Method and device for combined ion mobility and tandem mass spectroscopy analysis

Abstract

A method of combined liquid chromatography (LC) or capillary electrophoresis (CE), ion mobility and tandem mass spectroscopy analysis, comprising the following steps: introducing precursor ions resulting a TIMS separator, sequentially releasing precursor ions according to their ion mobility, introducing the precursor ions into a mass filter in a first mass dimension (M-1), fragmenting the precursor ions transmitted through said mass filter to generate fragment ions, carrying out a mass spectroscopy measurement on said fragment ions in a second mass dimension (M-2), and associating detected fragments with precursor ions, wherein said TIMS separator and said mass filter are scheduled in a synchronized manner, and wherein for associating detected fragments with their corresponding precursor ions, the isotope distribution of the precursor ions and of the fragments thereof are taken account of, and wherein at least two isotope signals of a corresponding precursor ion in the first mass dimension are taken account of.

Description

[0001] TITLE

[0002] METHOD AND DEVICE FOR COMBINED ION MOBILITY AND TANDEM MASS SPECTROSCOPY ANALYSIS

[0003] TECHNICAL FIELD

[0004] The present invention relates to methods of combined liquid chromatography (LC) or capillary electrophoresis (CE), ion mobility and tandem mass spectroscopy analysis, taking account of isotope information, and corresponding devices.

[0005] PRIOR ART

[0006] US 7,838,826 B1 (M. A. Park, 2008) and the corresponding patent family members presents a small ion mobility analyzer / spectrometer which has become known under the acronym “TIMS” analyzer / spectrometer (TIMS = trapped ion mobility spectrometry). The terms ion mobility analyzer and ion mobility spectrometer are used interchangeably here. A TIMS analyzer comprises a gas flow that drives ions against a counter-acting electric field barrier such that the ions are at first trapped along the axis of the TIMS analyzer. The ions are confined in the radial direction by an electric RF field. After transferring ions from an ion source to the electric field barrier, the height of the electric field barrier or the gas velocity is adjusted such that ion species are released from the electric field barrier in the sequence of their mobility.

[0007] Commonly, the length of the ion mobility separation unit of a TIMS analyzer amounts to about five centimeters only. In a small tube with an inner diameter of about eight millimeters, a radial RF quadrupole field is generated to hold ions near to the axis. A gas flow inside a tube drives ions entrained in the gas flow against a ramped counter-acting electric DC field barrier where the ions are trapped and separated according to their mobilities at locations on the field ramp at which the friction force of or rather from collisions with the moving gas equals the counter-acting force of the electric DC field on the ramp. After loading the TIMS with ions, the height of the electric DC field barrier is decreased; this scan releases the ion species in the sequence of their mobility. Unlike many other trials to build small ion mobility spectrometers, the small device by M. A. Park has already achieved, with reduced scan speeds, ion mobility resolutions up to RmOb = 400, which is extraordinarily high.

[0008] Regarding the theoretical basis of TIMS, see the research article “Fundamentals of Trapped Ion Mobility Spectrometry”, K. Michelmann, J. A. Silveira, M. E. Ridgeway and M. A. Park, J. Am. Soc. Mass Spectrom., (2015) 26: 14-24.

[0009] US 8,766,176 B2 proposes improvements of the scan modes for TIMS analyzers by application of non-linear scans to achieve a linear mobility scale, a constant resolution along the mobility scale, or a temporal zoom. Furthermore, US 9,984,864 B2 describes a spatial zoom.

[0010] A TIMS analyzer with parallel ion accumulation is described in US 9,683,964; it improves the utilization of the ions from the ion source to nearly 100%. Importantly, many ion sources, such as electrospray ion sources produce ions continuously. TIMS with parallel accumulation can also be operated to collect and analyze ions continuously - that is, TIMS can operate at 100% duty cycle. Barring pseudopotential or space charge effects, substantially all ions of the ion source are collected and analyzed without loss. TIMS with parallel ion accumulation further provides the possibility to prolong the ion accumulation and scan duration, thereby increasing the ion mobility resolution so as to separate and detect more ion species.

[0011] The ions are collected in an accumulator unit, preferably almost identical to the scanning unit, at a ramp of an electric DC field barrier such that they get spatially separated by their ion mobility along the ramp. Therefore, the accumulated ions are less influenced by space charge than in other types of accumulator units. Of greatest importance, however, is the unique feature of a TIMS analyzer that a longer accumulation period permits to increase the mobility resolution by choosing correspondingly longer mobility scan durations, e.g. 100 milliseconds scan duration with an ion mobility resolution of RmOb = 75 instead of 20 milliseconds scan duration with RmOb = 30. As a consequence of the higher number of ions collected and the better ion mobility resolution, more ion species can be detected and measured. Once an ion mobility scan is completed (optionally after twenty to some hundred milliseconds), the accumulated ions are transferred (in about a millisecond) from the accumulation unit to the scanning unit, and the next ion mobility scan can be started. In total, a skilled practitioner will appreciate that it will be possible to achieve a measurement rate of 300 to 450 ion species per second. If TIMS with parallel ion accumulation is installed in tandem mass spectrometer (MS / MS instrument) an MS-MS instrument, 300 to 450 characteristic fragment ion spectra per second may be measured quantitatively.

[0012] Some improvements for higher amounts of stored ions in selected regions of ion mobility, particularly for ions of low ion mobility, are given in US 9,304,106 B1. The higher loading capacity is based on non-linear electric DC field ramps, with flatter field ramps for ion species of interest, in order to diminish the effect of space charge for these ion species. But for precise ion mobility analyses of low abundant ion species in complex mixtures the influence of the space charge is still high.

[0013] TIMS extends conventional liquid chromatography-mass spectrometry (LC-MS) and bioanalytical workflows, such as proteomic, lipidomic, metabolomic, drug metabolism, structure elucidation or other methods, with an additional ion mobility dimension. In addition to the general benefits of the additional signal separation, the implementation of TIMS in the Bruker timsTOF family of instruments has demonstrated that collisional cross section (CCS) values of analytes, i.e. peptides, lipids, or metabolites, are highly reproducible. The additional separation dimension already allows in data dependent acquisition (DDA) methods to identify precursors of isomeric compounds to be scheduled for fragmentation individually. As omics workflows aim at covering as much of the chemical complexity of a sample with tandem-MS spectra by DDA, TIMS can already greatly improve the analytical coverage of complex samples.

[0014] WO-A-2019096852 relates to use of an isobaric label in mass spectrometry (MS) analysis using data-independent acquisition (DIA), wherein said isobaric label comprises or consists of a group which fragments in the mass spectrometer (i) at an energy below the energy required for fragmenting analyte-derived precursor ions and / or a higher conversion rate than said precursor ions; and (ii) at said energy according to (i) and when coupled to a precursor ion, at a single site within said group, to yield a first moiety and a second moiety, said second moiety being coupled to said precursor ion. It proposes the use of a trapped ion mobility spectrometry- time of flight (timsTOF) instrument, equipped with parallel / serial fragmentation (PASEF); see, e.g. Meier et al. 2015, doi: 10.1021 / acs.jproteome.5b00932. US-A-2019371585 relates to selection of precursors from a measured mobility-mass map for tandem mass spectrometry and is based on processing a peak list from measured signals and clustering these peaks in the mobility-mass space.

[0015] US-A-2022034840 discloses an apparatus and a method of data independent combined ion mobility and mass spectroscopy analysis which includes introducing precursor ions into an ion mobility spectrometer (IMS), sequentially releasing precursor ions from said IMS according to their ion mobility, introducing said released precursor ions into a mass filter, fragmenting the precursor ions transmitted through said mass filter to generate fragment ions, and carrying out a mass spectroscopy measurement on said fragment ions. The IMS and mass filter are controlled in a synchronized manner to carry out a plurality of IM scans, wherein adjacent mass windows in said IM scan that are associated with consecutive mass spectroscopy measurements of fragment ions overlap, such that precursor ions transmitted through said mass filter during said IM scan are located in at least one continuous scan region in an m / z-IM plane which extends in a generally diagonal direction in said m / z-IM plane.

[0016] Targeted selection of peptides from complex protein digests subjected to fragmentation is a performance-limiting factor in tandem mass spectrometry (MS). The problem is mainly attributed to the large number of co-eluting high abundance peptides and limitations in the duty cycle of the mass spectrometer arranged to operate in data-dependent acquisition (DDA) mode whereby precursor ions are selected sequentially for fragmentation in order of decreasing intensity. Furthermore, the depth of proteomic analyses is also limited by the overwhelming proportion of high abundance background ions that compromise the identification and quantification of low abundance peptides. Whether low-intensity precursors are selected for fragmentation is primarily dictated by the speed at which the mass analyzer can perform tandem MS analysis. The duty cycle of the MS remains a problem even in data-independent acquisition (DIA) mode where all ions within a selected mass-to-charge range are selected for fragmentation. The issue of sample complexity is routinely addressed by fractionating the sample.

[0017] Liquid chromatography coupled to Mass Spectrometry (LC-MS) has now been used for many years in the proteomic community for the identification and quantification of peptides (and thus proteins) from complex sample mixtures. In proteomics, the analytes are typically peptides generated by tryptic digestion of protein samples. The commonly most used approaches are variants of the so-called LC-MS / MS or “shotgun” MS approach that is based on the generation of fragment ions from precursor ions that are automatically selected based on the precursor ion profiles (data dependent analysis, DDA). A main shortcoming of these methods is poor reproducibility which results in only partially overlapping protein sets in repeated analysis of substantially similar samples. Several new approaches have recently been developed that address these limitations and which can conceptually be described as targeted proteomics approaches.

[0018] The most mature technology is called Selected Reaction Monitoring (SRM), frequently also referred to as Multiple Reaction Monitoring (MRM). The targets for MRM experiments are defined on a rational basis and depend on the hypothesis to be tested in the experiment. Selected combinations of precursor ions and fragment ions (so called transitions, the set of transitions for one target precursor is called MRM assays) for these targets are programmed into a mass spectrometer, which then generates measurement data only for the defined targets. Thus, in SRM, all transitions are monitored one at a time.

[0019] Lesur et al in "Highly Multiplexed Targeted Proteomics Acquisition on a TIMS-QTOF" (Anal Chem 2021 Jan 26;93(3):1383-1392; doi: 10.1021 / acs.analchem.0c03180) report, that targeted proteomics allows the highly sensitive detection of specific peptides and proteins in complex biological samples. They describe a methodology for targeted peptide quantification using a trapped ion mobility quadrupole time-of-flight mass spectrometer (timsTOF Pro). The prm-PASEF method (prm=parallel reaction monitoring) exploits the multiplexing capability provided by the trapped ion mobility separation, allowing more than 200 peptides to be monitored over a 30 min liquid chromatography separation. Compared to conventional parallel reaction monitoring (PRM), precursor ions are accumulated in the trapped ion mobility spectrometry (TIMS) cells and separated according to their shape and charge before eluting into the quadrupole time-of-flight (QTOF) part of the mass spectrometer. The ion mobility trap allows measuring up to six peptides from a single 100 ms ion mobility separation with the current setup. Using these improved mass spectrometric capabilities, they detected and quantified 216 isotope-labeled synthetic peptides (AQUA peptides) spiked in HeLa human cell extract with limits of quantification of 17.2 amol for some peptides. The acquisition method is reported to be highly reproducible between injections and to enable accurate quantification in biological samples, as demonstrated by quantifying KRas, NRas, and HRas as well as several Ras mutations in lung and colon cancer cell lines on fast 10 min gradient separations.

[0020] SUMMARY OF THE INVENTION

[0021] One may in a first approach consider individual isotope peaks when discussing the way signals in the fragment m / z, mobility plane are affected by the scanning quadrupole. The precursor m / z prediction obtained from characteristic fingerprints of these individual isotope peaks can be in the range of 1 Da if the signal intensity and data quality is sufficient and systematic deviations from the calibration are under control.

[0022] This achievable precision also illustrates that different isotope peaks of the same precursor molecule are projected in a significantly different way onto the space of fragment m / z, mobility and e.g. the midiaPASEF frame number which specifies the m / z mobility slice used for a certain MS / MS timsTOF frame (for midiaPASEF it is referred to Distler et al. in "midiaPASEF maximizes information content in data-independent acquisition proteomics", BioRxiv; htps: / / doi.Org / 10.1101 / 2023.01.30.526204, the content of which is included into this disclosure as it concerns this technology).

[0023] However, such approaches do not take into account the fact that non-monoisotopic peaks which are composed of different isotopes like 13-C, 15-N, ... contribute to different fragment m / z peaks. E.g. a “M+2” peak which has a mass of roughly 2 Da above the monoisotopic precursor mass can contribute to three fragment m / z values m, m+1 and m+2 depending on the number of heavy isotopes and whether the isotope has one or two additional neutrons.

[0024] Such a contribution from M+2 to the m+1 fragment peak has a different distribution in mobility and midia frame number as the contribution from M+1 since for a certain scan number in a certain frame, e.g. a midia frame, M+2 might be transmitted by the quadrupole but M+ 1 not yet, which will only be transmitted for larger scan numbers, or will be transmitted with a corresponding dampening transfer function.

[0025] The effects coming from this isotope distribution are limiting for a precursor m / z determination and a holistic modeling as part of the feature finding might also improve the selectivity and - with a certain acceptable false positive rate - achievable sensitivity.

[0026] Such a kind of analysis is also vital for digital synthesis scanning quadrupoles with arbitrary multi window and non-binary transmission functions beyond the current ones aiming for a single rectangular transfer window.

[0027] According to a first aspect of the present invention, for associating detected fragments with their corresponding precursor ions, the isotope distribution of the constituting atoms of the precursor ions and of the constituting atoms of the fragments thereof are taken account of, and at least two isotope signals of a corresponding precursor ion in the first mass dimension are taken account of for the association with corresponding fragment distributions in the second mass dimension.

[0028] More generally speaking, the present invention according to the first aspect relates to a method of combined liquid chromatography (LC) or capillary electrophoresis (CE), ion mobility and tandem mass spectroscopy analysis, comprising the following steps: introducing precursor ions resulting from LC or CE separation into at least one trapped ion mobility spectrometry (TIMS) separator, and separating the precursor ions according to mobility in the trapped ion mobility spectrometry (TIMS) separator, sequentially releasing precursor ions from said trapped ion mobility spectrometry (TIMS) separator according to their ion mobility, introducing said released precursor ions into a mass filter which selectively transmits precursor ions having m / z values falling within a controllable mass window in a first mass dimension (M-1), fragmenting the precursor ions transmitted through said mass filter to generate fragment ions, carrying out a mass spectroscopy measurement on said fragment ions in a second mass dimension (M-2), wherein each fragment ion is associated with a mass window and an ion mobility (IM) range.

[0029] Associating detected fragments with corresponding precursor ions.

[0030] Preferably, said trapped ion mobility spectrometry (TIMS) separator and said mass filter are scheduled in a synchronized manner such as to carry out a plurality of ion mobility (IM) scans, during which precursor ions of increasing or decreasing IM are successively released from said second ion mobility separator (IMS), and during which the mass window of said mass filter is shifted continuously or stepwisely towards lower or higher m / z values, respectively.

[0031] As mentioned for associating detected fragments with their corresponding precursor ions, the isotope distribution of the constituting atoms of the precursor ions and of the constituting atoms of the fragments thereof are taken account of, and at least two isotope signals of a corresponding precursor ion in the first mass dimension are taken account of for the association with corresponding fragment distributions in the second mass dimension. Typically data independent acquisition is used.

[0032] According to a first preferred embodiment of that first aspect, for associating detected fragments with their corresponding precursor ions, a cluster of precursor signals in the first mass dimension specified by a distribution in at least one of mass to charge ratio (m / z), retention time and ion mobility is taken account of.

[0033] Different algorithms can be used to consider the isotope patterns. One approach “A” can start with clusters of raw peaks determined for the MS-1 data which are required anyway for the overall processing pipeline to minimize the MS-1 precursor tolerance for data base search and de novo strategies.

[0034] These MS-1 clusters are specified by distributions in m / z, retention time and especially ion mobility. The isotope pattern of each peptide charge state (or any other kind of molecule to be considered) is composed of a certain (small) number of individual clusters with a very similar RT and mobility distribution and m / z distances according to the charge state.

[0035] For associating detected fragments with their corresponding precursor ions, expected distributions in the fragment space, including at least one of mass to charge ratio (m / z), retention time, ion mobility and parameter of the first mass filter, can be calculated starting out from at least two isotope signals of target or decoy precursor ions using the isotope distribution of the constituting atoms of the target or decoy precursor ions.

[0036] So in a peptide centric variant “A1” one can simulate the expected distributions in the fragment space (m / z, mobility, RT, frame number) for those fragments which are expected based on the target or decoy sequences. This may include a detailed, correct simulation of the isotope intensity patterns which are folded with the quadrupole transfer function. These simulated distributions can then be matched against the experimental data, e.g. by typical correlation metrics. A further approach can be a variation of the precursor m / z value, analyzing a scoring metric as a function of this m / z value, checking the quality of the match. Multiple scores can be combined with traditional or neural network machine learning approaches.

[0037] For associating detected fragments with their corresponding precursor ions, clusters of fragment signals detected in the second mass dimension specified by a distribution in at least one of mass to charge ratio (m / z), retention time and ion mobility can be taken account of for calculating corresponding precursor ion distributions in terms of the isotope distribution of the constituting atoms.

[0038] So in a spectrum centric variant “A2” one can start from individual isotope peak MS-2 clusters or correlate the MS-2 signals with the MS-1 single isotope peak distributions for “fishing” matching signals which can then be used as starting points for an analysis as described for the peptide centric variant of “A”.

[0039] In a fully spectrum centric variant “B” one can start from a MS-2 clustering + isotope pattern recognition and do precursor m / z predictions based on the different isotope cluster. Since one does not know the exact distribution of the MS-1 signals leading to these fragment peaks one can use a parametric model and if need be vary the width in mobility and RT to simulate the MS-2 signals assuming different precursor m / z values in the neighborhood of the precursor m / z value predicted from the fragments. Again, scoring models can be applied to decide about the isotopic pattern features one wants to accept for further analysis, especially matching with the MS-1 features determined independently.

[0040] Preferably for the calculation of an intensity distribution of a prediction in the fragment space or of a prediction in the precursor space, the transfer function of the mass filter is taken account of, in particular when summing up and / or averaging and / or combining fragment spectra originating from different filtering in the mass dimension, preferably by folding or unfolding, wherein preferably the mass filter is a quadrupole mass filter, and wherein the transfer function is the quadrupole transfer function of that quadrupole mass filter.

[0041] In said IM scans, according to a preferred embodiment adjacent mass windows that are associated with consecutive mass spectroscopy measurements of fragment ions overlap, such that the precursor ions transmitted through said mass filter during one IM scan are located in at least one continuous scan region in an m / z-IM plane which extends in a generally diagonal direction in said m / z-IM plane, wherein adjacent scan regions associated with different IM scans overlap in the m / z-direction.

[0042] Preferably in said IM scans, adjacent mass windows that are associated with consecutive mass spectroscopy measurements of fragment ions overlap by at least 30% of their width, and / or wherein adjacent scan regions associated with different IM scans overlap in the m / z- direction by at least 33% of their width in m / z direction.

[0043] Preferably the occurrence said fragment corresponds to a relative or absolute intensity of the fragment in the mass spectrometry measurement.

[0044] Normally, at the beginning of one LC or CE observation retention time window a MS-1 survey scan is taken, wherein in that survey scan a full ion mobility width of interest and a full m / z width of interest is scanned.

[0045] From that survey scan automatically a list of detectable signals is determined, that from that list of detectable signals at least one cluster of precursor signals in the first mass dimension specified by a distribution in at least one of mass to charge ratio (m / z), retention time and ion mobility is automatically selected for scheduling at least once in a synchronized manner in that LC or CE observation retention time window, and is taken account of for associating detected fragments with their corresponding precursor ions.

[0046] The LC or CE observation retention time window is typically in the range of 0.2-15 seconds, preferably in the range of 0.3-10 seconds or 0.5 - 2 seconds.

[0047] For associating detected fragments with their corresponding precursor ions further the isotope patterns, especially the isotope peak distances and relative intensities of precursor ions and / or fragments are used. If one starts from hypothetical target or decoy peptide sequences one can calculate the exact isotope patterns based on the known sum formula. Or it can be good enough for this purpose to use an averagine model (see e.g. Goldfarb et al in "Approximating Isotope Distributions of Biomolecule Fragments", ACS Omega. 2018 Sep 30; 3(9): 11383-11391 , the disclosure of which is included into this disclosure as concerns such an averagine model) or something similar for non proteomics use cases.

[0048] For associating detected fragments with their corresponding precursor ions at least three or at least four or at least five, or in the range of 2-10, or in the range of 3-8 isotope signals of a corresponding precursor ion in the first mass dimension can be taken account of for the association with corresponding fragment distributions in the second mass dimension.

[0049] Preferably, said trapped ion mobility spectrometry (TIMS) separator is a TIMS analyzer with parallel accumulation and separation, in which the precursor ions are accumulated in an accumulation section in an RF ion trap, in which the ions are radially confined by an RF field and pushed by a gas flow against a rising edge of an axial electric DC field barrier such that the transferred ions are spatially separated along the rising edge according to ion mobility, and wherein the height of the electric DC field barrier is chosen such as to retain only ions having an ion mobility higher than a threshold value, followed by transferring at least a subset of the accumulated ions into a trapping ion mobility separator in a separation section, radially confined by an RF field and pushed by a gas flow against a rising edge of an axial electric DC field barrier such that the transferred ions are spatially separated along the rising edge according to ion mobility, and wherein the height of the electric DC field barrier at the beginning of the release is preferably chosen such as to retain only ions having an ion mobility higher than said threshold value.

[0050] According to a further aspect of the present invention it relates to the use of such a method for the analysis of a complex sample, including samples in proteomics and metabolomics. According to yet another aspect of the present invention, it relates to an apparatus for combined liquid chromatography (LC) or capillary electrophoresis (CE), ion mobility and mass spectroscopy analysis, for carrying out the method according to any of the preceding claims, said apparatus preferably comprising: a liquid chromatography (LC) or capillary electrophoresis (CE) unit, an ionization unit ionizing precursors eluting as a function of time from said liquid chromatography unit, an ion mobility separator (IMS) in the form of at least one trapped ion mobility spectrometry (TIMS) separator for receiving and sequentially releasing precursor ions according to their ion mobility, a mass filter arranged to receive said released precursor ions and to selectively transmit precursor ions having m / z values falling within a controllable mass window, a fragmentation device for fragmenting the precursor ions transmitted through said mass filter to generate fragment ions, an apparatus for carrying out a mass spectroscopy measurement on said fragment ions, wherein each fragment ion is associated with a mass window and an ion mobility (IM) range, and a control system, wherein said control system is configured to control said IMS and said mass filter in a synchronized manner such as to carry out a plurality of IM scans, during which precursor ions of increasing or decreasing IM are successively released from said IMS, and during which the mass window of said mass filter is shifted continuously or stepwisely towards lower or higher m / z values, respectively, wherein said control system is preferably configured to control said IMS and said mass filter in a synchronized manner such that, in said IM scans, adjacent mass windows that are associated with consecutive mass spectroscopy measurements of fragment ions overlap, such that the precursor ions transmitted through said mass filter during said IM scan are located in at least one continuous scan region in an m / z-IM plane which extends in a generally diagonal direction in said m / z-IM plane, wherein further preferably said control system is configured to control said IMS and said mass filter in a synchronized manner such that adjacent scan regions associated with different IM scans overlap in the m / z-direction.

[0051] The trapped ion mobility spectrometry (TIMS) separator also in this case preferably is a TIMS analyzer with parallel accumulation and separation, in particular operating using a method comprising the steps:

[0052] (a) accumulating ions from the first ion mobility separator in an RF ion trap;

[0053] (b) transferring at least a subset of the accumulated ions into a trapping ion mobility separator, in which the transferred ions are radially confined by an RF field and pushed by a gas flow against a rising edge of an axial electric DC field barrier such that the transferred ions are spatially separated along the rising edge according to ion mobility;

[0054] (c) successively releasing the transferred ions according to their ion mobility by decreasing the height of the electric DC field barrier while ions from the ion source are further accumulated in the RF ion trap; and

[0055] (d) restoring the height of the electric DC field barrier which triggers a consecutive transfer of the accumulated ions from the RF ion trap into the trapping ion mobility separator. Further embodiments of the invention are laid down in the dependent claims.

[0056] BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,

[0058] Fig. 1 shows a schematic of the basic concept starting from experimental mobilograms for the isotope peaks in MS-1 ;

[0059] Fig. 2 shows a schematic of the basic concept starting from simulated isotopic patterns for MS-1 ;

[0060] Fig. 3 shows the transfer function of the first mass filter (quadrupole);

[0061] Fig. 4 shows the folding of an isotope distribution by the transfer function;

[0062] Fig. 5 shows an example of and isotope pattern of a peptide;

[0063] Fig. 6 shows how the pattern changes as a function of the frame;

[0064] Fig. 7 shows another schematic of a peptide centric approach A1 ;

[0065] Fig. 8 shows a schematic of a spectrum centric centric approach A2;

[0066] Fig. 9 shows another schematic of a fully spectrum centric centric approach B.

[0067] DESCRIPTION OF PREFERRED EMBODIMENTS

[0068] Fig. 1 shows a schematic of the basic concept starting from experimental MS-1 data for mobilograms of the different isotopic peaks of a peptide, leading to the fragment isotope patterns as a function of the slice and mobility scan number, "scan nr" stands for the number of the TOF scan within a "frame" produced by the ramp of mobility (IM) . "slice" stands for a number or other identifier for one of a series of different sliding quadrupole m / z + 1 / K0 scans forming e.g. a midia frame group with one kind of slice producing one of the frames of such a frame group.

[0069] An experimental mobilogram for the MS-1 isotope peaks is taken and using the quadrupole transmission function as a function of slice and scan number, the quadrupole isolated isotopes are calculated or simulated. The result is a modulated isotope pattern as a function of the slice and the scan number. This is then used for the calculation of fragment isotope patterns considering the isotope composition in the first dimension MS-1 , and this is used with known, hypothetical or representative sum formulae of fragments. The expected fragment isotope pattern is thereby calculated as a function of slice and scan number and can be used for associating detected fragments with corresponding precursor ions.

[0070] Fig. 2 shows a schematic of an approach starting from a sum formula of the precursor ion. This sum formula can be either based on a hypothetical target or decoy sequence of the peptide under consideration by the so called proteomics data base or spectral library search or an averagine model is used to get a representative sum formula of a peptide of a certain mass.

[0071] Fig. 3 shows an exemplary schematic transfer function of the first mass filter (quadrupole). For this particular quadrupole setting there is essentially constant transfer only for about a window of + / -4 m / z, with a decaying transfer function outside of this window. When scanning the quadrupole across a signal in M-1 and in particular across an isotope distribution of the corresponding precursor, for the analysis therefore the resulting spectra have to be folded (in a forward view) or unfolded (in a backward view) with such a transfer function for the analysis if the isotope distribution is to be used for the fingerprinting / analysis.

[0072] Fig. 4 illustrates the effect of the dampening of the signal intensity at the flanks of the transfer function when traveling through such a distribution by shifting the quadrupole window along the MZ dimension. As one can see, there is a significant variation of the isotope pattern at the declining and rising edge of the quadrupole transfer function.

[0073] Fig. 5 shows an example of and isotope pattern of a peptide (top) and corresponding hypothetical first and second part fragments (bottom), in each case illustrating also the associated isotope pattern of the precursor and the respective fragments. The shown isotope patterns of the fragments are calculated under the assumption that the isotope pattern of the precursor is fragmented without a modification by the quadrupole transfer function.

[0074] Fig. 6 for the example illustrated in Fig. 5, illustrates how the isotope pattern of this precursor (a) for the first (left) fragment would appear in the corresponding fragment spectra when only a single isotopic peak M(0) in 6b to M(3) in 6e would contribute.

[0075] During the scan of the quadrupole, the isotopes of the peptide will be selected and will contribute to the fragment ion pattern in a similar way. When the peptide is isolated, first the contributions from the low m / z precursor isotope peaks will dominate, when the peptide gets out of the isolation window the high m / z precursor isotopes will contribute most (scanning from large 1 / K0 and m / z values to low ones).

[0076] Fig. 7 shows a more comprehensive schematic of a peptide centric approach A1. In a variant of this, the m / z value of the simulated MS-1 isotope peaks can be slightly varied by up to a few Th to evaluate whether the best matching is obtained at the theoretical m / z value.

[0077] According to this approach, a known target or decoy sequence of a precursor is taken as starting point. The sum formula is calculated and the corresponding isotope distribution. So the MS-1 isotope pattern is simulated. Using the quadrupole transmission function as a function of slice and scan number the quadrupole isolated isotopes of this distribution are simulated, resulting in a modulated isotope pattern as a function of slice and scan number. This is the real distribution as a function of slice and scan number that arrives behind the mass filter. Then, if desired by using known sequence and sum formulae of fragments / hypotheses, fragment isotope patterns are calculated considering this MS-1 isotope composition. The result is then the expected fragment isotope patterns as a function of slice and scan number. This can be convoluted with the mobilogram, wherein the mobilogram can be taken from individual precursor models or experimental data. The result is a simulated fragment isotope pattern in the mass to charge and mobility dimension. The corresponding result can be used in a correlation analysis or another method as part of the overall scoring.

[0078] Fig. 8 shows a schematic of a spectrum centric centric approach A2. Again, in a variant of this, the m / z value of the simulated MS-1 isotope peaks can be slightly varied by up to a few Th to evaluate whether the best matching is obtained at the theoretical m / z value.

[0079] According to this approach, the experimentally detected MS-1 features in the mass to charge, mobility and retention time dimension are used as a starting point. Averagine formulae can be used for the detected mass or directly the simulated MS-1 isotope pattern of a known or hypothetical formula can be calculated. Using the quadrupole transmission function as a function of slice and scan number, the quadrupole isolated isotopes are simulated. The result is a modulated isotope pattern as a function of slice and scan number. This is used for the calculation of fragment isotope patterns considering the MS-1 isotope composition, wherein averagine sum formulae of the fragments can be used. The expected fragment isotope pattern as a function of slice and scan number can be convoluted with mobilogram information, which can be obtained from individual precursor data or models. The result is a simulated fragment isotope pattern in the mass to charge and mobility dimensions. This can again be used for correlation analysis or another method as part of the overall scoring.

[0080] Fig. 9 shows another schematic of a fully spectrum centric centric approach B. In this variant the MS-2 data is evaluated without prior knowledge of the precursor leading to the MS-2 signals. For Ml DI A, a prediction of the precursor m / z is established and the m / z value can be varied for optimal match of the predicted and observed MS-2 isotope pattern, leading to quality scores and especially a refined predicted m / z value.

[0081] According to this approach, a predicted precursor m / z, z from known Ml DI A processing pipeline, is used as a starting point. Averagine formulae can be used for the detected mass or directly the simulated MS-1 isotope pattern can be calculated. Using the quadrupole transmission function as a function of slice and scan number, the quadrupole isolated isotopes are simulated. The result is a modulated isotope pattern as a function of slice and scan number. This is used for the calculation of fragment isotope patterns considering the MS-1 isotope composition, wherein averagine sum formulae of the fragments can be used. The expected fragment isotope pattern as a function of slice and scan number can be convoluted with mobilogram information, which can be obtained from individual precursor data or models. The result is a simulated fragment isotope pattern in the mass to charge and mobility dimensions. This can again be used for a correlation analysis or other method to be used with a refined assignment of relationships between MS-1 and MS-2.

[0082] LIST OF REFERENCE SIGNS

[0083] LC liquid chromatograpy

[0084] CE capillary electrophoresis

[0085] DDA Data dependent acquisition

[0086] DIA Data independent acquisition

[0087] RT retention time

[0088] IM ion mobility m / z mass to charge ratio

[0089] 1 / k0 ion mobility value

[0090] CCS collision cross section

[0091] PASEF parallel accumulation serial fragmentation

[0092] M-1 first mass dimension (quadrupole)

[0093] M-2 second mass dimension (TOF) midiaPASEF Maximizing Information content in DIA-PASEF

[0094] TOF time of flight

Claims

CLAIMS1. A method of combined liquid chromatography (LC) or capillary electrophoresis (CE), ion mobility and tandem mass spectroscopy analysis, comprising the following steps: introducing precursor ions resulting from LC or CE separation into at least one trapped ion mobility spectrometry (TIMS) separator, and separating the precursor ions according to mobility in the trapped ion mobility spectrometry (TIMS) separator, sequentially releasing precursor ions from said trapped ion mobility spectrometry (TIMS) separator according to their ion mobility, introducing said released precursor ions into a mass filter which selectively transmits precursor ions having m / z values falling within a controllable mass window in a first mass dimension (M-1), fragmenting the precursor ions transmitted through said mass filter to generate fragment ions, carrying out a mass spectroscopy measurement on said fragment ions in a second mass dimension (M-2), wherein each fragment ion is associated with a mass window and an ion mobility (IM) range, and associating detected fragments with corresponding precursor ions, wherein said trapped ion mobility spectrometry (TIMS) separator and said mass filter are scheduled in a synchronized manner such as to carry out a plurality of ion mobility (IM) scans, during which precursor ions of increasing or decreasing IM are successively released from said second ion mobility separator (IMS), and during which the mass window of said mass filter is shifted continuously or stepwisely towards lower or higher m / z values, respectively, wherein for associating detected fragments with their corresponding precursor ions, the isotope distribution of the constituting atoms of the precursor ions and of the constituting atoms of the fragments thereof are taken account of, and wherein at least two isotope signals of a corresponding precursor ion in the first mass dimension are taken account of for the association with corresponding fragment distributions in the second mass dimension.

2. Method according to claim 1 , using data independent acquisition.

3. Method according to any of the preceding claims, wherein for associatingdetected fragments with their corresponding precursor ions, a cluster of precursor signals in the first mass dimension specified by a distribution in at least one of mass to charge ratio (m / z), retention time and ion mobility is taken account of.

4. Method according to any of the preceding claims, wherein for associating detected fragments with their corresponding precursor ions, expected distributions in the fragment space, including at least one of mass to charge ratio (m / z), retention time, ion mobility and parameter of the first mass filter, are calculated starting out from at least two isotope signals of target or decoy precursor ions using the isotope distribution of the constituting atoms of the target or decoy precursor ions.

5. Method according to any of the preceding claims, wherein for associating detected fragments with their corresponding precursor ions, clusters of fragment signals detected in the second mass dimension specified by a distribution in at least one of mass to charge ratio (m / z), retention time and ion mobility are taken account of for calculating corresponding precursor ion distributions in terms of the isotope distribution of the constituting atoms.

6. Method according to any of the preceding claims, wherein for the calculation of an intensity distribution of a prediction in the fragment space or of a prediction in the precursor space, the transfer function of the mass filter is taken account of, preferably by folding, wherein preferably the mass filter is a quadrupole mass filter, and wherein the transfer function is the quadrupole transfer function of that quadrupole mass filter.

7. Method according to any of the preceding claims, wherein in said IM scans, adjacent mass windows that are associated with consecutive mass spectroscopy measurements of fragment ions overlap, such that the precursor ions transmitted through said mass filter during one IM scan are located in at least one continuous scan region in an m / z-IM plane which extends in a generally diagonal direction in said m / z-IM plane, wherein adjacent scan regions associated with different IM scans overlap in the m / z-direction, wherein preferably in said IM scans, adjacent mass windows that are associated with consecutive mass spectroscopy measurements of fragment ions overlap by at least 30% of their width, and / or wherein adjacent scan regions associated with different IM scans overlap in the m / z-direction by at least 33% of their width in m / z direction, and / or wherein preferably the occurrence said fragment corresponds to a relativeor absolute intensity of the fragment in the mass spectrometry measurement.

8. Method according to any of the preceding claims, wherein at the beginning of one LC or CE observation retention time window a survey scan is taken, wherein in that survey scan a full ion mobility width of interest and a full m / z width of interest is scanned, and wherein from that survey scan automatically a list of detectable signals is determined, that from that list of detectable signals at least one cluster of precursor signals in the first mass dimension specified by a distribution in at least one of mass to charge ratio (m / z), retention time and ion mobility is automatically selected for scheduling at least once in a synchronized manner in that LC or CE observation retention time window, and is taken account of for associating detected fragments with their corresponding precursor ions.

9. Method according to any of the preceding claims, wherein the LC or CE observation retention time window is in the range of 0.2 - 15 seconds, preferably in the range of 0.3-10 seconds or 0.5 - 2 seconds.

10. Method according to any of the preceding claims, wherein for associating detected fragments with their corresponding precursor ions further the isotope patterns, in particular the isotope peak distances and relative intensities, of precursor ions and / or fragments are used.

11. Method according to any of the preceding claims, wherein for associating detected fragments with their corresponding precursor ions at least three or at least four or at least five, or in the range of 2-10, or in the range of 3-8 isotope signals of a corresponding precursor ion in the first mass dimension are taken account of for the association with corresponding fragment distributions in the second mass dimension.

12. Method according to any of the preceding claims, wherein said trapped ion mobility spectrometry (TIMS) separator is a TIMS analyzer with parallel accumulation and separation, in which the precursor ions are accumulated in an accumulation section in an RF ion trap, in which the ions are radially confined by an RF field and pushed by a gas flow against a rising edge of an axial electric DC field barrier such that the transferred ions are spatially separated along the rising edge according to ion mobility, and wherein the height of the electric DC field barrier is chosen such as to retain only ions having an ion mobilityhigher than a threshold value followed by transferring at least a subset of the accumulated ions into a trapping ion mobility separator in a separation section, radially confined by an RF field and pushed by a gas flow against a rising edge of an axial electric DC field barrier such that the transferred ions are spatially separated along the rising edge according to ion mobility, and wherein the height of the electric DC field barrier at the beginning of the release is preferably chosen such as to retain only ions having an ion mobility higher than said threshold value.

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

14. An apparatus for combined liquid chromatography (LC) or capillary electrophoresis (CE), ion mobility and mass spectroscopy analysis, for carrying out the method according to any of the preceding claims, said apparatus preferably comprising: a liquid chromatography (LC) or capillary electrophoresis (CE) unit, an ionization unit ionizing precursors eluting as a function of time from said liquid chromatography unit, an ion mobility separator (IMS) in the form of at least one trapped ion mobility spectrometry (TIMS) separator for receiving and sequentially releasing precursor ions according to their ion mobility, a mass filter arranged to receive said released precursor ions and to selectively transmit precursor ions having m / z values falling within a controllable mass window, a fragmentation device for fragmenting the precursor ions transmitted through said mass filter to generate fragment ions, an apparatus for carrying out a mass spectroscopy measurement on said fragment ions, wherein each fragment ion is associated with a mass window and an ion mobility (IM) range, and a control system, wherein said control system is configured to control said IMS and said mass filter in a synchronized manner such as to carry out a plurality of IM scans, during which precursor ions of increasing or decreasing IM are successively released from said IMS, and during which the mass window of said mass filter is shifted continuously or stepwisely towards lower or higher m / z values, respectively, wherein said control system is preferably configured to control said IMS and said mass filter in a synchronized manner such that, in said IM scans, adjacent mass windows that are associated with consecutive mass spectroscopy measurements of fragment ionsoverlap, such that the precursor ions transmitted through said mass filter during said IM scan are located in at least one continuous scan region in an m / z-IM plane which extends in a generally diagonal direction in said m / z-IM plane, wherein further preferably said control system is configured to control said IMS and said mass filter in a synchronized manner such that adjacent scan regions associated with different IM scans overlap in the m / z-direction.

15. An apparatus according to claim 13, wherein the trapped ion mobility spectrometry (TIMS) separator is a TIMS analyzer with parallel accumulation and separation, in particular operating using a method comprising the steps:(a) accumulating ions from the first ion mobility separator in an RF ion trap;(b) transferring at least a subset of the accumulated ions into a trapping ion mobility separator, in which the transferred ions are radially confined by an RF field and pushed by a gas flow against a rising edge of an axial electric DC field barrier such that the transferred ions are spatially separated along the rising edge according to ion mobility;(c) successively releasing the transferred ions according to their ion mobility by decreasing the height of the electric DC field barrier while ions from the ion source are further accumulated in the RF ion trap; and(d) restoring the height of the electric DC field barrier which triggers a consecutive transfer of the accumulated ions from the RF ion trap into the trapping ion mobility separator.

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