IPRM-pasef with correlation analysis

Abstract

A method for the combined identification and localization of substances of a sample surface, the method comprising: acquiring a mass spectrometric image; wherein for each pixel for the generation of said mass / mobility maps of masses and ion mobility values the method comprises the following steps: introducing precursor ions into at least TIMS separator, sequentially releasing precursor ions from said TIMS separator, introducing said released precursor ions into a mass filter, fragmenting the precursor ions transmitted, carrying out a mass spectroscopy measurement on said fragment ions, wherein said TIMS separator and said mass filter are scheduled in a synchronized manner, and wherein for the identification of substances for the identification of substances at least three data points for given spatial pixel coordinates of at least two fragments or at least one fragment and a precursor are correlated in a correlation map.

Description

[0001] TITLE

[0002] IPRM-PASEF WITH CORRELATION ANALYSIS

[0003] TECHNICAL FIELD

[0004] The present invention relates the field of mass spectrometry imaging, in particular to a method for the identification and localization of substances of a sample surface involving tandem mass spectroscopy and ion mobility determination, in particular with a view to discriminating isobaric and / or isomeric substances present in / on said sample surface, as well as uses of such methods and devices for implementing such methods.

[0005] PRIOR ART

[0006] 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.

[0007] 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.

[0008] 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.

[0009] 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.

[0010] 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 ion 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 possibility to prolong the ion accumulation and scan duration, thereby increasing the ion mobility resolution so as to separate and detect more ion species.

[0011] 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] TIMS extends conventional liquid chromatography-mass spectrometry (LC-MS) and bioanalytical workflows, such as proteomic, lipidomic, metabolomic, drug metabolism, structure elucidation or other methods, with an additional ion mobility dimension. In addition to the general benefits of the additional signal separation, the implementation of TIMS in the Bruker timsTOF family of instruments has demonstrated that collisional cross section (CCS) values of analytes, i.e. peptides, lipids, or metabolites, are highly reproducible. The additional separation dimension already allows in data dependent acquisition (DDA) methods to identify precursors of isomeric compounds to be scheduled for fragmentation individually. As omics workflows aim at covering as much of the chemical complexity of a sample with tandem-MS spectra by DDA, TIMS can already greatly improve the analytical coverage of complex samples.

[0014] 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.

[0015] 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.

[0016] 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 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.

[0017] 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.

[0018] 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. Thus, in SRM, all transitions are monitored one at a time.

[0019] In addition to the DIA methods mentioned above, a novel targeted proteomics technique was developed which can be considered a successor of SRM. This method, called parallel reaction monitoring (PRM, see Peterson et al in Mol Cell Proteomics. 2012 Aug 3; 11(11): 1475-1488. doi: 10.1074 / mcp.0112.020131), relies on a quadrupole mass filter which is combined with a high-resolution mass analyzer, such as e.g. in a quadrupole-equipped bench-top orbitrap MS instrument. Replacing the last quadrupole of a triple quadrupole with a high-resolution mass analyzer allows the parallel detection of all fragment ions at once. In principle it would also be possible to combine a linear ion trap with the orbitrap instead of the quadrupole. The advantage of PRM over SRM is that less prior knowledge about the target molecules is required. In terms of dynamic range PRM performs even better than SRM under some conditions due to its high selectivity.

[0020] Mass spectrometry imaging (MSI) is a technique used to visualize the spatial intensity distributions of molecules, such as pharmaceuticals, metabolites, lipids, peptides or proteins, by their molecular masses. After locally ionizing the molecules on the surface of a sample and collecting a mass spectrum at one position in a Cartesian grid, the sample is moved to reach the next spot, and so on, until the entire sample is scanned. By choosing a peak, that corresponds to a compound of interest, in the resulting representative spectrum, the MS data is used to map its intensity distribution across the sample. This results in pictures of the spatially resolved distribution of a compound pixel by pixel. Each data set contains a set of pictures because any peak in each spectrum can be spatially mapped. MSI has been generally considered a qualitative method, the signal generated by this technique is however proportional to the relative abundance of the analyte. Therefore, quantification is possible. Most common known ionization technologies in the field of MSI are desorption electrospray ionization (DESI) imaging, matrix-assisted laser desorption / ionization MALDI imaging, secondary ion mass spectrometry imaging (SIMS imaging) and Nanoscale SIMS (NanoSIMS).

[0021] EP-A-2455759 relates to the mass spectrometry imaging of histologic thin tissue sections, where a mass spectrum is acquired for every image point. The invention provides a method for the identification and localization of proteins or other biomolecules of a histologic thin tissue section, comprising the following steps: a) the biomolecules of two similar thin tissue sections are enzymatically digested, substantially preserving the biomolecule positions in the tissue; b) a mass spectrometric image of the digest products in the first thin tissue section is acquired; c) the digest products of the second tissue section are extracted and separated with the aid of a separation method, and then introduced into a tandem mass spectrometer, which acquires mass spectra and daughter ion spectra of all the digest products present; d) a list of all identifiable biomolecules of the tissue is created by comparison of the mass spectra and daughter ion spectra of the digest products with spectra derived from biomolecule structure databases or correspondent spectral libraries; and e) the biomolecules in the list are assigned, on the basis of the masses of their digest products, to the digest products of the same mass in the mass spectra of the mass spectrometric image of the thin tissue section, after intelligent filtering, where necessary. With the aid of correlations of the mass-selective images, digest products can be identified as being wrongly assigned. The summation of well-correlating mass-selective images of the digest products of the biomolecules produces biomolecule-selective images, i.e. images of the distribution of the individual biomolecules in the thin tissue section.

[0022] EP-A-3832310 discloses a method for the identification and localization of small molecule species in a histologic thin tissue section comprises the steps of: a) acquiring a mass / mobility image of the tissue section and generating a mass / mobility map of the small molecule species of interest for each pixel of the image; b) providing a second sample of the same tissue and extracting the small molecules of interest, separating them, and acquiring mass and ion mobility spectra from the separated small molecules; c) identifying the small molecules of interest using corresponding reference databases; and d) assigning identified small molecules to entries in the mass / mobility maps of the first tissue section by comparison of ion masses and mobilities of the identified species to those of the second thin tissue section.

[0023] Ellis et al (Nat Methods 2018; vol. 15(7):515-518; doi: 10.1038 / s41592-018-0010-6) report a method that enables automated data-dependent acquisition of lipid tandem mass spectrometry data in parallel with a high-resolution mass spectrometry imaging experiment. The method does not increase the total image acquisition time and is combined with automatic structural assignments. This lipidome-per-pixel approach automatically identified and validated 104 unique molecular lipids and their spatial locations from rat cerebellar tissue.

[0024] Han et al (TRAC TRENDS IN ANALYTICAL CHEMISTRY, vol. 112, pages 13-28, 2019) report how recent development of mass spectrometry imaging (MSI) technology allowed to obtain extremely detailed images of the spatial distribution of proteins in tissue at high spatial resolution reaching cell dimensions, high target specificity and a large dynamic concentration range. This review focusses on the development of two main MSI principles, namely targeted and untargeted detection of protein distribution in tissue samples, with special emphasis on the improvements in analyzed mass range and spatial resolution over the last 10 years. Untargeted MSI of in situ digested proteins with matrix-assisted laser desorption ionization is the most widely used approach, but targeted protein MSI technologies using laser ablation inductively coupled plasma (LA-ICP) and photocleavable mass tag chemical labeling strategies are gaining momentum. Moreover, this review also provides an overview of the effect of sample preparation on image quality and the bioinformatic challenge to identify proteins and quantify their distribution in complex MSI data.

[0025] Heuckeroth et al (Nature Communications vol. 14, Art. Nr. 7495 (2023)) report how trapped ion mobility spectrometry (TIMS) adds an additional separation dimension to mass spectrometry (MS) imaging, but that the lack of fragmentation spectra (MS2) impedes confident compound annotation in spatial metabolomics. They describe spatial ion mobility- scheduled exhaustive fragmentation (SIMSEF), a dataset-dependent acquisition strategy that augments TIMS-MS imaging datasets with MS2 spectra. The fragmentation experiments are systematically distributed across the sample and scheduled for multiple collision energies per precursor ion. Extendable data processing and evaluation workflows are implemented into the open-source software MZmine. The workflow and annotation capabilities are demonstrated on rat brain tissue thin sections, measured by matrix-assisted laser desorption / ionisation (MALDI)-TIMS-MS, where SIMSEF enables on-tissue compound annotation through spectral library matching and rule-based lipid annotation within MZmine and maps the (un)known chemical space by molecular networking. The SIMSEF algorithm and data analysis pipelines are open source and modular to provide a community resource.

[0026] EP-A-2356668 discloses atmospheric pressure mass spectrometry (MS), and more specifically a process and apparatus which combine infrared laser ablation with electrospray ionization (ESI). A method for three-dimensional imaging of a sample having a native water content by mass spectrometry is proposed, the method comprising: subjecting the sample to infrared laser ablation electrospray ionisation mass spectrometry (LAESI-MS), wherein the LAESI-MS is performed using a LAESI-MS device directly on the sample, wherein the sample does not require chemical / physical pre-treatment an wherein the LEASI-MS is performed at atmospheric pressure, wherein the LAESI-MS device is equipped with a 3D scanning apparatus for lateral and depth scanning of multiple points following the cellular pattern defined on the sample, and for depth profiling of each point following the cellular pattern defined on the sample by performing multiple ablations at each point, each laser pulse of said ablations ablating a deeper layer of the sample than a prior pulse, wherein each laser pulse has a laser energy that is absorbed by the native water in the sample, characterised in that the combination of lateral scanning and depth profiling provides three- dimensional molecular distribution imaging data, and in that the LAESI-MS device comprises an automated feedback mechanism to correct for variances in water content of the sample by continuously adjusting laser energy and / or laser wavelength while recording the depth of ablation for each pulse.

[0027] SUMMARY OF THE INVENTION

[0028] The above-mentioned mass spectrometry imaging techniques can be combined with the above-mentioned tandem mass spectrometry techniques, and in particular with tandem mass spectrometry techniques which include, as a further dimension, collision cross-section (CCS) measurements, e.g. using the above-mentioned TIMS technology. To further increase the analytical power of these techniques, the corresponding methods can be refined by using parallel accumulation serial fragmentation (PASEF) and PRM approaches. This leads to a particularly high discriminative analytical power even though desorption from the surface in particular in case of biological samples leads to a complex mix of precursors or, in case of in source fragmentation (ISF), of already fragmented analyte molecules, in combination with high spatial resolution.

[0029] Preferably a PRM approach is used, which simultaneously detects and quantifies one, if present normally two or more fragment ions resulting from fragmentation of a (normally one) common precursor ion. Generally, PRM entails the accumulation and subsequent mass analysis of different fragment ion species. In one non-limiting example, precursor ions of a specified m / z are selectively transmitted by a quadrupole mass filter to a collision cell, wherein they collide at relatively high energies with molecules or atoms of a neutral gas and consequently undergo collision-induced dissociation to form fragment ions. The collected product ions, which include different ion species having various m / z's, are then delivered to an orbital electrostatic trap (Orbitrap) mass analyzer or a time-of-flight mass analyzer or an FT-ICR / MRMS mass analyzer, which mass analyzes the fragment ions to generate a mass spectrum representing the individual abundances (intensities) of each of a plurality of product ion species.

[0030] This allows for surprising new bioinformatics approaches to further increase the analytical power on the spatial resolution side as well as on the molecular identification side. This is particularly powerful in case of significant overlap of differing systems in one or both analytical dimensions (spatial, spectroscopic, the latter depending on the technique including MS as well as CCS dimensions).

[0031] So, for example in case of isomeric or isobaric molecules leading to identical or chimeric MS1 spectra, differences in the spatial distribution pattern can be used for more reliably and unambiguously separating such molecules in the data analysis.

[0032] In the context of the present application the synonymously used terms isomers, isomer molecules or isomeric molecules are considered to represent at least two molecules which have the same atomic composition (molecular formula) and thus the same molecular weight but either have a different atomic connectivity (so-called constitutional isomers) or which are stereoisomers. The latter stereoisomers however are only included in as far as they are not mirror images of each other, so in the present context the term isomeric molecules does not include enantiomers, since these cannot be separated in a mass spectroscopic protocol leading to discriminative fragments. However, stereoisomers include diastereomers, including also cis-trans isomers, conformers, rotamers. Generally speaking, isomeric molecules according to this invention are molecules which lead to essentially identical mass spectroscopy signals before fragmentation in MS1 , but which can give rise to different fragment ions subsequent to fragmentation in the MS2 dimension.

[0033] In the context of the present application the synonymously used terms isobars, isobar molecules or isobaric molecules are considered to represent at least two molecules which have the same nominal mass but different exact masses, and which are molecules with different molecular formula. In particular, the term relates to at least two molecules where the differences in exact mass are too small for a mass analyzer to resolve them, leading to chimeric MS1 spectra. These partially overlapping species are usually difficult if not impossible to separate, normally the differences in exact mass for isobaric molecules according to this invention are smaller than 25 mDa, preferably smaller than 10 mDa or smaller than 5 mDa.

[0034] The mass-to-charge ratio is represented as (m / z), where (m) is the mass of the ion (typically in atomic mass units, amu) and (z) is the charge number of the ion (the number of elementary charges, e). This ratio is dimensionless and crucial for identifying and characterizing ion. The m / z value is calculated by dividing the mass of the ion by its charge. For example, an ion with a mass of 100 amu and a charge of +1 will have an m / z value of 100. If the same ion has a charge of +2, its m / z value will be 50. In a mass spectrometer, ions are separated based on their m / z using electric and magnetic fields. This separation allows for the detection and analysis of ions with different masses and charges. The m / z values help in identifying the molecular structure and composition of the sample. By analyzing the mass spectrum, which plots ion intensity against m / z, chemists can determine the presence of specific ions and infer the structure of the molecules.

[0035] Mass spectrometry involves ionizing chemical compounds to generate charged particles (ions). These ions are then separated and detected based on their mass-to-charge ratio (m / z). The resulting data, presented as a mass spectrum, provides valuable information about the molecular structure and composition of the sample. An ionization source ionizes the sample molecules, meaning it converts them into ions by adding or removing electrons. The mass analyzer separates the ions based on the mass-to-charge ratio, and ions are detected by the detector. The instrument computer converts the detector signal into a mass spectrum. A mass spectrometer typically operates under vacuum conditions to ensure a collision-free flight path between ionization source and detector.

[0036] A mass spectrum is a plot that displays the ion signal as a function of the mass-to-charge ratio (m / z) of the ions present in a sample. This spectrum is typically generated using a mass spectrometer and provides valuable information about the molecular composition and structure of the sample. The x-axis of a mass spectrum represents the mass-to-charge ratio, while the y-axis shows the intensity of the ion signal, which correlates with the abundance of each ion. By analyzing the peaks in a mass spectrum, chemists can identify and quantify different molecules within the sample.

[0037] Tandem mass spectrometry (MS / MS) involves two or more stages of mass spectrometry separated by a fragmentation step. The first mass analyzer selects and isolates ions of a specific m / z (MS1 dimension), the so-called precursor ions, which are then fragmented, for example by collision-induced dissociation (CID). The resulting fragment ions are separated based on their m / z in a second mass analyzer, and detected (MS2 dimension) to form a MS / MS spectrum. Like a regular mass spectrum, an MS / MS spectrum has peaks that represent detected ions. In a typical MS / MS spectrum, peaks representing both the (unfragmented) precursor ion, as well as fragment ions can be found. The fragment ions can be used to identify the chemical structure of the selected and isolated precursor.

[0038] Matrix-assisted laser desorption / ionization (MALDI) is an ionization technique used in mass spectrometry to analyze biomolecules with minimal in-source fragmentation. In MALDI, the sample molecules are mixed with a matrix compound that absorbs laser energy. When a laser pulse is directed at the sample-matrix mixture, the matrix absorbs the energy and desorbs, carrying the sample molecules into the gas phase and ionizing them. The sample, a mixture of sample molecules and matrix, is introduced into the ion source where it is illuminated with a laser. The energy of the laser is absorbed by the matrix, which causes ablation and desorption of the molecules on the sample surface. This forms a so-called MALDI plume is formed in which charges are transferred from matrix to sample molecules, forming ions. The ions are transferred to the mass analyzer where they are separated based on their m / z.

[0039] Mass Spectrometry Imaging (MSI) is an analytical technique that combines the molecular specificity of mass spectrometry with spatial information, allowing for the visualization of the intensity distribution of molecules within a sample. The intensity distribution of a specific ion within a sample is commonly referred to as an “ion image”, or “spatial distribution”. MSI involves collecting mass spectra at multiple points across a sample surface, typically following a Cartesian coordinate system. Each point, also referred to as “pixel”, provides a mass spectrum that represents the molecular composition at that specific location. The spatially correlated mass spectra are stored in a virtual data cube. The three axes of the cube represent the x- and y-coordinates of the Cartesian coordinate system, and the m / z axis of the mass spectrum. Each voxel (i.e. 3D pixel) of the data cube contain the intensity value of an m / z value at a given position in the coordinate system. Typically, an MSI data set is represented by a representative mass spectrum (e.g., the overall average mass spectrum) and a collection of ion images. The data point for given spatial pixel coordinates of one specific fragment is the intensity value of the signal of that fragment in the spectrum of these spatial pixel coordinates. Trapped ion mobility spectroscopy (TIMS) involves using an electric field to hold ions stationary against a moving gas. This setup allows the drift force to be compensated by the electric field, effectively separating ion packages based on their size-to-charge ratio, commonly referred to as ion mobility or collisional cross section (CCS).

[0040] A mass-mobility heatmap is a visual representation used in ion mobility spectrometry (IMS) coupled with mass spectrometry (MS) to analyze complex samples. This heatmap plots the mass-to-charge ratio (m / z) of ions on one axis and their ion mobility on the other, creating a two-dimensional map that helps in identifying and characterizing compounds based on their mass and mobility. In practical terms, the mass-mobility heatmap allows to visualize how ions are separated not only by their mass but also by their shape and size, which affects their mobility through a gas. This is particularly useful for distinguishing between isomeric compounds (those with the same mass but different structures) and for identifying isobaric compounds with similar masses but different mobilities. One heatmap is the intensity values of one peak or feature (e.g. one precursor of one fragment) in all of the spectra as a function of the spatial coordinates. The x-axis of the heatmap represents the mass dimension of the analysis, the y-axis represents the mobility dimension of the analysis, and the feature intensity is displayed as a color scale.

[0041] The concept of ion mobility-enabled mass spectrometry imaging (IM-MSI) is very similar to that of “regular” MSI. The key difference is that in IM-MSI with mobility a further dimension is added and a mass-mobility heatmap, or frame, is recorded at each coordinate in the Cartesian coordinate system, compared to a mass spectrum in MSI. This consequently results in a representative mass-mobility heatmap that represents the full data set, and from which mass-mobility features can be selected in order to display their intensity distributions, or ion images.

[0042] Parallel Accumulation SErial Fragmentation (PASEF) leverages trapped ion mobility spectrometry (TIMS) to accumulate ions in parallel before fragmenting them in series. This process involves holding ions stationary in a tunnel using opposing forces of gas flow and an electric field. The ions find an equilibrium point based on their collisional cross-section, which is a function of their size and shape. Once the desired number of ions have accumulated, they are eluted by gradually lowering the electric field strength, allowing them to enter the mass spectrometer for fragmentation. This method improves the signal-to-noise ratio by concentrating ions of the same mass-to-charge ratio, which enhances the detection of low-abundance species.

[0043] Parallel Reaction Monitoring (PRM) is a targeted mass spectrometry technique used for the precise quantification and characterization of specific proteins or peptides in complex samples. In PRM, the process begins with the isolation of target precursor ions using a quadrupole mass filter. These isolated ions are then fragmented in a collision cell, and the resulting fragment ions are detected and quantified using a high-resolution mass analyzer, such as an Orbitrap or a time-of-flight (TOF) mass spectrometer.

[0044] In prm-PASEF, the process begins with the targeted selection of precursor ions using Parallel Reaction Monitoring (PRM). These ions are then fragmented in the collision cell, and the resulting fragments are analyzed with high mass resolution. The unique aspect of prm-PASEF is that it leverages the trapped ion mobility spectrometry (TIMS) device to separate ions based on their size, shape, and charge before fragmentation. This separation allows for the mobility-resolved fragmentation of analytes, which significantly increases the number of precursors that can be targeted in a single acquisition without compromising sensitivity or selectivity.

[0045] Similar to MSI and IM-MSI, iprm-PASEF involves recording a mass-mobility frame at each position in a Cartesian coordinate system. In iprm-PASEF, each mass-mobility frame consists of a prm-PASEF measurement, in which one or more (max. 25) predefined precursor ion is fragmented. Since the same (set of) precursors is fragmented in every pixel, the spatial distributions of the fragment ion images can be visualized in an iprm-PASEF data set.

[0046] The proposed scheme can also be used to identify and handle in source fragmentation, so to avoid false analytical interpretation results due to fragmentation occurring already at the source side and not only on the fragmentation side of the tandem mass spectroscopy analysis.

[0047] According to a first aspect of the present invention it correspondingly relates to a method for the combined identification and localization of substances of a sample surface, the method comprising acquiring a mass spectrometric image of said substances of the sample with a mass spectrometer having a built-in ion mobility spectrometer, and generating mass / mobility maps of masses and ion mobility values for sample ions for each pixel of the image. According to the proposed approach, the sample surface is systematically scanned using a grid, normally a cartesian grid, overlaid over the tissue for MS imaging acquisition for the generation of the data pixels, and this not only for the initial MS1 scan for the selection of precursors, but also for the MS / MS measurements including fragmentation.

[0048] The number of MS / MS pixels in a single acquisition (i.e. obtained from a single tissue) depends on both the size of the tissue (or the measurement area) and the Cartesian grid spacing (i.e. spatial resolution). Typically, between 20.000 and 2.000.000 pixels are acquired in single acquisition. Typically, a minimum spacing of the grid is around 5 x 5 micrometer. However, also a minimum spatial resolution starting at 500 x 500 nm, so e.g. 650 x 650 nanometer is possible for the proposed approach. As to a maximum for the proposed approach, a maximum spacing of the grid is typically 100 x 100 micrometer. Most commonly, and specifically for the MS / MS application, grid spacings of 20 x 20 to 50 x 50 micrometer balance spatial detail with sufficient detection sensitivity.

[0049] Mass / mobility maps are maps for (precise) masses and ion mobilities obtained for each single pixel in an image. So, there are mass values (MS1 and MS2 dimensions, in case of a survey scan mass / mobility map only MS1 , see further below) and ion mobility values (IM dimension) for sample ions (defined by a specific MS1 or MS2 window and a IM window, the above mentioned feature). The spatially-correlated visualization of the intensity values extracted from a collection of mass / mobility maps of a given feature, results in the display of a mass / mobility map of masses and ion mobility values, or termed differently, an ion image, for the given feature. For each pixel for the generation of said mass / mobility maps of masses and ion mobility values, the method comprises the following steps: introducing precursor ions resulting from ionization from the sample surface into at least one trapped ion mobility spectrometry (TIMS) separator, and separating the precursor ions according to mobility in the trapped ion mobility spectrometry (TIMS) separator, sequentially releasing precursor ions from said trapped ion mobility spectrometry (TIMS) separator according to their ion mobility for a (preferably single) precursor ions falling within an ion mobility window, introducing said released precursor ions into a mass filter which selectively transmits precursor ions having m / z values falling within a controlled mass window, fragmenting the precursor ion(s) 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 optionally associating detected fragments with its corresponding precursor ion.

[0050] Said trapped ion mobility spectrometry (TIMS) separator and said mass filter are scheduled 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. So preferably the above PASEF approach is used.

[0051] It is noted that already this part of the approach differs from the above-mentioned SIMSEF approach as disclosed in Heuckeroth et al. One essential difference between the SIMSEF method and the presented method lies in the structured acquisition of spectra across the entire sample surface following a grid, normally a Cartesian grid (i.e. imaging) versus the sparse acquisition of mobility-resolved MS / MS spectra at pre-determined molecular hotspots in the SIMSEF approach (i.e. profiling). So, in particular the MS / MS part of the proposed workflow is what differentiates in addition to the correlation below. SIMSEF performs a discontinuous acquisition of a series of pixels scattered over a (sub)region, also called profiling. These positions are based on the original cartesian grid from the MS1 acquisition. This makes that the SIMSEF approach may in some cases separate and fragment isomeric / isobaric precursors. However, it cannot correlate, group and validate fragments based on MS / MS data image correlations as are possible in the proposed approach. The approach presented here performs a continuous and systematic MS / MS acquisition of a series of pixels contained within a (sub)region, also called imaging.

[0052] For the identification and / or discrimination of substances, according to the invention, at least three data points for given spatial pixel coordinates of at least two fragments or at least one fragment and a precursor are correlated in a correlation map.

[0053] The proposed method is therefore a fundamentally different approach compared with the one proposed by Heuckeroth et al, so compared with the SIMSEF approach, in the sense that the proposed method identifies compounds not just on the basis of an incidental presence of a precursor and (several) fragment(s) at a single location, but on a statistically evaluated pattern across a full sample surface.

[0054] In conventional MS / MS profiling (SIMSEF) the skilled person knows they are not generating MS / MS images, but only mobility-resolved MS / MS profiles. As such, making use of correlations to identify sets of fragment and / or precursor images to identify a compound makes absolutely no sense, as there are no images available to correlate. The skilled person operating the SIMSEF workflow, therefore does not consider the proposed correlations as a reasonable and successful approach.

[0055] Typically, at least three data points for given spatial pixel coordinates of at least two fragments or at least one fragment and a precursor are correlated in a correlation map, in which the intensity of a respective data point of given spatial pixel coordinates of one fragment is given along a first axis (x-axis) and the intensity of the respective data point of the same given spatial pixel coordinates of the other fragment or the precursor is given along a second axis (y-axis, preferably orthogonal to x-axis).

[0056] Put differently, two or more fragment or fragment and precursor ion images can each be converted into a one-dimensional vector (signal intensity vs. pixel index) and then subjected to a correlation analysis.

[0057] So preferably two or more fragment ion images are each converted into a one-dimensional vector (signal intensity vs. pixel index). The vectors can be subjected to a Pearson correlation (the ratio between the covariance of two vectors and the product of their standard deviations). The resulting ratio (R) has a range between -1 and 1. A ratio of 1 represents a perfect correlation, a ratio of -1 represents anti-correlation, and a value of 0 represents a lack of correlation. The ratio is often displayed as a R2, which ranges between 0-1 .

[0058] It is noted that the mention of using Pearson product-moment correlation coefficients in the above mentioned EP-A-2 356 668 is not in the context of compound identification as in this present context. EP-A-2 356 668 refers to finding specific signals in a dataset that share the same spatial localization. As such, the application of the Pearson Correlation Coefficient in EP-A-2 356 668 has nothing to do with the calculation of correlations between fragments and precursors specifically, and it also has no relation to MS / MS identification of molecules. EP-A-2 356 668 furthermore does not mention that data from three or more pixels is used to calculate the Pearson Correlation Coefficient.

[0059] Said correlation can be used to separate and / or identify fragment contribution to isobaric and / or isomeric precursor substances.

[0060] The correlation can also be used to identify in source fragmentation resulting from ionization from the sample surface.

[0061] To first identify a feature as a precursor of interest, in a survey scan a mass / mobility map of precursor masses and ion mobility values is determined so the process is carried out without fragmentation. To this end, so for the determination of said controlled mass window and of said ion mobility window for selecting a chosen precursor, in a preceding step the following steps are preferably carried out: acquiring a mass spectrometric image of said substances of the sample with said mass spectrometer having a built-in ion mobility spectrometer, and generating mass / mobility maps of masses and ion mobility values for sample ions for each pixel of the image; wherein for each pixel for the generation of said mass / mobility maps of masses and ion mobility values the method comprises the following steps (the mass spectrometer generates a mass / mobility map for each pixel of the image, so that a mass / mobility map of masses and ion mobility values can be created over the entire image): introducing precursor ions resulting from ionization from the sample surface into at least one trapped ion mobility spectrometry (TIMS) separator, and separating the precursor ions according to mobility in the trapped ion mobility spectrometry (TIMS) separator, sequentially releasing precursor ions from said trapped ion mobility spectrometry (TIMS) 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, carrying out a mass spectroscopy measurement on said precursor ions. After picking a chosen precursor, around this said controlled mass window and said ion mobility window are determined to select said chosen precursor for the above fragmentation process. For the separation and / or identification of isobaric and / or isomeric substances and / or for the identification of fragments resulting from in-source fragmentation according to a first preferred embodiment, a low correlation between two fragments or a fragment and a precursor are used to identify discriminating fragments and for separating and / or identifying isobaric and / or isomeric substances and / or in-source ionization, and / or a high correlation of two fragments or a fragment and a precursor are used to identify fragments common to one single substance for separating and / or identifying isobaric and / or isomeric substances.

[0062] A (relatively) low correlation between the fragment signals or a fragment signal and a precursor signal is preferably used to attribute those fragments or that fragment and the corresponding precursor to different substances, in particular isobaric and / or isomeric substances. There can however also be different reasons for having ion images with low correlation. For example, when one of two compared ion images (with the same spatial distribution) has a very low intensity. These effects having different origin can also be taken account of in the evaluation and use of the observed correlations for the attribution.

[0063] A (relatively) high correlation between the fragment signals or a fragment signal and a precursor signal is preferably used to attribute those fragments or that fragment and the corresponding precursor to the same substances, in particular isobaric and / or isomeric substances.

[0064] When talking about a low correlation and a high correlation this is to be seen in particular in a relative sense and not so much in absolute numbers; so normally one can identify two or more groups of signals, one with a correlation higher than of the other group, and these relative differences are meant when talking about (relatively) low correlations and (relatively) high correlations.

[0065] A medium correlation between the fragment signals or a fragment signal and precursor signal is preferably used to identify these fragments to be discriminative, in particular between isobaric and / or isomeric substances.

[0066] 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.

[0067] Said method preferably involves fragmenting the precursor ions transmitted through said mass filter to generate fragment ions.

[0068] The ionization from the sample surface is preferably taking place in the form of matrix- assisted laser desorption / ionization or in the form of desorption electrospray ionization.

[0069] Said trapped ion mobility spectrometry (TIMS) separator is preferably a TIMS analyzer with parallel accumulation and separation, in which the precursor ions are accumulated in an accumulation section 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 a 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 threshold value.

[0070] 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 may 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 measurement.

[0071] Carrying out said mass spectroscopy measurement on said fragment ions is preferably implemented by simultaneously detecting and quantifying two or more fragment ions resulting from fragmentation of a common precursor ion, wherein preferably the fragment ions, which include different ion species having various m / z's, are delivered to an orbital electrostatic trap (Orbitrap) mass analyzer or a time-of-f light mass analyzer or an FT- ICR / MRMS mass analyzer, which mass analyzes the fragment ions to generate a mass spectrum representing the individual abundances (intensities) of each of a plurality of fragment ion species.

[0072] According to yet another aspect of the present invention, it relates to the use of the method for the image analysis of a surface with a complex sample distribution, including biological samples such as tissue samples, in particular for spatially resolved lipid analysis, in particular for the discrimination of isobaric and / or isomeric substances and / or for the detection of in source fragmentation.

[0073] According to another aspect of the present invention, it relates to an apparatus for mass spectrometry imaging with ion mobility and mass spectroscopy analysis, for carrying out the method as described above, said apparatus preferably comprising: a unit for moving the sample surface relative to the ionization unit for the generation of said image pixels, an ionization unit locally ionizing substances of a sample surface as precursors from said sample surface, an ion mobility separator (IMS) in the form of at least one trapped ion mobility spectrometry (TIMS) separator for receiving and sequentially releasing precursor ions 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, preferably using PRM, 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 controlling said unit for moving the sample surface relative to the ionization unit configured and, in the same or a different control system, to control said IMS and said mass filter in a synchronized manner taking account of said priority list 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, 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 are adjacent or 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.

[0074] The trapped ion mobility spectrometry (TIMS) separator is preferably a TIMS analyzer with parallel accumulation and separation, in particular operating using a method comprising the steps:

[0075] (a) accumulating ions from the first ion mobility separator in an RF ion trap;

[0076] (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;

[0077] (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

[0078] (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.

[0079] Further embodiments of the invention are laid down in the dependent claims.

[0080] BRIEF DESCRIPTION OF THE DRAWINGS

[0081] 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,

[0082] Fig. 1 shows an overview over the current methods for molecular identification in MSI and the corresponding advantages / disadvantages;

[0083] Fig. 2 shows a schematic representation of the iprm-PASEF workflow as a targeted, multiplexed MALDI-MS / MS imaging method;

[0084] Fig. 3 in a) shows the principle of parallel reaction monitoring and in b) shows the principle of parallel accumulation serial fragmentation;

[0085] Fig. 4 shows an overview over the full iprm-PASEF workflow, wherein in the top row the acquisition of CCS-enabled MS1 data is shown to generate a list with target precursors and in the lower row the acquisition in iprm-PASEF mode of MS2 data and the annotation of the target precursors is shown;

[0086] Fig. 5 shows exemplary CCS-enabled MALDI imaging of lipids in the first quadrant of a sample areas (or pixel) in a) and in a second quadrant of the same sample areas (or pixel) in b) and fragment ion images of the annotated precursors in c);

[0087] Fig. 6 shows in a) how two isomeric compounds cannot be separated in an MS1 spectrum and produce a chimeric MS2 spectrum in b) how two isobaric compounds cannot be separated in an MS1 spectrum and produce a chimeric MS2 spectrum;

[0088] Fig. 7 shows a schematic overview of the results of an iprm-PASEF acquisition of a pure compound, wherein in a) from left to right a spectrum after detection of the precursor ion in Tl MS-enabled MS1 is shown, isolation of the precursor in mass and mobility dimensions and fragmentation of mass-mobility-isolated precursor ion and in b) shows a schematic overview of the sample surface analyzed using MS imaging and MS / MS imaging, in c) precursor ion after MS imaging and in d) a selected fragment after MS / MS imaging, showing strong correlation;

[0089] Fig. 8 shows a schematic overview of the results of an iprm-PASEF acquisition of an isomeric set of compounds, wherein in a) (left-to-right) spectra after detection of the precursor ion in TIMS-enabled MS1 , isolation of the precursor in mass and mobility dimensions, and fragmentation of mass-mobility-isolated precursor ion are shown, b) shows a schematic overview of a sample surface analyzed using MS imaging and MS / MS imaging; c) shows the precursor ion image after MS imaging and how it is effectively a sum of the two diagnostic fragment images after MS / MS imaging (d), the fragment images will have low correlation values when compared in a correlation analysis;

[0090] Fig. 9 shows in a) a schematic representation of (left-to-right) in-source fragmentation (ISF) results in TIMS-enabled MS1 data; isolation and fragmentation of the suspected ISF precursor, b) shows a schematic overview of a sample surface analyzed using MS imaging and MS / MS imaging; c) shows the image of the intact analyte, and d) suspected ISF product, which likely have a strong correlation; in the MS / MS Imaging experiment, the MS / MS fragment with identical m / z to suspected ISF product (e), shall also have a high correlation to both images in (c) and (d), and can be used to confirm the ISF origin of the peak visualized in (d);

[0091] Fig. 10 gives another exemplary representation of principles underlying the present invention, wherein in a) the MS1 image from a feature showing two distinct pixel populations is given, in b) the situation if the corresponding structure is the result of a single molecular species so if there is no isobaric or isomeric molecule involved, and in c) the situation if the distribution is the result of an isobaric or isomeric superposition;

[0092] Fig. 11 in a) shows two isobaric species detected in a CCS-enabled MS1 experiment; these ions are inseparable in an MS2 experiment, and will result in a chimeric MS2 spectrum; their corresponding MS1 ion images are shown in b), c) (second group) and d) (first group); the mass-mobility heatmap display after iprm-PASEF MALDI-MS / MS Imaging shows multiple fragment series in the single isolation window; the two series, displayed in f) (second group) and g) (first group) produce fragment ion images with similar spatial distributions, as evidenced by their Pearson correlation scores, h) and i) show two ion correlation plots or maps, wherein in h) negative correlation between two fragment ion images (2 and 5 in e) specific for the two different isobars, and in i) positive correlation between two fragment ion images (2 and 3 in e) obtained from the fragmentation of the same isobar

[0093] Fig. 12 further illustrates the differences of the proposed approach compared with prior art approaches, in particular the SIMSEF protocol, wherein (A) shows a schematic illustration of the SIMSEF approach, (B) a Cartesian grid overlaid over the surface for the MS1 scan, (C) MS imaging survey scan result of the surface showing two distinct populations with a high intensity, (D) a schematic overview over Cartesian grid overlaid over the tissue for MS / MS imaging using iprm-PASEF according to the invention, (E) MS / MS imaging results, (F) a schematic overview of the acquisition positions for the profiling method and (G) two mobility resolved MS / MS spectra obtained.

[0094] DESCRIPTION OF PREFERRED EMBODIMENTS

[0095] Fig. 1 is an overview over the most common protocols for molecular identification in mass spectroscopy imaging.

[0096] Top left the database matching approach is illustrated. Molecular identification is accomplished by matching mass, isotope pattern and ion mobility MALDI imaging data to an in silico database, potentially constructed using molecular extraction and LC-MS / MS analysis of a comparable sample. The advantage is comparably little effort but at the cost of low confidence of identification.

[0097] Top right the manual MALDI-MS / MS profiling approach is shown. In this approach hotspots are identified, and for each individual precursor MALDI-MS / MS is manually targeted on these hotspots for data analysis. The advantage is fragmentation-based identification at the cost of low throughput and lack of fragment ion images.

[0098] Single-precursor MALDI MS / MS imaging is illustrated on the bottom left. Here tissues are prepared and MALDI MS / MS is carried out on these using targeted MALDI imaging. The advantage is that fragment ion images are provided, at the cost of low throughput.

[0099] On the bottom right automated MALDI MS / MS profiling is illustrated, which involves automated pixel detection, provision of automated target lists and corresponding MALDI MS / MS profiling. The advantage is high coverage at the cost of again no fragment ion images being available.

[0100] Fig. 2 illustrates the protocol used for iprm-PASEF, the main protocol under which the present invention can be implemented. This is a full workflow solution to acquire, analyze and annotate and which allows to analyze up to 25 precursors or even more in a single acquisition. Furthermore, the approach generates fragment ion images (MS2) and not only precursor images (MS1).

[0101] As further advantage not only the two mass dimensions precursor (MS1) and fragment ions (MS2) are provided, but also collision cross-section (1 / K0, CCS) is added as a further dimension, allowing for the above-mentioned large number of precursors open to analysis and identification as well as quantification (at least relative quantification) and spatial location.

[0102] CCS-enabled MALDI imaging is carried out as broadband analysis and enables selecting precursors in the m / z (MS1) and collision cross-section two-dimensional plane by using TIMS technology. The selected precursors are analyzed using a parallel reaction monitoring PASEF protocol for fragmentation and MS2 analysis for each pixel, in a so-called iprm- PASEF approach. The corresponding fragment ions in the m / z (MS2) and collision crosssection (CCS) two-dimensional plane can be individually identified, used for analysis and visualized as a function of their location.

[0103] Fig. 3 in a) more generally illustrates the PRM approach underlying the above-mentioned protocol. In this parallel reaction monitoring approach, a targeted ion population is isolated from a larger ion population, generated by ionization, in a first quadrupole, the corresponding isolated population is fragmented in a collision cell and the resulting fragments are analyzed in parallel using a high mass resolution device, for example an reflectron time-of-flight device or an Orbitrap device.

[0104] Fig. 3 in b) specifically illustrates the parallel accumulation serial fragmentation (PASEF) approach combined with the above-mentioned PRM approach. An ion population generated by ionization is first separated by ion mobility (CCS dimension) in a TIMS device, involving parallel accumulation and allowing for sequential release and separation as a function of time. The sequential release from the corresponding TIMS device is scheduled in a targeted and synchronized way with the first quadrupole (MS1) to switch isolation masses, so that from a single acquisition multiple precursors can be sequentially released, isolated in MS1 in the first quadrupole, subsequently fragmented and the resulting multiple fragment ions for each precursor be detected in the high-resolution time-of-flight mass analyzer using the PRM approach.

[0105] In a corresponding imaging experiment, such a single acquisition (or a series for one pixel) corresponds to a single pixel, which means that one can record mobility separated fragmentation spectra for the specified set of precursors, in each pixel of an imaging acquisition, allowing to visualize the fragment ion intensities distributions over the measured sample surface.

[0106] This allows, in a highly multiplexed and highly sensitive approach, for each pixel, to generate a multidimensional data matrix opening up new possibilities for data analysis, i.e. visualization, identification, quantification, separation as well as a combination thereof.

[0107] So, as further illustrated in Fig. 4, the proposed protocol starts off with acquiring a regular CCS-enabled MALDI imaging data set only involving MS1 analysis and information, which is used to find and select appropriate fragmentation precursors. The precursors can be exported into a corresponding software for feature finding and selecting relevant fragmentation precursors (if desired recurring to databases or simulation data or both), for the generation of an export precursor list, which contains precursor details such as m / z centroids, 1 / K0 ranges, etc.

[0108] This precursor list can be used for setting up the corresponding parameters for the iprm- PASEF method and for scheduling the corresponding second-round imaging measurement on the sample, now including the full protocol with PASEF, fragmentation and PRM fragment ion measurement.

[0109] Based on the resulting fragment information rule-based annotation is possible or target list annotation with a spectral library. The protocol can for example be used for lipid annotation. Fig. 5 a) gives an experimental example of the corresponding iprm-PASEF approach. Represented on top is the MS1 spectrum of a survey scan without spatial resolution, in the bottom the heat maps without fragmentation for some of the peaks are given. The figure illustrates CCS-enabled MALDI imaging of lipids on a sagittal rat brain section. A smartbeam 3D laser is used to precisely define the position and ablation area for each pixel. For the shown data, the pixel size was 40 by 40 micrometers, but the ablation area was only set to 20 by 20 micrometers, allowing reanalysis of the same tissue for complementary datasets, by offsetting the position of the ablation area for the consecutive measurements. Here for example, the first sub-pixel was used to generate the CCS-enabled lipid MALDI imaging MS1 dataset from a sagittal rat brain section. The T-ReX algorithm was used to find the top 200 most intense mass mobility features, and that was filtered down to a selection of fragmentation precursors.

[0110] As illustrated on the bottom, for each selected precursor in the corresponding CCS / MS1 plane a two-dimensional spatial image can be generated after measurement of all the pixels.

[0111] Fig. 5 b) shows how the iprm-PASEF dataset from the second subpixel is analyzed, for one selected precursor, so one isolation window which means for one controlled mass window and one ion mobility window, resulting in the illustrated mass mobility heatmap. Here one can see the precursor isolation windows, defined by the precursor mobility interval, and the quadrupole m / z isolation width, which here was set to 1 m / z. Feature finding was then employed to pick features in the mass-mobility space. Software then automatically characterizes these features as either precursors or fragments. Precursor features are those found within the mass and mobility bounds of an isolation window, and the fragment features are those found within the mobility bounds of an isolation window, but outside the quadrupole m / z isolation window. Software can be used to compute a fragmentation spectrum, connected to each of the detected precursors in the isolation window. Then a molecular annotation workflow was used to identify the precursors. To do so a rule-based lipid species annotation tool was used, which is able to interpret lipid MS / MS spectra. And this informed that the most likely identity for this particular was a phosphatidylethanolamine with 18:0 and a 20:4 acyl chains.

[0112] Finally, as illustrated in Fig. 5 c) one can measure and study the fragment ion images of the annotated precursors, and compare their spatial distributions. Also, one can compare the fragment ion distributions to that of the original precursor in the MS1 dataset (given to the very right). For this particular lipid one can see that the headgroup fragment had a poor distribution, due to it being a very low intensity feature, but the two acyl chains clearly show a similar spatial distribution to the MS1 precursor.

[0113] Fig. 6 illustrates, independent of the spatial imaging dimension, the inherent problems associated with discriminating between isomeric compounds and isobaric compounds or molecules.

[0114] As illustrated in a), two isomeric compounds cannot be separated in an MS1 spectrum, and isolation of the relevant m / z window (illustrated by the bar on the bottom of the peak in the middle representation) before fragmentation in the first quadrupole will result in a chimeric fragment ion spectrum, which however will (in most cases) include one or more fragment signals which are characteristic of only one of the isomeric compounds. Nevertheless, the MS2 fragment spectrum is a superposition of both isomeric molecules. The same is equally possible if there are more than two isomeric precursor molecules.

[0115] As illustrated in b), two isobaric compounds, if their mass difference is smaller than the resolution in the MS1 dimension, also cannot be fully separated. Equally here, isolation of the relevant m / z window will result in a mix of the two compounds and subsequent fragmentation will result in a chimeric spectrum being a superposition of the fragments of both isobaric compounds or molecules. Again (in most cases) the superposition will however include one or more discriminative fragment signals characteristic of only one of the isobaric compounds.

[0116] Fig. 7 gives a schematic overview of the results of an iprm-PASEF acquisition of a pure compound. In a), left-to-right, spectra after detection of the single precursor ion in TIMS- enabled MS1 , isolation of the precursor in mass and mobility dimensions (middle, bright grey), and fragmentation of mass-mobility-isolated precursor ion (right, including the bright grey unfragmented precursor as well as a dark grey fragment, b) shows a schematic overview of a sample surface analyzed using MS imaging and MS / MS imaging with two different molecules in different spatial regions, the left one the selected as the above- mentioned single compound for measurement. The selected precursor ion after MS1 imaging is shown in c) (bright grey) and a selected fragment (dark grey) after MS / MS imaging is shown in d) which show strong colocalization and correlation.

[0117] Fig. 8 gives a schematic overview of the results of an iprm-PASEF acquisition of an isomeric set of compounds. In a), left-to-right, spectra after detection of the selected isomeric precursor ion pair in TIMS-enabled MS1 is shown, isolation of the precursor ion in mass and mobility dimensions in the middle, and fragmentation of mass-mobility-isolated precursor ions on the left, b) shows a schematic overview of a sample surface analyzed using MS imaging and MS / MS imaging. The precursor ion image after MS imaging in c) is effectively a sum of the two diagnostic discriminative fragment images (bright grey, left; dark grey, right) after MS / MS imaging as can be seen in d), showing the corresponding spatial distribution of the two characteristic and discriminative fragment ions as also illustrated in the rightmost spectrum in a). The fragment images will have low correlation values when compared in a correlation analysis.

[0118] An analogous behavior is seen for two isobaric molecules.

[0119] Fig. 9 gives a schematic representation of a), left-to-right, in-source fragmentation results in TIMS-enabled MS1 data (left), isolation (middle) and fragmentation (right)of the suspected ISF precursor. b) shows a schematic overview of a sample surface analyzed using MS imaging and MS / MS imaging. The image of the intact analyte is given in c), and the one of a suspected ISF product in d), and these will likely have a strong correlation. In the MS / MS Imaging experiment, the MS / MS fragment with identical m / z to suspected ISF product as given in e) will also have a high correlation to both images in c) and d), and can be used to confirm the ISF origin of the peak visualized in d).

[0120] Fig. 10 in a) gives a schematic representation of an MS1 ion image from a mass-mobility feature showing two distinct pixel populations with a high intensity.

[0121] As illustrated in b), in case the isolated and fragmented mass-mobility feature is the result of a single molecular species, all fragments will have very similar or even identical spatial distributions, which are in turn highly similar to the original MS1 ion image.

[0122] Image correlation analysis of all fragment ion images, including the MS1 ion image will produce high correlation scores.

[0123] As illustrated in c), in case of isomeric or isobaric precursors being located in separate areas, isolation and fragmentation of the selected mass-mobility feature results in two (or more) fragment groups (here i: A+C and ii: B+D). These groups are the result of discriminatingly fragmenting two (or more) isomeric or isobaric ions. The ion images within one fragment group (dark grey OR bright grey) resulting from the same isomer or isobar will have a high spatial similarity (in other words, their ion images are similar) and as such these images will have high correlation scores in image correlation analysis. Correlation analysis on ion images from the different fragment groups resulting from different isomers or isobars will also yield low correlation scores, since the images are complementary. In relation to the MS1 ion image (precursor given in black in the MS2 representation on top), both groups will have intermediate correlation scores, since the ion images are only partly similar.

[0124] Using an experimental data set, this was verified as follows:

[0125] Two or more ion images were each converted into a one-dimensional vector (signal intensity vs. pixel index).

[0126] The data can be represented in a correlation map, examples of such correlation maps are given in Fig. 11 h) and i) discussed further below.

[0127] The vectors were subjected to a Pearson correlation (the ratio between the covariance of two vectors and the product of their standard deviations, as described by McDonnell et al. in J. Proteome Res 7, 3619 (2008), see in particular last row of Scheme 1 thereof), using the following correlation coefficient R determined from the covariance C of vectors A and B of the images (each image first having been converted to a one-dimensional vector of intensity as a function of pixel number):

[0128] The resulting ratio (R) has a range between -1 and 1. A ratio of 1 represents a perfect correlation, a ratio of -1 represents anti-correlation, and a value of 0 represents a lack of correlation.

[0129] Instead of the signed ratio R, the squared ratio R2is often used, which ranges between 0- 1.

[0130] The implementation used here relies on such Pearson correlation (described above). There are alternatives to this correlation algorithm (e.g. Spearman correlation, Kendall’ tau correlation), which could be used but have not been explored here.

[0131] Fig. 11 shows the measurements and the analysis results. In a) it shows two isobaric species detected in a CCS-enabled MS1 experiment. These ions are inseparable in an MS2 experiment, and result in a chimeric MS2 spectrum. Their corresponding MS1 ion images are shown in b), c) and d). The mass-mobility heatmap display after iprm-PASEF MALDI- MS / MS Imaging shows multiple fragment series in the single isolation window. The two series, displayed in f) and g) produce fragment ion images with similar spatial distributions, as evidenced by their Pearson correlation scores R given in the table below as a function the m / z values:

[0132] The negative correlation values indicate anti-correlation between the two series, and the positive correlation values indicate correlation within the two series of ion images. The relatively high correlation between the first group of signals at 744.6035, 868.5991 and 927.6734 (4-6 in Fig, 11 e), and of the second group of signals at 744.6327, 868.6172 and 927.7004 (1-3 in Fig, 11 e), in combination with the low inter-group correlations allows to associate the respective signals to the individual systems forming the chimeric twin inseparable in MS2.

[0133] Fig. 11 h) and i) illustrate the above-mentioned correlation maps for the situation wherein in h) the fragment peak at 868.6172 (2 in e) is correlated with the fragment peak at 868.5991 (5 in e), showing a low and negative correlation, which is also given in the above table, to be -0.1191.

[0134] This indicates that these two fragments are of different origin so result from different precursors, the two fragments are therefore discriminative for example for two isobaric precursors which in MS1 cannot be distinguished.

[0135] In i) the fragment peak at 927.7004 (3 in e) is correlated with the fragment peak at 868.6172 (2 in e), showing a high positive correlation, which is also given in the above table, to be 0.3371. These two fragments therefore stem from the same precursor and are not discriminative. Fig. 12 serves to illustrate the differences between the prior art approaches as e.g. disclosed in the Heuckeroth et al publication and the proposed approach.

[0136] Fig. 12 A shows a schematic overview of a sample surface analyzed using MS imaging, MS / MS imaging (iprm-PASEF) and MS / MS profiling (SIMSEF) according to the prior art. No systematic measurement of the full surface in a grid is carried out in the SIMSEF approach but only hotspots are measured, or areas around hotspots. There is no actual systematic grid of MS / MS data pixels allowing for correlation or suggesting correlation.

[0137] Fig. 12 B illustrates how a Cartesian grid can be overlaid over the tissue surface and used for MS1 imaging acquisition.

[0138] Fig. 12 C illustrates an MS imaging (MS1) ion survey scan image from mass-mobility features showing two distinct pixel populations with a high intensity, and Fig. 12 D illustrates how the Cartesian grid can be overlaid over such a tissue surface and used for MS / MS imaging (iprm-PASEF) acquisition.

[0139] Fig. 12 E shows the MS / MS imaging, resulting in a MS / MS spectrum containing multiple fragments (shown as average mass-mobility heatmap representing the entire region acquired by MS / MS imaging). Upon visualizing the distributions of the resulting fragment ions two sets of fragment ions with distinctly different spatial distributions become apparent (ions 1+3 and 2+4), which confirms the presence of isomeric or isobaric precursor contributions. By calculating the Pearson correlation coefficients between the individual fragment ion images, the subsets of fragment images can be statistically confirmed (i.e. the correlation coefficient between ions within the same subset (e.g. 1 & 3 or 2 & 4) will be high, whereas the correlation coefficient of ions different subsets will be low (e.g. 1 & 2, 1 & 4 or 3 & 4). Only by using the systematic MS / MS imaging approach, where the MS / MS sampling involves systematically acquiring pixels following a Cartesian grid, the method described in the present invention allows unambiguously identifying specific isomeric or isobaric contributions.

[0140] Fig. 12 F shows a schematic overview of the acquisition positions for the MS / MS profiling method as proposed here. Isomeric contributions can only be detected if the analyst / user decides to include two sample positions based on the MS1 ion image or if the isomeric ions share the same positions. With three or more MS / MS profile spectra calculating correlations would theoretically be possible, however, due to a lack of systematic acquisition and the limited number of spectra, the statistics derived from this approach would have limited power.

[0141] Fig. 12 G shows two mobility resolved MS / MS spectra obtained from the MS / MS profiling acquisition as proposed. Note that the mobility resolved MS / MS spectra here exclusively represent the precursor and fragments analyzed at the indicated sampling positions in Fig. 12 F. In comparison, the MS / MS imaging results in Fig 12 E display the MS / MS results as average mass-mobility heatmap that captures the entire region acquired in the MS / MS imaging analysis. The exact spectral data from the MS / MS profiling analysis (Fig. 12 F) can be extracted from the MS / MS imaging data. It is however impossible to extract the spatial distribution of the fragment ions from the MS / MS profiling data according to the SIMSEF approach.

[0142] LIST OF REFERENCE SIGNS

[0143] CCS collision cross section

[0144] DDA data dependent acquisition

[0145] DESI desorption electrospray ionization

[0146] DIA data independent acquisition

[0147] EIC extracted ion chromatogram

[0148] EIM extracted-ion mobilogram

[0149] MALDI matrix-assisted laser desorption / ionization

[0150] MSI mass spectrometry imaging

[0151] RT retention time m / z mass-to-charge ratio

[0152] 1 / K0 (inverse) ion mobility value

[0153] CCS collision cross section

[0154] PASEF parallel accumulation serial fragmentation

[0155] SIMSEF spatial ion mobility-scheduled exhaustive fragmentation TIMS trapped ion mobility spectrometry

Claims

CLAIMS1 . A method for the combined identification and localization of substances of a sample surface, the method comprising: acquiring a mass spectrometric image of said substances of the sample with a mass spectrometer having a built-in ion mobility spectrometer, and generating mass / mobility maps of masses and ion mobility values for sample ions for each pixel of the image; wherein for each pixel for the generation of said mass / mobility maps of masses and ion mobility values the method comprises the following steps: introducing precursor ions resulting from ionization from the sample surface into at least one trapped ion mobility spectrometry (TIMS) separator, and separating the precursor ions according to mobility in the trapped ion mobility spectrometry (TIMS) separator, sequentially releasing precursor ions from said trapped ion mobility spectrometry (TIMS) separator according to their ion mobility for precursor ions falling within an ion mobility window, introducing said released precursor ions into a mass filter which selectively transmits precursor ions having m / z values falling within a controlled 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 optionally associating detected fragments with its corresponding precursor ion, wherein said trapped ion mobility spectrometry (TIMS) separator and said mass filter are scheduled 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, wherein for the identification of substances at least three data points for given spatial pixel coordinates of at least two fragments or at least one fragment and a precursor are correlated in a correlation map.

2. The method according to claim 1 , wherein at least three data points for given spatial pixel coordinates of at least two fragments or at least one fragment and a precursorare correlated in a correlation map, in which the intensity of a respective data point of given spatial pixel coordinates of one fragment is given along a first axis and the intensity of the respective data point of the same given spatial pixel coordinates of the other fragment or the precursor is given along a second axis.

3. The method according to claim 1 , wherein said correlation is used to separate and / or identify isobaric and / or isomeric substances.

4. The method according to any of the preceding claims, wherein the correlation is used to identify in source fragmentation resulting from ionization from the sample surface.

5. The method according to any of the preceding claims, wherein for the determination of said controlled mass window and of said ion mobility window for selecting a chosen precursor, in a preceding step the following steps are carried out: acquiring a mass spectrometric image of said substances of the sample with said mass spectrometer having a built-in ion mobility spectrometer, and generating mass / mobility maps of masses and ion mobility values for sample ions for each pixel of the image; wherein for each pixel for the generation of said mass / mobility maps of masses and ion mobility values the method comprises the following steps: introducing precursor ions resulting from ionization from the sample surface into at least one trapped ion mobility spectrometry (TIMS) separator, and separating the precursor ions according to mobility in the trapped ion mobility spectrometry (TIMS) separator, sequentially releasing precursor ions from said trapped ion mobility spectrometry (TIMS) 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, carrying out a mass spectroscopy measurement on said precursor ions.

6. The method according to any of the preceding claims, wherein for the separation and / or identification of isobaric and / or isomeric substances and / or for the identification of fragments resulting from in-source fragmentation a low correlation between two fragments or a fragment and a precursor are used to identify discriminating fragments and for separating and / or identifying isobaric and / or isomeric substances and / or in-source ionization, and / ora high correlation of two fragments or a fragment and a precursor are used to identify fragments common to one single substance for separating and / or identifying isobaric and / or isomeric substances.

7. The method according to any of the preceding claims, wherein the ionization from the sample surface is taking place in the form of matrix-assisted laser desorption / ionization or in the form of desorption electrospray ionization.

8. 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.

9. Method according to any of the preceding claims, wherein said trapped ion mobility spectrometry (TIMS) separator is a TIMS analyzer with parallel accumulation and separation, in which the precursor ions are accumulated in an accumulation section 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 a 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 threshold value.

10. 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 them / 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 measurement.11 . Method according to any of the preceding claims, wherein carrying out said mass spectroscopy measurement on said fragment ions is implemented by simultaneously detecting and quantifying two or more fragment ions resulting from fragmentation of a common precursor ion, wherein preferably the fragment ions, which include different ion species having various m / z's, are preferably delivered to an orbital electrostatic trap (Orbitrap) mass analyzer or a time-of-f light mass analyzer or an FT-ICR / MRMS mass analyzer, which mass analyzes the fragment ions to generate a mass spectrum representing the individual abundances (intensities) of each of a plurality of fragment ion species.

12. Use of the method of any of the preceding claims for the image analysis of a surface with a complex sample distribution, including biological samples such as tissue samples, in particular for spatially resolved lipid analysis.

13. Use according to claim 12 for the discrimination of isobaric and / or isomeric substances and / or for the detection of in source fragmentation.

14. An apparatus for mass spectrometry imaging with ion mobility and mass spectroscopy analysis, for carrying out the method according to any of the preceding claims, said apparatus preferably comprising: a unit for moving the sample surface relative to the ionization unit for the generation of said image pixels, an ionization unit locally ionizing substances of a sample surface as precursors from said sample surface, an ion mobility separator (IMS) in the form of at least one trapped ion mobility spectrometry (TIMS) separator for receiving and sequentially releasing precursor ions 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, preferably using PRM, 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 controlling said unit for moving the sample surface relative to the ionization unit configured and, in the same or a different control system, to control said IMS and said mass filter in a synchronized manner taking account of said priority list 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, 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 are adjacent or 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.

15. An apparatus according to claim 14, wherein the trapped ion mobility spectrometry (TIMS) separator 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; 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.

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