090] Fig. 2 shows how in a set of four PASEF frames in the IM (y-axis) and m / z (x-axis) plane, using a possible implementation of (single) knockout PASEF, the mass filter (quadrupole) is operated as a selective band-stop with synchronized travelling mass filter window over the course of the ramp of the TIMS device;

[0091] Fig. 3 illustrates how the ion utilization (y-axis) changes as a function of the chosen number of PASEF frames (x-axis) for the different approaches, in particular pointing out the most sensitive and the most selective knockout approaches and the range of possibilities in between;

[0092] Fig. 4 illustrates how a mass range of interest can be divided into some number of m / z bands and can be encoded in a decimal or binary way, and on the right side a possible subset of four frames for a six band knockout PASEF scheme;

[0093] Fig. 5 shows an example for a 4 frame, 6 band encoding knockout PASEF scheme, wherein in a) the underlying table is given, and in b) - e) extracted mobilograms are given for different m / z values as seen in the four frames;

[0094] Fig. 6 shows how in a set of four PASEF frames in the IM (y-axis) and m / z (x-axis) plane, using a possible implementation of knockout PASEF (which is a complement of the scheme used in Fig. 5), the mass filter (quadrupole) is only in some frames operated with at least one band stop with synchronized travelling mass filter window over the course of the ramp of the TIMS device;

[0095] Fig. 7 shows further possibilities with 6-bit encoding and possible six frame patterns on the right side;

[0096] Fig. 8 shows the number of mass bands as a function of the sampling factor A for different bit encodings;

[0097] Fig. 9 shows the number of mass bands as a function of the sampling factor A for different number n of frames and where the different methods are located;

[0098] Fig. 10 shows the mass bin width as a function of the PASEF frames for the different techniques;

[0099] Fig. 11 shows how encoding may be different between mobility steps and / or over the F07355 11.9.2025

[0100] 14 course of a chromatographic run and frame dependent;

[0101] Fig. 12 shows how encoding may be different for each m / z-mobility cell;

[0102] Fig. 13 shows how encoding may be different in any of the areas of the m / z-mobility plane;

[0103] Fig. 14 in a) shows the mask scheme as also illustrated in Fig. 4 and a corresponding matrix representation for the evaluation of the correlation on the right side; in b) and c) shows examples from simulated data;

[0104] Fig. 15 illustrates how decoding is also possible across the whole EIM for one fragment m / z in a single matrix operation; and

[0105] Fig. 16 shows normalizing each encoding vector to unit length before calculating the correlation matrix.

[0106] DESCRIPTION OF PREFERRED EMBODIMENTS

[0107] Fig. 1 illustrates a typical PASEF loop with 4 frames in a synchro PASEF experiment. In this matrix representation, 1 indicates, that corresponding precursors being located in the corresponding area in the ion mobility (IM, unit 1 / K, y-axis) - m / z (x-axis) plane are transmitted by the TIMS device and the quadrupole located downstream thereof, while 0 indicates that a corresponding precursor is not passing through.

[0108] The trapezoidal transmission window is traveling, over the four frames of the experiment, from the low m / z region to the high region without overlap, from left to right, so the corresponding frames, illustrated by the respective trapezoidal white areas, are not overlapping.

[0109] Over the course of 4 TIMS ramps, the total ion utilization is 25%, and fragment ions may be assigned to a single precursor mass band, each of which covers 1 / 4 the total mass range. Finer fragment-precursor correlation down to ~1 Da may be achieved by 'Slicing' (see e.g. slice-PASEF, Szyrwiel L. et al. “Slice-PASEF: fragmenting all ions for maximum sensitivity in proteomics”; bioRxiv. 2022).

[0110] Existing PASEF experiments ramp the quadrupole isolation window synchronously with the TIMS ramp. This moving mass cut-off often 'slices' though mobility peaks, causing the precursor ions to transition between adjacent frames. The slope of the slicing line (typically 1-2 Da per TIMS step) is set such that most mobility peaks will be sliced. This is a function of: the number of Frames in the experiment; peak width along the mobility axis; the general mass-mobility trend line. Slices also help align data from several frames in post-processing. The approach proposed here, also termed Knockout PASEF, is the photo negative of Synchro PASEF. One (or several) narrow mass bands are ejected in each Frame instead of transmitting one narrow mass band. Each m / z is transmitted in most Frames so the full F07355 11.9.2025

[0111] 15 mass range is analyzed repeatedly. Each m / z is ejected in at least one Frame to ensure cover coverage of fragment-precursor correlation. Knockout PASEF applies the quadrupole selection via Resonant Ejection wherein a low amplitude supplemental waveform is applied to multipole rods to selectively eject ions. Supplemental waveform is e.g. <5 Vp-p and may be generated with off-the-shelf 16 kS, 1 MHz arbitrary waveform generators.

[0112] As for quadrupole operation the following:

[0113] Mathieu parameters define stable operating parameters in a linear quadrupole device; both parameters scale with the RF frequency squared and ion charge / mass; VDC scales linearly with the amplitude of DC potential applied between rod pairs; VRF scales linearly with the amplitude of RF potential applied to all rods; ions outside of this stable region quickly gain energy until they are ejected from the device; trapped ions exhibit periodic secular motion with frequency dependent on each ion’s mass-to-charge ratio and their VDC, RF coordinates.

[0114] As for quadrupole DC isolation the following:

[0115] A range of ions can be isolated by adjusting the ratio of the confining RF and resolving DC potentials. This ratio may be represented by a diagonal working line in VDC, VRF space. The ions in the stable central region of the working line are transmitted, but ions with m / z above or below this stable band are ejected.

[0116] As for quadrupole resonant ejection the following:

[0117] Ions may be ejected at normally stable VDC, VRF coordinates by applying a low voltage excitation waveform in addition to the confining RF.

[0118] This excitation creates an unstable iso-beta line in VDC, VRF space.

[0119] Ions are ejected where the working line intersects the excitation line. The ejected m / z may be selected by changing the RF voltage, DC voltage, and / or the frequency(s) of the excitation waveform.

[0120] Physically, excitation occurs when the excitation frequency matches an ion's secular frequency, and all ions with the corresponding m / z gain energy until they are no longer trapped.

[0121] This resonant excitation ejects a narrow notch in the range of transmitted ions, as small as ~5 Da.

[0122] The proposed Knockout PASEF has much in common with Synchro and Midia PASEF, but several big differences: Method of ion selection; AC Resonant Ejection in quadrupole or transfer multipole; much higher ion utilization; number of mass bands: more than 1 ejection band per Frame and scales factorially with Frame count; width of mass bands; precision of Slices; wealth of possible encoding schemes. F07355 11.9.2025

[0123] 16

[0124] Fig. 2 illustrates a typical single knockout PASEF loop with 4 frames. Again, 1 indicates, that corresponding precursors being located in the corresponding area in the ion mobility (IM, unit 1 / K, y-axis) - m / z (x-axis) plane are transmitted by the TIMS device and the quadrupole located downstream thereof, while 0 indicates that a corresponding precursor is not passing through.

[0125] The trapezoidal blocking window in this case is traveling, over the four frames of the experiment, from the low m / z region to the high m / z region without overlap, from left to right, so the corresponding blocking frames, illustrated by the respective trapezoidal grey areas, are not overlapping.

[0126] So, in contrast to the situation of conventional synchro PASEF, where so to speak the band of a bandpass is traveling in the ion mobility-m / z plane, in the single knockout PASEF experiment, the band of a band stop is traveling in the ion mobility-m / z plane, so in each Frame the majority of the ions, in this specific case, 75%, are transmitted.

[0127] Over the course of 4 TIMS ramps, total ion utilization is thus 75%. Fragment ions may nevertheless be assigned to a single precursor mass band, by differential data handling, each of which covers 1 / 4 the total mass range.

[0128] As illustrated in Fig. 3, showing the ion utilization in % as a function of the number of PASEF frames in one Loop or RT window, knockout-PASEF comprises a class of operating modes which provide higher ion utilization and more precise precursor selection windows than all existing mass-selective PASEF techniques.

[0129] Synchro- and Midia-PASEF transmit each m / z in a fixed number of frames per PASEF cycle: Ion utilization and sensitivity decrease with the number of PASEF frames.

[0130] In contrast, knockout-PASEF ejects each m / z in a tunable number of frames per PASEF cycle: Ion utilization and sensitivity increase with the number of PASEF frames; the number of times each m / z is ejected may be chosen to prioritize sensitivity or precursor mass selectivity.

[0131] Knockout PASEF divides the mass range of interest into some number of m / z bands and assigns each band a unique number which may be represented in either decimal or binary notation.

[0132] In each frame, the ions in a single m / z band are either transmitted (1) or rejected (0).

[0133] One can treat this pattern of 1s and Os as an n-bit binary number.

[0134] Because each mass band has a unique pattern of 1s and Os, the ions transmitted in each band may be decoded from the data set. I.e.: If a fragment of 200.0 m / z appears in only frames 2 and 4, then its precursor was transmitted in band b0101.

[0135] For a four bit encoding scheme an example is given in Fig. 4, illustrating how a mass range of interest can be divided into some number of m / z bands and can be encoded in a decimal F07355 11.9.2025

[0136] 17 or binary way, wherein on the right side a possible subset of four frames for a six band knockout PASEF scheme is given.

[0137] Each Frame provides one additional bit of data for encoding each mass channel.

[0138] In a four Frame experiment, one can unambiguously encode 2A4 = 16 mass bands. However, each mass bands will not be transmitted in the same number of Frames.

[0139] To ensure uniform ion utilization across the full mass range, one can sort the 16 binary values by their number of 1s (or sum of bits), then use only those values which transmit in the desired number of Frames.

[0140] Synchro PASEF (lower rectangle) and single Knockout PASEF (as given in Fig. 2, upper rectangle) frames are immediately visible. Another set of frames with 6 mass bands also appears, in which: each mass band is sampled exactly 2 times; each mass band has a unique, decodable binary pattern over 4 frames.

[0141] The corresponding pattern as given on the right can be used in a four frame six band knockout PASEF loop.

[0142] Fig. 5 shows an example for such a 4 frame, 6 band encoding knockout PASEF scheme. In a) the underlying table is given. Each band is transmitted 2 times over the 4 frames. The table given was used to filter simulated MS1 species. Extracted Ion Mobilograms for simulated fragments may be compared to the table to decode the fragment-precursor relationships. In b) - e) extracted mobilograms are given for different m / z values as seen in the four frames.

[0143] In b), showing the Extracted Ion Mobilogram for m / z = 538.0, this fragment appears only in frames 0 and 2. Reading its intensity over the 4 frames, one can deduce its precursor was transmitted in band 1010 (m / z 500-550).

[0144] In c), showing the Extracted Ion Mobilogram (EIM) for m / z = 637.0, this Extracted Ion Mobilogram contains several isobaric fragments. The precursor of the fragment at IM 1.4 was transmitted in band 0011 (m / z 300-350). The precursor(s) of the fragments covering 0.7-1.1 were transmitted in band 1001 (m / z 450-500).

[0145] In d), showing the Extracted Ion Mobilogram for m / z = 969.0, isobaric fragments appear only in frames 2 and 3. Thus their precursors were both transmitted in band 0011 (m / z 300- 350). These two precursors may be differentiated based on other, non-isobaric fragments. In e), showing the Extracted Ion Mobilogram for m / z = 428.0, the Extracted Ion Mobilogram contains two isobaric fragments. The precursor of the fragment at 1.4 was transmitted in band 0110 (m / z 400-450). The precursor of the fragment at 0.5 was transmitted in band 1001 (m / z 450-500).

[0146] Fig. 6 schematically illustrates a corresponding knockout PASEF loop with 4 frames. Again, 1 indicates, that corresponding precursors being located in the corresponding area in the F07355 11.9.2025

[0147] 18 ion mobility (IM, unit 1 / K, y-axis) - m / z (x-axis) plane are transmitted by the TIMS device and the quadrupole located downstream thereof, while 0 indicates that a corresponding precursor is not passing through.

[0148] In this case not all frames are using a band stop characteristic, but at least one of the frames has a band stop characteristic (frames 2-4). Again, differential data analysis allows to isolate systems located in six bands.

[0149] In this 4 Frame Encoded Knockout PASEF experiment, over the course of 4 TIMS ramps, the total ion utilization is 50%. Fragment ions may be assigned to a single precursor mass band, each of which covers 1 / 6 the total mass range.

[0150] The number of available mass bands grows exponentially with the number of bits, n. For a six bit encoding scheme an example is given in Fig. 7, illustrating how a mass range of interest can be divided into a number of 15 or 20 m / z bands and can be encoded in a binary way manner, wherein on the right side the possible subsets of six frames for knockout PASEF schemes are given. Two possible 6-bit encoding schemes with differing ion utilization and band counts are shown. All bands need not be used in final encoding.

[0151] For an n-Frame experiment where each band is sampled A times, the maximum number of mass bands BA is defined by

[0152] One can verify this by noting

[0153] Fig. 8 shows the number of mass bands as a function of the sampling factor A for different bit encodings. Depending on the specific requirements, the number of bands which are sampled can be adapted and tuned.

[0154] All encoded PASEF techniques exist in a class of experiments where each mass is sampled A times across n frames. Synchro PASEF transmits each m / z band once and is equivalent to any Knockout PASEF experiment with A=1.

[0155] The most sensitive Knockout PASEF transmits each m / z band almost every frame (A=n- 1) but provides a limited number of mass bands.

[0156] The most selective Knockout PASEF maximizes the number of mass bands and samples each band in -50% of frames (A=n / 2).

[0157] This disclosure covers all previously undescribed members of the class.

[0158] Fig. 9 correspondingly shows the number of mass bands as a function of the sampling factor A for different number n of frames and where the different methods are located. F07355 11.9.2025

[0159] 19

[0160] Sampling and band count are not usual figures of merit, this shall be translated: Ion utilization is A / n and directly relates to sensitivity which should increase as (A / n)-2. Fragment-precursor correlation precision scales linearly with the number of mass bands.

[0161] Knockout thus provides 10x improvement in ion utilization for 12 frame experiments when compared to Synchro PASEF.

[0162] Knockout further provides >25x improvement in correlation precision for 12 frame experiments when compared to Synchro PASEF. Fig. 10 shows the mass bin width as a function of the PASEF frames for the different techniques, wherein the compromise knockout schemes are given by isolated dots; see also Fig. 3 in this respect.

[0163] Fig. 11 shows how encoding may be different between mobility steps and / or over the course of a chromatographic run and frame dependent. Encoding at each mobility step may be assigned with or without survey scan data. Mobilities with few, low-intensity precursors might benefit from a more sensitive encoding. Mobilities with many precursors might benefit from narrower mass bands at lower sensitivity. For example in frame 4 shown in the right column on top, the upper and lower area represent six bands with five times sampling, while the central more patterned portion represents 20 bands with three times sampling.

[0164] Fig. 12 shows how encoding may be different for each m / z-mobility cell. Encoding at each m / z may be assigned with or without survey scan data. Mass ranges with few, low-intensity precursors might benefit from a more sensitive encoding. Mass ranges with many precursors might benefit from narrower mass bands at lower sensitivity. In this case frame 5 given in the right column in the middle represents two tilted frames, one on the left (more patterned) with 20 bands and three times sampling, and the one on the right having a single central band stop feature, with six bands and five times sampling.

[0165] Fig. 13 shows how encoding may be different in any of the areas of the m / z-mobility plane. Unwanted precursors may be undersampled to reduce interferences. As opposed to Fig. 11 , where different horizontal stripes with different bands / sampling characteristics are given, and Fig. 12, where diagonal stripes with different bands / sampling characteristics are given, this figure represents the possibility of having arbitrary areas in the plane with different bands / sampling characteristics as needed.

[0166] Slices may take advantage of small changes in the quadrupole RF amplitude band within a single PASEF frame to localize precursors more precisely.

[0167] Scanning the RF amplitude slowly and synchronously with the TIMS moves the quad highland low-mass cutoffs diagonally across the m / z-mobility plane. The slope of slices can be determined by: Width of the mass isolation window; peak width along the mobility axis; the general mass-mobility trend line.

[0168] The slope in Da / TOF Pulse determines precision of precursor assignment. F07355 11.9.2025

[0169] 20

[0170] Generally, a set of slices in a PASEF experiment should be angled to cross each m / z once every -25-50 TIMS steps (TOF pulses).

[0171] For a 30 Da Synchro isolation window, slope is -1 Da / Pulse. This limits the precursor mass range at each mobility to the number of frames times the isolation window (eg: 4 Frames * 25 Da = 100 Da precursors at each mobility step).

[0172] For a 5 Da Knockout isolation window, slope is -0.2 Da / Pulse.

[0173] As for slicing, the proposed knockout approach enables more mass bands which allows to change the slope of slices without sacrificing mass range.

[0174] A comparison of the different techniques is given in the following table:

[0175] Fig. 14 in a) shows the mask scheme as also illustrated in Fig. 4 discussed above and a corresponding matrix representation for the evaluation of the correlation on the right side. Data is decoded by computing the correlation between the MS2 Extracted Ion Mobiligram (EIM) and the MS1 encoding matrix described in the context of in Fig. 4 (4 frames, 6 bands). The resulting correlation matrix provides a weighted score for each mass band B reflecting the likelihood that the fragment’s precursor was in each mass band. The fragment is assigned to the correlation matrix element with the highest score.

[0176] In Fig. 14 b) and c) examples from simulated data are given for that encoding matrix illustrating the assignment of peaks to the respective band by using the respective computed correlation matrix.

[0177] Decoding is also possible across the whole EIM for one fragment m / z in a single matrix operation:

[0178] • Precursor transmission band B varies between 0 and b

[0179] • Mobility index K varies between 0 and k

[0180] • Frame index F varies between 0 and f

[0181] The maximum value in each column of the correlation matrix indicates precursor transmission in band Bnat mobility Kj. F07355 11.9.2025

[0182] 21

[0183] This generalizes to higher dimensions along the m / z and / or LC retention time axes, which is illustrated in Fig. 15. Decoding may be performed in the same manner along one or several orthogonal axes, including mobility (shown in figure), m / z, chromatographic retention time, etc. This permits decoding of high-dimensional Knockout datasets in a single matrix operation or in multiple lower-dimensional operations.

[0184] For mixed-sampling encoding, the encoding vectors (matrix rows) for more highly sampled bands are longer and may give erroneously high correlation values. This may incorrectly assign some fragments to these highly sampled bands. This may be fixed by normalizing each encoding vector to unit length before calculating the correlation matrix, which is illustrated in Fig. 16.

[0185] LIST OF REFERENCE SIGNS

[0186] DC direct current

[0187] DDA data dependent acquisition

[0188] DIA data independent acquisition

[0189] DMS differential mobility spectrometry

[0190] DTI MS drift tube ion mobility spectrometry

[0191] EIM extracted ion mobilogram

[0192] IM ion mobility

[0193] IMS ion mobility spectrometry

[0194] FAIMS field asymmetric ion mobility spectrometry m / z mass to charge ratio

[0195] MRM multiple reaction monitoring

[0196] MS1 first spectral dimension in LC-MS / MS experiment

[0197] MS2 second spectral dimension in LC-MS / MS experiment

[0198] PASEF parallel accumulation serial fragmentation midia PASEF maxmizing information content in DIA-PASEF RF radio frequency

[0199] RT retention time

[0200] SRM Selected Reaction Monitoring

[0201] TIMS trapped ion mobility spectrometry

[0202] TWIMS travelling wave ion mobility spectrometry

Claims

1. F07355 11.9.202522CLAIMS1. A method of combined ion mobility and mass spectrometry analysis, comprising the following steps: introducing precursor ions into an ion mobility separator, preferably a trapped ion mobility spectrometry (TIMS) separator, separating the ions according to mobility in the ion mobility separator, sequentially releasing precursor ions from said 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 one or several controllable mass windows, fragmenting the precursor ions transmitted through said mass filter to generate fragment ions, 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, and associating detected fragments with its corresponding precursor ion, wherein said ion mobility separator and said mass filter in at least one, preferably all mass spectrometry measurement frames 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 ion mobility separator, and during which the mass window of said mass filter is shifted continuously or stepwisely towards lower or higher m / z values, respectively, wherein in a least one mass spectrometry measurement frame said mass filter is operated in a mode in which, within an overall filter width thereof, it acts as a band stop filter having at least one band stop mass range, and on both sides thereof band pass mass ranges, wherein precursor ions having m / z values falling within the band stop mass range are not transmitted, while precursor ions having m / z values falling within any the band pass mass ranges are transmitted.

2. Method according to claim 1 , wherein overall filter widths of the mass filter that are associated with consecutive mass spectrometry measurement frames of target ions are adjacent to each other wherein preferably in said IM scans, adjacent mass windows that are associated with consecutive mass spectrometry measurements of fragment ions are not overlapping,F07355 11.9.202523 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.

3. Method according to claim 1 , wherein overall filter widths of the mass filter that are associated with consecutive mass spectrometry measurement frames of target ions overlap with each other, wherein preferably in said IM scans, adjacent mass windows that are associated with consecutive mass spectrometry 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, and / or 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 spectrometry measurements of fragment ions overlap by at least 30% of their width, and / or wherein adjacent scan regions associated with different IM scans overlap in the m / z-direction by at least 33% of their width in m / z direction.

4. Method according to any of the preceding claims, wherein the area covered by the overall filter widths of the mass spectrometry measurement frames in the m / z-IM plane in one IM scan is arranged generally diagonally.

5. Method according to any of the preceding claims, wherein within the overall filter width there is provided one single band stop mass range, which, in consecutive measurement frames, preferably shifts its position, preferably sequentially, along the m / z dimension, preferably leading to different mass filter patterns, to preferably cover the full m / z range of the overall filter width of the mass filter in a full set of mass spectrometry measurement frames within one LC observation retention time window.

6. Method according to any of the preceding claims 1-4, wherein, within the overall filter width there are provided more than one individual band stop mass ranges, preferably 2, 3, 4 or 5 individual band stop mass ranges within the overall filter width, which, in consecutive measurement frames, preferably shift their position, preferably sequentially, along the m / z dimension, preferably leading to different mass filter patterns, to preferably, both or each alone, cover the full m / z range of the overall filter width of the mass filter in aF07355 11.9.202524 full set of mass spectrometry measurement frames within one LC observation retention time window.

7. Method according to any of the preceding claims, wherein in a set of mass spectrometry measurement frames within one LC observation retention time window frames with different mass filter patterns are combined, including at least one mass spectrometry measurement frame with at least one band stop mass range or at least two band stop mass ranges, preferably in combination with at least one mass spectrometry measurement frame with the mass filter being structured as a high pass filter, and / or as a low-pass filter, and / or as a bandpass filter.

8. Method according to any of claims 4 or 5 or 6, wherein the mass filter patterns of the mass filter are associated with a code, preferably a binary code, uniquely identifying the patterns used in each mass spectrometry measurement frame at least within the respective LC observation retention with time window, and wherein the corresponding code is used for the data analysis for associating detected fragments with its corresponding precursor ion.

9. Method according to any of the preceding claims 4-7, wherein the mass filter pattern is selected to be different between different LC observation retention time windows, and / or between different frames within one LC observation retention time window and / or for different areas in the ion mobility-m / z plane.

10. Method according to any of the preceding claims, wherein 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, 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 correspondingF07355 11.9.202525 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, and wherein further preferably a mass filter pattern is selected as a function of the survey scan for the different frames within the LC observation retention time window.

11. Method according to any of the preceding claims, 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.

12. Method according to any of the preceding claims, wherein said mass filter is a quadrupole mass filter, and wherein preferably the band stop is implemented by applying a respective excitation waveform in addition to the confining radiofrequency to lead to resonant ejection, wherein preferably for the implementation of several band stop ranges, a corresponding number of different excitation frequencies are applied, if need be in combination with DC isolation techniques.

13. Method according to any of the preceding claims, wherein a liquid phase separation device, preferably a liquid chromatography (LC) device is provided, loading a sample on the liquid phase separation device, in particular a column, (chromatographically) separating sample components; and providing the separated sample components as ion source for ionization of said precursor ions.

14. Method according to any of the preceding claims, wherein the TIMS analyzer is 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; andF07355 11.9.202526(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.

15. An apparatus for combined ion mobility and mass spectrometry analysis, in particular for carrying out the method according to any of the preceding claims, said apparatus preferably comprising: an ion mobility separator, preferably a trapped ion mobility spectrometry (TIMS) separator for receiving and sequentially releasing precursor ions from said ion mobility separator, preferably trapped ion mobility spectrometry (TIMS) separator, 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 spectrometry 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 ion mobility separator, preferably said trapped ion mobility spectrometry (TIMS) separator, and said 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 ion mobility separator, preferably said trapped ion mobility spectrometry (TIMS) separator, 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 mass filter can be operated to act as a bandstop filter, which, within an overall filter width, it acts as a band stop filter having at least one band stop mass range, and on both sides thereof band pass mass ranges, wherein precursor ions having m / z values falling within the band stop mass range are not transmitted, while precursor ions having m / z values falling within any the band pass mass ranges are transmitted.

16. Apparatus according to claim 15, which, upstream of the ion mobility separator, preferably said trapped ion mobility spectrometry (TIMS) separator, a liquid phase separation device, preferably a liquid chromatography (LC) device is provided and downstream thereof an ionization device, preferably an electrospray ionization device, for providing said precursor ions.

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