Combined DIA DDA approach for LC ms / ms experiments

Abstract

A method of liquid chromatography (LC) coupled combined ion mobility and mass spectroscopy analysis, wherein at the beginning of one LC observation retention time cycle, preferably in the range of 0.5-4 seconds, particularly preferably in the range of 0.8-3 seconds, a survey scan (MS1) is taken, wherein in that survey scan a full ion mobility width of interest and a full m / z width of interest is scanned without fragmentation, wherein this survey scan is followed by at least one DIA frame, preferably at least 2 or at least 3 DIA frames, followed by at least two DDA frames, preferably in the range of 3-15 DDA frames or in the range of 5-10 DDA frames. According to the proposed approach for the scheduling of the DDA frames, precursors are selected using a combined analysis on the basis of peak picking of precursors identified in the survey scan, as well as on the basis of peak picking of precursors and / or fragments identified in the at least one DIA frame, preferably in addition using advanced knowledge, from databases and / or in silico sources, for that combined analysis, wherein preferably fragments identified in the at least one DIA frame are used for selecting to be scheduled precursors identified from the survey scan.

Description

[0001] F07395 15.1.2026

[0002] 1

[0003] TITLE COMBINED DIA DDA APPROACH FOR LC MS / MS EXPERIMENTS

[0004] TECHNICAL FIELD

[0005] The present invention relates to methods as well as devices for LC MS / MS experiments, in particular for the analysis of complex mixtures, especially mixtures of compounds of biological origin which e.g. vary widely in abundance. In particular it relates to trapped ion mobility spectrometry with optimized mobility window filtering, accumulation, and mobilitymass analysis and optimized scheduling.

[0006] PRIOR ART

[0007] The motion of molecular ions in gases as compared to their motion in a condensed phase has established ion mobility as a powerful separation tool, especially in conjunction with mass spectrometry. Ion mobility spectrometry (IMS), such as drift tube ion mobility spectrometry (DTIMS), travelling wave ion mobility spectrometry (TWIMS), and differential mobility spectrometry (DMS), also known as field asymmetric ion mobility spectrometry (FAIMS), act as a post-ionization method for gas-phase filtering of ions in a controlled atmosphere, fractionating complex samples, suppressing chemical noise, separating isobaric mixtures, and characterizing molecular ions based on their mobility coefficients. These are methods that separate gas phase ions based on their interaction with a collision gas and their masses. In the first step, the ions are separated according to their mobility through a buffer gas on a millisecond timescale using an ion mobility spectrometer. The separated ions are then introduced into a mass analyzer in a second step where their mass-to-charge ratios can be determined on a microsecond timescale. The effective separation of analytes achieved with this method makes it widely applicable in the analysis of complex samples such as in proteomics and metabolomics.

[0008] 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 sequenceF07395 15.1.2026

[0009] 2

[0010] of their mobility.

[0011] 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.

[0012] A TIMS analyzer with parallel ion accumulation is described in US 9,683,964; it improves the utilization of the ions from the ion source to nearly 100%. Importantly, many ions sources, such as electrospray ion sources produce ions continuously. TIMS with parallel accumulation can also be operated to collect and analyze ions continuously - that is, TIMS can operate at 100% duty cycle. Barring pseudopotential or space charge effects, substantially all ions of the ion source are collected and analyzed without loss. TIMS with parallel ion accumulation further provides the unique possibility to prolong the ion accumulation duration to find more detectable ion species, thereby even increasing the ion mobility resolution by a corresponding prolongation of the scan time. The ions are collected in an accumulator unit, preferably almost identical to the scanning unit, at a ramp of an electric DC field barrier such that they get spatially separated by their ion mobility along the ramp. Therefore, the accumulated ions are less influenced by space charge than in other types of accumulator units. Of greatest importance, however, is the unique feature of a TIMS analyzer that a longer accumulation period permits to increase the mobility resolution by choosing correspondingly longer mobility scan durations, e.g. 100 milliseconds scan duration with an ion mobility resolution of RmOb = 75 instead of 20 milliseconds scan duration with Rmob = 30. As a consequence of the higher number of ions collected and the better ion mobility resolution, more ion species can be detected and measured. Once an ion mobility scan is completed (optionally after twenty to some hundred milliseconds), the accumulated ions are transferred (in about a millisecond) from the accumulation unit to the scanning unit, and the next ion mobility scan can be started. In total, a skilled practitioner will appreciate that it will be possible to achieve a measurement rate of 300 to 450 ion species per second. If TIMS with parallel ion accumulation is installed in tandem mass spectrometer (MS / MS instrument) an MS-MS instrument, 300 to 450 characteristic fragment ion spectra perF07395 15.1.2026

[0013] 3

[0014] second may be measured quantitatively.

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

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

[0017] Targeted selection of peptides from complex protein digests subjected to fragmentation is a performance-limiting factor in tandem mass spectrometry (MS). The problem is mainly attributed to the large number of co-eluting high abundance peptides and limitations in the duty cycle of the mass spectrometer arranged to operate in data-dependent acquisition (DDA) mode whereby precursor ions are selected sequentially for fragmentation in order of decreasing intensity. Furthermore, the depth of proteomic analyses is also limited by the overwhelming proportion of high abundance background ions that compromise the identification and quantification of low abundance peptides. Whether low-intensity precursors are selected for fragmentation is primarily dictated by the speed at which the mass analyzer can perform tandem MS analysis. The dynamic range of the analysis in DDA mode can be extended by excluding previously analyzed precursor ions, but this strategyF07395 15.1.2026

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[0019] is no longer efficient for highly complex samples with an extended dynamic range in concentration. The duty cycle of the MS remains a problem even in data-independent acquisition (DIA) mode where all ions within a selected mass-to-charge range are selected for fragmentation. The issue of sample complexity is routinely addressed by fractionating the sample.

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

[0021] Another variant of measurement is data independent acquisition (DIA). Contrary to MRM or PRM, this approach does not require any preliminary method design prior to the sample injection. Since the LC-MS acquisition covers the complete analyte contents of a sample through the entire mass and retention time (RT) ranges the data can be mined a posteriori for any peptide / precursor of interest. Data is acquired in a data independent manner, on the complete mass range (e.g. 200-2000 Thomson) and through the entire chromatography, disregarding of the content of the sample. This is commonly achieved by stepping the wide selection window of the mass analyzer step by step through the complete mass range. In effect, this data acquisition method generates a complete fragment ion map for all the analytes present in the sample and relates the fragment ion spectra back to the precursor ion selection window in which the fragment ion spectra were acquired. This is achieved by widening the precursor isolation windows on the mass analyzer and thus accounting a priori for multiple precursors co-eluting and concomitantly participating to the fragmentation pattern recorded during the analysis. Such a precursor window is called a precursor selection window. The result is complex fragment ion spectra from multiple precursor fragmentations, that require a more challenging data analysis.

[0022] SUMMARY OF THE INVENTION

[0023] Usually DDA experiments are based only on MS-1 signals. This is severely limiting the information available from an analysis. Having additional, maybe low quality, informationF07395 15.1.2026

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[0025] about the fragments from a large variety of precursors of potential interest can be be helpful to make better decisions about the fragmentation experiments and the scheduling of DDA frames to focus on, especially for cutting edge experiments as in the field of glyoproteomics and cross linking experiments.

[0026] The new approach presented here is therefore to offer a DDA, in particular DDA-PASEF measurement mode, which combines a MS-1 frame (also termed survey scan), a bunch of (diagonal scanning) DIA frames and finally a series of DDA, in particular DDA-PASEF frames. The DIA frames can be measured with a shorter ramp time if that is good enough. The MS-1 and DIA frames get peak picked in real time to allow for a real time decision about the targets for the following DDA frames and can e.g. get offered via interfaces to scripts running either on the acquisition PC or if more demanding on a special box.

[0027] This feature can be offered both as a toolbox for users and for “mainstream” acquisition modes. Thinking about future hardware variants which offer MS-3 capabilities either based on Omnitraps or multi TIMS devices with the option to select precursors prior to the quadrupole it will become interesting to get the full picture for the MS-2 fragment space. As for the precursor selection the targets derived from MS-1 and DIA of cycle n can be used for cycle n but also only or in addition used for cycle n+1 or later in particular if the processing is too time consuming, but that is optional if the time between measuring the MS-1 + DIA frames and the DDA frames should be minimized and a dead time between the DIA and DDA frames is acceptable.

[0028] If the complexity of the sample is low enough a full bbCID (or other fragmentation mechanism) can be used, saving time.

[0029] As with normal DDA experiments the knowledge about prior precursor selections can be used to control the repeated fragmentation of a molecular feature with the same m / z and 1 / K0 value (dynamic exclusion). E.g. it can be allowed to be measured twice within a certain time interval or a second time only if the intensity has increased by a certain factor.

[0030] An additional optional step can be a continuous quality check of the obtained DDA spectra to optimize parameters for the precursor selection, e.g. a threshold for the minimal intensity of a MS-1 precursor. Quality criteria can the number of peaks above some threshold, the presence of expected peak distances between the dominant peaks or application specific criteria which are calculated by a Python script defined by skilled users.

[0031] The processing of the MS-1 and DIA frames and optionally DDA frames used for parameter optimizing can be done in an asynchronous way, started as soon as the data is available to make best use of multicore computers. A peaklist gathering step takes care to collect the results and forward the data to the precursor selection function which is either a hard coded part of the acquisition software (timsControl e.g.) or is completely or partially done by userF07395 15.1.2026

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[0033] specific scripts, typically implemented in the Python language.

[0034] For the peak picking of the MS-1 and DIA frames existing algorithms used already for MS-1 frames for dda-PASEF- or variants being at least prototyped - can be applied. In principle, there can also be an interface for a customer supplied peak picking script to gain further flexibility.

[0035] Examples of such peak picking algorithms can be the search for local maxima after an appropriate smoothing, the use of wavelet transformations or second derivatives to deal with overlapping signals or clustering algorithms which are also appropriate due to the sparsity of the data.

[0036] For the smoothing of the sparse data of timsTOF MS frames, defined by intensities as a function of the time-of-f light index and the number of the mobility scan within the frame, each data point can for example be replaced by the sum of a gaussian kernel functions multiplied by the intensity of the detection events in the neighbourhood. Using a Mexican Hat function instead would give a smoothed second derivative, helpful for the separation of overlapping peaks.

[0037] Experiments where the proposed approach is of particular interest are e.g.:

[0038] • PRM experiments triggering quant measurements by the onset of fragments of interesting targets seen in the DIA frames. For complex samples with large target lists this might be more reliable than just going for MS-1 and a kind of real time RT correction. • For glyco- and cross linking the fragments should be helpful to classify precursors to be selected for DDA-PASEF measurements to get clean spectra which are then easier to interpret than DIA (the arguments for the MIDIA spectrum centric approach).

[0039] • Also for phosphor-proteomics the detection of neutral losses is valuable to filter for MOMA (mobility offset mass aligned) pairs which really stand for different site localizations.

[0040] • Maybe also for MS-3 TMT (tandem mass tag) labelling quantification experiments (in MS-3 first a peptide ion is isolated and fragmented, followed by the selection of a fragment ion that is again isolated and fragmented to produce an MS3 spectrum) with additional fragmentation / isolation capabilities and fine structure analysis of the reporter region of our TOF spectra.

[0041] According to a first aspect of the present invention, it relates to a method of liquid chromatography (LC) coupled combined ion mobility and mass spectroscopy analysis, wherein at the beginning of one LC observation retention time cycle, preferably in the range of 0.5-4 seconds, particularly preferably in the range of 0.8-3 seconds, a survey scan (MS1) is taken, wherein in that survey scan a full ion mobility width of interest and a full m / z width of interest is scanned without fragmentation.

[0042] This survey scan is followed by at least one DIA frame, preferably at least 2 or at least 3F07395 15.1.2026

[0043] 7

[0044] DIA frames.

[0045] These are followed by at least two DDA frames, preferably in the range of 3-15 DDA frames or in the range of 5-10 DDA frames.

[0046] Note that there may be LC observation retention time cycles within one LC run, where one DDA frame will be enough if there are not enough interesting candidates, or there may be observation retention time cycles where no DDA frame is carried out if there is nothing of specific interest in the survey frames and / or DIA frame(s) and nothing else should be fragmented to save time and check for candidates with the next survey scans as soon as possible. However, within at least one LC observation retention time cycle of an LC run (normally a significant proportion of the cycle of an LC run, so e.g. at least one quarter, or at least one third, or at least one half of all cycles) the survey scan is followed by at least one DIA frame, and then followed by at least two DDA frames.

[0047] For the scheduling of the following DDA frames, withing the same LC observation retention time cycle or a following LC observation retention time cycle (typically within one same LC peak), according to the invention precursors are selected using a combined analysis on the basis of peaks (peak picking) of precursors identified in the survey scan, as well as on the basis of peaks (peak picking) of precursors and / or fragments identified in the at least one DIA frame. In addition, using advanced knowledge, from databases and / or in silico sources, for that combined analysis is possible.

[0048] Preferably characteristic fragments in the at least one DIA frame, characteristic fragment patterns in the at least one DIA frame, characteristic differences between fragments in the at least one DIA frame and associated precursors, or combinations thereof are used for selecting to be scheduled precursors identified from the survey scan.

[0049] Definitions:

[0050] 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 absolute charge of the ion (which can be positive or negative depending on the measurement mode; typically and especially for proteomics one is in positive mode, having less electrons than protons in the ion if one goes for the elementary particles of the atoms; molecules are usually getting charged by attaching H+so protons). 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 (If it is the same ion it will be roughly 50.5 because of the additional proton, assumed as the charge carrier: MH+ : (99+1 ) / 1 = 100 Da, MH2A2+: (99+2) / 2 = 50.5 Da. Other formulation: If an ion with a massF07395 15.1.2026

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[0052] of 100 amu 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.

[0053] 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 charge carriers, typically H+for proteomics applications with electrospray ion sources. 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.

[0054] 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.

[0055] 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.

[0056] 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).F07395 15.1.2026

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[0058] 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. A heatmap shows the intensity values of raw signals, peaks or features (e.g. a fragment of a precursor) 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 signal intensity is displayed using color coding and / or equipotential lines.

[0059] 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 typically 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-f light (TOF) mass spectrometer.

[0060] 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 been 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.

[0061] LC observation retention time cycle: is a measurement cycle within a given time span. The measurement cycle comprises or consists of several measurement frames. Such an LC observation retention time cycle is typically part of an LC observation time window for one LC peak (the retention time window during which a certain peptide - or analyte in general - elutes from the chromatographic column and is detectable by the MS instrument). In such an LC observation retention time cycle, after an initial survey scan as a first measurement frame, a series of individual DDA frames, and in accordance with the presentF07395 15.1.2026

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[0063] invention at the beginning at least one DIA frames or at least two or a series of DIA frames, are carried out. This is based on a scheduling scheme which is determined at least partly by the initial survey scan. An LC observation retention time cycle is terminated by starting a next initial survey scan in the next LC observation retention time cycle. Typically, a LC observation retention time cycle has a length in the range of 0.5-4 seconds, preferably in the range of 0.8-3 seconds or 1-1.5 seconds.

[0064] In typical experiments, an LC observation time window covering one LC peak has a duration of about 10 seconds, allowing to place 5-7 LC observation retention time cycles to be taking place in that window to measure what is within that LC peak.

[0065] Within one LC observation retention time cycle typically 5-15 measurement frames are collected, one of which is the above-mentioned survey scan as the initial measurement frame. One measurement frame, whether a survey scan, DIA frame or a DDA frame, typically takes 50-100 ms.

[0066] A frame is forming a data structure, e.g. a data structure which is the result of a tims ramp and the tof scans done during that ramping of the tims cell. A scan can be both the process and the data obtained as the result of this process.

[0067] When a sample is analyzed, the full LC analysis of the sample is called an LC run, and the overall time of such an LC run is split into a large number of such LC observation retention time cycles. The LC observation retention time cycles can be chosen to be always the same within one LC run or can be adapted dynamically in length and / or scheduling, so also the number of DIA and / or DDA frames within such LC observation retention time cycles can be dynamically adapted, this normally as a function of the presence of LC peak(s) and / or signals within such peaks as seen in the survey scans and / or DIA scans.

[0068] Survey scan: also called MS1 frame, is a scan of a full ion mobility range of interest and a full m / z range of interest without fragmentation, just for the identification of the precursors of interest in the respective LC observation retention time cycle. In the figures given below a survey scan is indicated by a box with "MS1".

[0069] The terms “data-dependent” and “data-dependent acquisition” (abbreviated “DDA”) have their art-established meaning (Hu et al. 2016, doi: 10.12688 / f1000research.7042.1.). As such, precursor ions determined and picked in a survey scan in MS1 without fragmentation are collected based on (a) their intensities, whereby normally precursor ions are selected sequentially for fragmentation, and measurement of the associated fragments takes place in the following DDA frames in MS2 in order of decreasing intensity; and / or (b) prior knowledge.

[0070] In one individual DDA frame within one LC observation retention time cycle (in the figures given below one such frame is indicated by a box designated with DDA), one, or in case ofF07395 15.1.2026

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[0072] PASEF a group of precursors is selected for measurement within that DDA frame. While scanning or ramping the ion mobility continuously from a minimum value to a maximum value of the upstream ion mobility separator (e.g. 1 / K0 = 1.4 to 0.6 Vs / cm2), the precursors of that group are sequentially measured. That measurement of the group of precursors takes place in that, when arriving at an ion mobility value of the respective precursor during the IM scan, the mass filter upstream of fragmentation is chosen to be in a very narrow m / z window (typically a narrow window spanning a range of only 2-3 Da) around the respective m / z value of that precursor to be measured. Said isolating by mass filtering is preferably performed with a quadrupole. Accordingly, preferred mass spectrometers are those equipped with a quadrupole. So, while the ion mobility is continuously ramped from a minimum value to a maximum value, the mass filter jumps to the respective places of where a corresponding precursor is to be measured. Fragmenting takes place downstream of the filter and the MS2 spectra of only the associated fragments of that precursor are measured for each for the precursors of that group in one DDA frame. In a series of such individual DDA frames within one LC observation retention time cycle therefore a corresponding number of groups of precursors are measured. The precursors to be measured in these DDA cycles are scheduled, according to prior art approaches, based on information of interesting precursors determined only in the above-mentioned survey scan (normally based on intensities) of the same or a preceding LC observation time cycle.

[0073] Typically, and this applies to DDA frames as well as DIA frames, within one frame several hundred mass scans, typically TOF scans, are carried out.

[0074] Within one DDA frame normally 10-15 precursors are individually mass selected and measured, so that for each precursor there are 20-50 mass scans, in particular TOF scans. The term “data-independent acquisition”, abbreviated “DIA”, in conjunction with MS has its art-established meaning (Gillet et al. 2012, doi: 10.1074 / mcp.0111.016717). Typically, it refers to the acquisition of MS2 scans of m / z ranges in MS1 having breadths which are much wider and generally comprise multiple analytes / precursors. Number or type of peaks in the MS1 scan are typically of no influence on the choice of the m / z range. All ions within a selected MS1 mass-to-charge range (typically a window spanning a range of 10-200 Da, typically 20-30 Da) are selected for fragmentation. In case of DIA, the precursors are not specifically isolated but isolated together because they are in a certain m / z range that is isolated as a whole. Said isolating is preferably performed with a quadrupole. Accordingly, preferred mass spectrometers are those equipped with a quadrupole.

[0075] In one individual DIA frame for one given broad MS1 window all precursor ions within that window are selected for fragmentation and all fragments thereof are collectively measured in the MS2 dimension. In the figures given below one such scan is indicated by a boxF07395 15.1.2026

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[0077] designated with DIA. Also in a DIA frame the ion mobility is scanned, or ramped, and within one ramp in a diaPASEF DIA frame the wide m / z window can jump between 2 or 3 different ranges to cover several parts of a diagonal in MS1. Alternatively, in the so-called MIDIA approach, to capture stripes in a corresponding MS1 diagonal it is possible to successively shift the broad m / z window while ramping the ion mobility from the above-mentioned minimum value to the maximum value. MIDIA is a special case of diagonal-PASEF with overlapping stripes. For diaPASEF and midiaPASEF see Distler et al in https: / / doi.org / 10.1101 / 2023.01.30.526204.

[0078] If a series of DIA frames is carried out within one LC retention time cycle, correspondingly different (preferably non-overlapping) MS1 windows are selected forthose DIA frames. Mobilogram: Trace of an ion in the ion mobility dimension at a specific value of the retention time. It is generated by tracing an ion. The x-axis is a metric for ion mobility (e.g.

[0079] 1 / K0, reversed ion mobility, collision cross section) and y-axis is intensity. Mobilograms from multiple retention time points can be summed up into a new mobilogram.

[0080] According to a first preferred embodiment of the above-mentioned method, the DDA frames, the DIA frames, or both are comprising the following steps:

[0081] introducing precursor ions into an ion mobility separator (IMS), preferably a trapped ion mobility spectrometry (TIMS) separator,

[0082] separating the precursor ions according to mobility in the ion mobility separator, sequentially releasing precursor ions from said ion mobility separator according to their ion mobility,

[0083] introducing said released precursor ions into a mass filter which selectively transmits precursor ions having m / z values falling within a controllable mass window, fragmenting the precursor ions transmitted through said mass filter to generate fragment ions,

[0084] carrying out a mass spectroscopy measurement on said fragment ions, wherein each fragment ion is associated with a mass window and an ion mobility (IM) range, and associating detected fragments with its corresponding precursor ion,

[0085] wherein preferably said second ion mobility separator and said mass filter are controlled in a synchronized manner preferably 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 stepwise towards lower or higher m / z values, respectively, and wherein preferably said step of associating a detected fragment with its corresponding precursor ion is based on determining or utilizing the corresponding mass windows and ionF07395 15.1.2026

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[0087] mobility (IM) ranges associated with various occurrences of said fragment in said mass spectrometry measurement.

[0088] For analyzing the survey scan, information about LC retention time, ion mobility and m / z for precursors not of interest and / or for precursors of interest, and / or for analyzing the at least one DIA frame information about LC retention time, ion mobility and m / z for fragments not of interest and / or for fragments of interest, a database containing respective information can be consulted and / or a list, which is preferably supplemented successively during one or a series of LC experiments, comprising precursors to be excluded, is consulted.

[0089] The results of the combined analysis of the survey scan and of the at least one DIA frame can be used not only for scheduling of the corresponding LC observation retention time cycle, but also for scheduling of at least the following LC observation retention time cycle. The combined analysis preferably involves peak picking of precursor ions in the survey scan, peak picking of individual fragment ions and / or precursors in the at least one DIA frame, in case of several DIA frames merging of the respective fragment ion and precursor peak lists, gathering the survey scan precursor list and the DIA frame fragment and precursor list and selecting precursors for scheduling of the following DDA frames based on that gathered list, wherein preferably the selection is based on correlating and associating fragments and / or precursors, and / or is based on database and / or in silico information about precursors and / or fragments and / or precursors and associated fragments and / or dynamic exclusion bookmarks and / or information from previous LC retention time windows and / or neutral loss information and / or cross-linker information.

[0090] A characteristic mass difference between a precursor and an associated fragment or characteristic mass difference between at least two fragments of an associated precursor, at least one characteristic fragment or a characteristic group of fragments of an associated precursor, or a characteristic fragment pattern of an associated precursor, or relative intensities of precursors and associated fragments or between fragments, or a combination of these criteria, is preferably used for identifying precursors of interest and for scheduling the following DDA frames for measurement of these precursors of interest.

[0091] According to yet another preferred embodiment, said ion mobility separator (IMS) is a TIMS analyzer with parallel accumulation and separation,

[0092] 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 valueF07395 15.1.2026

[0093] 14

[0094] 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.

[0095] Said ion mobility separator (IMS) and said mass filter are preferably 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,

[0096] wherein in said IM scans, in case of DIA frames in particular, 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,

[0097] wherein adjacent scan regions associated with different IM scans overlap in the m / z-direction.

[0098] In said IM scans, in case of DIA frames, preferably 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. For DIA frames preferably a mass filter window width in the range of 10-100 Dalton, preferably in the range of 20-40 Dalton is applied.

[0099] For DDA frames preferably a mass filter window width in the range of 1-4 Dalton, preferably in the range of 2-3 Dalton is applied.

[0100] At least two DIA frames are preferably measured within one LC observation retention time cycle.

[0101] 5-15 DDA frames, preferably 7-10 DDA frames are preferably measured subsequent to measurement of the DIA frames within one LC observation retention time cycle.

[0102] For the selection of precursor peaks to be scheduled for measurement in the DDA frames, preferably an analysis of at least one mobilogram is used, preferably a distortion analysis, preferably for determining overlap and / or correlations of signals.

[0103] The present invention also relates to the use of such a method for the analysis of a complex sample, including samples in proteomics and metabolomics.

[0104] Also the present invention relates to an apparatus for combined liquid chromatography (LC),F07395 15.1.2026

[0105] 15

[0106] ion mobility and mass spectroscopy analysis, for carrying out the method according to any of the preceding claims, said apparatus preferably comprising:

[0107] a liquid chromatography (LC) unit,

[0108] an ionization unit ionizing precursors eluting as a function of time from said liquid chromatography unit,

[0109] 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,

[0110] 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,

[0111] a fragmentation device for fragmenting the precursor ions transmitted through said mass filter to generate fragment ions,

[0112] an apparatus for carrying out a mass spectroscopy measurement on said fragment ions, wherein each fragment ion is associated with a mass window and an ion mobility (IM) range, and a control system,

[0113] wherein said control system is configured to control said IMS and said mass filter in a synchronized manner for the scheduling of the DDA frames, by selecting precursors from a combined analysis on the basis of peak picking of precursors identified in the survey scan, as well as on the basis of peak picking of precursors and / or fragments identified in the at least one DIA frame, preferably in addition using advanced knowledge, from databases and / or in silico sources, for that combined analysis, wherein preferably fragments identified in the at least one DIA frame are used for selecting to be scheduled precursors identified from the survey scan,

[0114] such as to carry out a plurality of IM scans in the DDA 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,

[0115] 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, in particular of DIA frames, adjacent mass windows that are associated with consecutive mass spectroscopy measurements of fragment ions overlap, such that the precursor ions transmitted through said mass filter during said IM scan are located in at least one continuous scan region in an m / z-IM plane which extends in a generally diagonal direction in said m / z-IM plane,

[0116] wherein further preferably said control system is configured to control said IMS and said mass filter, in particular of DIA frames, in a synchronized manner such that adjacent scan regions associated with different IM scans overlap in the m / z-direction.F07395 15.1.2026

[0117] 16

[0118] 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:

[0119] (a) accumulating ions from the first ion mobility separator in an RF ion trap;

[0120] (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;

[0121] (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

[0122] (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.

[0123] Further embodiments of the invention are laid down in the dependent claims.

[0124] BRIEF DESCRIPTION OF THE DRAWINGS

[0125] 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,

[0126] Fig. 1 schematically shows two adjacent LC observation retention time cycles Fig. 2 shows schematically two adjacent LC observation retention time cycles with a minimal flow diagram of the scheduling and data flow for mixed mode cycles, minimal version;

[0127] Fig. 3 shows schematically two adjacent LC observation retention time cycles with a flow diagram of the scheduling, in this case data flow for mixed mode cycles under inclusion of dynamic exclusion;

[0128] Fig. 4 shows schematically two adjacent LC observation retention time cycles with a flow diagram of the scheduling, in this case the data flow for mixed mode cycles, dynamic exclusion, and delayed scheduling;

[0129] Fig. 5 shows schematically two adjacent LC observation retention time cycles with a flow diagram of the scheduling, in this case data flow for mixed mode cycles, full version, including dynamic exclusion, delayed scheduling and continuous optimization of parameters;

[0130] Fig. 6 illustrates the typical steps of the peak picking;

[0131] Fig. 7 illustrates a visualization for neutral loss detection;

[0132] Fig. 8 illustrates a flow chart for MS1 / MS2 peak pair detection for the example ofF07395 15.1.2026

[0133] 17

[0134] neutral losses, in particular for phosphopeptides;

[0135] Fig. 9 illustrates a flow chart for MS1 I MS2 peak pair detection for the example of cross-linkers, in particular for DSBll;

[0136] Fig. 10 illustrates a visualization for characteristic fragment detection (Trigger Mass Detection), namely oxonium ion detection,

[0137] Fig. 11 illustrates a visualization for Trigger Mass Detection with signals at the border of the isolation range.

[0138] DESCRIPTION OF PREFERRED EMBODIMENTS

[0139] Fig. 1 schematically shows two adjacent LC observation retention time cycles. In this case each comprising the above-mentioned initial survey scan (indicated as MS1), then follows a series of four DIA frames (indicated by DIA), followed by 8 DDA frames (indicated by DDA). At the end of the cycle 1 , cycle 2 starts by its survey scan MS 1 and the sequence is continued. As discussed above, the combination of the survey scan MS1 and of the DIA frames is used to make decisions for the scheduling of the DDA frames of the same cycle, or one of the following cycles, or both. More specifically, the peak picking from the survey scan and from the DIA scans is used in a combined manner to identify precursors of interest, and these precursors of interest are then taken for the scheduling of the following DDA frames.

[0140] Fig. 2 schematically shows two adjacent LC observation retention time cycles, in this case including a schematic flow diagram. The survey scan MS1 is analyzed using peak picking for precursors of interest, and the same is applied with the spectra obtained in the four DIA frames, by using individual MS2 frame peak picking, identifying precursor as well as in particular fragment signals. The peak picking from the DIA frames is merged into an MS2 peak list, and both peak lists are typically gathered into one common structured peak list. Using the combined information in that gathered peak list, precursors are selected, and then only those selected precursors or at least some of those selected precursors are used for scheduling the following DDA frames in the same LC observation retention time cycle. This happens in a combined way, in the sense that peak information from the survey scan as well as from the DIA frames is used for the targeted selection of precursors for the scheduling of the following DDA frames.

[0141] Fig. 3 schematically shows two adjacent LC observation retention time cycles like the one as illustrated in the preceding figure, in this case additionally dynamic exclusion bookmarks are applied for the acquisition scheduling. Dynamic exclusion means, that precursors are only selected if they have not been selected already within a certain time frame or there are other good reasons to repeat the measurement, e.g. if the intensity is rising significantly inF07395 15.1.2026

[0142] 18

[0143] a new cycle. For that purpose, a list of m / z values, 1 / K0 values, intensities and assigned charges are logged together with a time stamp for the selection of the precursor.

[0144] Fig. 4 schematically shows two adjacent LC observation retention time cycles like the one as illustrated in the preceding figure, but in this case it is illustrated that the scheduling not within the same LC observation retention time cycle is controlled by the respective survey scan and the sequence of DIA frames, but the scheduling of the following cycle.

[0145] Notably, this can be combined, so the results of the analysis of the survey scan and the DIA frames of the first cycle can be used for the scheduling of the DDA frames within the same cycle and also of the DDA frames of the next cycle, and also a combined analysis is possible using survey scan and DIA scan information from two adjacent cycles.

[0146] Fig. 5 schematically shows this namely two adjacent LC observation retention time cycles like the one as illustrated in the preceding figure, but in this case the gathered peak list of the first cycle is used for continuous evaluation of the data quality and the dynamic parameters and for the next iteration of the precursor selection.

[0147] Fig. 6 illustrates the typical steps of the peak picking, The shown scheme is applied for the spectra in the survey scan as well as of the DIA frames. Typically smoothing / Mexican hat filtering is followed by 2D maximum detection, charge and monoisotopic peak assignment, and the results are filled into a peak list.

[0148] Fig. 7 schematically shows the situation where a characteristic difference between the precursor and an associated fragment is used as a criterion for precursor selection for DDA frame scheduling, as for example the case for neutral loss detection of phosphopeptides in a workflow for phosphoproteomics.

[0149] Workflow example for phosphoproteomics:

[0150] In the case of phosphoproteomics we are looking for a neutral loss of H3PO4 in the fragment spectrum showing a signal shifted by a delta mass of 97.977 Da relative to the mass of the precursor ion. Neutral loss means, that the remaining fragment has the same charge as the precursor ion.

[0151] For the workflow this means that we have to analyze the mass range mz-f1 to mz-f2 of the DIA frames ranging from approximately 100 Da below the lower end of the DIA isolation window (mz-isol1 for the lower end of a diagonal DIA isolation window) up to 100 Da below the upper end of the DIA isolation window (mz-isol2) to be able to detect the different charge states (down to charge 1 with the largest m / z difference) of the potential neutral losses. The charge state of the precursor and the putative neutral loss fragment will be determined from the isotope peaks occurring in a distance of approximately 1 / z Da. The charge states have to be the same for precursor and fragment and the m / z distance between both has to beF07395 15.1.2026

[0152] 19

[0153] 97.977 / z Da. To further validate the neutral loss process, the connection between a precursor and a fragment the mobility information will be analyzed in the same way as for the glycopeptide case further below.

[0154] To find those potentially matching pairs we check for each precursor peak within the isolation window used for DIA whether there is a fragment peak within the given distance + tolerance range and a similar average mobility and identical charge state (in case of neutral losses).

[0155] Considering the capability of the timsTOF to (at least partially) separate different phosphopeptides with the same composition but different positions of the phosphorylation especially pairs of phosphopeptides with the same mass but slightly different mobility are of special interest to get insights into the distribution of phosphorylation site localizations. The method is helpful to find such phosphorylation site related MOMA pairs (mobility offset mass aligned). Without evidence of a phosphorylation given by the observed neutral loss the only indication for two peaks with the same m / z but different mobility is a maybe only partial splitting of the mobilogram in MS1. With the new method we get additional evidence for a phosphorylation, excluding other options leading to a very similar m / z and nearby mobility values, e.g. different conformation isomers of the peptides with a different mobility due to a different shape of the two or more conformers.

[0156] Going back to the schematic illustration of Fig. 2, for this scenario in the step of peak picking of the MS2 frames in this case of neutral loss of H3PO4 the search can be limited to min(isolation m / z)- 102 Da to max(isolation m / z) - 102 Da for neutral losses of around 98 Da.

[0157] In addition to that, in the MS1 frame for peak picking, the search can be limited to the m / z & mobility area covered by the set of DIA isolation windows.

[0158] In the step of precursor selection, there can be a selection of precursors for which we find evidence for neutral loss based on lower m / z fragments at expected distance and with identical charge state.

[0159] Going back now to the schematic illustration of Fig. 7, for this scenario, wherein the filled ellipsoid (arrow 1) shown in the upper right area represents an example of an MS1 peak in the survey scan of a z=2 phosphopeptide with

[0160]

[0161] The arrow 2 to the left represents the characteristic m / z difference to a corresponding fragment (empty ellipsoid, arrow 3) as seen in a corresponding DIA frame, giving the associated neutral loss signal observed in the left DIA frame at ~ mz - 97.98 / 2 Da also with charge state 2 and same mobility as the precursor. The tilted trapezoid overlapping frames are illustrating to overlapping diagonal isolation ranges in a diagonal DIA measurement, more specifically they represent midia PASEF frames. As illustrated by arrow 4, there is one diagonal isolation rangeF07395 15.1.2026

[0162] 20

[0163] for a diagonal-DIA measurement.

[0164] Fig. 8 illustrates a flow chart for MS1 I MS2 peak pair detection for the example of neutral losses, in particular for phosphopeptides. In this case one iterates over the DIA frames by carrying out the following steps:

[0165] • Detect MS1 features in isolation windows of the current DIA-Frame i;

[0166] • Search for MS2 features in DIA-Frame i, optionally limited to relevant region for neutral loss of known mass;

[0167] • Search for MS-2 features in DIA-Frame i, limited to relevant region for neutral loss of known mass;

[0168] • Search for matching pairs of MS1 and MS2 features: same charge, m / z and mobility distance within tolerance limits;

[0169] • In case of shared features between those pairs, check for best match of mobilograms to select partners;

[0170] • Collect MS1 features of finally selected pairs, offering m / z value and mobility range for precursor selection.

[0171] Workflow example for cross linking:

[0172] With cross linking experiments (see Piersimoni et. al., Chem. Rev. 2022, 122, 8, 7500-7531; https: / / doi.org / 10.1021 / acs.chemrev.1c00786) two peptides of a protein are connected by means of a chemical reaction of a cross-linker reagent which reacts for example with amine side chains of certain peptides. The cross linkers can have different distances between the two reactive sites which allows for the determination of distance constraints for the peptides within one protein, giving insights into the conformation. Another application is the linkage between different proteins which gives insights into the proximity and interactions of proteins in cells. After linking the peptides, the proteins are digested and analyzed in the usual way.

[0173] From the analytical side it’s a challenge that only a small fraction of the signals comes from cross linked peptides. The majority of signals are generated by peptides which did not react or only one peptide was reacting with the linker agent (called single linkage).

[0174] This can be overcome to some extent with linkers which allow for an enrichment of the products, but an efficient selection of the single linkage and especially of the cross linked products is nevertheless important to optimize the information obtained from such experiments.

[0175] Using the DSBU crosslinker (disuccinimidyl dibutyric urea) as an example the fragment spectra show two pairs of peaks in a distance of 26 Da each. From the lowest and highest mass of the four peaks it is possible to derive the mass of the precursor. For single linkedF07395 15.1.2026

[0176] 21

[0177] only one pair of peaks in 26 Da distance will occur.

[0178] This signature can be used to detect such single and especially cross linked peptides from the DIA data. For single linked peptides a matching MS1 precursor has to be found to get the m / z and mobility range for the DDA experiment. For cross linked peptides with this chemistry it would in principle be enough to determine these ranges from the MS2 data. A matching peak from the MS-1 data can be used as a further confirmation.

[0179] Fig. 9 illustrates a flow chart for MS1 I MS2 peak pair detection for the example of cross-linkerst, in particular DSBLI. In this case one iterates over the DIA frames by carrying out the following steps:

[0180] • Detect MS1 features in isolation windows of the current DIA-Frame i;

[0181] • Search for MS2 features in DIA-Frame i;

[0182] • Search for two matching pairs of fragment features for a certain mass distance of e.g. 26 Da for DSBLI and same charge;

[0183] • Calculate expected MS-1 precursor m / z from the two pairs;

[0184] • Validate the hypothesis of cross linking by checking the MS1 features for a matching precursor (m / z, mobility);

[0185] • Collect MS1 features of finally selected pairs, offering m / z value and mobility range for precursor selection.

[0186] Optionally, the measurement and / or analysis of MS-1 frames can be skipped and the precursor m / z values and the mobility range for fragmentation can be taken from the MS-2 signals, if MS-1 is not requested for the offline postprocessing of the data.

[0187] Workflow example for the selective fragmentation of glycopeptides:

[0188] The algorithm in that case is configured to detect oxonium ions, relatively low-mass ions derived from monosaccharide and disaccharide fragmentation (see Bagdonaite et al, Nat Rev Methods Primers 2, 48 (2022). https: / / doi.orq / 10.1038 / s43586-022-00128-4). Measuring of DDA spectra is reported using HCD, higher-energy collision dissociation, a variant of CID, collision-induced dissociation, which is used as the default fragmentation method in electrospray TOF instruments like the timsTOFs. Checking in real time for oxonium-ions in the HCD spectra they could limit the time-consuming measurement of ETD, electron transfer dissociation, spectra to those precursors which according to the detection of a signal for the defined list of trigger ions, are glycopeptides. This was an improvement compared to doing ETD in addition to HCD for all precursors.

[0189] With the proposed method one is able to detect the presence of glycopeptides for all precursors which give rise to a peak in the DIA data for a listed oxonium ion and won’t miss glycopeptide precursors which are not selected among all peptides for fragmentationF07395 15.1.2026

[0190] 22

[0191] because of the selection rules required to limit the number of precursors which can be fragmented in a DDA experiment.

[0192] Making use of a DIA-PASEF method has the additional advantage compared to a DIA method without ion mobility that one can check for correlations of oxonium ion mobilograms generated from the DIA-PASEF frames against the mobilograms of the peaks in the MS1 data which are in the m / z and mobility range of the DIA windows of the DIA-PASEF measurement.

[0193] One can, as an example, check for four different oxonium ions and two fragments of one of the oxonium ions (see also Saba et al, Int J Proteomics 2012:2012:560391. doi: 10.1155 / 2012 / 560391):

[0194]

[0195] To limit the effort for the data processing one can only analyze small m / z regions of the DIA-PASEF data frames around the list of these 5 masses. From the raw signals which are leading to a large enough peak (according to given threshold criteria for intensity, area and maybe signal / noise) in these regions of the DIA data one can create mobilograms.

[0196] Alternatively, one could directly create mobilograms from the raw data from within a window of for example m / z 163.060 + / - 0.01 for the Hex oxonium ion. These mobilograms can then be searched for peaks in a one-dimensional manner. If multiple glycopeptides are present within the isolation window of the DIA experiment this can lead to more than one peak for each specific kind of oxonium ion.

[0197] For each found peak of each specified oxonium ion one creates a mobilogram which is then used for a correlation analysis against the mobilograms of the MS1 peaks found in the DIA isolation window for which one has analyzed the DIA fragment signals.

[0198] To find potential candidates for the observed oxonium ions among the MS1 peaks in the simplest way one can check for the average mobility value of both the observed MS1 and DIA peak and whether they are within a given tolerance range or not or just going for the best match if no candidate can be found within the tolerance ranges, maybe because of overlaps. In the more likely case that there are many precursors within the tolerance range a correlation analysis of the mobilograms or a statistical distribution test like Kolmogorow-Smirnow can be used to reduce the list of precursors which remain as candidates for the DDA experiment, especially DDA-PASEF which allows to measure a very large number of precursors in a given time window. Note: since the mobility separation is done prior to the enforced fragmentation (ignoring unintended in source fragmentation) the mobilograms ofF07395 15.1.2026

[0199] 23

[0200] precursors and fragments can be expected to be strongly correlated, limited by the signal to noise ratio if there are no overlapping signals.

[0201] Fig. 10 schematically shows such situation where a characteristic fragment, in this case an oxonium ion, can be used for identifying precursors of interest, namely by identifying a corresponding characteristic fragment (empty ellipsoid on the left with arrow 5, oxonium ion observed for the putative glycopeptide), and by identifying the associated precursor signal (uppermost filled ellipsoid on the right side with arrow 6, representing the MS1 peak of a putative glycopeptide). Again, the tilted trapezoid indicates one diagonal isolation range for a diagonal DIA measurement (midiaPASEF). The filled ellipsoid (arrow 7) slightly lower down from the above-mentioned glycopeptide of interest, is an MS1 peak with similar ion mobility but lower correlation with the oxonium ion. This peak will not be selected for the scheduling of the DDA frames. The lowermost MS1 peak (arrow 8) is too far off in ion mobility and also will not be selected for scheduling of the DDA frames. As illustrated by arrow 9, there is one diagonal isolation range for a diagonal-DIA measurement.

[0202] Fig. 11 schematically shows such situation where a characteristic fragment, in this case an oxonium ion, can be used for identifying precursors of interest, and where edge effects are used for identifying the precursor signal of interest. In this case what is noted in a DIA scan is an oxonium ion observed for the putative glycopeptide, with distorted ion mobility distribution due to the precursor at the edge of isolation window, this is the non-filled half ellipsoid illustrated on the left with arrow 11. If in addition to that, as illustrated on the same height to the right, there is another MS1 peak (arrow 12) with similar ion mobility but not justifying the distorted fragment ion distribution. Further to the right there is another MS1 peak (arrow 13) of a putative glycopeptide which is at the edge of the isolation window and this one justifies the distorted fragment ion distribution. So, this graphic illustrates that the characteristics of fragment signals and the corresponding correlation with a signal in the survey scan taking account of the isolation range for the corresponding diagonal DIA measurement for the fragment can be used for identifying precursor signals of interest. As illustrated by arrow 14, there is one diagonal isolation range for a diagonal-DIA measurement.

[0203] Workflow example for PRM:

[0204] PRM, parallel reaction monitoring, is a targeted fragmentation method where predefined precursors are fragmented in predefined retention time windows and the fragments are measured in parallel using mass spectrometers like orbitraps or time of flight analyzers instead of triple quad instruments where the fragments have to be scanned one after the other in a serialized manner. The purpose is a very sensitive and selective quantification ofF07395 15.1.2026

[0205] 24

[0206] known molecules. Especially with prm-PASEF a very large number of targets can be measured in that way and the use of so called retention time standards helps to reduce the size of the required retention time windows over which these targets have to be measured. The presence of these retention time standards is detected also by fragmentation experiments and their retention time maxima are used to calibrate the retention time which can shift slightly from one HPLC run to the next, especially for longer measurement campaigns. Unfortunately, not all peptides are shifted to the same amount which means that the retention time windows still have to be larger than the chromatographic peak width to make sure they are not missed or only measured partially.

[0207] Using the presented method allows a sensitive detection of the onset of the fragments of a certain target by analyzing the DIA data. Checking the DIA fragments is more selective than going with the MS1 data only due to potentially overlapping signals. For very complex samples it still makes sense to do targeted fragmentations with narrow isolation windows instead of just using DIA data for the quantification. Detecting the putative presence of a target within a reasonable time window will than trigger the targeted fragmentation for a certain amount of time. The presence of the target can be validated by checking for the known expected fragments in the DDA data. After a successful detection it can be removed from the list of targets of interest giving free capacity for additional measurements using a dynamic scheduling.

[0208] The smaller the required retention time windows, the more targets can be measured within one HPLC run and / or it is possible to trigger the fragmentation of targets more often which leads to a better quality of the quantification results.

[0209] Being able to detect the onset of targets with DIA data also reduces the need to predefine the retention time windows in a precise way. This allows the use of machine learning approaches which can predict the retention time, mobility and the relative intensities of fragment ions of the target molecules with increasing confidence, especially peptides. This helps a lot to reduce the effort of designing good PRM methods based on prior studies where the retention time, mobilities and fragmentation patterns have to be determined.

Claims

F07395 15.1.202625CLAIMS1. A method of liquid chromatography (LC) coupled combined ion mobility and mass spectroscopy analysis,wherein at the beginning of one LC observation retention time cycle, preferably in the range of 0.5-4 seconds, particularly preferably in the range of 0.8-3 seconds, a survey scan (MS1) is taken, wherein in that survey scan a full ion mobility width of interest and a full m / z width of interest is scanned without fragmentation,wherein this survey scan is followed by at least one DIA frame, preferably at least 2 or at least 3 DIA frames,followed by at least two DDA frames, preferably in the range of 3-15 DDA frames or in the range of 5-10 DDA frames,and wherein for the scheduling of the following DDA frames, withing the same LC observation retention time cycle or a following LC observation retention time cycle, precursors are selected using a combined analysis on the basis of peak picking of precursors identified in the survey scan, as well as on the basis of peak picking of precursors and / or fragments identified in the at least one DIA frame, preferably in addition using advanced prior knowledge, from databases and / or in silico sources, for that combined analysis, wherein preferably fragments identified in the at least one DIA frame are used for selecting to be scheduled precursors identified from the survey scan.

2. Method according to claim 1, wherein the DDA frames, the DIA frames, or both are comprising the following steps:introducing precursor ions into an ion mobility separator (IMS), preferably a trapped ion mobility spectrometry (TIMS) separator,separating the precursor ions according to mobility in the ion mobility separator, sequentially releasing precursor ions from said ion mobility separator according to their ion mobility,introducing said released precursor ions into a mass filter which selectively transmits precursor ions having m / z values falling within a controllable mass window, fragmenting the precursor ions transmitted through said mass filter to generate fragment ions,carrying out a mass spectroscopy measurement on said fragment ions, wherein each fragment ion is associated with a mass window and an ion mobility (IM) range, and associating detected fragments with its corresponding precursor ion,F07395 15.1.202626wherein preferably said second ion mobility separator and said mass filter are controlled in a synchronized manner preferably such as to carry out a plurality of ion mobility (IM) scans, during which precursor ions of increasing or decreasing IM are successively released from said second ion mobility separator (IMS), and during which the mass window of said mass filter is shifted continuously or stepwisely towards lower or higher m / z values, respectively,and wherein preferably 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.

3. Method according to claim 1 or 2, wherein for analyzing the survey scan information about LC retention time, ion mobility and m / z for precursors not of interest and / or for precursors of interest, and / or for analyzing the at least one DIA frame information about LC retention time, ion mobility and m / z for fragments not of interest and / or for fragments of interest, a database containing respective information is consulted and / or a list, which is preferably supplemented successively during one or a series of LC experiments, comprising precursors to be excluded, is consulted.

4. Method according to any of the preceding claims, wherein the results of the combined analysis of the survey scan and of the at least one DIA frame is used not only for scheduling of the corresponding LC observation retention time cycle, but also or exclusively for scheduling of at least the following LC observation retention time cycle.

5. Method according to any of the preceding claims, wherein the combined analysis involves peak picking of precursor ions in the survey scan, peak picking of individual fragment ions and / or precursors in the at least one DIA frame, in case of several DIA frames merging of the respective fragment ion and precursor peak lists, gathering the survey scan precursor list and the DIA frame fragment and precursor list and selecting precursors for scheduling of the following DDA frames based on that gathered list, wherein preferably the selection is based on correlating and associating fragments and / or precursors, and / or is based on database and / or in silico information about precursors and / or fragments and / or precursors and associated fragments and / or dynamic exclusion bookmarks and / or information from previous LC retention time windows and / or neutral loss information and / or cross-linker information.F07395 15.1.2026276. Method according to any of the preceding claims, wherein a characteristic mass difference between a precursor and an associated fragment or characteristic mass difference between at least two fragments of an associated precursor, at least one characteristic fragment or a characteristic group of fragments of an associated precursor, or a characteristic fragment pattern of an associated precursor, or relative intensities of precursors and associated fragments or between fragments, or a combination of these criteria, is used for identifying precursors of interest and for scheduling the following DDA frames for measurement of these precursors of interest.

7. Method according to any of the preceding claims 2 - 6, wherein said ion mobility separator (IMS) 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 valuefollowed 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.

8. Method according to any of the preceding claims 2 -7, wherein said ion mobility separator (IMS) 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 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 theF07395 15.1.202628m / z-direction.

9. Method according to claim 10, wherein 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.

10. Method according to any of the preceding claims wherein for DIA frames a mass filter window width in the range of 10-100 Dalton, preferably in the range of 20-40 Dalton is applied,and / or wherein for DDA frames a mass filter window width in the range of 1-4 Dalton, preferably in the range of 2-3 Dalton is applied.

11. Method according to any of the preceding claims, wherein at least two DIA frames are measured within one LC observation retention time cycle,and / or wherein 5-15 DDA frames, preferably 7-10 DDA frames are measured subsequent to measurement of the DIA frames within one LC observation retention time cycle.

12. Method according to any of the preceding claims, wherein for the selection of precursor peaks to be scheduled for measurement in the DDA frames, an analysis of at least one mobilogram is used, preferably a distortion analysis, preferably for determining overlap and / or correlations of signals.

13. Use of the method of any of the preceding claims for the analysis of a complex sample, including samples in proteomics and metabolomics.

14. An apparatus for combined liquid chromatography (LC), ion mobility and mass spectroscopy analysis, for carrying out the method according to any of the preceding claims, said apparatus preferably comprising:a liquid chromatography (LC) unit,an ionization unit ionizing precursors eluting as a function of time from said liquid chromatography unit,an ion mobility separator (IMS) in the form of at least one trapped ion mobility spectrometry (TIMS) separator for receiving and sequentially releasing precursor ionsF07395 15.1.202629according to their ion mobility,a mass filter arranged to receive said released precursor ions and to selectively transmit precursor ions having m / z values falling within a controllable mass window, a fragmentation device for fragmenting the precursor ions transmitted through said mass filter to generate fragment ions,an apparatus for carrying out a mass spectroscopy measurement on said fragment ions,wherein each fragment ion is associated with a mass window and an ion mobility (IM) range, and a control system,wherein said control system is configured to control said IMS and said mass filter in a synchronized manner for the scheduling of the DDA frames, by selecting precursors from a combined analysis on the basis of peak picking of precursors identified in the survey scan, as well as on the basis of peak picking of precursors and / or fragments identified in the at least one DIA frame, preferably in addition using advanced knowledge, from databases and / or in silico sources, for that combined analysis, wherein preferably fragments identified in the at least one DIA frame are used for selecting to be scheduled precursors identified from the survey scan,such as to carry out a plurality of IM scans in the DDA 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, in particular of DIA frames, adjacent mass windows that are associated with consecutive mass spectroscopy measurements of fragment ions overlap, such that the precursor ions transmitted through said mass filter during said IM scan are located in at least one continuous scan region in an m / z-IM plane which extends in a generally diagonal direction in said m / z-IM plane, wherein further preferably said control system is configured to control said IMS and said mass filter, in particular of DIA frames, 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 mobilityF07395 15.1.202630separator, 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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