WO2025040781A1 - System and method for targeted analysis of diagonal family of dia-pasef acquisition methods - Google Patents

Abstract

The present invention determines how to extract data for a peptide precursor and its fragments in an efficient manner when processing the so-called diagonal family of dia- PASEF acquisition methods, such as synchro-PASEF or midia-PASEF. In one implementation of the invention, the entire ion cloud is treated as if it were acquired by a single diagonal DIA window.

Description

TITLE

SYSTEM AND METHOD FOR TARGETED ANALYSIS OF DIAGONAL FAMILY OF DIA- PASEF ACQUISITION METHODS

TECHNICAL FIELD

Analysis of compounds in mass spectrometry and more particularly to instruments, and methods for polypeptide analysis.

PRIOR ART

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

DIA requires a spectral library which contains peptide precursor ions to be used for the targeted extraction in DIA data. This targeted extraction could be over multiple dimensions, such as retention time, ion mobility, etc. The existing methods do not speedily or efficiently analyze the data in multiple dimensions in order to generate quantification of all data (for as many peptides as possible). One way of achieving the targeted extraction is to use the full range of available data when searching for a given peptide precursor ion. However, this degrades the overall analysis due to increased occurrence of interferences in the data. Furthermore, it also leads to significantly longer analysis times as more data needs to be processed. Another way of achieving the targeted extraction is by using only the retention time dimension. However, this does not take into account the ion mobility dimension. Therefore, a system and method are needed that can achieve the targeted extraction in the ion mobility dimension in addition to the retention time dimension.

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.

Ion mobility-based data independent acquisition has recently had a revival with the timsTOF Pro™.

EP-A-3054473 discloses such operation of trapping ion mobility spectrometers based on pushing ions by a gas flow against a counter-acting electric DC field barrier, preferably in combination with a mass analyzer as ion detector. The invention provides an additional RF ion trap upstream of the trapping ion mobility spectrometer, wherein the RF ion trap is operated as an accumulation unit in parallel with the trapping ion mobility spectrometer such that a first group of ions can be analyzed in the trapping ion mobility spectrometer while a second group of ions from an ion source are simultaneously collected in accumulation unit. Due to its parallel accumulation-serial fragmentation (PASEF) strategy, it achieves a high duty cycle and has shown promise in biological applications. However, the early acquisition methods that were proposed for this type of an instrument were not efficient in sampling the ion cloud (Fig. 1).

Recently, researchers have proposed new DIA acquisition methods which sample the ion cloud more efficiently; namely synchro-PASEF (Skowronek P. et al. "Synchro-PASEF allows precursor-specific fragment ion extraction and interference removal in data- independent acquisition"; Mol. Cel. Prot. 2022; 22(2)) and midia-PASEF (Distler U. et al. "midiaPASEF maximizes information content in data-independent acquisition proteomics"; bioRxiv. 2023). The latter distinguishes from the former by using overlapping mass (DIA) windows, and is described in US-A-2022034840, disclosing an apparatus and a method of data independent combined ion mobility and mass spectrometry analysis including introducing precursor ions into an ion mobility spectrometer (IMS), sequentially releasing precursor ions from said IMS according to their ion mobility, introducing said released precursor ions into a mass filter, fragmenting the precursor ions transmitted through said mass filter to generate fragment ions, and carrying out a mass spectrometry measurement on said fragment ions. The IMS and mass filter are controlled in a synchronized manner to carry out a plurality of IM scans, wherein adjacent mass windows in said IM scan that are associated with consecutive mass spectrometry measurements of fragment ions overlap, such that precursor ions transmitted through said mass filter during said IM scan are located in at least one continuous scan region in an m/z-IM plane which extends in a generally diagonal direction in said m/z-IM plane.

Both methods, synchro-PASEF and midia-PASEF, work by seamlessly and continuously following the natural shape of the ion cloud in ion mobility or collisional cross section (CCS) and peptide precursor mass dimensions (Fig. 2a). In this process, the DIA windows take the shape of diagonal slices instead of vertical boxes overlaid on top of the ion cloud. The immediate benefit of these types of acquisition methods is improved sampling of the ion cloud as it is more efficient than vertical boxes.

However, analyzing such data is still an open challenge and is generally quite complex. Each diagonal window slice is made up of hundreds of individual scans with continuously changing m/z and ion mobility ranges. So far efforts have focused on deconvolution of such DIA data to create data dependent acquisition (DDA) type of data which has a relatively non-chimeric MS2 spectra due to narrow isolation widths. This is achieved by making use of the so-called concept of precursor slices wherein the knowledge of the expected slice of a precursor and its relationship with the fragments is used to assign fragments belonging to a specific peptide precursor. Thereby a cleaner DDA type of MS2 spectra is realized.

However, these methods are computationally slow and assume that DIA data can be cleaned up to be like DDA data without a significant loss of information. Additionally, this method can suffer for peptides without a high quality MS1 signal if it relies on MS1 for the deconvolution step. In such methods, by design, most peptide precursor ions are expected to be sliced by 2 or more diagonal DIA windows. This makes the data analysis more complex while also providing for new type of scores that make use of this acquisition scheme.

SUMMARY OF THE INVENTION

While current efforts have focused on trying to deconvolute DIA data to make it resemble narrow window DDA data, one potential approach is to analyze such data in a manner that embraces its complexity.

The present invention determines how to extract data for a peptide precursor and its fragments in an efficient manner when processing the so-called diagonal family of dia- PASEF acquisition methods, such as synchro-PASEF or midia-PASEF. In one implementation of the invention, the entire ion cloud is treated as if it were acquired by a single diagonal DIA window (Fig. 2b). In one approach of this implementation, the data is extracted for a precursor ion that is measured by multiple diagonal DIA windows. Then a novel approach is used that allows processing this type of DIA data in a standard targeted manner. Additionally, a novel category of scores can be used that utilizes the slicing phenomenon. In one implementation of this approach, the invention seamlessly queries the mass spectrometry data in a manner that allows efficient extraction of both the complete (across all diagonal DIA windows, Fig. 3a, 3b) and partial (diagonal DIA window specific, Fig. 3c, 3d) ion traces of fragment ions and/or precursor ions in retention time (RT) (Fig. 4d, 4e) and ion mobility (IM) dimensions (Fig. 4f, 4g). One advantage of this invention is that it is significantly faster than the present solutions and should lead to higher identifications as it queries the data in all possible ways in both RT and IM dimensions. Additionally, it does not rely on the presence of a high quality MS1 signal for deconvolution. A person with ordinary skill in the art will understand that in addition to proteomics, this invention can also be applicable to other mass spectrometer-based omics data such as metabolomics, etc. Also, a person with ordinary skill in the art will understand that this invention can be used for other similar methods in addition to synchro-PASEF or midia-PASEF. For example, the shape of the diagonal slices does not need to be described by a linear function but can also be described by a non-linear function such as a polynomial function. Additionally, this method can also be used to analyze rudimentary diagonal acquisition schemes such as slice-PASEF (Szyrwiel L. et al. “Slice-PASEF: fragmenting all ions for maximum sensitivity in proteomics”; bioRxiv. 2022). The specific mention of synchro-PASEF and midia-PASEF in this description are for illustrative purposes only and not intended as a limitation.

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

Definitions:

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

MS1, MS2: The molecules of a given sample in an LC-MS/MS experiment are ionized and their mass-to-charge ratio (often given as m/z or m/Q) is measured/selected by the mass analyzer (designated MS1). Ions of a particular m/z-ratio coming from MS1 are selected and then made to split into smaller fragment ions, e.g. by collision-induced dissociation, ion- molecule reaction, or photo-dissociation. These fragments are then introduced into the mass analyzer (MS2), which in turn measures the fragments by their m/z-ratio. The fragmentation step makes it possible to identify and separate ionized molecules that have very similar m/z-ratios but produce different fragmentation patterns in MS2. The unfragmented peptide ion that dissociates to a smaller fragment ion, usually as a result of collision-induced dissociation in an MS/MS experiment, is typically referred to as precursor. Data dependent acquisition (DDA): LC- MS/MS or “shotgun” MS approach that is based on the generation of fragment ions from precursor ions that are automatically selected in the first (MS1) dimension based on the precursor ion profiles in that dimension. The window for the second (MS2) dimension is chosen as a function MS1 output (single precursor peak) automatically by the machine. This means that in this mode the MS2 dimension is not continuously sampled but only selectively as a function of the MS1 signal. In a typical shotgun acquisition method, top 10 precursor ions are selected for fragmentation per MS1 scan by the MS for measurement in MS2 with a relatively narrow isolation width of 1-2 Thomson. Precursor ions that have been selected for fragmentation are also typically ignored by the MS in the subsequent scans to allow fragmentation of new precursor ions.

Data independent acquisition (DIA): LC-MS/MS approach, in this mode, all ionized compounds of a given sample that fall within a specified mass range in the first MS1 dimension are fragmented in a systematic and unbiased fashion resulting in corresponding spectra in the MS2 dimension. In contrast to DDA, in this case the MS2 space is continuously sampled. This not only leads to a larger data volume, but also has the effect that the spectra measured in the MS2 space comprise fragments not just from one precursor in the MS1 dimension but potentially from several such precursors. The common feature of DIA methods is that instead of selecting and sequencing a single precursor peak, wider m/z windows are fragmented resulting in complex spectra containing fragment ions of several precursors. This avoids the missing peptide ID data points typical for shotgun methods and potentially allows sequencing whole proteomes within one run, which offers a clear advantage over the small number of peptides that can be monitored per run by SRM. Furthermore, DIA have excellent sensitivity and a large dynamic range. To identify the peptides present in a sample, the fragment ion spectra can be searched against theoretical spectra or can be mined using SRM-like transitions. The detected fragments are subsequently arranged in SRM-like peak groups. In DIA acquisition, windows size in MS2 dimension is often more than 30 Thomson. This means that a typical MS2 scan in DIA is more complex than in DDA because of significantly more precursor ions being cofragmented.

Protein database: a database, preferably selectively just for the organism of which the sample originates, comprising peptide and protein data from that organism, which means sequence information but no spectral information.

Spectral library: a database which contains information about peptide and protein systems as well as about fragments thereof, and which specifically associates to these peptides, proteins and fragments spectral information from an LC-MS/MS experiment, including (indexed) retention time, ion mobility, m/z ratios and expected fragment ion relative intensities. A spectral library can be a predicted library, an empirical library, or an intermediate output of library-free search analysis.

Empirical (spectral) library (also termed In-silico spectral library): is a spectral library obtained based on an LC-MS/MS experiment typically using DDA and analysis of the data using a protein database and a spectrum-centric analysis.

Predicted (spectral) library (also called in-silico spectral library): is a spectral library obtained using computer simulation results, in particular by the prediction of pre-existing machine learning models (like but not limited to deep-learning neural networks). These models are usually trained on a large set of empirical data that allows them to predict aspects of a spectral library (like but not limited to fragmentation, retention time or ionmobility).

Deconvolution: The process of resolving complex MS2 spectra to determine the underlying precursors that made up those spectra.

Calibration: Calibration is used to detect shifts between a theoretical quantity and its empirical counterpart and is a process of reducing the influence thereof. For example, an untuned MS can lead to a relatively large shift in the measured m/z in MS2 of precursors. A calibration step detects this shift and corrects for it usually based on some of a regression analysis. Calibration is typically done for m/z, ion mobility, and retention time (or iRT).

Library-free search analysis: In the field, library-free search analysis of DIA refers to the process where you do not need to acquire MS measurements for the specific purpose of creating a spectral library only.

Spectrum-centric analysis: data analysis of data obtained in an LC-MS/MS experiment, which can be DDA or DIA data, in which the search is spectrum centric. This means that the spectra in the MS2 dimension are scanned for possible matches with all theoretical peptides and their fragments derived from a protein database typically with no or limited prior spectral information. Typically, the parent precursor ion for a MS2 spectrum is matched with a certain m/z tolerance to the theoretical m/z 508 (see also Fig. 5 and the corresponding description further below) for all precursors in the search space 506 giving a set of candidate peptides 509. Then the candidate peptide which best explains the spectra in terms of theoretical fragment ions 511 is considered as the peptide spectrum match (PSM). No further prior information on the fragments is required.

Peptide-centric analysis/peptide centric search: data analysis of data obtained in an LC- MS/MS experiment, which can be DDA or DIA data, in which the search is precursor centric. The predicted possible peptides and their fragments derived from a predicted spectral library or an empirical spectral library 601 (see also Fig. 6 and the corresponding description further below) are queried against the spectra in the MS1 and MS2 dimension 604. In this analysis, spectral information of the peptides is required, in particular retention time, ion mobility, and likely to be observed fragment ions with relative fragment intensities. This information is used to narrow the search space of the peptide by querying only the spectrum that falls within a certain m/z, iRT or IM tolerance 605 and for scoring of matches. Having this additional information greatly improves the sensitivity of the analysis by leading to more powerful scores 608.

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 in (normally) a single 5D scan. The x-axis is a metric for ion mobility (e.g. 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.

Extracted ion chromatogram (XIC): Trace of an ion in the retention time dimension. It is generated based on 5D scans corresponding to multiple retention time points. A single data point in the XIC for a specific retention time can be based on the apex intensity or the area under the curve or summing up of all data points in the mobilogram. The x-axis is the retention time dimension and y-axis is intensity. The reason why it is informative to look at both dimensions, so at mobilograms as well as extracted ion chromatograms is that at XIC level, the ion mobility level is collapsed.

Ion mobility (IM) index: In the context of this invention, the ion mobility index refers to an index within a master-table that contains all unique ion-mobility dimension values of the given LC-MS measurement. So it is an index that maps to a reference or master table (see example 425 further below) that contains all unique ion-mobility values for this scan.

Isolation index: In the context of this invention, the isolation index refers to an index within a master-table (see example 418 further below) that contains all unique m/z isolation ranges that were applied in the given LC-MS measurement for isolating MS1 signals for subsequent MS2 fragmentation.

DIA window: the scan region, i. e. the area covered in the m/z-IM plane in one IM scan, it is preferably arranged generally diagonally. The term "generally diagonal" is to be interpreted in a broad manner, and in particular does not require that the window extends along a straight line. Instead, the term "generally diagonal" mainly reflects the fact that in the continuous scan region, both the m/z range and the IM range concurrently varied.

Target peptide: A peptide originating from the query search-space. Those could be, for example, all peptides known to exist in the human proteome.

Decoy peptide: Artificially constructed peptides that are added to the search-space. These are primarily used for machine-learning and false discovery estimation to separate the true target peptides (peptides that are actually present in the sample) from the false target peptides (peptides that are not in the sample and therefore behave like the artificially constructed decoy peptides) in the search-space.

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

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

The present invention according to a first aspect thereof relates to a method for the data independent acquisition (DIA) targeted peptide precursor identification from sample mass spectrometry intensity data acquired as a function of mass to charge ratio (m/z), of retention time (RT) as well as of ion mobility (IM).

This is done using data having been acquired by introducing precursor ions from said sample into an 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 m/z window, fragmenting the precursor ions transmitted through said mass filter to generate fragment ions, carrying out a mass spectrometry measurement on said fragment ions, wherein each fragment ion is associated with a mass window and an ion mobility (IM) range, and associating detected fragments with its corresponding precursor ion.

Said ion mobility separator and said mass filter are controlled in a synchronized manner such as to carry out a plurality of ion mobility scans, during which precursor ions of increasing or decreasing ion mobility (IM) are successively released from said ion mobility separator, and during which the mass window of said mass filter is shifted continuously or stepwisely towards lower or higher m/z values, respectively.

For a given value of RT, the resulting data comprise at least two mass windows (DIA windows) of said mass filter, each attributable with a DIA window index, and each mass window comprising a multitude of micro scans performed with a specific precursor m/z isolation width at a specific ion mobility, each attributable with an isolation index.

In order to allow the querying of fragment ions of a given precursor ion, all ions of the MS2 scans at every retention time point are associated with their ion mobility and isolation indices and stored in a (at least) 5D data matrix comprising of or consisting of the dimensions m/z, intensity, ion mobility index, isolation index and DIA window. This 5D data scan may act as a function that takes m/z tolerance, ion mobility tolerance, and precursor m/z to return an intensity.

For associating detected fragments with its corresponding precursor ion, the data of the at least two mass windows are combined into an (at least) 5D data set (for the given value of RT) as a function of mass to charge ratio (m/z), of intensity, of ion mobility (index) (IM), of DIA window (index) and of isolation index.

According to a first preferred embodiment of this method, for associating detected fragments with its corresponding precursor ion, from the 5D data set, for each target (and decoy) peptide precursor from a spectral library, ion traces are extracted, scores are calculated, preferably for a plurality of scores, and preferably for at least one of or all of complete extracted ion chromatograms (XIC) and partial extracted ion chromatogram and mobilograms, targets are separated from decoys, preferably by using calculated scores, including based on at least one of correlation, peak shape, intensity, in particular by using machine learning, precursor ions and/or peptides are identified, preferably by target/decoy based false discovery rate analysis.

Mass windows that are associated with consecutive mass spectrometry measurements of target ions do preferably not overlap with each other.

The area covered in the m/z-IM plane in one IM scan is normally arranged generally diagonally.

Again, the term "generally diagonal" is to be interpreted in a broad manner, and in particular does not necessarily require that the frame extends along a straight line. Instead, the term "generally diagonal" mainly reflects the fact that in the continuous scan region, both the m/z range and the IM range concurrently varied. For example, the shapes of the diagonal slices do not need to be linear but can be polynomial.

From the 5D data set, preferably for each target and decoy peptide precursor from a spectral library, preferably ion traces are extracted in the form of complete extracted ion chromatograms, by iterating over all 5D IM scans that were measured within a given RT range of interest for a given ion starting from first to last scan in the RT dimension, wherein during the iteration process, for each 5D IM scan a first module determines a start index of peak to iterate over by performing a preferably binary search of the lower bound of the m/z window to be used for the fragment ion of pre-sorted peaks by m/z in the 5D IM scan, till it either encounters the last peak in the array or a peak whose m/z is higher than the upper bound of the m/z window, wherein at each iteration step, a module checks if the current peak’s IM index falls within the IM window and if the m/z of a parent peptide precursor is within the isolation range of the micro scan in which this peak was measured, and if yes, then the intensity of the peak is added to a running sum and then the peak index is incremented, and if false, then directly the peak index is incremented, then it goes back to the first module and keeps repeating till the iteration is finished.

The procedure preferably involves a step of ensuring that only peaks measured in a specific DIA window are used for making the extracted ion chromatograms (XIC) in the RT dimension.

According to yet another preferred embodiment, from the 5D data set, preferably for each target and decoy peptide precursor from a spectral library, ion traces are extracted in the form of complete mobilograms, by looking up the IM scan that corresponds to a specific RT for which said mobilogram is to be created, preferably this is done for the apex RT point of a extracted ion chromatograms wherein in the resulting 5D IM scan, a preferably binary search is performed to find the peak index such that it is closest to the lower bound of the m/z window but still within its range, then exit criteria are checked for the current peak index, wherein the exit criteria is true if either the current peak index is larger than the index of the last peak in the 5D IM scan or the m/z of the peak at the current peak index is not within the m/z window, and if the exit criteria are not met, then it is checked if the current peak’s IM index falls within the IM window and if the m/z of the parent peptide precursor is within the isolation range of the micro scan in which this peak was measured, wherein if true, then the intensity value of the current peak is added to the mobilogram array for the IM index of the current peak and then the current peak index is incremented, if false, then the current peak index is incremented, again checking the exit criteria given the new peak index.

From the 5D data set, preferably for each target and decoy peptide precursor from a spectral library, ion traces can be extracted in the form of partial mobilograms, by looking up the IM scan that corresponds to a specific RT for which said mobilogram is to be created, preferably this is done for the apex RT point of an extracted ion chromatograms wherein in the resulting 5D IM scan, a preferably binary search is performed to find the peak index such that it is closest to the lower bound of the m/z window but still within its range, then exit criteria are checked for the current peak index, wherein the exit criteria is true if either the current peak index is larger than the index of the last peak in the 5D IM scan or the m/z of the peak at the current peak index is not within the m/z window, and if the exit criteria are not met, then it is checked if the current peak’s IM index falls within the IM window and if the m/z of the parent peptide precursor is within the isolation range of the micro scan in which this peak was measured, wherein if true, then the intensity value of the current peak is added to the mobilogram array for the IM index of the current peak and then the current peak index is incremented, if false, then the current peak index is incremented, again checking the exit criteria given the new peak index wherein the procedure involves a step of ensuring that only peaks measured in a specific DIA window are used for making the mobilogram.

For the analysis of the 5D data set, data can be loaded by virtual batches of arbitrary m/z width.

The data is preferably a set of data independent acquisition data obtained from a sample, preferably a digestive proteomic sample, in an LC-MS/MS experiment.

The data preferably is in the form of sample mass spectrometry intensity data acquired as a function of mass to charge ratio (m/z), of retention time (RT) as well as of ion mobility (IM) determined using an LC tandem mass spectrometry method, preferably LC-DIA.

Further preferably the data is a set of data independent acquisition data obtained from a sample in an LC-MS/MS experiment and wherein the sample is a complex mixture of at least one protein of interest and further proteins and/or other biomolecules in the form of a complex native biological matrix which has been digested prior to LC-MS/MS analysis.

The at least one protein of interest can be a protein based exclusively on proteinogenic amino acids, or is based on proteinogenic amino acids and carries post-translational modifications.

The ion mobility separator is preferably a TIMS analyzer, preferably a TIMS analyzer with parallel accumulation and separation, in particular operating using a method comprising the steps:

(a) accumulating ions 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.

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.

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 has already achieved, with reduced scan speeds, ion mobility resolutions up to Rmob = 400, which is extraordinarily high.

At the beginning of one LC observation retention time window, preferably in the range of 1- 15 seconds, particularly preferably in the range of 3-10 seconds, preferably a survey scan is taken, and wherein in that survey scan a full ion mobility width of interest and a full m/z width of interest is scanned, and wherein as a function of that survey scan for the remainder of said LC observation window said second ion mobility separator and said mass filter are controlled in a synchronized manner such as to carry out a plurality of IM scans, during which precursor ions of increasing or decreasing IM are successively released from said IMS, and during which the mass window of said mass filter is shifted continuously or stepwisely towards lower or higher m/z values, respectively, to avoid peptides not of interest identified in the survey scan, and wherein said step of associating a detected fragment with its corresponding precursor ion is based on determining or utilizing the corresponding mass windows and IM ranges associated with various occurrences of said fragment in said mass spectrometry measurement

According to yet another aspect of the present invention, it relates to the use of a method according to any of the above aspects for the determination of at least one of the composition of the sample including quantitative information about the constituents, or a medically relevant conformation of the constituents, for the determination or the influence of protein-based drugs, for the influence of drugs or other ligands on proteins, or for quality control of protein-based pharmaceutical preparations.

Finally, the present invention relates to a computer program product to analyse data using the method as detailed above or a computer-readable medium having stored thereon such a computer program product.

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

BRIEF DESCRIPTION OF THE DRAWINGS

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,

Fig. 1 illustrates a schematic depiction of the ion cloud (ellipse) with ion mobility on the y-axis and m/z in the x-axis using standard dia PASEF measurement schemes, where the vertical boxes depict DIA windows;

Fig. 2 illustrates in a) a schematic depiction of the ion cloud (ellipse) with ion mobility on the y-axis and m/z on the x-axis using synchro-PASEF schemes, where DIA windows are represented by diagonal boxes, in b) a schematic depiction of how the present invention conceptually treats the method depicted in Fig. 1 ;

Fig. 3 illustrates in a) an example ion trace of fragments belonging to peptide in the RT dimension across all DIA windows, in b) an example ion trace of fragments belonging to peptide in the IM dimension by using merged diagonal scan at the retention time when the peptide has apex intensity, in c) an example ion trace of fragments belonging to peptide in the RT dimension per each DIA window in which it was measured, in d) an example ion trace of fragments belonging to peptide in the IM dimension by using merged diagonal scan at the retention time when the peptide has apex intensity and individual DIA windows in which it was measured separately;

Fig. 4 illustrates in a) the 5D IM scan 400 and its relationship with the raw data coming from the mass spectrometer, in b) describes the input that may be required in one implementation, to query the 5D IM scan in an efficient manner, in c) describes a high-level flowchart depicting one implementation^ in d) describes one implementation of the flowchart used to create the complete XIC, in e) describes one implementation of the flowchart which is used to create a partial XIC, in f) describes one implementation of the flowchart for module which is used to create a complete mobilogram, in g) describes one implementation of the flowchart used to create a partial mobilogram;

Fig. 5 in a) illustrates an example to construct a complete XIC for a fragment ion from a 5D IM scan, in b) illustrates one implementation as a method to construct an ion trace for a fragment ion from a 5D IM scan;

Fig. 6 illustrates in a) - c) examples to construct a partial mobilogram for a fragment ion from a 5D IM scan;

Fig. 7 illustrates one implementation of a method to load data by virtual batches to improve memory footprint of data processing pipeline.

DESCRIPTION OF PREFERRED EMBODIMENTS

In one implementation of the present invention, it operates by first converting the mass spectrometry data into a series of 5D ion mobility (IM) scans with dimensions m/z [401], intensity [402], IM index [403], isolation index [404], and DIA window [405], In order to produce a 5D scan from the measurements for one value of RT (or rather RT window, or single time-point), the collective m/z-intensity pairs from all individual MS2 DIA windows

204, 205, 206 are combined into one list. Each data-point in this list will be annotated with the parent DIA window index 405 (identifying from which of the original DIA windows 204,

205, 206 the data point originated), the actual isolation this m/z-intensity pair was subjected to (encoded as an isolation index referring to the master table (depicted in Fig. 4a) and the corresponding ion-mobility (also encoded as IM index 402 referring to a master-table of all ion-mobility values. The resulting list is then sorted by m/z (401) and stored in a matrix of dimensions n times 5. Fig. 4a, illustrates an example 5D IM scan at a specific RT point 406. Then during the targeted analysis with a spectral library (predicted or empirical), for each peptide precursor the data is queried (Fig. 4d-4g). The data is queried in two ways.

First, the data is queried by treating the data as if it were acquired by a single diagonal DIA window (Fig. 4d, 4f). This is achieved by not using any DIA window filter in the query. Second, the data is queried by using a DIA window filter to acquire ion traces per diagonal DIA window (Fig. 4e, 4g). These queries are for creating ion traces in both the retention time (RT) and in the ion mobility (IM) dimensions, giving four different types of ion traces, each can be with partially different information. Further, the four types of ion traces can also be combined. The resulting ion traces can be used for scoring in different manners (e.g., correlation score, shape score, etc.) as it is typically done in targeted analysis (Fig. 4c). Additionally, we also describe a novel method to process this type of data by virtual blocks for an efficient usage of memory (Fig. 7).

Fig. 1 illustrates a schematic depiction of the ion cloud (ellipse) with ion mobility on the y- axis 101 and m/z in the x-axis 102. The vertical boxes depict DIA windows which are measured in one cycle consisting of 8 full TIMS ion mobility scans. DIA windows with contiguous boxes in the ion mobility dimension depict one full TIMS scan 104a, 104b. Each full TIMS scan is a collection of micro scans as shown for 104a and 104b and can be considered as an array of ion mobility indices 103 when m/z dimension is ignored. The m/z isolation range for all micro scans in a DIA window is the same and unique to that DIA window. An illustration of a peptide 105 that is measured in DIA window 104a is shown. The vertical DIA window boxes are geometrically “awkward” in following the ion cloud and leads to inefficient sampling of the ion cloud.

Fig. 2a illustrates a schematic depiction of the ion cloud (ellipse) with ion mobility on the y- axis 201 and m/z on the x-axis 202. Unlike in figure 1 , DIA windows are represented by diagonal boxes 204, 205, 206. These diagonal DIA windows can be overlapping or nonoverlapping in m/z dimension. Each diagonal DIA window represents a full TIMS scan and is a collection of micro scans. However, in this schema, each micro scan in a DIA window can have different isolation ranges in both ion mobility 201 and m/z dimensions 202. This allows efficient sampling of the ion cloud by following its natural shape which is not possible with the vertical boxes with the same m/z isolation range as depicted in Fig. 1. In this type of schema, a peptide is typically expected to be measured by multiple DIA windows. An illustration of a peptide 200 that is measured in all three DIA windows, 204, 205, and 206, is shown. In one variation, a small number of consecutive micro scans in a diagonal DIA window can have the same m/z range, but different IM range.

In a classical dia-PASEF method (Fig. 1), the acquisition software will in each DIA window measure a fixed m/z isolation window 106 over a certain range in ion mobility 101. This results in several rectangular regions for which MS2 ions were acquired, illustrated by the boxes 104a 1 104b. In contrast to that, a simple flavour of diagonal dia-PASEF moves the isolation window with each increment in the ion mobility dimension 201 by a fixed amount. This results in the signature rhomboid shape of the acquired MS2 windows (Fig. 2a).

Fig. 2b illustrates a schematic depiction of how the present invention conceptually treats the method depicted in Fig. 2a. Multiple diagonal DIA windows 204, 205, 206 are merged as if there was only one DIA window 207 per MS1 cycle, but this is done in a manner that there is no information loss. This allows seamless extraction of fragment ion traces for a peptide precursor ion that is measured by multiple diagonal DIA windows in both RT and ion mobility dimensions. An illustration of peptide 200 is shown which can now be treated as if it was measured in a single DIA window 207. Additionally, we can also extract fragment ion traces for peptide 200 per DIA windows, 204, 205, 206, that it was measured in, as we still have this information on hand.

Fig. 3a illustrates an example ion trace of fragments belonging to peptide 200 in the RT dimension across all DIA windows. It is built by extracting data from several merged diagonal scans 207 that were measured between time point 13.25 mins and 13.50 mins. We refer to this as a complete XIC 301 as it is based on all measurements related to this peptide across both retention time and ion mobility dimensions.

Fig. 3b illustrates an example ion trace of fragments belonging to peptide 200 in the IM dimension by using merged diagonal scan 207 at the retention time when the peptide has apex intensity (13.375 min). Similar mobilograms can be created from other measurement time points in the RT dimension. We refer to this as complete mobilogram 302 as it is based on all measurements related to this peptide at a given retention time point.

Fig. 3c illustrates an example ion trace of fragments belonging to peptide 200 in the RT dimension per each DIA window in which it was measured. Partial XIC 303 is built by extracting data from several merged diagonal scans by only selecting data that was measured by a specific diagonal DIA window. 301 can be thought of as a sum of all these individual partial XICs. However, there may be additional information available by looking at individual partial XIC as they may potentially have different interferences. In another implementation, they can also be concatenated across DIA windows, and additionally across RT dimension, as two additional variations of mobilograms that can also be used for scoring.

Fig. 3d illustrates an example ion trace of fragments belonging to peptide 200 in the IM dimension by using merged diagonal scan 207 at the retention time when the peptide has apex intensity and individual DIA windows in which it was measured separately. Similar mobilograms can be created from other measurement time points in the RT dimension. We refer to this as partial mobilogram 304 as it is based on measurements related to this peptide at a given retention time point and specific DIA window.

Fig. 4a illustrates the 5D IM scan 400 and its relationship with the raw data coming from the mass spectrometer. In one implementation, at every given RT position, the mass spectrometer can separate the compounds by their ion mobility. The data representing a full IM scan is recorded in multiple micro scans where each micro scan corresponding to a specific ion mobility 403 (the actual measured ion mobility value 419 is used in an indexed form, indexed as given in the corresponding master table 425) is a collection of measured peaks with an associated m/z dimension 401 and intensity 402. Additionally, for each micro scan, a specific m/z range is used for isolating the precursor ions 407 which can be encoded as an isolation index 404 referring back to the actual range isolation used based on a global isolation master table 418. Finally, we can encode the DIA window 405 in which a specific micro scan was measured. These dimensions can be implemented as one single multidimensional array with 5 dimensions as shown here. In one embodiment, a single full IM scan can consist of hundreds of micro scans, each with hundreds of data points. In another embodiment, the micro scans can also be merged (array for data handling keeping the individual attribution information). In yet another embodiment, each micro scan in a full scan can be represented by its ion mobility scan index 403 as a placeholder for the actual ion mobility value, precursor isolation index 404 as a placeholder for the actual parent precursor isolation m/z range 407, and DIA window 405. In one implementation, different micro scans are converted into a single 5D IM Scan 400 by merging all of the individual micro scans in the full IM scan into a single array with five dimensions, m/z 401 , intensity 402, IM index 403, precursor isolation index 404, and DIA window 405. This 5D IM scan can be sorted by m/z 401 for fast m/z-based access.

Fig. 4b describes the input that may be required in one implementation, to query the 5D IM scan to generate 301 , 302, 303, and 304 in an efficient manner. The windows (ranges) 410 (m/z window), 411 (RT window), and 412 (IM window) are typically determined empirically based on a pre-analysis to determine the most optimal tolerance windows.

Fig. 4c describes a high-level flowchart depicting one implementation of the role that the modules 420, 421 play in the context of the other modules which are typically used in targeted analysis of DIA data 422, 423, and 424. The modules described in this implementation are responsible for converting the raw data into 5D IM scans 420 and then querying these scans to create ion traces of different types 301 , 302, 303 and 304 using module 421 for each target and decoy peptide precursors. In order to produce a single 5D scan from a single time-point, the collective m/z-intensity pairs from all individual MS2 frames 204, 205, 206 are combined into one list. Each data-point in this list will be annotated with the parent DIA window index 405, the actual isolation this m/z-intensity pair was subjected to (encoded as an isolation index referring to the master table) and the corresponding ion-mobility (also encoded as IM index 402 referring to a master-table of all ion-mobility values). The resulting list is then sorted by m/z (401) and stored in a matrix of dimensions n x 5. Thereafter, these ion traces are scored using a variety of different scores based on correlation, peak shape, etc. and used for identification of peptide precursors in a manner that is already known in the field. Without the encoded information about the parent isolation for each data-point, the query for extracting XIC data would also include datapoints that could not have come from the query ion in question. This results in cleaner (less noisy) data for subsequent data analysis.

Fig. 4d describes one implementation of the flowchart for module 421a (for an example of 421a see Fig. 5a) which is used to create the complete XIC 301. The novel method iterates over all 5D IM scans that were measured within the RT range of interest for a given ion 411 starting from first to last scan in the RT dimension. During the iteration process 430, for each 5D IM scan, module 431 determines the start index of peak to iterate over by performing a binary search of the lower bound of the m/z window to be used for the fragment ion 410 of the pre-sorted peaks by m/z in the 5D IM scan 401. 432 will iterate till it either encounters the last peak in the array or a peak whose m/z is higher than the upper bound of the m/z window 410. At each iteration step, module 433 checks if the current peak’s IM index 403 falls within the IM window 412 and if the m/z of the parent peptide precursor 408 is within the isolation range of the micro scan in which this peak was measured 407. If yes, then the intensity of the peak is added to a running sum 434 and then it increments the peak index 435. If false, then it directly increments the peak index 435. Then it goes back to module 431 and keeps repeating till the iteration is finished. At this point, module 436 sets current running sum as the intensity value for the current RT in the XIC array and resets the running sum to zero. Module 437 and 430 repeats this entire process till the last 5D IM scan is processed.

Fig. 4e describes one implementation of the flowchart for module 421 b which is used to create a partial XIC 303. One difference from 421a is that it introduces a submodule 440 which ensures that only peaks measured in a specific DIA window are used for making the XIC in the RT dimension.

Fig. 4f describes one implementation of the flowchart for module 421c which is used to create a complete mobilogram 302. Module 441 looks up the 5D IM scan that corresponds to a specific RT for which mobilogram is to be created. Typically, this is done for the apex RT point of a XIC, but it can be done for all or another subset of measurement points in the RT dimension. In the 5D IM scan, a binary search is performed to find the peak index such that it is closest to the lower bound of the m/z window 410 but still within its range. Then in module 443, the exit criteria are checked for the current peak index. Exit criteria is true if either the current peak index is larger than the index of the last peak in the 5D IM scan or the m/z of the peak at the current peak index is not within the m/z window 410. If the exit criteria are not met, then module 444 checks if the current peak’s IM index 403 falls within the IM window 412 and if the m/z of the parent peptide precursor 408 is within the isolation range of the micro scan in which this peak was measured 407. If true, then the intensity value of the current peak is added to the mobilogram array for the IM index of the current peak 445 and then the current peak index is incremented 446. If false, then the current peak index is incremented 446. Module 443 again checks for the exit criteria given the new peak index and the entire process 444-446 is repeated. When exit criteria is met, the method comes to stop.

Fig. 4g describes one implementation of the flowchart for module 421 d which is used to create a partial mobilogram 304 (for an example of 421 d see Fig. 6a). One difference from 421 is the module 447 which ensures that only peaks measured in a specific DIA window are used for making the mobilogram.

Fig. 5a illustrates an example of 421a to construct a complete XIC for a fragment ion from a 5D IM scan 520 using an example. Similar to the other implementations and examples mentioned in this disclosure, this example is intended to be non-limiting and for illustrative purposes only. In one implementation, for each full IM scan at a given RT, a 5D IM scan 520 is created. An ion trace 518, 519 is built for a fragment belonging to a parent precursor ion with the specified expected m/z 500, retention time window 503, and ion mobility window 504. The example fragment has a specific expected m/z 501 and m/z window 502. Each 5D IM scans within the specified RT window 503 is processed in an iterative manner. Here we walk through an example 5D IM scan 520 at RT index of 30. The inventive method will first find the starting position corresponding to the lower bound of the m/z window using a binary search in the pre-sorted m/z dimension 512. Then it will iterate through each position, starting from the first index, in the array till the ending position 513 where the m/z 506 is still within the m/z window 502. For each position it iterates, it will sum up all intensity values where the IM index 508 is within the expected IM window 504 and isolation index 509 corresponds to the parent isolation range 511 that encompasses the targeted precursor’s m/z 500. Even though in this example, for simplicity, only one isolation index corresponds to the targeted precursor (index 2). In practice, there can be several isolation indices where the parent precursor was measured. In this example, cells at position [i,3] and [i,5] match all the criteria and their intensities 507 are summed up 514 to give a single data point 515 for the fragment ion’s intensity 517 at the current RT position 516. Tracing such data points over the entire range of the RT window 501 for all the fragment ions belonging to a peptide precursor will create the complete XIC for 301 .

Fig. 5b illustrates one implementation as a method to construct an ion trace for a fragment ion from a 5D IM scan using an example. Continuing from the example in Fig. 5a, we iterate to the next 5D IM scan 527 at RT index of 31. Following the same process as described in Fig. 5a, three data points at [i,4], [i,5] and [i,6] are summed up 523 to get the intensity of the fragment ion 524 at RT index 516 of 31 . The updated XIC figure with trace derived thus far based on RT index of 30 and 31 is shown as 526.

Fig. 6a illustrates an example of 421 d to construct a partial mobilogram 304 for a fragment ion from a 5D IM scan. Initially, a two-dimensional mobilogram trace array 606 is initialized with a length based on the range of IM window 605. IM index 607 in the array is initialized to the range of the IM window (12 to 19) with intensity values set to 0. Next, we find the starting position corresponding to the lower bound of the m/z window using a binary search in the pre-sorted m/z dimension 512. Then it will iterate through each position, starting from the starting index, in the array till it meets the exit criteria 443. At the starting position ([i, 1] in this example), it checks the acceptance criteria 444 and 447 are true. In this example, this translates to 1) isolation index 614 equals to 2 because target precursor 600 is encompassed by the precursor isolation 617 of 520-580, 2) IM index 613 value in the range of 12 to 19, and 3) DIA window 615 equal to 1 604. At the starting position, the acceptance criteria are not met 619 which means the intensity value 608 of 0 is set for [i, j] where j is calculated by the equation 609 (in this example, this is j = 5).

Fig. 6b illustrates an example of 421 d to construct a partial mobilogram 304 for a fragment ion from a 5D IM scan. Continuing from Fig. 6a, the current position 621 is incremented by 1. The acceptance criteria are passed 622 and intensity value 608 for [i,3] 623 is set to the intensity value 612 at current position.

Fig. 6c illustrates an example of 421 d to construct a partial mobilogram 304 for a fragment ion from a 5D IM scan. Continuing from Fig. 6b, we skip few iteration points and consider the last position 631 before the exit criteria is met. The acceptance criteria are passed 632 and intensity value 608 for [i,0] 633 is set to the intensity value 612 at current position. The resulting mobilogram 635 is the partial ion trace of a single fragment ion for DIA window equal to 1 .

Fig. 7 illustrates one implementation of a method to load data by virtual batches to improve memory footprint of data processing pipeline. There is a higher memory cost associated with processing the data as depicted in Fig. 3. We present a novel method of batchwise loading which is facilitated by the 5D IM scan data structure as described in Fig. 4a. Instead of loading all micro scans belonging to a 5D IM scan, depicted by rectangular blocks here, in memory, we can load them by virtual blocks of arbitrary m/z width. Two examples of such virtual blocks are shown in the figure 803, 804. Each block has micro scans associated to it which fall fully or partially within the boundaries of the block. To process block 803, all micro scans of type 805 and 807 would have to be loaded. Similarly, to process block 804, all micro scans of type 806 and 807 would need to be loaded. Micro scans belonging to block 807 would have to be loaded at least twice as they are associated with both blocks. The benefit of this approach is that not all micro scans must be loaded in memory at the same time.

Terminology:

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

2. Micro scan: A MS1 or MS2 scan performed with a specific precursor isolation width at a specific ion mobility and retention time. The x-axis is m/z and y-axis is intensity.

3. Mobilogram: Trace of an ion in the ion mobility dimension. It is usually generated by tracing an ion in a single 5D scan. The x-axis is a metric for ion mobility (e.g. reversed ion mobility, collision cross section) and y-axis is intensity.

A person with ordinary skill in the art will understand that in addition to proteomics, this invention can also be applicable to other mass spectrometer-based omics data, including metabolomics. Also, a person with ordinary skill in the arty will understand that this invention can be used for other similar methods in addition to synchro-PASEF or midia-PASEF. The specific mention of synchro-PASEF and midia-PASEF in this description are for illustrative purposes only and not intended as a limitation. A person skilled in the art will also understand that the present system and method can be extended or applied to any additional dimension.

While certain aspects of the present invention have been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims. It will also be understood that the components of the present disclosure may comprise hardware components or a combination of hardware and software components. The hardware components, methods, and workflows may comprise any suitable tangible components that are structured or arranged to operate as described herein. Some of the hardware components may comprise processing circuitry (e.g., a processor or a group of processors) to perform the operations described herein. The software components may comprise code recorded on tangible computer-readable medium. The processing circuitry may be configured by the software components to perform the described operations. It is therefore desired that the present embodiments be considered in all respects as illustrative and not restrictive.

LIST OF REFERENCE SIGNS

DDA data dependent acquisition

DIA data independent acquisition

IM ion mobility iRT indexed retention time m/z mass to charge ratio

MRM multiple reaction monitoring

MS1 first spectral dimension in LC-MS/MS experiment

MS2 second spectral dimension in LC-MS/MS experiment

PASEF parallel accumulation serial fragmentation

RT retention time

SRM Selected Reaction Monitoring

TIMS trapped ion mobility spectrometry

XI C extracted ion chromatogram

101 y-axis, ion mobility

102 x-axis, mass to charge ratio

103 ion mobility index

104 micro scan

105 illustrated peptide

106 m/z isolation window for one DIA window

200 illustrated peptide

201 y-axis, ion mobility

202 x-axis, mass to charge ratio

203 ion mobility index

204-206 diagonal DIA windows

207 merged diagonal DIA windows/scans for analysis

301 complete XIC

302 complete mobilogram

303 partial XIC

304 partial mobilogram 400 5D IM scan example

401 m/z dimension

402 intensity

403 specific ion mobility

404 isolation index

405 DIA window

406 RT point

407 precursor isolation (m/z)

408 m/z of the parent peptide precursor

409 m/z of fragment ion

410 m/z window (range)

411 RT window (range)

412 IM window (range)

413 DIA window filter

418 isolation master table

419 ion mobility value

420 merge to 5D data

421 extract ion traces

422 calculate scores

423 separate using scores

424 peptide identification

425 ion mobility master table

430 iteration process

431 find start peak index

432 junction

433 junction

434 add intensity

435 increment index

436 set intensity value

437 junction

440 sub module

441 look up 442 find index

443 junction

444 junction

445 set intensity

446 increment index

447 junction

500 targeted precursor

501 extract fragment

502 m/z window

503 RT window

504 IM window

505 DIA window filter

506 m/z

507 intensity

508 IM index

509 isolation index

510 DIA window

511 precursor isolation

512 starting position

513 ending position

514 sum

515 data entry intensity

516 RT index

518 XI C array

519 XIC

520 5D IM scan

523 sum

524 data entry intensity

525 XIC array

526 XIC

527 5D IM scan

600 target precursor

601 extract fragment

602 m/z window 603 IM window

604 DIA window

605 mobilogram trace length

606 mobilogram trace

607 IM index

608 intensity

609 index

610 5D IM scan

611 m/z

612 intensity

613 IM index

614 isolation index

615 DIA window

616 index

617 precursor isolation

618 starting position

619 fail

620 mobilogram

621 current position

622 pass

623 index

624 mobilogram trace

625 mobilogram

631 current last position

632 pass

633 index

634 mobilogram trace

635 mobilogram

801 ion mobility

802 m/z

803-804 virtual blocks

805-807 micro scans

Claims

1. Method for the data independent acquisition (DIA) targeted peptide precursor identification from sample mass spectrometry intensity data acquired as a function of mass to charge ratio (m/z), of retention time (RT) as well as of ion mobility (IM), using data having been acquired by introducing precursor ions from said sample into an 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 m/z window, fragmenting the precursor ions transmitted through said mass filter to generate fragment ions, carrying out a mass spectrometry measurement on said fragment ions, wherein each fragment ion is associated with a mass window and an ion mobility (IM) range, and associating detected fragments with its corresponding precursor ion, wherein said ion mobility separator and said mass filter are controlled in a synchronized manner such as to carry out a plurality of ion mobility scans, during which precursor ions of increasing or decreasing ion mobility (IM) are successively released from said ion mobility separator, and during which the mass window of said mass filter is shifted continuously or stepwisely towards lower or higher m/z values, respectively, wherein, for a given value of RT, the resulting data comprise at least two mass windows (204-206) of said mass filter, each attributable with a DIA window index (405), and each mass window (204-206) comprising a multitude of micro scans (104) performed with a specific precursor m/z isolation width at a specific ion mobility, each attributable with an isolation index (404), wherein for associating detected fragments with its corresponding precursor ion, the data of the at least two mass windows (204-206) are combined into an at least 5D data set (207) as a function of mass to charge ratio (m/z), of intensity, of ion mobility index (IM), of DIA window index (405) and of isolation index (404).

2. Method according to claim 1 , wherein for associating detected fragments with its corresponding precursor ion, from the 5D data set (207), for each target and decoy peptide precursor from a spectral library, ion traces are extracted (421), scores are calculated (422), preferably for a plurality of scores, and preferably for at least one of or all of complete extracted ion chromatograms (XIC) and partial extracted ion chromatogram (XIC) and mobilograms, targets are separates from decoys (423), preferably by using calculated scores, including based on at least one of correlation, peak shape, intensity, in particular by using machine learning, precursor ions and/or peptides are identified, preferably by target/decoy based false discovery rate analysis.

3. Method according to any of the preceding claims, wherein mass windows that are associated with consecutive mass spectrometry measurements of target ions do not overlap with each other.

4. Method according to any of the preceding claims, wherein the area covered in the m/z-IM plane in one IM scan is arranged generally diagonally.

5. Method according to any of the preceding claims, wherein from the 5D data set (207), preferably for each target and decoy peptide precursor from a spectral library, ion traces are extracted (421) in the form of complete extracted ion chromatograms (XIC), by iterating over all 5D IM scans that were measured within a given RT range of interest for a given ion starting from first to last scan in the RT dimension, wherein during the iteration process, for each 5D IM scan a first module (431) determines a start index of peak to iterate over by performing a preferably binary search of the lower bound of the m/z window to be used for the fragment ion (410) of pre-sorted peaks by m/z in the 5D IM scan (401), till it either encounters the last peak in the array or a peak whose m/z is higher than the upper bound of the m/z window (410), wherein at each iteration step, a module (433) checks if the current peak’s IM index (403) falls within the IM window (412) and if the m/z of a parent peptide precursor (408) is within the isolation range of the micro scan in which this peak was measured, and if yes, then the intensity of the peak is added to a running sum (434) and then the peak index (435) is incremented, and if false, then directly the peak index (435) is incremented, then it goes back to the first module (432) and keeps repeating till the iteration is finished.

6. Method according to claim 5, wherein the procedure involves a step of ensuring that only peaks measured in a specific DIA window are used for making the extracted ion chromatograms (XIC) in the RT dimension.

7. Method according to any of the preceding claims, wherein from the 5D data set (207), preferably for each target and decoy peptide precursor from a spectral library, ion traces are extracted (421) in the form of complete mobilograms (302), by looking up the IM scan that corresponds to a specific RT for which said mobilogram is to be created, preferably this is done for the apex RT point of a extracted ion chromatograms wherein in the resulting 5D IM scan, a preferably binary search is performed to find the peak index such that it is closest to the lower bound of the m/z window (410) but still within its range, then exit criteria are checked for the current peak index, wherein the exit criteria is true if either the current peak index is larger than the index of the last peak in the 5D IM scan or the m/z of the peak at the current peak index is not within the m/z window (410), and if the exit criteria are not met, then it is checked if the current peak’s IM index (403) falls within the IM window (412) and if the m/z of the parent peptide precursor (408) is within the isolation range of the micro scan in which this peak was measured (407), wherein if true, then the intensity value of the current peak is added to the mobilogram array for the IM index of the current peak (445) and then the current peak index is incremented (446), if false, then the current peak index is incremented, again checking the exit criteria given the new peak index.

8. Method according to any of the preceding claims, wherein from the 5D data set (207), preferably for each target and decoy peptide precursor from a spectral library, ion traces are extracted (421) in the form of partial mobilograms (302), by looking up the IM scan that corresponds to a specific RT for which said mobilogram is to be created, preferably this is done for the apex RT point of a extracted ion chromatograms wherein in the resulting 5D IM scan, a preferably binary search is performed to find the peak index such that it is closest to the lower bound of the m/z window (410) but still within its range, then exit criteria are checked for the current peak index, wherein the exit criteria is true if either the current peak index is larger than the index of the last peak in the 5D IM scan or the m/z of the peak at the current peak index is not within the m/z window (410), and if the exit criteria are not met, then it is checked if the current peak’s IM index (403) falls within the IM window (412) and if the m/z of the parent peptide precursor (408) is within the isolation range of the micro scan in which this peak was measured (407), wherein if true, then the intensity value of the current peak is added to the mobilogram array for the IM index of the current peak (445) and then the current peak index is incremented (446), if false, then the current peak index is incremented, again checking the exit criteria given the new peak index wherein the procedure involves a step of ensuring that only peaks measured in a specific DIA window are used for making the mobilogram.

9. Method according to any of the preceding claims, wherein for the analysis of the 5D data set (207), data are loaded by virtual batches of arbitrary m/z width.

10. Method according to any of the preceding claims, wherein the data is a set of data independent acquisition data obtained from a sample, preferably a digestive proteomic sample, in an LC-MS/MS experiment.

11. Method according to any of the preceding claims, wherein the data is in the form of a sample mass spectrometry intensity data acquired as a function of mass to charge ratio (m/z), of intensity as well as of ion mobility index (IM) determined using an LC tandem mass spectrometry method, preferably LC-DIA, wherein preferably the data is a set of data independent acquisition data obtained from a sample in an LC-MS/MS experiment and wherein the sample is a complex mixture of at least one protein of interest and further proteins and/or other biomolecules in the form of a complex native biological matrix which has been digested prior to LC-MS/MS analysis, and/or wherein the at least one protein of interest is a protein based exclusively on proteinogenic amino acids, or is based on proteinogenic amino acids and carries post- translational modifications.

12. Method according to any of the preceding claims, wherein the ion mobility separator is a TIMS analyzer, preferably a TIMS analyzer with parallel accumulation and separation, in particular operating using a method comprising the steps:

(a) accumulating ions 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.

13. Method according to any of the preceding claims, wherein at the beginning of one LC observation retention time window, preferably in the range of 1-15 seconds, particularly preferably in the range of 3-10 seconds, a survey scan is taken, and wherein in that survey scan a full ion mobility width of interest and a full m/z width of interest is scanned, and wherein as a function of that survey scan for the remainder of said LC observation window said second ion mobility separator and said mass filter are controlled in a synchronized manner such as to carry out a plurality of IM scans, during which precursor ions of increasing or decreasing IM are successively released from said IMS, and during which the mass window of said mass filter is shifted continuously or stepwisely towards lower or higher m/z values, respectively, to avoid peptides not of interest identified in the survey scan, and wherein said step of associating a detected fragment with its corresponding precursor ion is based on determining or utilizing the corresponding mass windows and IM ranges associated with various occurrences of said fragment in said mass spectrometry measurement

14. Use of a method according to any of the preceding claims for the determination of at least one of the composition of the sample including quantitative information about the constituents, or a medically relevant conformation of the constituents, for the determination or the influence of protein-based drugs, for the influence of drugs or other ligands on proteins, or for quality control of protein-based pharmaceutical preparations.

15. A computer program product to analyse data using the method according to any of the preceding claims 1-13 or a computer-readable medium having stored thereon such a computer program product.