Mass spectrometry method of data-dependent quadrupole operation

Abstract

The invention relates to a method for analyzing a sample comprising one or more molecules of interest by a mass spectrometer, comprising ionizing a multitude of molecules and / or fragments thereof comprised within a sample to obtain a multitude of molecule ions (source ions), and performing mass spectrometry (MS) analysis comprising a. analyzing the molecule ions and / or fragments thereof in a MS1 measurement, thereby acquiring ion mass spectral data of at least a fraction of the molecule ions and / or fragments thereof (source ions) comprising their mass to charge ratio (m / z), b. / c. isolating a first fraction and a second of the molecule ions (precursor ions) having a m / z within a first m / z range (mz1; isolation window) and a second m / z range (mz2; isolation window), using a mass filter device, wherein the first m / z range is selected based on the ion mass spectral data acquired in step a. or one or more iterations of step a. and / or e., and wherein the second m / z range (mz2) overlaps with the first m / z range (mz1), d. fragmenting at least a fraction of the molecule ions (precursor ions) isolated in steps b. and c., separately, to obtain a multitude of biomolecule fragment ions from each of the respective fragmented molecule ions (precursor ions), and e. analyzing the multitude of biomolecule fragment ions in one or more MS2 measurement, thereby acquiring ion mass spectral data of the analyzed biomolecule fragment ions.

Description

[0001] The invention lies in the field of biochemistry and analytical chemistry, particularly in the field of mass spectrometry.

[0002] The invention relates to a method for analyzing molecules of interest from a sample using mass spectrometry, wherein ionized molecules are analyzed in a first MS scan and a plurality or even all obtained precursor ions are subsequently isolated for fragmentation and MS2 analysis by defining overlapping pairs of isolation windows in a data-dependent fashion, thereby enabling deconvolution of the obtained MS / MS data.

[0003] In a preferred embodiments the invention relates to a method for analyzing a sample comprising one or more molecules of interest by a mass spectrometer, comprising ionizing a multitude of molecules and / or fragments thereof comprised within a sample to obtain a multitude of molecule ions (source ions), and performing mass spectrometry (MS) analysis comprising a. analyzing the molecule ions and / or fragments thereof in a MS1 measurement, thereby acquiring ion mass spectral data of at least a fraction of the molecule ions and / or fragments thereof (source ions) comprising their mass to charge ratio (m / z), b. / c. isolating a first fraction and a second fraction of the molecule ions (precursor ions) having a m / z within a first m / z range (mz1; isolation window) and a second m / z range (mz2; isolation window) respectively, using a mass filter device, wherein the first m / z range is selected based on the ion mass spectral data acquired in step a. or one or more iterations of step a. and / or e., and wherein the second m / z range (mz2) overlaps with the first m / z range (mz1), d. fragmenting at least a fraction of the molecule ions (precursor ions) isolated in steps b. and c., separately, to obtain a multitude of biomolecule fragment ions from each of the respective fragmented molecule ions (precursor ions), and e. analyzing the multitude of biomolecule fragment ions in one or more MS2 measurement, thereby acquiring ion mass spectral data of the analyzed biomolecule fragment ions.BACKGROUND OF THE INVENTION

[0004] Mass spectrometry (MS) allows to identify and quantify the compounds present in a sample. Different mass spectrometry methods are available for the analysis of different types of compounds of interest, such as proteomics for the analysis of proteins (e.g., via their tryptic digestion to peptides), metabolomics for the analysis of small molecules and lipidomics for the analysis of lipids. All of these different methodologies are widely applied in research, e.g., MS proteomics allows to gain proteome-wide view of the cell, and with reliable quantification, as opposed to tracking just the levels of view proteins with classical semi-quantitative methods such as Western blotting. For example, modern mass spectrometry allows comprehensive measurement of up to 10,000 proteins in human cell lysates or hundreds to thousands of proteins from human blood plasma, depending on the workflow. Thus, mass spectrometry methods have become invaluable both in basic research, but also in translational approaches. In the latter case, mass spectrometry allows to achieve, e.g., improved comprehensive analysis of patient samples, compared to the standard clinical biomarker assays. For example, upon the onset of the COVID-19 pandemic, the inventors were able to use MS proteomics to rapidly measure hundreds of hospital COVID-19 inpatient's blood plasma samples, to establish highly accurate predictors of the disease outcome in critical patients and gain further insights in COVID-19 progression [PMID: 34139154, PMID: 36812516].

[0005] The highlight of mass spectrometry as a technology is rapid development, driven by new faster and more sensitive mass spectrometers, but equally important, new MS technologies and data processing methods. MS technology advances (i) boost throughput and reduce costs, (ii) improve the data quality and (iii) eventually enable new applications. For example, compared to the state of the art in 2015, it is at present possible to measure samples about 20 times quicker, with the respective reduction in costs, while achieving significantly higher data quality. At the same time, such applications as sub-single-cell proteomics are being established, allowing to, e.g., create spatial proteomic maps (>1000 proteins quantified) of a cancer tissue, with single cell spatial resolution. Such advances open new applications both in basic research, helping to understand the progression of the disease, but also in the clinics, and are one of the potential pillars for personalized medicine in the future.

[0006] In terms of the global MS market a rapid growth is fueled by the emerging applications, including in clinical research and drug discovery, enabled by the new technology developments. The rapidly growing market for mass spectrometry is creating a highly competitive environment, driving the constant need for advancing MS technologies, data processing and analytics.

[0007] In general, two main MS / MS (also referred to as MS2) approaches are commonly used for proteomics, namely data-dependent acquisition (DDA) and data-independent acquisition (DIA). DDA uses narrow (~ m / z 1.0, typically m / z 0.4 - m / z 3.0) isolation windows, determined in a data-dependent manner, wherein preferably the mass spectrometer analyses the data acquired so far and determines the optimal positions (m / z bounds) of the isolation windows. DIA, in contrast, repeatedly cycles through a set of pre-defined wider isolation windows (typically m / z 2.0 - m / z 200.0). DIA has many significant advantages, such as that all peptide ions falling in the m / z range of interest (e.g. m / z 400 to m / z 1000) are consistently fragmented and thus can potentially be identified, provided the signals generated are strong enough. Another advantage is significantly more reliable quantification. However, DIA commonly results in 'complex multiplexed fragment spectra', wherein multiple precursor ions are fragmented simultaneously and share fragment ions, which increases the complexity of data processing and analysis.

[0008] Moreover, while methods to obtain precursor mass information for fragment signals using DIA exist, they lack sensitivity and face technical difficulties, namely precursor ion information can only be obtained through computational deconvolution that relies on the cognate signals being of sufficiently high quality, e.g., using the method Scanning SWATH [PMID: 33767396]. Other modern DIA technologies, e.g., for trapped ion mobility-enabled mass spectrometers, currently do not possess fully established implementations allowing for reliable assignment of precursor masses for fragment signals with high sensitivity. In light of the fast development of the field of mass spectrometry and the persistent interest in increasing sensitivity and specificity of sample analysis, there exists an ongoing need for new mass spectrometry methodologies enabling an advance in target detection, analyte characterization and the processing and analysis of acquired MS-data.SUMMARY OF THE INVENTION

[0009] In light of the prior art the technical problem underlying the present invention is to provide means for mass spectrometry-based target molecule analysis with high sensitivity in the analysis of precursor ions and their fragmentation spectra, including precursor ions generating only moderate or weak intensity signals or generating no signals during MS1 measurements, preferably combined with the ability to determine with high confidence the matching precursor ion species for some of the fragment ions recorded by the mass spectrometer.

[0010] A further aim of the present invention is to provide means and methods that enable the identification and sensitive quantification of a large number of target molecules, e.g., peptides of interest within a sample, independently of the limitations of prior art methods such as DIA or DDA. In other words, an aim of the present invention is to provide means and methods for analyzing and quantifying both, target peptides generating strong precursor ion signals during MS1 measurements, and target peptides generating weak(er) intensity or no precursor ion signals during MS1 measurements, in each case with an improved data quality compared to either of the state of the art DDA or DIA methods.

[0011] This problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

[0012] Therefore the invention relates to a method for analyzing molecules of interest from a sample using mass spectrometry, wherein ionized molecules are analyzed in a first MS1 scan and a plurality or even all obtained precursor ions are subsequently isolated for fragmentation and MS2 analysis by defining overlapping pairs of isolation windows in a data-dependent fashion, thereby enabling deconvolution of the obtained MS / MS data. It was entirely surprising that the present method enables, through the synergistic interaction of its features, the collection of significantly more precise precursor mass information for a proportion of the observed fragment ions, compared to prior art DIA methods, whilst maintaining all the benefits of DIA, compared to prior art DDA methods.

[0013] In one aspect the invention therefore relates to a method for analyzing a sample comprising one or more molecules of interest by a mass spectrometer, comprising ionizing a multitude of molecules and / or fragments thereof comprised within a sample to obtain a multitude of molecule ions (source ions), and performing mass spectrometry analysis comprising a. analyzing the molecule ions and / or fragments thereof in a first mass spectrometry (MS) measurement (MS1), thereby acquiring ion mass spectral data of at least a fraction of the molecule ions and / or fragments thereof (source ions) comprising their mass to charge ratio (m / z), b. isolating a first fraction of the molecule ions (precursor ions) having a m / z within a first m / z range (mz1; isolation window), using a mass filter device, preferably a quadrupole device, wherein the first m / z range is selected based on the ion mass spectral data acquired in step a. or one or more iterations of step a. and / or e., c. isolating a second fraction of the molecule ions (precursor ions) having a m / z within a second m / z range (mz2; isolation window), wherein the second m / z range is selected based on the ion mass spectral data acquired in step a. or one or more iterations of step a. and / or e., wherein the second m / z range (mz2) overlaps with the first m / z range (mz1), d. fragmenting at least a fraction of the molecule ions (precursor ions) isolated in steps b. and c., separately, to obtain a multitude of biomolecule fragment ions from each of the respective fragmented molecule ions (precursor ions), e. analyzing the multitude of biomolecule fragment ions in one or more second mass spectrometry measurements (MS2), thereby acquiring ion mass spectral data of the analyzed biomolecule fragment ions.

[0014] In preferred embodiments steps b.-e. or steps c.-e. are repeated at least once.

[0015] In embodiments, steps b.-e. or steps c.-e. are repeated at least once, wherein in repetitions of steps c.-e. the range mz1 may refer to any of the ranges previously acquired in a step b. or step c.

[0016] In embodiments, step b. and / or c. comprise theoretical considerations with respect to the expected physical properties of precursor ions of interest. In exemplary embodiments comprising the measurement of a large number of peptides of interest, e.g., 1000 peptides, e.g., stored within an inclusion list, the mass spectrometer may align or evaluate m / z values in real-time, thereby considering the already identified peptides, for selecting isolation windows to measure the fraction of peptides of interest that is expected to elute from the column next.

[0017] In preferred embodiments of the step (b.) isolating a first fraction of the molecule ions (precursor ions) having a m / z within a first m / z range (mz1; isolation window), the first m / z range is selected based on the ion mass spectral data (i) acquired in step a., or one or more iterations of step a. and / or e., and optionally (ii) an exclusion list and / or an inclusion list, and wherein in step (c.) isolating a second fraction of the molecule ions (precursor ions) having a m / z within a second m / z range (mz2; isolation window), the second m / z range is selected based on the ion mass spectral data (i) acquired in step a., or one or more iterations of step a. and / or e., and optionally (ii) an exclusion list and / or an inclusion list, and wherein the second m / z range (mz2) overlaps with the first m / z range (mz1). In other words, in embodiments the selection of a first m / z range in step b. and / or a second m / z range in step c. is based (respectively) on ion mass spectral data acquired in a first mass spectrometry (MS) measurement (MS1) and optionally the data comprised within an exclusion list (wherein the data (temporarily or permanently) comprised within the exclusion list preferably indicates masses or data to be ignored or excluded from analysis by the mass spectrometry) and / or an inclusion list.

[0018] In embodiments certain m / z ranges (isolation windows) selected and isolated in steps b. and c. overlap with their neighboring m / z range(s). In embodiments two selected and isolated m / z ranges are referred to as neighboring if (i) they either overlap or the distance between the upper m / z bound of one range is within m / z 2.0 from the lower m / z bound of the other range and (ii) they are selected and isolated in a time period comparable to the time a particular species of molecule ions of interest is present among source ions or an average such time. In embodiments, a second m / z range (mz2) isolated in c. overlaps with a first m / z range (mz1) isolated in b.

[0019] The inventors herein propose a method wherein in preferred embodiments the boundaries of some or all isolation windows may be set in a 'data-dependent' fashion. In preferred embodiments, similar to DIA (data independent acquisition) methods, all peptide ions falling in a m / z range of interest, or more generally, a range defined by boundary conditions imposed on separately or jointly on the physical properties of peptide ions such as the m / z value, the ion mobility and the collisional cross section, are consistently fragmented. In embodiments, the m / z bounds are selected in such a way as to enable deconvolution of the data, to generate in silico 'clean' DDA (data dependent acquisition)-like fragment spectra by processing with suitable software (algorithms). Hence, in embodiments the herein described new acquisition method combines the advantages of DDA and DIA. However, the design of the present method was not obvious or straight forward derivable from a simple combination of DDA and DIA analysis. The synergistic effects achieved by the present method would not be obtained by simply acquiring the sample with DIA and then with DDA instead; or scheduling the determination of isolation window boundaries in DDA-like and DIA-like fashion, for different isolation windows within the same mass spectrometry acquisition. First, such a strategy would not be financially or economically feasible, as this simple combination would not just require twice the time of simple DIA and DDA, but also require double amounts of sample (which is often a critical factor for LC-MS / MS analysis). Most importantly, a simple combination of a DDA and DIA measurement would introduce extra variability of quantities that would render the normalization between DIA and DDA data extremely difficult. Also, both software tools as well as instruments that are presently available are not suitable for performing the present method without further modification.

[0020] In preferred embodiments the present method comprises a MS / MS analysis (MS2), however, compared to classical DDA or DIA methods, the present method preferably employs a different and new approach for the MS2 operation, wherein a plurality or even all precursor ions are repeatedly isolated for fragmentation by controlling the isolation window of the mass filter device, e.g., quadrupole, in a data-dependent fashion, to enable deconvolution of the data. It was entirely surprising that the present method enables, through the synergistic interaction of its features, the collection of significantly more precise precursor mass information for some (or a proportion or portion) of the observed fragment ions, compared to prior art DIA methods, whilst simultaneously enabling the collection of fragment ion information for all precursor ions falling within a range of interest defined by boundaries on their m / z, ion mobility or collisional cross section characteristics, as opposed to prior art DDA methods. In summary, the present method achieves the advantageous effects of both DIA and DDA by a single acquisition method, as the present method enables the identification of most or all peptides which would have been identified with DIA, but at the same time also enables the identification of most or all peptides which would have been identified with DDA, such that the sum of these peptides may be quantified with the quality of quantification inherent to DIA.

[0021] In certain embodiments of the present invention, instead of or besides peptides any other (bio)molecule detectably by mass spectrometry measurement may be analyzed according to the present invention, such that embodiments of the present invention may also be used in / for, e.g., metabolomics measurements (instead of proteomics measurements). In such specific embodiments the term 'peptide' may refer to, or may be considered to mean any '(bio)molecule' (e.g., metabolites, small molecules etc.) to be analyzed, e.g., in the context of a respective mass spectrometry measurement.

[0022] In embodiments, the mass filter, e.g., quadrupole, isolation window boundaries are selected in such a way, as to repeatedly cover a precursor m / z range, such that every m / z value (or m / z and ion mobility value combination) within a respective m / z range is expected to be selected by the mass filter, e.g., quadrupole, at least once during the observed / expected elution of a typical peptide from the chromatography source.

[0023] In embodiments, in the context of the present invention collisional cross section value(s) may be determined and considered for subsequent calculations in addition or alternatively to ion mobility.

[0024] The present invention provides new means for a variety of MS-analysis workflows, e.g., those involving untargeted mass spectrometry, and has strong potential not only for basic research but also for translation, in applications such as, e.g., biomarker discovery, drug screens and analyses of population-scale sample banks.

[0025] In embodiments, during isolation window and / or target m / z value selection (e.g., in one or more steps of a.-e.), an exclusion list and / or a dynamic exclusion is used, wherein the exclusion list preferably comprises certain masses (ions) to be excluded from analysis, e.g., permanently (e.g., for contaminants) and / or for a certain period of time during dynamic exclusion, e.g. for the subsequent 2-100 s after their previous selection, isolation, fragmentation and / or MS2 analysis.

[0026] In some embodiments, an exclusion list and / or a dynamic exclusion is used, wherein the exclusion list preferably comprises certain masses (ions) to be excluded from analysis, e.g., permanently (e.g., for contaminants, throughout the whole of the acquisition or for a limited period of time of the expected elution; with the exclusion potentially limited to a specific range comprised by combinations of m / z, ion mobility and collisional cross section values).

[0027] In embodiments, during isolation window and / or target m / z value selection (e.g., in one or more steps of a.-e.), an inclusion list is used, preferably wherein the inclusion list comprises analytes of (particular) interest.

[0028] In embodiments, the first and / or second m / z range is selected (respectively) based on the ion mass spectral data: (i) acquired in step a. (the previous MS1 measurement), or (i) acquired in one or more iterations of step a, and optionally (ii) data comprised within an exclusion list and / or an inclusion list, wherein, for example, the exclusion list comprises contaminants and / or masses selected within past several seconds, and the inclusion list comprises analytes of interest.

[0029] In embodiments, the first m / z range is selected based on the ion mass spectral data acquired in step a. (MS1 scan) and optionally an exclusion list and / or an inclusion list (thereby determining the m / z values to target / analyze).

[0030] In embodiments the ion mass spectral data in step b. and / or c. is additionally filtered for m / z values which can be (precisely) targeted (e.g., preferably m / z values that are (more or less) evenly spaced within a respective m / z range of interest.

[0031] In embodiments, a multitude of biomolecule fragment ions comprised within said isolation windows, which cover the respective masses, are analyzed in one or more MS2 measurement.

[0032] In embodiments, a first MS1 scan is performed to determine a (very) broad list of m / z values to target. In embodiments, subsequently certain targeted values of interest are selected based on a database search of the so far acquired MS1 and / or MS2 data (e.g., DIA data) to obtain a final set of m / z values (e.g., 10 different m / z values) to be targeted in a range of, e.g., m / z 400-500, or alternatively the whole m / z range of interest. In embodiments, subsequently a number of isolation windows are acquired, wherein consecutive windows overlap with each other and the targeted m / z values are comprised within said overlaps or within the non-overlapping part / portions of the windows, preferably in whichever of the m / z ranges (overlap or non-overlap) is more narrow (has / spans a smaller / shorter m / z range) compared to the other. In embodiments, said procedure is repeated at least for a different m / z range, e.g., spanning in this example m / z 600-800, etc.

[0033] In embodiments, the analysis of ions located within the 'narrower' part / portion (overlaps or non-overlapping part / portion) between neighboring isolation windows and the controlling of the quadrupole in data-dependent fashion, as proposed in the present invention, can significantly empower the deconvolution of the obtained MS data, leading to cleaner in silico generated deconvoluted spectra and thereby to an improved quality of data analysis, target detection and target identification.

[0034] In embodiments, the first and / or second m / z range is selected based on the ion mass spectral data acquired in (i) the last / previous MS1 measurement and / or (ii) an inclusion list and / or an (dynamic) exclusion list that comprises, for example, previously selected masses acquired within the past several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 90, 120, 180 etc.) seconds. In embodiments an (dynamic) exclusion list may (further) comprise contaminants to be excluded from analysis / to be ignored by the mass spectrometer.

[0035] In embodiments, the information / data from the last acquired MS1 scan(s) (from one or more iterations of step a.) are (additionally or separately) used for the selection of the first m / z range in step b. (isolating a first fraction of the molecule ions having a m / z within a first m / z range) and / or c. (isolating a second fraction of the molecule ions having a m / z within a second m / z range).

[0036] In embodiments, the overlap or the non-overlapping window-part of a m / z range (isolation window) selected and isolated in steps b. and c. that is more narrow than the other part preferably comprises at least one target(ed) ion. In embodiments, a m / z range (e.g., from two or more m / z ranges) is `more narrow', if it has / spans a smaller / shorter m / z range compared to the other m / z range(s).

[0037] Preferably, a m / z range (e.g., mz1 or mz2) may be divided or considered to be divided into its 'overlapping' part(s) / overlap(s) (overlapping with one or more neighboring m / z ranges) and the non-overlapping part (i.e., not overlapping with neighboring m / z ranges or a particular neighboring m / z range). In embodiments, the non-overlapping part can be on an one or both 'ends' (terminus) of pair of a m / z ranges. A non-limiting example may be for two windows where the non-overlapping part (i) is only on one terminus: m / z 400-430 and m / z 400-431; or (ii) where the non-overlapping part is on both sides, such that the overlap is located 'central': m / z 400-430 and m / z 410- 420). In embodiments, m / z range(s) (isolation window(s)) selected and isolated in steps b. and c. constitute either the range / part that is overlapping or non-overlapping with its neighboring m / z range(s).

[0038] In embodiments a molecule ion (source ion) and / or fragment ion of a molecule of interest may be referred to as `target ion' or `molecule ion of interest'.

[0039] In preferred embodiments the m / z range / part (either overlapping or non-overlapping with neighboring m / z ranges), comprising one or more target ion, is narrower (regarding its m / z range; having the smaller range) than the range / part that does not comprise the one or more target ion. Preferably the 'narrower' of said parts of the m / z ranges comprises at least one molecule ion of interest (target ion).

[0040] In embodiments of the invention, ions are physically isolated (by the mass spectrometer) from (within) each of the isolation windows, e.g., mz1, mz2..., whereas the consideration if ions are either comprised within the non-overlapping or the overlapping parts of m / z ranges is only performed (in silica) in the context of subsequent in silico analysis.

[0041] In embodiments, the overlap or the non-overlapping window-part of a m / z range (isolation window) selected and isolated in steps b. and / or c. comprising the one or more m / z values characteristic for a molecule ion (source ion) and / or fragment ion of a molecule of interest, is more narrow (e.g., has / spans a smaller / shorter range) than the other window-part, preferably spanning less than 30% of the entire respective m / z range (isolation window). In other words, in embodiments one or more m / z values characteristic for a molecule ion (source ion) and / or fragment ion of a molecule of interest are comprised either within the overlap (overlapping part) or the non-overlapping part (non-overlap) of a m / z range (isolation window), whichever is more narrow, preferably spanning less than 30% of the entire respective m / z range (isolation window), and less than the other part.

[0042] In embodiments, the overlap or the non-overlapping window-part of a m / z range (isolation window) selected and isolated in steps b. and c. comprises a target ion and is more narrow than the other (remaining) part of the m / z range. Preferably the selected and isolated part of the m / z range is narrow, e.g., less than 30%, 25%, 20%, 15%, 10%, or 5% of the total window (m / z range) size / range and / or less than 80%, 75%, 70%, 75%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the other (remaining) part of the m / z range.

[0043] The inventors recently introduced a new data-independent method of controlling the quadrupole depending on the ion mobility, termed Slice-PASEF (Szyrwiel et al., 2022), which contents are incorporated by reference herein. In Slice-PASEF, the precursor ions are fragmented using m / z isolation windows that depend on the ion mobility or collisional cross-section characteristic of said precursor ions, with a single fragmentation scan across an ion mobility range termed frame. Conceptually, Slice-PASEF is a generalization of the earlier introduced diaPASEF concept, with Slice-PASEF lifting the restriction of the originally acquired diaPASEF data [PMID: 33257825], that is that the consecutive isolation windows within a frame must not overlap in the m / z dimension. The free control of isolation window boundaries depending on the ion mobility within the frame, characteristic of Slice-PASEF, enabled acquisition schemes that result in significant advantages compared to diaPASEF, but are characterized by more challenging to process data. In their work on Slice-PASEF, the inventors managed to solve the computational problem of dealing with such data, and reported significant benefits for single cell-level proteomics. Therefrom, the inventors presently developed methods to conceptually improve on this idea. Surprisingly, they found that by controlling a mass filter, e.g., quadrupole, in a data-dependent fashion they can significantly improve data deconvolution, such that basically all or numerous ions within a m / z and ion mobility range of interest (e.g. spanning m / z 400 to m / z 1000) may be (consistently) fragmented and analyzed and thus can potentially be identified by MS / MS analysis, similar to Slice-PASEF and other state-of-the-art DIA methods, but also significantly more precise precursor mass information can be obtained for some of the fragment ions, enhancing confidence in identification of the precursor ions from which these fragment ions have originated - similar to state-of-the-art DDA methods.

[0044] One embodiment of the Slice-PASEF methods developed by the inventors controls the mass filter, e.g. quadrupole, in such a way, that a pair of frames, termed cycle, covers the range (preferably that is m / z and ion mobility region of interest) of precursor ions of interest. In the first frame, for a particular ion mobility value, the quadrupole is set to the mass window [start m / z, x]. In the second frame, it's set to [x, stop m / z]. The start m / z and stop m / z as well as 'x' depend on the ion mobility value, and 'x' is also varied between the cycles, allowing for later computational deconvolution of the data, which nevertheless only has a limited positive effect on eventual data quality, given that the variation of the position of x between the cycles consists of large fixed steps, typically greater than m / z 10 or even m / z 50, and does not allow for confident matching of precursor masses to fragment signals.

[0045] While a highly sensitive method compared to the state-of-the-art DIA alternatives, Slice-PASEF thus suffers from poor separation of precursor ions being selected for fragmentation by their m / z values, exasperating the DIA-typical problem of complex and difficult to deconvolute spectra. This problem originates from the inability of data-independent acquisition in general and Slice-PASEF in particular to adapt the selection of isolation windows to the actual data being observed. In the present invention, this problem is addressed in preferred embodiments by the mass spectrometer adapting the isolation window boundaries placement to allow optimized deconvolution based on the information acquired so far, including based on the MS1 and MS2 data acquired in one or more of the preceding frames and cycles.

[0046] In embodiments of the present invention, the value of x is selected as a function of ion mobility in a data-dependent manner for all or some ion mobility values, for all or some frames, for all or some cycles, to allow data deconvolution for spectra corresponding to all or some ion mobility values.

[0047] In embodiments, the isolation windows mz1 and mz2 that facilitate deconvolution for a particular ion mobility value belong to the same or different cycles and are of the form [start m / z, x1] and [x2, stop m / z], where x1 > x2 and the part x1 - x2 is narrow, preferably less than 0.5, 1.0, 2.0, 3.0, 5.0, 10.0 m / z.

[0048] In embodiments, the isolation windows mz1 and mz2 that facilitate deconvolution for a particular ion mobility value belong to the same or different cycles and are of the form either (i) [start m / z, x1] and [start m / z, x2] or (ii) [x1, stop m / z] and [x2, stop m / z], where the isolation window part with x1 and x2 as boundary values is preferably less than m / z 0.5, 1.0, 2.0, 3.0, 5.0, 10.0.

[0049] In embodiments, the isolation windows mz1 and mz2 that facilitate deconvolution for a particular ion mobility value belong to the same or different cycles and are of the form [x1, y1] and [x2, y2] where at least one of the boundaries of each of the windows is determined in a data-dependent manner and either the overlapping or non-overlapping part, whichever is narrow, contains at least one analyte of interest.

[0050] In embodiments, two or more mass isolation windows are acquired for a given ion mobility value within the time comparable to the elution time of a particular or an average analyte of interest, such that some or both of the boundaries of at least two of those windows are selected in data-dependent fashion. In embodiments, two or more mass isolation windows are acquired for a given ion mobility value within the time comparable to the elution time of a particular or an average analyte of interest, such that (i) some or both of the boundaries of at least two of those windows are selected in data-dependent fashion, and (ii) the partition of the m / z space by the boundaries of the said acquired windows is optimized and / or adapted (accordingly), preferably to achieve the best possible deconvolution, preferably given (in light of) the purpose of the respective experiment. In embodiments, such optimization and / or adaption involves, for example, (1) achieving comparable total MS1 signal within the said parts at the given ion mobility and / or (2) achieving comparable numbers of well-detectable MS1 peaks within the said parts at the given ion mobility and / or (3) achieving comparable numbers of targeted m / z values falling within different parts, from the list of targeted m / z values, preferably narrowed down depending on the relevant retention time or the ion mobility value or (4) achieving a desirable balance between (1)-(3).

[0051] This way, the present invention enables in embodiments window boundaries placement in a way that facilitate deconvolution for mass spectral data that needs it most. For example, if at a particular point in time and at a particular ion mobility value a large number of intense peaks is observed in MS1 spectrum in the m / z range 600-650, whereas few such peaks are observed in the m / z range 650-700, deconvolution will likely have a more prominent effect on the ability of the processing software to identify and accurately quantify the analytes if more isolation window boundaries fall within the range 600-650 than within the range 650-700.

[0052] The present invention was surprisingly able to overcome the shortcoming of Slice-PASEF, that the window placement (in Slice-PASEF) is highly sensitive to ion mobility calibration. In case of poor instrument calibration, the optimal placement of isolation windows in Slice-PASEF is often difficult, cumbersome or even not possible, necessitating frequent and time-intensive recalibration. On the contrary, in the present method this problem is solved by considering MS1 spectra for determining the optimal isolation window placement (which is preferably performed automatically by the mass spectrometer in the context of the present method). This proceeding is also beneficial, when the ion cloud changes with retention time.

[0053] In embodiments, the control of isolation window positioning depending on the ion mobility value can differ or be the same within each frame. In embodiments, the control of isolation window positioning depending on the ion mobility value can differ or be the same within each frame and may comprise less than 10 distinct m / z windows per frame. In embodiments, the control of isolation window positioning depending on the ion mobility value can differ or be the same within each frame and may comprise more than 10, 15 or 25 windows, or more than 100 windows. In embodiments, the control of isolation window positioning depending on the ion mobility value can differ or be the same within each frame and may in embodiments comprise a continuous adjustment of the window boundaries, preferably using a so-called scanning quadrupole or a similar mass filter operation mode, preferably implemented via a continuous change in mass filter configuration, preferably resulting in continuous and not stepped adjustment of isolation window boundaries across the frame. In embodiments of such a 'scanning' method, the present invention allows to combine the benefits of the scanning quadrupole as described in EP3945314A1 with either or both of (i) the generation of high quality deconvoluted spectral data for targeted analytes and (ii) enabling dynamic optimization of isolation window boundary positioning based on the observed data.

[0054] In embodiments, molecule ions (precursor ions) are selected for isolation based on the ion mass spectral data acquired in a MS1 measurement, e.g., in above step a., and / or an inclusion list and / or an exclusion list, wherein in some embodiments, ions are selected that are preferably more or less evenly spaced, such that the m / z range of interest can be covered by a number of windows not exceeding a selected number, whilst the size of said windows does not exceed a selected size limit, with said number and said limit potentially determined dynamically in a data-dependent fashion with expectation to achieve the optimal performance. In embodiments, the isolation windows for fragmentation and subsequent MS2 measurement(s) (e.g., in above step e.), preferably cover one or more or all (expected) masses of interest (e.g., of a molecule, protein or peptide (ion) of interest).

[0055] In embodiments, the isolation windows selected for fragmentation and MS2 measurement (e.g., in one or more of above steps b.-e.) are selected for isolation (e.g., in steps b. and c.) to comprise rather broad m / z range(s), e.g., m / z ≥ 10, or between m / z 2-10, 5-20, 10-100, 50 or 100.

[0056] In embodiments, one or more ions of interest are identified from one or more cycles of MS1 and / or MS / MS measurements (e.g., one or more repetition of above steps a.-e. or b.-e.), wherein rather wide isolation windows, e.g., m / z ≥ 10, or between m / z 4-100, are selected for isolation, and wherein the obtained MS2 or MS / MS data is subjected to a database search (comparison to data in one or more database). Preferably said data base search reveals one or more m / z values of interest for the one or more ions of interest that may be targeted in subsequent cycles of MS2 or MS / MS measurements (e.g., one or more repetition of above steps a.-e. or b.-e.).

[0057] In embodiments, specific m / z values of interest for the one or more ions of interest (e.g., of the one or more molecule of interest) are identified or selected, e.g., either from prior MS1, MS / MS or MS2 data or an inclusion list and / or an exclusion list, and used for the selection of isolation windows for subsequent fragmentation and MS2 measurement(s) (e.g., one or more repetition of above steps b.-e), wherein the isolation windows are selected to comprise one or more of said specific m / z values of interest. In preferred embodiments said isolation windows comprise a wide m / z range, e.g., such as m / z ≥ 10, or between m / z 4-100. In embodiments, the isolation windows (mz1, mz2, ...) comprise specific m / z values of interest within their overlaps (or non-overlaps, whichever range has the more narrow m / z range). Preferably such analysis procedures are repeated for one or more (subsequent) and preferably 'wide' m / z range, e.g., in a first cycle covering m / z 400-500, in a second cycle covering m / z 500-600 etc.

[0058] Such embodiments would resemble a new combination of data-independent mass spectrometry combined with data-dependent mass spectrometry measurements, wherein rather 'wide' isolation windows, as used in DIA (e.g., between m / z 4-200), may be analyzed with higher precision (e.g., sensitivity and / or specificity) than known from DIA, due to the analysis of isolation window overlaps (or non-overlaps) and a certain (intermittent) 'preselection' of ions of interest, which may be 'targeted' or comprised by said overlaps (or non-overlaps).

[0059] In one example of certain embodiments, from MS1 measurement data, e.g., step a., one or more isolation windows for fragmentation and MS2 measurement (e.g., one or more of steps b.-e.) are selected to comprise rather broad m / z range(s), comparable to wide DIA isolation windows, such as, e.g., m / z 400-450 and / or, 450-500. In some of said embodiments the ions of interest are then identified and selected for subsequent isolation using overlapping windows, fragmentation and MS2 measurements (e.g., subsequent repetitions of one or more steps b.-e.) based on a data base search of the MS2 or MS / MS data obtained from said 'wide' isolation windows (wide m / z ranges, e.g., m / z > 30), such that subsequently MS2 data is acquired, e.g., for specific m / z values within a wide m / z range, e.g., m / z 400-500, preferably wherein the isolation windows (mz1, mz2, ...) comprise specific m / z values of interest within their overlaps (or non-overlaps, whatever has the more narrow m / z range), followed by optional repetition of said procedure for the next wide m / z range, e.g., m / z 500-600.

[0060] In embodiments of the present method, one or more m / z ranges (isolation windows) selected and isolated in steps b. and / or c of the method according to the invention are selected to comprise one or more m / z values characteristic for a molecule ion and / or fragment ion (source ions) of a molecule of interest.

[0061] In embodiments of the present method, the m / z range(s) (isolation window(s)) that constitute either the range / part that is overlapping or non-overlapping with its neighboring m / z range(s) and that are selected and isolated in steps b. and c. comprise one or more m / z values characteristic for a molecule ion and / or fragment ion (source ions) of a molecule of interest.

[0062] In embodiments, the second m / z range (mz2) overlaps with the first m / z range (mz1), wherein the second m / z range comprises the first m / z range at least partially or entirely and / or wherein the second m / z range is wider (spans a greater m / z range) than the first m / z range, or vice versa. Non-limiting examples would be mz1 spanning m / z 429.5 - 470.5 and mz2 spanning m / z 439.5 - 465.5; or and mz1 spanning m / z 439.5 - 465.5 and mz2 spanning m / z 419.5 - 490.5; or and mz1 spanning m / z 439.5 - 465.5 and mz2 spanning m / z 440.5 - 464.5.

[0063] In preferred embodiments of the present method the quadrupole isolation window boundaries are selected in such a way, as to ensure that in a short period of time a pair of different isolation windows (mz1, mz2) are acquired. In embodiments a 'short period of time' is, for example a timespan less than or comparable to the time within which elution of a typical peptide ion from a chromatography source is observed / expected, preferably no more than 5 seconds, or, in case of direct infusion MS, for the whole analysis time.

[0064] In embodiments, said isolation windows (mz1, mz2) preferably satisfy one or more of the following criteria: 1. the two (first and second) isolation windows partially overlap with each other, 2. the precursor ions selected (comprised) by each of the two overlapping isolation windows (mz1, mz2) are subjected to fragmentation, 3. either the difference between the isolation windows (the area where they don't overlap) or their overlap constitute one or more target intervals, out of which at least one is 'narrow' and contains at least one of the m / z values of interest for MS / MS targeting. The width of the interval is referred to below also as 'precursor selection precision', 4. the resulting data is deconvoluted by a raw data processing software. The deconvolution ensures that for strong fragment ion signals it is possible to determine the respective precursor mass with the precision which is defined by the 'precursor selection precision' and the mass filter device error.

[0065] In embodiments, 'narrow' refers to an m / z interval that is narrow in comparison to the entire span of each isolation window (mz1, mz2) and / or to either (i) an interval that is the overlap of the windows that is narrow in comparison to the non-overlapping part(s) of the windows or (ii) to any of the interval(s) that constitute the non-overlapping region with respect to the two windows, with said interval being narrow in comparison to the overlapping region of the windows.

[0066] In embodiments, a preferred size / span of an overlapping interval may be within the range m / z 0.5 - m / z 3.0, and / or 1-5 Da. In embodiments, a preferred size / span of an overlapping interval may be within the range m / z 0.5 - m / z 10.0 or m / z 0.5 - m / z 5.0, or m / z 0.5 - m / z 50.0.

[0067] In embodiments, at least one or each m / z range (isolation window) selected and isolated in steps b. and c. overlaps with at least one of its neighboring m / z ranges by less than m / z 10, less than m / z 5 or less than m / z 3.0, preferably with an overlap of m / z 0.5 or m / z 1.0, or between m / z 0.5 and m / z 1.5. In embodiments, said overlap has a range of less than: m / z 10.0, m / z 5.0, m / z 4.5, m / z 4, m / z 3.5, m / z 3, m / z 2.5, m / z 2, m / z 1.5, m / z 1, m / z 0.75, m / z 0.5, m / z 0.25, m / z 0.1 or less than m / z 1.0. In embodiments the overlap (overlapping part / portion) of a m / z range (with its neighboring m / z range) is more narrow (spans a smaller m / z range) than the non-overlapping part / portion of the m / z range.

[0068] In embodiments each m / z range (isolation window) selected and isolated in steps b. and c. overlaps with its neighboring m / z range(s) by less than m / z 3.0, preferably with an overlap between m / z 0.5 and m / z 1.5. In embodiments at least one m / z range (isolation window) selected and isolated in steps b. and c. overlaps with its neighboring m / z range(s) by less than m / z 3.0, preferably with an overlap between m / z 0.5 and m / z 1.0.

[0069] In embodiments, at least one or each m / z range (isolation window) selected and isolated in steps b. and c. overlaps with its neighboring m / z ranges, wherein the non-overlapping m / z range is less than m / z 10, less than m / z 5 or less than m / z 3.0, preferably 0.5 m / z or 1.0 m / z, or between m / z 0.5 and m / z 1.5, or between m / z 0.5 - m / z 10. In embodiments, said non-overlapping m / z range (part / portion) has a range of less than m / z 10.0, m / z 5.0, m / z 4.5, m / z 4, m / z 3.5, m / z 3, m / z 2.5, m / z 2, m / z 1.5, m / z 1, m / z 0.75, m / z 0.5, m / z 0.25, m / z 0.1 or m / z 1.0. In embodiments the non-overlapping (with its neighboring m / z range) part / portion of a m / z range is more narrow (spans a smaller m / z range) than the overlapping part / portion of the m / z range (with its neighboring m / z range).

[0070] In embodiments each m / z range (isolation window) selected and isolated in steps b. and c. comprises a non-overlapping part (m / z range) of less than m / z 3.0, preferably between m / z 0.5 and m / z 1.5. In embodiments at least one m / z range (isolation window) selected and isolated in steps b. and c. comprises a non-overlapping part (m / z range) of less than m / z 3.0, preferably between m / z 0.5 and m / z 1.5. In other words, at least one or each m / z range (isolation window) selected and isolated in steps b. and c. comprises a part (m / z range) not overlapping with its neighboring m / z range that preferably has a range of less than m / z 3.0, more preferably between m / z 0.5 and m / z 1.5.

[0071] In embodiments the non-overlapping part / portion of a m / z range is more narrow (spans a smaller m / z range) than the part(s) / portion(s) of the m / z range overlapping with one or more neighboring m / z ranges.

[0072] In embodiments, at least one or each m / z range(s) (isolation window) selected and isolated in steps b. and c. comprises a non-overlapping part with its neighboring m / z ranges, wherein the range of the overlap if any is less than m / z 3.0. In preferred embodiments the overlap with neighboring m / z ranges is between m / z 0.5 and m / z 1.5. In embodiments one or each m / z range / part (either overlapping or non-overlapping with neighboring m / z ranges) comprises at least one molecule of interest.

[0073] In some embodiments, at least one or each m / z range(s) (isolation window) selected and isolated in steps b. and c. has / have only one overlapping part (overlap) with the neighboring window(s) (on one side). In other words, in such embodiments a m / z range only overlaps with one neighboring window, instead with two distinct neighboring windows on both sides.

[0074] In embodiments one or more m / z range (isolation window) selected and isolated in steps b. and c. comprises only one non-overlapping part with the neighboring window (on one side).

[0075] In embodiments, the one or more m / z values characteristic for a molecule ion (source ion) and / or fragment ion of a molecule of interest are selected / identified based on: the ion mass spectral data acquired in one or more iterations of step a. and / or e., and / or a database comprising m / z ratios characteristic for one or more molecule ion (source ion) and / or fragment ion of molecules of interest as well as the given retention time and ion mobility values, if any, associated with the target ion(s) or the isolation windows to be selected and isolated as in steps b. and c.

[0076] In embodiments, the m / z ranges representing isolation windows (mz1, mz2,... mzX) in the context of the present methods may exhibit a range of m / z 4 - 100, preferably of m / z 10-50, m / z 20-60 or m / z 25-75. In the context of 'mzX', X represents herein any natural number, such as e.g., 2, 3, 4, 5, 6, ... etc.

[0077] Any variation of the above-defined particular m / z ranges may also be envisaged herein, such as a variation of ± 5 %, ± 10 %, ± 20 % and / or ± m / z 1, ± m / z 2.5, ± m / z 5, ± m / z 7.5, ± m / z 10, ± m / z 15, ± m / z 20, ± m / z 25. Any particular value encompassed by the above ranges is also envisaged herein, and new ranges may be defined from any of the endpoints of the above-defined ranges.

[0078] In embodiments, the isolation according to steps b. and c. is repeated for multiple iterations (e.g., mz1-mzX), such that a total m / z range of interest is covered by overlapping m / z ranges (mz1, mz2, ...mzX), wherein each m / z range overlaps with at least one neighboring m / z range.

[0079] In embodiments, the isolation according to steps b. and c. is repeated, or in other words, performed in iterations in such a way that the window selected in either of steps b. or c. in a particular iteration is considered to be the window selected in step b. during the subsequent iteration. In embodiments, the total number of iterations is preferably between 1-2, 2-10, 5-20, 10-50, 20-200 or 50-1000. It is preferred that individual b. or c. steps can be shared between iterations.

[0080] In embodiments the entire acquisition takes more than one minute, e.g., at least 5, 10, 15, 20, 30, 35, 40, 45, 50, 55, 60, 65, 70 ,75, 80, 85, 90, 95, 100, 110 or 120 minutes, such that the isolation according to steps b. and c. is repeated for multiple iterations.

[0081] In embodiments, the isolation according to steps b. and c. is repeated such that the m / z ranges cover only a fraction of the total m / z range of interest and multiple repetitions of steps b.-e. are required to cover the total m / z range of interest.

[0082] In embodiments, the total m / z range of interest spans (covers) a m / z range of between m / z 300.0 (mz1) - 2000.0 (mzX), m / z 400-1200, m / z 450-850, m / z 400-850, m / z 400-900, m / z 400-950, m / z 450-950 m / z 300-1000, m / z 300-850, or m / z 350-950 or any m / z range in between. In embodiments, the total m / z range of interest of between m / z 300-2000 is preferred, wherein in embodiments a m / z range of between m / z 400-1200 may be preferred for timsTOF experiments.

[0083] The particular m / z ranges defined above may exhibit a certain variation of ± 5 %, ± 10 %, ± 20 % and / or ± m / z 1, ± m / z 2.5, ± m / z 5, ± m / z 7.5, ± m / z 10, ± m / z 15, ± m / z 20, ± m / z 25, ± m / z 50, ± m / z 100. Any particular value encompassed by the above ranges is also envisaged herein, and new ranges may be defined from any of the endpoints of the above-defined ranges.

[0084] In the context of 'mzX', X represents herein any natural number, such as e.g., 2, 3, 4, 5, 6, ... etc.

[0085] In embodiments, the ion mass spectral data acquired in step a. (MS1) comprises m / z and ion mobility data and / or collisional cross section (CCS) data.

[0086] In embodiments, in step b. one or more m / z range(s) are selected based on the m / z data (acquired in step a) and the ion mobility and / or collisional cross section (CCS) data acquired in step a.

[0087] In embodiments, in steps b. and c. one or more of the m / z ranges selected for mass filter isolation and subsequent fragmentation are selected independently of the ion mass spectral data acquired in step a. or any previous iteration of step a. In some of said embodiments the one or more m / z ranges selected for mass filter isolation and subsequent fragmentation are selected based on predefined isolation windows (e.g., with predefined width, like regular DIA) or depending on the retention time of one or more analyte(s) of interest.

[0088] In embodiments, the present method employs liquid or gas chromatography, or electrophoresis coupled to mass spectrometry, wherein molecules are subjected to chromatographic (or any other kind) separation and then directed towards the mass spectrometer, and each molecule of interest elutes from the chromatography column within a certain period of time.

[0089] In embodiments, prior to performing mass spectrometry analysis the sample is processed by chromatography, and the molecules comprised within the sample elute over a timespan from the chromatography or electrophoresis source and are processed according to the steps a.-e. during an elution timespan of said molecules, thereby acquiring ion mass spectral data of the molecules comprised within the sample in relation to their elution time (retention time (RT)) from the chromatography or electrophoresis source.

[0090] In embodiments the `average elution time (retention time (RT))' of an analyte of interest may be considered to be the approximate measurement time period during which an analyte of interest is (e.g., eluting from a chromatography source and) detectable for the mass spectrometer. In embodiments the average elution time (retention time (RT)) of an analyte of interest may be determined based on the average time period during which an analyte of interest (time period averaged across all analytes of interest) is detectable.

[0091] In embodiments, the method further comprises f. analyzing the ion mass spectral data acquired in one or more iterations of steps a. and e., thereby identifying and / or characterizing at least one molecule of interest comprised within the sample. In embodiments, the method comprises f. analyzing the (ion mass spectral) data acquired in one or more iterations of steps a. to e., thereby identifying and / or characterizing at least one molecule of interest comprised within the sample.

[0092] In embodiments, said analysis according to f. comprises identifying one or more molecules (of interest) by comparing the signal intensity determined in one or more MS1-measurements at a specific mass to charge ratio (m / z) to a database comprising m / z ratios characteristic for one or more molecule ion (source ion) of respective molecules (of interest). In embodiments, identifying one or more molecule ions (source ions) further comprises comparing m / z values determined in one or more MS1- and corresponding MS2-measurements to a database comprising m / z values characteristic for one or more molecule ion (source ion) and one or more fragment ion of respective molecules (of interest).

[0093] In embodiments, the obtained MS / MS data is deconvoluted, preferably by a raw data processing software or appropriate algorithms or equivalent computational means. In general, deconvolution is the process of computationally separating co-eluting molecules to obtain a 'pure' / 'clean' in silico spectrum for each molecule. One advantage of the deconvolution of the presently obtained MS data is that it ensures that for strong fragment ion signals the respective precursor mass is determined with a precision which is in embodiments defined by the 'precursor selection precision' (e.g., the width of the interval overlapping between two isolation windows containing at least one of the m / z values of interest) and the mass filter error.

[0094] In a non-limiting and merely illustrative example, the spectra for the targeted m / z values are deconvoluted by a software or suitable algorithm such that, for example, if the mass spectrometer targeted m / z 440, and used as isolation windows mz1 400.0 - 440.5 and mz2 439.5 - 465.5 in the respective cycle, the deconvolution may consider only MS / MS fragments detected for both isolation windows as the `spectrum of the precursor' at m / z 440, thus significantly simplifying the spectrum and preferably solving the data deconvolution problem specifically for this precursor, similar to DDA methods. Thereby, the present method would identify most or all the peptides which would have been identified with either a comparable DIA method or with a comparable DDA method and would be able to quantify all these peptides with the superior quality of quantification inherent to DIA, such that the advantages of both DIA and DDA are combined in a single acquisition.

[0095] In embodiments, the mass (and / or mass bound or m / s bounds) of a molecule ion (precursor ion) is calculated during ion mass spectral data analysis in step f. (with precision defined by precursor selection precision and mass filter error) based on the deconvoluted data of one or more of the corresponding molecule fragments.

[0096] In embodiments, two or more MS1 and / or MS2 measurements and / or MS1 and MS2 spectra are combined (or merged) to generate a single spectrum. In embodiments, two or more MS1 or MS2 measurements and / or MS1 or MS2 spectra are combined to generate a single spectrum.

[0097] In embodiments collision energy is modulated to also include a large proportion of unfragmented precursors in the same spectrum.

[0098] In embodiments, an MS1 measurement of step a. is acquired as part of one or more MS2 measurements, such as but not limited to the measurements of step b. and c., e.g. by collision energy modulation that produces also unfragmented precursors.

[0099] In embodiments, MS1 and MS2 spectra are merged, for example using a mass spectrometer equipped with a TOF (time of flight) mass analyzer or Astral mass analyzer or any other mass analyzer the mode of operation of which comprises separation of ions by their m / z values by means of different flight time of said ions. In a non-binding exemplary embodiment, the mass spectrometer measures spectra, e.g., at a frequency of 10 kHz, wherein said spectra are merged (automatically) by an acquisition software (e.g., to obtain spectra corresponding to tens of milliseconds of time). In another non-binding example, two or more spectra, which might or might not correspond to the same isolation windows or ion mobility values, are merged before being processed via peak picking and / or centroiding, wherein one peak or a group of peaks in the merged spectrum may be replaced with one or more different peaks, this transformation applied to multiple individual peaks or groups within the merged spectrum.

[0100] In embodiments, one or more MS1 measurement(s) involve(s) isolation of molecule ions (source ions) using a mass filter. In embodiments, one or more MS2 measurement(s) involve(s) no isolation of source ions using a mass filter.

[0101] In embodiments, steps a. to e. or c. to e. are repeated for multiple iterations over the elution time (retention time (RT)) of at least one molecule of interest.

[0102] In embodiments the ionization of the molecules in step a. comprises electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI).

[0103] In embodiments prior to the transfer of the biomolecule ions to a mass spectrometer in step a. the sample is processed by chromatography, such as gas chromatography (GC), capillary electrophoresis or liquid chromatography (LC), preferably High-performance liquid chromatography (HPLC).

[0104] In embodiments, the invention is also applicable to direct infusion mass spectrometry (e.g., constituting a simplified application, as no separation of analytes occurs prior to MS / MS analysis).

[0105] In embodiments the invention relates to the present method for use in direct infusion mass spectrometry, gas chromatography mass spectrometry (GC-MS), capillary electrophoresis or liquid chromatography mass spectrometry (LC-MS).

[0106] In embodiments, an ion source is selected from the group comprising an electrospray ionization (ESI) ion source, a matrix assisted laser desorption ionization (MALDI) ion source, a laser desorption ionization (LDI) ion source; an atmospheric pressure ionization (API) ion source, a chemical ionization (CI) ion source. In embodiments, the ionization of the molecules in step a. comprises electrospray ionization (ESI), matrix-assisted laser desorption ionization (MALDI), atmospheric pressure ionization (API) and / or chemical ionization. In embodiments, the ionization of the molecules in step a. comprises electrospray ionization (ESI). In embodiments, the ionization of the molecules in step a. comprises matrix-assisted laser desorption ionization (MALDI). In embodiments, the ionization of the molecules in step a. comprises atmospheric pressure ionization (API). In embodiments, the ionization of the molecules in step a. comprises chemical ionization.

[0107] In embodiments the present method may be performed either using a (modified) trapped ion mobility (TIMS) device mass spectrometer, e.g., a timsTOF-series instrument (Bruker), or a mass spectrometer equipped with a SLIM (structures for lossless ion manipulation) device (MOBILion).

[0108] In embodiments, the term ion mobility should be understood as the ion mobility or collisional cross section or a similar ion characteristic.

[0109] In embodiments, the selection and isolation of precursor ions may be based on ion mobility values obtained by a TIMS or SLIM device and / or the m / z values acquired in a MS1 measurement in step a.

[0110] In embodiments the ion mass spectral data acquired in a MS1 measurement in step a. comprises ion mobility and / or m / z values.

[0111] In embodiments, the operation of the mass filter device, preferably a quadrupole device, can be tied / linked to the ion mobility data obtained by a TIMS or SLIM device. That is, in embodiments for one or more or each ion mobility value, the boundaries of the m / z range of a respective isolation window can be set separately, either in data-independent or data-dependent manner. One beneficial effect of such embodiments is that this linking of ion mobility data (TIMS or SLIM device) and Q1 operation leads to a fold-change boost in the numbers of precursor ions that can be targeted in data-dependent fashion, in comparison to non-TIMS and non-SLIM mass spectrometers. This boost originates from the ability to set the isolation windows independently for different ion mobility values and thus enable to target multiple ions with different ion mobility values within a single frame of acquisition that spans the ion mobility range of interest.

[0112] In some of such embodiments, in addition to the extra separation of ions, which leads to 'cleaner' spectra, the TIMS device has further the following benefit: Without being bound by theory, a TIMS device' operation is general characterized by two configurable time periods: accumulation time (time spent accumulating ions) and ramp time (time spent releasing ions depending on their ion mobility). These are usually set to the same value, e.g. 100 ms. In embodiments, during the ramp time, the quadrupole can be switched between different settings multiple times, e.g. 10 or 25 times. Commonly, the acquisition during the ramp time is called a 'frame'. In embodiments, different frames can feature different isolation windows. Commonly, the property of TIMS acquisition is such, that the sensitivity is similar to the hypothetical sensitivity of a non-TIMS acquisition, if each of those isolation windows were acquired with the specified accumulation time, e.g. 100 ms.

[0113] In one aspect the present invention relates to a computer-implemented method for analyzing ion mass spectral data.

[0114] In embodiments, the computer-implemented method comprises for analyzing ion mass spectral data acquired during one or more iterations of steps a. and e. (of the method of the invention): a. receiving acquired ion mass spectral data of a MS1 analysis according to step a. (of the method of the invention), b. identifying one or more m / z range(s) (isolation window(s)) of interest comprising m / z data of a molecule of interest, c. selecting molecule ions from one or more m / z range(s) (isolation window(s)) of interest for fragmentation and inducing the mass filter device of the mass spectrometer to transmit said molecule ions to a fragmentation and / or reaction device, d. receiving acquired ion fragment mass spectral data of a MS2 analysis according to step e. (of the method of the invention), e. deconvoluting and analyzing the received ion (fragment) mass spectral data thereby identifying and / or characterizing at least one molecule of interest comprised within a sample.

[0115] In embodiments the computer-implemented method for analyzing mass spectral data comprises: A. receiving mass spectrometry data, preferably comprising mass spectral data of a MS1 and optionally MS2, analysis, and one or more of the following steps: B. merging multiple MS1 or MS2 spectra (derived from the data received in A.) and / or subjecting them to peak picking or centroiding, and / or C. combining data corresponding to (derived from) a window mz1 isolated in step b. (of the method of the invention), and window mz2 isolated in step c. (of the method of the invention), and / or D. selecting m / z values that are either (i) simultaneously comprised within (shared by the data recorded for) at least two overlapping windows of the windows mz1, mz2, ... mzX, or (ii) unique for at least one of the windows, preferably wherein similar m / z values (i.e. m / z values are `similar' if their difference is comparable to the mass accuracy of the mass spectrometer), are considered identical, and / or E. selecting the data matching (corresponding to) the m / z values selected in D. and one or more of the isolation windows mz1, mz2, ... mzX, as defined in D., and / or F1. generating, based on D. or E., a set of peaks, preferably wherein each peak is a numerical representation characterizing the likelihood distribution for the fragment ion m / z value, the respective precursor ion m / z value, the intensity value and / or (if applicable) the ion mobility value, and / or F2. generating, based on D. or E., a set of peaks, preferably wherein each peak comprises a m / z value or a m / z range, putative precursor m / z value or an m / z range, an intensity value or an intensity range, optionally an ion mobility value or an ion mobility range, optionally score(s) characterizing the likelihood distribution within any or all of the aforementioned range(s), and / or G. selecting a peak from the data matching a window mz1 and a peak from the data matching a window mz2, with the m / z values of the two peaks and the ion mobility values of the peaks, if recorded, being close, and applying a mathematical operation (or calculation) to the recorded intensities of the said peaks, preferably wherein the mathematical operation is or comprises the minimum, the maximum, the sum or the difference, and / or H. selecting a group of peaks from the data matching a window mz1 and a group of peaks from the data matching a window mz2, preferably, if recorded, with neighboring m / z values of all selected peaks and / or the ion mobility values of all selected peaks, and generating a single peak based on the data recorded for the selected peaks, and / or I. calculating the sum of (summarizing) the intensities of peaks in each group or in both groups selected in H., optionally using a mathematic operation applied to the sums of peaks in two groups generated, preferably wherein the mathematical operation is the minimum, the maximum, the sum or the difference, and / or J. subjecting a set of peaks generated by any one or combination of steps B.-I. to a database search, the database containing theoretical information on analytes of interest comprising any combination of: molecular structure, physical or chemical properties, precursor m / z values, fragment ion m / z values, reference retention times, reference ion mobility values, optionally scoring the degree of correspondence between the theoretical information in a database for a particular analyte and a subset of peaks generated by any combination of steps B.-I. based on likelihood distribution information obtained in F1. or scores obtained in F2., and / or K. extracting chromatograms based on the data from any combination of B.-I. for one or more fragment ion m / z values corresponding to the analyte of interest, optionally integrating one or more of the extracted chromatograms, in full or in the vicinity of the presumed elution apex of the respective analyte, to generate estimated quantities of the fragment ions of the analyte corresponding to the extracted chromatograms, and optionally using the calculated quantities of fragment ions, and optionally also the MS1 information recorded for the analyte of interest, to quantify this analyte.

[0116] The steps B.-K. of the afore-described embodiment of the computer-implemented method may be performed in embodiments in a different order, or in no particular order. In embodiments, any of steps B.-K. may be optional and / or combined with any other of the afore-described steps of the computer-implemented method.

[0117] In the context of 'mzX', X represents herein any natural number, such as e.g., 0, 1, 2, 3, 4, 5,... etc.

[0118] In embodiments, a likelihood distribution may generally be characterized in different ways, e.g., as a precise value, as an indication "likelihood distribution unknown" or as a "normal distribution with mean M and standard deviation S".

[0119] In another aspect the invention relates to a computer-implemented method or a computer program comprising instructions to cause a mass spectrometer or mass spectrometry system to execute the steps of the method according to the invention.

[0120] In embodiments, the computer-implemented method or computer program comprises instructions to cause a mass spectrometer or mass spectrometry system to execute the steps of: (i) cause the ion source to ionize a multitude of molecules and / or fragments thereof comprised within a sample to obtain a multitude of molecule ions (source ions) and cause the mass spectrometer to analyze the molecule ions and / or fragments thereof in a first mass spectrometry (MS) measurement (MS1), thereby acquiring ion mass spectral data of at least a fraction of the molecule ions and / or fragments thereof (source ions) comprising their mass to charge ratio (m / z), (ii) cause the mass filter device to isolate a first fraction of the molecule ions (precursor ions) having a m / z within a first m / z range (mz1; isolation window), wherein the first m / z range is selected based on the ion mass spectral data (i) acquired in step (i), or one or more iterations of step (i), and optionally an exclusion list and / or an inclusion list. (iii) cause the mass filter device to isolate a second fraction of the molecule ions (precursor ions) having a m / z within a second m / z range (mz2; isolation window), wherein the second m / z range is selected based on the ion mass spectral data (i) acquired in step (i), or one or more iterations of step (i), and optionally an exclusion list and / or an inclusion list, and wherein the second m / z range (mz2) overlaps with the first m / z range (mz1), (iv) cause the fragmentation device or cell of the mass spectrometer to fragment at least a fraction of the molecule ions (precursor ions) isolated in steps (ii) and (iii), separately, to obtain a multitude of biomolecule fragment ions from each of the respective fragmented molecule ions (precursor ions), (v) cause the mass spectrometer or system to analyze the multitude of biomolecule fragment ions in one or more second mass spectrometry measurement (MS2), thereby acquiring ion mass spectral data of the analyzed biomolecule fragment ions, (vi) optionally cause the mass spectrometer or system to compare the so far acquired MS1 and / or MS2 data or its subset to a database of analytes to identify analytes in the so far acquired data, (vii) optionally cause the mass spectrometer or system to use the data from (vi) to align the current retention time to a reference retention time scale, (viii) optionally cause the mass spectrometer or system to use the result of (vii) to select the list of analytes likely to be observed at a current retention time or within a short period, preferably within < 1s, < 3s, < 10s, < 20s, < 60s from the current retention time, (ix) optionally cause the mass spectrometer or system to target one or more of the m / z values of the selected in (viii) analytes, (x) optionally cause the mass spectrometer or system, from the analytes identified in (vi), to select analytes likely detectable at the current retention time or within a short period of time in the past, preferably < 0.1 s, < 0.2s, < 0.5s, < 1 s, < 2s, < 5s, (xi) optionally cause the mass spectrometer or system to filter the list of analytes selected in (x) to retain analytes of particular interest, such as peptides bearing phosphates or peptides from a pre-defined list of analytes of particular interest for the experiment, (xii) optionally cause the mass spectrometer or system to target one or more of the m / z values of the selected in (x) or (xi) analytes.

[0121] In embodiments the mass spectrometer (instrument) or system, comprises an ion source, a mass filter device configured to receive ions from the ion source, optionally an ion storage device, a fragmentation device or cell configured to receive ions from the mass filter device (or the optional storage device), a mass analyzer, and preferably at least one computer or computational unit and / or electronic controller (electrically), preferably coupled to the mass filter device, optionally to the ion storage device, the fragmentation device or cell and / or the mass analyzer.

[0122] In another aspect the invention relates to a computer-readable medium having stored thereon the computer program according to the invention.

[0123] In one aspect the present invention relates to a mass spectrometer instrument or system, comprising an ion source, a mass filter device configured to receive ions from the ion source, optionally an ion storage device, a fragmentation device or cell configured to receive ions from the mass filter device (or the optional storage device), a mass analyzer, and preferably a computer or electronic controller (electrically) coupled to the mass filter device, optionally to the ion storage device, the fragmentation device or cell and / or the mass analyzer.

[0124] Mass spectrometry instruments or systems suitable for measuring ion mobility spectra of selected ion species may in embodiments comprise an ion mobility separation (IMS) device coupled to a mass spectrometer (MS), in detail, in embodiments such spectrometers may comprise an ion source, an ion beam modulation device, an ion drift region and a mass spectrometer downstream of the ion drift region.

[0125] Each optional or preferred feature of the invention that is disclosed or described in the context of one aspect of the invention is herewith also disclosed in the context of the other aspects of the invention described herein. All features disclosed in the context of the method for analyzing a sample according to the invention also relate to, and are herewith disclosed also in the context of, the computer-implemented method according to the invention, the mass spectrometry device and the data analysis method disclosed herein, and vice versa.

[0126] The various aspects of the invention are unified by, benefit from, are based on and / or are linked by the common and surprising finding of the unexpected advantageous effects of the present method to identify any target peptides commonly identifiable either only by DIA or DDA and to quantify said peptides with the quality of quantification inherent only to DIA. Accordingly, the method according to the invention combines the advantages of both DIA and DDA in a single acquisition method.DETAILED DESCRIPTION OF THE INVENTION

[0127] All cited documents of the patent and non-patent literature are hereby incorporated by reference in their entirety.

[0128] The present invention is directed to a method for analyzing a sample comprising one or more molecules of interest by a mass spectrometer. In embodiments the method of the invention relates to a method for analyzing molecules of interest from a sample using mass spectrometry, wherein ionized molecules are analyzed in a first MS1 scan and a plurality or even all obtained precursor ions are subsequently isolated for fragmentation and MS2 analysis by defining overlapping pairs of isolation windows in a data-dependent fashion, thereby enabling deconvolution of the obtained MS / MS data. It was entirely surprising that the present method enables, through the synergistic interaction of its features, the collection of significantly more precise precursor mass information for the observed fragment ions, compared to prior art DIA and DDA methods. It may even be concluded that the present method combines the advantages of both DIA and DDA in a single acquisition method, as the present method enables the identification of peptides which would have been identified with DIA, but at the same time also the identification of peptides which would have been identified with DDA such that the sum of these peptides may be quantified with the quality of quantification inherent to DIA.

[0129] A "sample" may comprise herein, without limitation thereto, a suspension comprising proteins, peptides, cellular components, fatty acids, fats, metabolites, nucleic acids, cell lysate, cells, living cells, fixated cells, body fluids, organ samples, tissue samples and / or subcellular extracts (e.g., nuclei). In embodiments a sample may be taken from a subject, a (human) patient, a mammal, a cell culture of patient cells or cell lines, an animal, or a cell culture of human cells, animal cells, bacterial cells or yeast cells or cell lines thereof, a biopsy, a tissue sample, a blood sample, or an environmental sample. Basically, any kind of sample that is suspected to contain biochemical information of interest.

[0130] In embodiments before a mass spectrometry measurement proteins to be analyzed, e.g., comprised in or extracted from a sample, are subjected to one or more of the steps of: removal or dilution of agents, which may interfere with subsequent steps of the procedure (e.g., chaotropic reagents, detergents, solvents, salts), the proteolytic digestion of proteins into peptides preferably with sequence-specific proteases trypsin or LysC, the desalting and concentration of peptides, and the elution of peptides from a solid phase prior to the analysis by mass spectrometry, preferably combined with liquid chromatography (LC) previous to mass spectrometry (LC-MS). Elution from a solid support can occur, e.g., in embodiments by acetonitrile (ACN) and / or trifluoroacetic acid (TFA). A proteolytic digestion is preferably performed before the mass spectrometry measurement of enriched / isolated proteins. In embodiments, the proteolytic digestion may be performed in solution or "on-bead". "On bead" digestion commonly refers to the digestion of proteins while still bound to a solid enrichment-phase or solid support, such as stationary solid phases, for example, a column, a surface, a resin or (magnetic) beads.

[0131] The term "mass spectrometry" (MS) generally refers to a technology for the separation of electrically charged molecules (ions) in the gas phase. In general, a `mass spectrometer' ionizes the molecules and records mass / charge values (m / z values) of the ions that enter it (MS1 acquisition). The obtained ions are preferably generated in an ion source, e.g., electrospray ionization (ESI) or nano ESI (for higher analytic sensitivity due to higher ionization frequency), that allows the transfer and ionization of analytes from a solid or liquid phase (e.g., from liquid chromatography; LC) into the gas phase. The gas-phase ions are then analyzed by a mass analyzer that sorts the ions in space or time according to their mass-to-charge ratio (m / z). In embodiments a mass spectrometer can also, at any given time point, isolate ions falling within a particular m / z range (isolation window), fragment those ions into smaller ions and record the m / z values of those smaller ions (MS / MS or MS2 acquisition). Herein, ions that are being fragmented are preferably termed 'precursor ions' or 'precursors'.

[0132] In general, mass spectra are depicted as histograms of intensity versus mass-to-charge ratio (m / z), wherein each bar representing an ion having a certain m / z indicates the relative abundance of the respective ion. A 'm / z range' preferably refers to a range of m / z (mass-to-charge ratio) values, wherein `neighboring m / z ranges' defines m / z ranges that comprise numeric ranges next to each other (one starts, where the other ends) or overlapping with each other. Non limiting examples of neighboring m / z ranges are m / z 400-450 and m / z 445-500 (overlapping by m / z 5) or m / z 400-450 and m / z 450-500.

[0133] In embodiments a mass spectrometer may comprise a liquid chromatography (LC) or gas chromatography (GC) or electrophoresis or pre-fractionation to achieve a separation of analytes from a sample by their physical and chemical properties. Such separation commonly occurs / is performed before the ionized sample enters the mass spectrometer.

[0134] Commonly, liquid chromatography (LC) refers to a separation method wherein the components of a liquid mixture are distributed between two immiscible phases, e.g., a mobile and a stationary phase. In general, liquid chromatography can be divided into five categories, such as ionexchange chromatography, size-exclusion chromatography, adsorption chromatography, partition chromatography and affinity chromatography. The most commonly employed method is the reverse-phase (RP) mode of the partition chromatography technique, which uses a nonpolar (hydrophobic) stationary phase and a polar mobile phase. High-performance liquid chromatography (HPLC), refers to a method applied to separate, quantify, and identify the components of a sample. In HPLC a pressurized liquid solvent containing the sample is forced through a column comprising a solid adsorbent material, wherein each component in the sample interacts differently with the adsorbent column material, such that each component comprises a different flow rate and is thereby separated from the other sample components while passing through the column. Accordingly, components of a sample elute from a column at a different time point.

[0135] In embodiments a mass spectrometer may comprise an ion source at the front-end of the mass spectrometer. The ionization of analytes (e.g., peptides are mostly ionized to charges 2-4), e.g., may occur via analyte protonation in the 'positive' mode. The resulting ions are charged and can be accelerated by electric fields, depending on their mass to charge (m / z) ratio.

[0136] Commonly, an `ion source' is a device for ionization of the chemical and / or biological, e.g., peptides or proteins, components of a sample. In embodiments an ion source is selected from the group comprising an electrospray ionization (ESI) ion source, a matrix assisted laser desorption ionization (MALDI) ion source, a laser desorption ionization (LDI) ion source; an atmospheric pressure ionization (API) ion source, a chemical ionization (CI) ion source.

[0137] In embodiments a mass spectrometer may comprise a mass filter device, such as e.g., a quadrupole device. A `mass filter' commonly facilitates the specifical selection of ions with specific m / z ratios to continue towards , e.g., a detector, while other, non-targeted species are removed or retained. A quadrupole allows for selective transmission of ions with masses falling in a particular m / z range.

[0138] In general a 'quadrupole' mass filter consists of four metal rods that serve as electrodes in a vacuum. Two diagonally opposite rods apply a constant current, while the other pair of rods applies an opposite high-frequency alternating current. This causes the forward moving ions to oscillate between the quadrupoles, such that by manipulating the alternating current, ions of a particular mass / charge (m / z) ratio can be oscillated until they either collide with one of the quadrupoles or are ejected and selectively transferred to the subsequent device in the mass spectrometer (Wilkinson, 2021).

[0139] In a triple quadrupole system, the first quadrupole (Q1) selectively filters the targeted precursor molecules (i.e. the protonated and / or deprotonated target analytes) through the quadrupole while removing all ions of other m / z ratios. The DC and AC currents required to stabilize the oscillation of the target ions are optimized during method development to minimize ultimate signal loss. T

[0140] Commonly a `Q1 quadrupole' or `Q1' stands for the first quadrupole in the ion path with a major function.

[0141] An `Isolation window' may be described as the setting of a mass filter, such as a (Q1) quadrupole mass filter, within the mass spectrometer at a given moment in time, aimed to selectively transmit ions within particular m / z boundaries. The transmitted (or 'selected' or 'isolated') precursor ions can then be subjected to fragmentation, with further detection of the m / z values of the resulting fragment ions, producing a fragmentation spectrum.

[0142] A `quadrupole error' arises from the phenomenon that the operation of a quadrupole is commonly imperfect. Thus, a proportion of precursor ions with m / z values that lie within the isolation window boundaries but are close to one of the boundaries might be lost, whilst a proportion of precursor ions with m / z values outside the boundaries but close to one of the boundaries might be transmitted. The maximum m / z deviation between the precursor ion m / z and one of the m / z boundaries, at which such effects are of practical significance, is referred to as the `quadrupole error'. An example of a typical quadrupole error may be ~m / z 0.5.

[0143] In embodiments a mass spectrometer may comprise a collision cell: a device where ions are fragmented, e.g., by being accelerated with an electric field and colliding with molecules of a gas.

[0144] In embodiments a mass spectrometer may comprise a high-resolution mass analyzer. The term high-resolution mass analyzer commonly refers to a device which measures the m / z values of ions with high precision, such as typically a time-of-flight (TOF), Astral or an Orbitrap device.

[0145] In general, a variant of common `mass spectrometry' (MS) analyses proteolytic digested proteins of a sample of interest. In embodiments the sample is separated before ionization and injection into the mass spectrometer by chromatography, most commonly (ultra or high performance) liquid-chromatography (LC-MS). The mass spectrometer then ionizes the chemical compounds of the sample and subsequently sorts the obtained ions based on their mass-to-charge (m / z) ratio.

[0146] In a common mass spectrometry set up a mass spectrometer (MS) is combined with an up-front chromatography system such as a liquid chromatograph (LC). In embodiments the mass spectrometer comprises a mass filter device (commonly an (radiofrequency, RF) quadrupole mass filter), an ion fragmentation device / cell, and a downstream time-of-flight mass analyzer.

[0147] Tandem mass spectrometry (termed 'MS / MS') involves the coupling of two or more mass analyzer steps in space or time to an additional gas-phase reaction step, thereby increasing the analysis resolution. In common embodiment of MS / MS pipelines, the molecules of a sample are in a first step ionized and separated by the first spectrometer (called MS1) according to their mass-to-charge ratio (m / z), in a second step, ions of a specific m / z ratio are then isolated in the same or a different mass analyzer and then fragmented, e.g., by collision-induced dissociation, higher-energy collision dissociation (HCD), electron capture dissociation (ECD), electron transfer dissociation (ETD), ion-molecule reaction or (ultraviolet) photodissociation. The (some or all) generated fragments are then commonly analyzed in a mass analyzer to separate and detect the fragments according to their m / z ratio (MS2). The separation and fragmentation increases the resolution of the detection by enabling the separation and identification of ions with very similar m / z ratios in single mass spectrometry (MS). MS / MS is commonly beneficial for correctly identifying peptide ions and may also be used for identifying metabolites.

[0148] For mass spectrometry, there exist relative and absolute quantification methods. Relative quantification comprises, for example, the stable isotope-labelling of samples, which facilitates to distinguish identical proteins in different samples. Relative quantification methods are, for example, stable isotope labelling with amino acids in cell culture (SILAC), dimethyl labelling, and isobaric tags for relative and absolute quantification (iTRAQ), isotope-coded affinity tags (ICAT), isobaric labelling (tandem mass tags (TMT), metal-coded tags for label-free quantification (MeCAT) and terminal amine isotope labelling of substrates (TAILS). Label-free approaches require the separate analysis of different samples followed by comparing their mass spectra to determine the relative abundance of peptides in each sample. Label-free quantification is commonly based on either spectral counting or precursor signal intensity. In spectral counting, the spectra of an identified protein are counted and standardized using an applicable normalization. In area under the curve (AUC) methods, the area under the spectral peak is calculated for each peptide spectrum of an LC-MS / MS run, which is linearly proportional to the concentration of protein in the analyzed sample.

[0149] In detail, the relative quantification method of tandem mass tags (TMT, tandem atomic mass tags) uses isobaric chemical tags, which enable multiplexed functions for relative quantitative proteomic analysis. In such experiments, different isobaric tags are used to label different conditions. Without being bound by theory, after tagging, all samples are mixed and analyzed in a single experiment using liquid chromatography-mass spectrometry (LC-MS). Commonly, after fragmentation, the tags generate a unique signature reporter from each individual systemic state in the lower m / z region of the MS / MS spectrum, such that peptide identification is achieved by matching the resulting ion peaks to values comprised within fragment databases. The final peptide quantification is performed by comparing the intensities of the reporter ions.

[0150] In detail, the relative quantification method of stable isotope labelling with amino acids in cell culture (SILAC) comprises labelling proteins in living cells and / or in vivo by replacing an isotopically heavy amino acid form with a naturally occurring light form. In SILAC labelled and unlabeled samples are combined during sample preparation such that SILAC enables both minimization of quantitative error and mixing of samples to for subsequent of enrichment procedures. Without being bound by theory, these procedures can improve the detection of abundance changes in both low abundance proteins and post-translational modifications.

[0151] In detail, the relative quantification method of label-free quantitation (LFQ) enables low-cost relative quantitation of protein samples, wherein samples are individually analyzed using advanced software with chromatographic capabilities prior to measurement. One advantage is that the number of sample comparisons is not limited. Further, peptide identification can be performed using any fragmentation method (CID, ETD, EThcD and / or HCD).

[0152] Commonly, targeted mass spectrometry methods comprise, for example, multiple reaction monitoring (MRM), also termed selected reaction monitoring (SRM), and parallel reaction monitoring (PRM).

[0153] One embodiment of mass spectrometry is mobility spectrometry, coupled to mass spectrometry. Mobility spectrometry may be performed with a MS system comprising an ion accumulation device and an ion mobility separation device, and further preferably a mass filter (usually an RF quadrupole mass filter), an ion reaction, collusion and / or fragmentation cell, optionally an ion trap or ion trapping region, and downstream a time-of-flight mass analyzer, preferably with orthogonal ion injection and optionally an ion detector. Non-limiting examples of ion mobility separators are drift tube ion mobility separator and trapped ion mobility spectrometry (TIMS) device.

[0154] A 'TIMS device' may be operated in a (basic) parallel accumulation mode, wherein ions, provided from the ion source, enter the ion mobility separator during a measurement cycle and accumulate parallel to the measurement cycles, such that ion accumulation duration is extended to enable the identification of more detectable ion species, or in using a spatial or temporal zoom, wherein in partial zoom mode, ions are again gathered in an accumulator unit at a rising edge of an electric field barrier. In other words, commonly a TIMS device is located between an ion source, which ionizes the analytes eluting from a chromatography system, and the Q1 quadrupole, which isolates ions based on their m / z values. The TIMS device accumulates ions and then gradually releases them, depending on their mobility in a gas. This way, additional separation of ions is achieved. Importantly, in embodiments the operation of the quadrupole can be tied to the ion mobility. That is, in embodiments for each ion mobility value, the m / z boundaries of the isolation window (m / z range) can be set separately, either in data-independent or data-dependent manner. In practice, this leads to a fold-change boost in the numbers of precursor ions that can be targeted in data-dependent fashion, in comparison to non-TIMS mass spectrometers.

[0155] In embodiments, a SLIM (structures for lossless ion manipulation) device can be used for ion mobility separation of analytes similarly to a TIMS device.

[0156] Examples for manufacturers of mass spectrometers for ion mobility measurements are Waters Technologies, MA, USA, MOBILion, USA, or Bruker Daltonik, DE.

[0157] Collision cross section (CSS) or collision cross section values are considered a robust and precise physiochemical property of an ion that corresponds to the chemical structure and 3D conformation of said ion.

[0158] Mass spectrometry ionization techniques can incur, especially for complex samples, overlapping isotopic patterns (MALDI) or isotopic clusters for each m / z value and multiple charge values per molecular mass (ESI), which commonly constitute obstacles for accurate mass determination. The preferred method to address said issues in MS proteomics is data deconvolution. MS data 'deconvolution' can improve the accuracy of signal intensity determination for analytes. Deconvolution of mass spectral data facilitates further the correction for instrumental mass bias and the identification of pure component spectra from a spectrum of a mixture of components. One example is that electro spray ionization of peptides / proteins typically results in multiple charged ions, such that determining ion masses requires the assignment of charge states. A deconvolution approach for determining charge states may consider the isotope distribution and utilizes the known mass distance between the different isotopes. In general mathematical terms 'deconvolution' of a signal or data refers to an algorithm-based reversion ('cleaning') of the effects of distortion, convolution or other impairing / adverse factors on recorded data.

[0159] In embodiments an ion reaction, collusion and / or fragmentation cell may be selected from the group comprising a collisional induced dissociation (CID) fragmentation device, a surface induced dissociation (SID) fragmentation device, an electron transfer dissociation (ETD) fragmentation device, and an electron capture dissociation (ECD) fragmentation device.

[0160] In embodiments a mass analyzer selected from the group comprising a quadrupole mass analyzer, a 2-D or 3-D a quadrupole mass analyzer, Penning trap mass analyzer, an ion trap mass analyzer and a time of flight (TOF) mass analyzer.

[0161] 'Time-of-flight' (TOF) mass spectrometry determines the m / z ratio of an ion based on its velocity after acceleration by an electric field of known strength. The mass-to-charge ratio of the ion may then be determined based on it's velocity, namely based on the time that the accelerated ion needs to reach a detector at a known distance. As ions having the same charge also have the same kinetic energy, heavier ions fly slower (reach the detector later) than lighter ions with a similar charge. TOF mass spectrometers therefore preferably comprise at least an ion source, a mass analyzer and a detector. In embodiments, fragmented ions are accumulated prior to the injection into a TOF mass analyzer. Generally, the time-of-flights themselves range from 700 Hz to 20000 Hz, wherein Astral may be e.g., 250 Hz-300 Hz, in embodiments DIA windows may be acquired with < 10 Hz also on TOFs.

[0162] An 'orbitrap' mass analyzer is a version of an ion trap that can function as both, an analyzer and detector. An orbitrap commonly comprises two outer and one central electrode, facilitating the separation of different ions, as they oscillate at different frequencies around the central electrode and between the outer electrodes. The measurement of the oscillation frequencies induced by ions on the outer electrodes facilities the acquisition of mass spectra using image current detection. Based on the design, an orbitrap mass analyzer may be considered a Fourier transform mass analyzer.

[0163] A mass spectrum indicates the abundance of ions for a particular m / z value of interest as a graph or table (or data array). Distinct peaks in the mass spectrogram (that do not overlap with each other) indicate the abundance of the ion or ion species at particular m / z values, hence, 'abundance' herein generally indicates the amount of a particular ion species. In embodiments, the whole spectrum as well as groups of peaks or individual peaks may be supplied with extra information, such as ion mobility (IM), collisional cross section (CCS), collision energy, fragmentation mode and the settings of the fragmentation device, m / z isolation window boundaries and mass filter settings, information on corrections applied to adjust the recorded m / z or intensities values, information on deisotoping, peak picking or centroiding applied.

[0164] Commonly, two main MS / MS data acquisition approaches are used comprising data-dependent acquisition (DDA) and data-independent acquisition (DIA).

[0165] During DDA MS / MS is performed on a subset of precursors ions acquired in a previous survey (MS1) scan. During DDA the acquisition alternates between MS1 survey scans and MS / MS (MS2) scans for the precursors of highest interest (e.g., with the highest intensity in MS1) acquired in the previous survey scan. Thereby DDA uses narrow (~ m / z 1.0) isolation windows, determined in a data-dependent manner, wherein preferably the mass spectrometer analyses the data acquired so far and determines the optimal positions (m / z bounds) of the isolation windows. Compared to DIA, it is easier to assign peptide sequences to DDA spectra, as they are less complex and better suited for database searches. However, DDA suffers regularly from interferences arising from co-eluting peptides, resulting in false negatives or positives. Moreover, in DDA only specific ions are selected for fragmentation and MS2 measurement, based on their intensity or abundance in a previous MS1 scan, such that low-abundance ions or ions with lower intensity in MS1 are potentially excluded from fragmentation, analysis and detection. Since the MS / MS data in a DDA experiment is stochastically sampled, it is commonly impossible to determine whether a peptide is undetectable without matching spectra or detectable but not sampled by MS / MS.

[0166] DIA, in contrast, repeatedly cycles through a set of pre-defined wide isolation windows (typically m / z 5.0 - m / z 50.0). DIA has many significant advantages, such as that all peptide ions falling in the m / z range of interest (e.g. m / z 400 to m / z 1000) are consistently fragmented and thus can potentially be identified. Another advantage is significantly more reliable quantification. DIA preferably enables the fragmentation and quantification of every analyte ion present in a sample, such that preferably all fragment ions within a predetermined m / z range are detected. However, DIA commonly results in complex multiplexed MS2 fragment spectra, wherein multiple precursor ions from a wide isolation window are fragmented simultaneously and share fragment ions, which increased the complexity of data processing and analysis. However, this enables the detection and quantification of each detectable analyte in the sample, such that even low-abundance peptides may be detected.

[0167] The `isolation window acquisition (or accumulation) time' commonly describes the time period within which the quadrupole is configured to particular m / z boundaries (e.g., a particular isolation window).

[0168] A 'precursor range' is a range of m / z values (e.g., in regular MS) or combinations of m / z and ion mobility values (e.g., in ion mobility-enabled MS) of interest for the identification of analyte ions (e.g., charged peptides or metabolites) in a particular MS experiment.

[0169] Targeted / scheduled m / z value - the m / z value of particular interest for MS / MS analysis, identified by mass spectrometer dynamically in a data-dependent fashion. For example, m / z values at which strong peaks are observed in MS1 spectrum can be targeted for MS / MS in a short period following the acquisition of that MS1 spectrum.

[0170] 'Isolation window difference' between two isolation windows is by definition the set of m / z values which belong to one of the windows but not the other. Such a difference can form an empty set, a single interval or two intervals.

[0171] 'Isolation window overlap' is by definition the set of m / z values which belong to both of the windows. Such an overlap can form an empty set or an interval.

[0172] In mass spectrometry, an exclusion list, or a dynamic exclusion, describes as list comprising certain masses (ions) that are excluded from analysis, e.g., for a certain period of time during dynamic exclusion (e.g. 5-100 s), such that the mass spectrometer can analyze other ions, e.g., less abundant ions and / or ignore, e.g., also contaminants. In other words, an exclusion list comprises / lists masses (e.g., undesired contaminants and / or just previously (a few ms or s before) analyzed masses) that the mass spectrometer is instructed to ignore. For example, during dynamic exclusion the mass spectrometer may first measure the most abundant ions (with the highest intensity), wherein said masses are subsequently added to a temporary / dynamic exclusion list (e.g., for a time period of 10-90 seconds), such that ions of the less abundant peptides may be measured in addition. In proteomics, dynamic exclusion therefore enables also the MS analysis of less abundant ions instead of repeatedly analyzing only the (peptide) ions with the highest abundance. In some embodiments an exclusion list may (permanently) comprise contaminants or other ions to be excluded from analysis. On the other hand, an inclusion list is commonly a list of analytes of (particular) interest.

[0173] The skilled person is familiar with suitable methods and preparations for performing (standard) mass spectrometric measurements and methods of sample preparation for mass spectrometric analysis and common MS data analysis methods. Exemplary workflows for performing the present method are described, in the examples herein.

[0174] The instant disclosure also includes kits, packages and multi-container units containing the herein described pharmaceutical compositions, active ingredients, and / or means for administering the same for use in the prevention and treatment of diseases and other conditions in mammalian subjects.

[0175] The term "at least one" may herein refer to at least one, more than one, at least two at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least twenty, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 500, 1000, 10.000.FIGURES

[0176] The invention is further described by the following figures. These are not intended to limit the scope of the invention, but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.

[0177] Figure 1: Extracted ion chromatograms were generated and plotted for selected fragment ions of a peptide with amino acid sequence NGGLGHMNIALLSDLTK (SEQ ID NO: 1), within 0.5 minutes of the elution (retention) time 35.63961 minutes, as recorded in the raw data file. The chromatograms were extracted from MS / MS spectra corresponding to the precursor ion isolation windows m / z 558-586 (top row) and m / z 585-614 (middle row). For each MS / MS cycle, the minimum of the signals recorded in each of the above chromatograms for this cycle was calculated, resulting in an in silico generated chromatogram corresponding to the overlap of the above isolation windows (bottom row). For visualization, the maxima of the chromatograms were scaled to 1, and y-axis annotation was omitted. Each point on each chromatogram plot corresponds to a single MS / MS cycle. The fragment ions to which the chromatograms correspond are annotated with the fragment ion series identifier ('b', i.e. N-terminal, or 'y', i.e. C-terminal) as well as the number of amino acid residues constituting the respective fragment ion. A dashed vertical line indicates the x-axis midpoint and corresponds to the MS / MS cycle with annotated elution (retention) time closest to 35.63961 minutes.EXAMPLES

[0178] The invention is further described by the following examples. These are not intended to limit the scope of the invention, but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.Example 1

[0179] In the first example the mass spectrometer operates part of its duty cycle in MS1 mode and part of it in MS2 (also called MS / MS) mode, however, compared to DDA or DIA methods, the present method may employ in the present example a different and new approach for the MS2 operation. The key property of the method according to the present example is that it repeatedly isolates all precursor ions for fragmentation as it is commonly performed in data independent acquisition (DIA), but does so by controlling the mass filter, e.g., quadrupole, isolation window in a data-dependent fashion, similar to classical DDA, to enable deconvolution of the data. Thereby the present method surprisingly obtains significantly more precise precursor mass information for the observed fragment ions, than it is possible with prior art DIA or DDA methods. This synergistic effect of the present method was entirely surprising.

[0180] In the present example of the method according to the invention, the mass spectrometer analyses the data acquired already (e.g., from the most recent, or from one or more previous MS1 cycles), to mark m / z values that might be of interest for MS / MS targeting (thereby selecting ions for fragmentation and subsequent MS2 detection) in the particular experiment. Different established mass spectrometric methods (from the context of DDA operation) may be used in the context of the present method for determining such m / z values of interest.

[0181] In contrast or in addition, the data from an inclusion lists representing analytes of a particular interest can be used to obtain the m / z values of particular interest for targeting.

[0182] In the present example of the proposed method, the mass filter, e.g., quadrupole, isolation window boundaries are selected in such a way, as to repeatedly cover the precursor m / z range, such that every m / z value (or m / z and ion mobility value combination) within the respective m / z range is expected to be selected by the quadrupole at least once during the observed elution from a chromatography source of a typical peptide from the chromatography system.

[0183] Moreover, the quadrupole isolation window boundaries are further selected to ensure that in a short period of time (preferably within a time period of less than or comparable to the time a typical peptide ion requires for the elution from a chromatography source or, in case of direct infusion, of the whole analysis time) a pair of different isolation windows are acquired (each for a certain amount of time, the principle is generally comparable to that typically used either for DDA or DIA) that satisfy the following properties: 1. the precursor ions selected by these two isolation windows are subjected to fragmentation, 2. the two isolation windows partially overlap with each other, 3. either the difference between the isolation windows (the area where they don't overlap) or their overlap constitute one or more intervals, out of which at least one is 'narrow' (in comparison to the entire span of each isolation window; in embodiments a preferred size / span of such overlapping interval may be within the range m / z 0.5 - m / z 3.0) and contains at least one of the m / z values of interest for MS / MS targeting. The width of the above 'narrow' interval is referred to below also as 'precursor selection precision', 4. The resulting data is deconvoluted by the raw data processing software. The deconvolution ensures that for strong fragment ion signals it is possible to determine the respective precursor mass with the precision which is defined by the 'precursor selection precision' and the quadrupole error.

[0184] In one specific example of conducting the present method some isolation window boundaries are selected data-dependent (e.g., based on MS1 or MS2 data acquired so far), while some isolation windows are defined by pre-selected criteria, such as a specific width / m / z range and not dependent on MS1.Example 2

[0185] In classical state of the art DDA experiment a DDA method can be envisioned to repeatedly go through the following cycle: 0) 0 ms - 50 ms: MS1 acquisition without precursor isolation or precursor ions fragmentation; 1) 50 ms - 75 ms: isolation of the 1 st scheduled m / z value with an m / z 1.0 window; 2) 75 ms - 100 ms: isolation of the 2nd scheduled m / z value with an m / z 1.0 window; 12) 300 ms - 325 ms: isolation of the 11 th scheduled m / z value with an m / z 1.0 window; 13) 325 ms - 350 ms: isolation of the 12th scheduled m / z value with an m / z 1.0 window;

[0186] The MS / MS spectra obtained are usually dominated by fragments of a single precursor and are thus easier to analyze. However, with this classical DDA proceeding many precursors are not fragmented at all and thus cannot be identified with confidence, which severely reduces the numbers of peptides and proteins detected and also introduces missing values between MS runs in the experiment.

[0187] In contrast, a DIA method can be envisioned to repeatedly go through the following cycle: 0) 0 ms - 50 ms - MS1 acquisition without precursor isolation or precursor ions fragmentation; 1) 50 ms - 75 ms - A first isolation window (mz1) of a range m / z 400.0 - 450.5 is applied to select precursors to fragmentation; 2) 75 ms - 100 ms - A second isolation window (mz2) of a range m / z 449.5 - 500.5 is applied to select precursors to fragmentation, wherein the overlap between the windows at m / z 450.0 is m / z 1.0 (this procedure accounts for the quadrupole error); 11) 300 ms - 325 ms - Another first isolation window (mz1) of a range m / z 899.5 - 950.5 is applied to select precursors to fragmentation; 12) 325 ms - 350 ms - A second isolation window (mz2) of a range m / z 949.5 - 1000.0 is applied to select precursors to fragmentation;

[0188] An embodiment of a suitable implementation of the present method is performed similar to a typical DDA method and repeatedly goes through the following cycle (all times in milliseconds (ms) from the beginning of the cycle): X) 0 ms - 50 ms - MS1 acquisition without precursor isolation or precursor ions fragmentation;

[0189] In the present exemplary embodiment the m / z values to be specifically targeted are determined as in DDA, e.g., the method described before, but only 11 (mzA,...,mzK) instead of 12 m / z values are selected and ordered not by the signal intensity in the MS1 spectrum but rather by the m / z value itself. Let mzA,...,mzK be their ordered masses. In a non-limiting example, the m / z values can be selected as the most abundant m / z peaks in the MS1 spectrum not contained in the (dynamic) exclusion list, or in any other way typical for DDA acquisition.

[0190] The present embodiment then defines pairs of isolation windows (mz1, mz2) that cover the m / z values to be specifically targeted as well as an m / z range upstream and / or downstream of said m / z value and that overlap with each other such that the m / z value to be specifically targeted is preferably covered by the overlap of said isolation windows. For example: A) 50 ms - 75 ms - A first isolation window (mz1) of a range m / z 400.0 - mzA +0.5< is used to direct precursors to fragmentation; B) 75 ms - 100 ms - A second isolation window (mz2) of a range of m / z mzA -0.5< - mzB +0.5< is used to direct precursors to fragmentation; ... wherein the last neighboring of isolation windows are selected as follows: K) 300 ms - 325 ms - An eleventh isolation window (mz11) of a range of m / z mzJ -0.5< - mzK +0.5< is used to direct precursors to fragmentation; L) 325 ms - 350 ms - A twelfth isolation window (mz12) of a range of: m / z mzK -0.5< - 1000.0 is used to direct precursors to fragmentation.

[0191] In such an experimental workflow for each m / z value to be covered by an isolation window, e.g., each target m / z value is comprised at least by a m / z range of + / - m / z 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3. Preferably one or more neighboring isolation windows covering neighboring target m / z values overlap with each other. Preferably said overlap covers a range of at least + / m / z 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3.

[0192] The data processing software then identifies peptides as follows, but in no particular order. First, the obtained mass spectral data is processed similar to regular DIA data, except for the consideration of the varied isolation window boundaries, which surprisingly were found to provide extra mass selectivity.

[0193] For example, a precursor ion with m / z 460 is eluting (from the chromatography source) for long enough time to be detectable in 3 consecutive MS / MS cycles of the workflow described above, in the first of these, the isolation window is 429.5 - 470.5, in the second it is 439.5 - 465.5, in the third it is 419.5 - 490.5. In this embodiment the target ion is comprised in the overlap of all three windows. Then the software may be programmed to recognize that the fragments from this precursor would be detectable in all three of these windows, but not in the adjacent windows like 400.0 - 439.5. Only precursor from the range m / z 439.5 - 465.5 will satisfy this condition. In contrast, in regular DIA, as illustrated above, the respective range could be, for example, 449.5 - 500.5, which is in fact wider. It is true that it is possible to also 'shift' isolation windows of regular DIA between cycles, gaining a bit of extra m / z selectivity (this is long known). The point here is that it is possible to analyze the data of our technology in exactly the same way as regular DIA data, with the same or even better performance.

[0194] Second, the data processing software also identifies peptides as follows: a raw-data analysis software deconvolutes the spectra for the targeted m / z values. This is indeed simple and requires less computation time and resources compared to a regular analysis of DIA data, e.g., when the mass spectrometer targeted m / z 440, and used the isolation windows 400.0 - 440.5 and 439.5 - 465.5 in the respective cycle. In this case the raw-data analysis software will consider only MS / MS fragments detected for both neighboring isolation windows (lying within their overlap) as the spectrum of the precursor at m / z 440, thus significantly simplifying the spectrum and solving the data deconvolution problem specifically for this precursor, similar to DDA.

[0195] In summary, the present method is able to identify the peptides which would have been identified with DIA and also identify the peptides which would have been identified with DDA. The method according to the invention is further capable of quantifying all these peptides with the quality of quantification inherent to DIA. Accordingly, the method according to the invention combines the advantages of both DIA and DDA in a single acquisition.

[0196] However, the design of the present method was not obvious or straight forward derivable from a simple combination of DDA and DIA analysis. The synergistic effects achieved by the present method would not be obtained by simply acquiring the sample with DIA and then with DDA instead. Such a strategy would not be financially or economically feasible, as this simple combination would not just require twice the time of simple DIA and DDA, but also require double amounts of sample (which is often a critical factor for LC-MS / MS analysis). Most importantly, a simple combination of a DDA and DIA measurement would introduce extra variability of quantities that would render the normalization between DIA and DDA data extremely difficult.

[0197] Also, both software tools as well as instruments that are presently available are not suitable for performing the present method without further modification.

[0198] In embodiments the present method may be performed using a (modified) trapped ion mobility (TIMS) device mass spectrometer, e.g., a TIMS-TOF instrument (Bruker), or a SLIM device (by MOBILion). In below a reference to a TIMS device should be understood as a reference to either TIMS or SLIM device.

[0199] In embodiments the selection and isolation of precursor ions is based on ion mobility values obtained by a TIMS device and / or the m / z values acquired in a MS1 measurement in step a. In embodiments the ion mass spectral data acquired in a MS1 measurement in step a. comprises ion mobility and / or m / z values.

[0200] In embodiments the operation of the mass filter device, preferably a quadrupole device, can be tied / linked to the ion mobility data obtained by a TIMS device. That is, in embodiments for one or more or each ion mobility value, the boundaries of the m / z range of a respective isolation window can be set separately, either in data-independent or data-dependent manner. One beneficial effect of such embodiments is that this linking of ion mobility data (TIMS device) and Q1 operation leads to a fold-change boost in the numbers of precursor ions that can be targeted in data-dependent fashion, in comparison to non-TIMS mass spectrometers.

[0201] In addition to the extra separation of ions, which leads to 'cleaner' spectra, the TIMS device has the following benefit. Without getting into technical detail, a TIMS device' operation is characterized by two configurable time periods: accumulation time (time spent accumulating ions) and ramp time (time spent releasing ions depending on their ion mobility). These are usually set to the same value, e.g. 100 ms is popular. During the ramp time, the quadrupole can be switched between different settings multiple times, e.g., 10 or 25 times. The acquisition during the ramp time is called a 'frame'. Different frames can feature different isolation windows.

[0202] Until recently, in the data-independent setting, the windows were designed in such a way so that within each frame they did not overlap in the m / z dimension. Importantly, the property of TIMS acquisition is such, that the sensitivity is similar to that of a non-TIMS acquisition, if each of those isolation windows were acquired with the specified accumulation time, e.g. 100 ms. That is TIMS allows for a fold-change boost in sensitivity.

[0203] The inventors recently introduced a new data-independent method of controlling the quadrupole depending on the ion mobility, termed Slice-PASEF (Szyrwiel et al., 2022). The method was the first working implementation of the idea that isolation windows within a frame may overlap in the m / z dimension, with lifting this restriction allowing the design of high-sensitivity methods. Therein, the inventors managed to solve the computational problem of dealing with such data, and reported significant benefits for single cell-level proteomics. Therefrom, the inventors now developed methods to improve and implement this initial idea. Surprisingly, they found that by controlling a mass filter, e.g., quadrupole, in a data-dependent fashion they can significantly improve data deconvolution, such that basically all or numerous ions within a m / z range of interest (e.g. m / z 400 to m / z 1000) may be (consistently) fragmented and analyzed and thus can be identified by MS / MS analysis, e.g., as significantly more precise precursor mass information can be obtained for fragment ions of interest.

[0204] Specifically, one of the Slice-PASEF methods developed by the inventors controls the quadrupole in such a way, that each DIA cycle consists of two frames. In the first frame, for a particular ion mobility value, the quadrupole is set to the mass window [start m / z, x]. In the second frame, it's set to [x, stop m / z]. The start m / z and stop m / z as well as 'x' depend on the ion mobility value, and 'x' is also varied between the cycles, allowing for later computational deconvolution of the data.

[0205] For example, the acquisition can look like this (for a particular ion mobility value): Cycle 1: 500 m / z - 600 m / z (frame 1), 600 m / z - 800 m / z (frame 2) [x = m / z 600 here] Cycle 2: 500 m / z - 650 m / z (frame 1), 650 m / z - 800 m / z (frame 2) [x = m / z 650 here] Cycle 3: 500 m / z - 700 m / z (frame 1), 700 m / z - 800 m / z (frame 2) [x = m / z 700 here]

[0206] This varied position of 'x' provides a slight benefit in terms of data deconvolution.

[0207] However, controlling the quadrupole in data-dependent fashion, as proposed in the present invention, can significantly improve and empower the deconvolution, leading to very 'clean' in silico deconvoluted spectra with reduced background noise.

[0208] In one exemplary embodiment, at a given ion mobility value m / z 670 is targeted, such that the MS cycles may look as follows (m / z bounds for the specific ion mobility): Cycle 1: 500 m / z - 670.5 m / z (first m / z range, mz1), 670.5 m / z - 800 m / z (second m / z range, mz2) [x = 670.5 m / z here] Cycle 2: 500 m / z - 669.5 m / z (first m / z range, mz1), 669.5 m / z - 800 m / z (second m / z range, mz2) [x = 669.5 m / z here]

[0209] This proceeding simultaneously allows for two levels of computational deconvolution, to obtain the spectrum of the precursor at 670 m / z: First, only fragment peaks detected in both mz1 of cycle 1 and mz2 of cycle 2 are considered. Second, from the above spectrum, the spectra of mz2 of cycle 1 and mz1 of cycle 2 are subtracted.

[0210] In embodiments the proceedings for one level of computational deconvolution are also possible, doubling the number of targeted precursors: Cycle 1: 500 m / z - 670.5 m / z (first m / z range, mz1), 669.5 m / z - 800 m / z (second m / z range, mz2). Also in this embodiment the m / z ranges mz1 and mz2 overlap at the m / z value of interest m / z 670), in other words the overlap between mz1 and mz2 comprises the m / z value of interest m / z 670.

[0211] For said examples, the computational deconvolution may thus consider only fragment peaks detected in both frames of the cycle, similar to the example for the non-TIMS mass spectrometer.

[0212] The above is one special case of the invention applied to TIMS acquisition, other methods featuring more frames per cycle and / or different deconvolution approaches are also possible, satisfying the same requirements as regular non-TIMS acquisition, but with m / z isolation controlled separately for different ion mobility values. The great benefit of TIMS here is the number of targeted m / z values. That is, in a regular non-TIMS method, it's roughly 1 m / z value per the accumulation time of an isolation window. With TIMS it can easily be 10+. That is vastly more precursors will get acquired with clean DDA-like fragmentation spectra, making the present invention particularly beneficial for TIMS acquisition.

[0213] Other ion mobility separation devices exist, e.g. FAIMS device from Thermo Fisher. These do not feature 'trapped' ion mobility, and thus the operation of mass spectrometers equipped with these is more similar to regular non-TIMS acquisition, with the proposed invention likewise applicable.Example 3

[0214] Extracted ion chromatograms were generated and plotted for selected fragment ions of a peptide with amino acid sequence NGGLGHMNIALLSDLTK (SEQ ID NO: 1), within 0.5 minutes of the elution (retention) time 35.63961 minutes, as recorded in the raw data file. The chromatograms were extracted from MS / MS spectra corresponding to the precursor ion isolation windows m / z 558-586 (top row) and m / z 585-614 (middle row). For each MS / MS cycle, the minimum of the signals recorded in each of the above chromatograms for this cycle was calculated, resulting in an in silico generated chromatogram corresponding to the overlap of the above isolation windows (bottom row). For visualization, the maxima of the chromatograms were scaled to 1, and y-axis annotation was omitted. Each point on each chromatogram plot corresponds to a single MS / MS cycle. The fragment ions to which the chromatograms correspond are annotated with the fragment ion series identifier ('b', i.e. N-terminal, or 'y', i.e. C-terminal) as well as the number of amino acid residues constituting the respective fragment ion. A dashed vertical line indicates the x-axis midpoint and corresponds to the MS / MS cycle with annotated elution (retention) time closest to 35.63961 minutes. The data is shown in Figure 1. The data demonstrates how computational deconvolution can be used to significantly reduce the numbers and intensity of non-cognate background peaks.

[0215] The raw data file was downloaded from the ProteomeXchange repository with identifier PXD005573 and converted to the mzML format with a tool MSConvert GUI, part of the Proteowizard software suite with version 3.0.23333.f17d5a0, with the settings: --32 --filter "peakPicking vendor msLevel=1-". The data analysis was carried out in R version 4.3.1. The mzML file was loaded using the readMSData() function of the MSnbase package (version 2.28.1). Chromatograms were extracted using the calculateFragments() function of the MSnbase package, with settings: 'tolerance' = 20.0 / 100000.0, 'relative' = T, 'method' = 'highest'.REFERENCES

[0216] Wilkinson, J., `High-performance liquid chromatography-tandem mass spectrometry for analysis of aquatic contaminants: A high-level introduction to the technique', Monitoring Environmental Contaminants, Elsevier, 2021, Pages 1-17, ISSN 24055654, https: / / doi.org / 10.1016 / B978-0-444-64335-3.00004-9. Szyrwiel, L., Sinn, L., Raiser, M, Demichev, V., 'Slice-PASEF: fragmenting all ions for maximum sensitivity in proteomics', bioRxiv 2022.10.31.514544; doi: https: / / doi.org / 10.1101 / 2022.10.31.514544

Claims

1. A method for analyzing a sample comprising one or more molecules of interest by a mass spectrometer, comprising ionizing a multitude of molecules and / or fragments thereof comprised within a sample to obtain a multitude of molecule ions (source ions), and performing mass spectrometry analysis comprising a. analyzing the molecule ions and / or fragments thereof in a first mass spectrometry (MS) measurement (MS1), thereby acquiring ion mass spectral data of at least a fraction of the molecule ions and / or fragments thereof (source ions) comprising their mass to charge ratio (m / z), b. isolating a first fraction of the molecule ions (precursor ions) having a m / z within a first m / z range (mz1; isolation window), using a mass filter device, preferably a quadrupole device, wherein the first m / z range is selected based on the ion mass spectral data (i) acquired in step a., or one or more iterations of step a. and / or e., and optionally (ii) an exclusion list and / or an inclusion list, c. isolating a second fraction of the molecule ions (precursor ions) having a m / z within a second m / z range (mz2; isolation window), wherein the second m / z range is selected based on the ion mass spectral data (i) acquired in step a., or one or more iterations of step a. and / or e., and optionally (ii) an exclusion list and / or an inclusion list, and wherein the second m / z range (mz2) overlaps with the first m / z range (mz1), d. fragmenting at least a fraction of the molecule ions (precursor ions) isolated in steps b. and c., separately, to obtain a multitude of biomolecule fragment ions from each of the respective fragmented molecule ions (precursor ions), e. analyzing the multitude of biomolecule fragment ions in one or more second mass spectrometry measurement (MS2), thereby acquiring ion mass spectral data of the analyzed biomolecule fragment ions.

2. The method according to claim 1, wherein steps b.-e. or steps c.-e. are repeated at least once.

3. The method according to claim 1 or 2, wherein each m / z range (isolation window) selected and isolated in steps b. and c. overlaps with its neighboring m / z range by less than m / z 3.0, preferably with an overlap between m / z 0.5 and m / z 1.5, or wherein each m / z range (isolation window) selected and isolated in steps b. and c. comprises a non-overlapping part with its neighboring m / z range of less than m / z 3.0, preferably between m / z 0.5 and m / z 1.5.

4. The method according to any one of the preceding claims, wherein one or more m / z ranges (isolation windows) selected and isolated in steps b. and c. comprise only one non-overlapping part with the neighboring window (on one side).

5. The method according to any one of the preceding claims, wherein one or more m / z ranges (isolation windows) selected and isolated in steps b. and / or c. are selected to comprise one or more m / z values characteristic for a molecule ion (source ion) and / or fragment ion of a molecule of interest, preferably wherein one or more m / z values characteristic for a molecule ion (source ion) and / or fragment ion of a molecule of interest are comprised either within the overlap or the non-overlapping part of a m / z range (isolation window), whichever is more narrow, preferably spanning less than 30% of the entire m / z range (isolation window), than the other part.

6. The method according to the preceding claim, wherein the one or more m / z values characteristic for a molecule ion (source ion) and / or fragment ion of a molecule of interest are selected / identified based on: the ion mass spectral data acquired in one or more iterations of step a. and / or e., and / or a database comprising m / z ratios characteristic for one or more molecule ion (source ion) and / or fragment ion of molecules of interest.

7. The method according to any one of the preceding claims, wherein the m / z ranges (isolation windows; mz1, mz2,... mzX) exhibit a range of m / z 5 - 100, preferably m / z 10-50, and / or wherein the total m / z range of interest spans a m / z range of between m / z 450.0 (mz1) - 800.0 (mzX).

8. The method according to any one of the preceding claims, wherein the ion mass spectral data acquired in step a. (MS1) comprises m / z and ion mobility data and / or collisional cross section (CCS) data.

9. The method according to the preceding claim, wherein in step b. one or more m / z range(s) are selected based on the m / z data and the ion mobility and / or collisional cross section (CCS) data acquired in step a.

10. The method according to any one of the preceding claims, wherein in steps b. and c. one or more of the m / z ranges selected for mass filter isolation and subsequent fragmentation are selected independently of the ion mass spectral data acquired in step a. or any previous iteration of step a.

11. The method according to any one of the preceding claims, wherein prior to performing mass spectrometry analysis, the sample is processed by chromatography, and the molecules comprised within the sample elute over a timespan from the chromatography or electrophoresis source and are processed according to the steps a.-e. during an elution timespan of said molecules, thereby acquiring ion mass spectral data of the molecules comprised within the sample in relation to their elution time (retention time (RT)) from the chromatography or electrophoresis source, optionally wherein steps a. to e. or c. to e. are repeated for multiple iterations over the elution time (retention time (RT)) of at least one molecule of interest.

12. The method according to any one of the preceding claims, wherein the method further comprises f. analyzing the ion mass spectral data acquired in one or more iterations of steps a. and e., thereby identifying and / or characterizing at least one molecule of interest comprised within the sample, and / or optionally wherein two or more MS1 or MS2 measurements and / or MS1 or MS2 spectra are combined to generate a single spectrum.

13. The method according to the preceding claim, wherein the analysis comprises identifying one or more molecules (of interest) by comparing the signal intensity determined in one or more MS1-measurements at a specific mass to charge ratio (m / z) to a database comprising m / z ratios characteristic for one or more molecule ion (source ion) of respective molecules (of interest).

14. The method according to the preceding claim, wherein identifying one or more molecule ions (source ions) further comprises comparing m / z values determined in one or more MS1-measurements and corresponding MS2-measurements to a database comprising m / z values characteristic for one or more molecule ion (source ion) and one or more fragment ion of respective molecules (of interest).

15. The method according to any one of claims 12-14, wherein the ion mass spectral data obtained in steps a. and e. is computationally deconvoluted prior to the data analysis according to step f., optionally wherein the mass of a molecule ion (precursor ion) is calculated during ion mass spectral data analysis in step f. based on the deconvoluted data of one or more of the corresponding molecule fragments.

16. A computer-implemented method for analyzing ion mass spectral data acquired during one or more iterations of steps a. and e. according to the method of any one of the preceding claims, comprising: a. receiving acquired ion mass spectral data of a MS1 analysis according to step a. of claim 1, b. identifying one or more m / z range(s) (isolation window(s)) of interest comprising m / z data of a molecule of interest, c. selecting molecule ions from one or more m / z range(s) (isolation window(s)) of interest for fragmentation and inducing the mass filter device of the mass spectrometer to transmit said molecule ions to a fragmentation and / or reaction device, d. receiving acquired ion fragment mass spectral data of a MS2 analysis according to step e. of claim 1, e. deconvoluting and analyzing the received ion (fragment) mass spectral data thereby identifying and / or characterizing at least one molecule of interest comprised within a sample.

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