Data independent combined ion mobility and mass spectrometry method and apparatus

Abstract

The present invention discloses an apparatus and method for data-independent combined ion mobility and mass spectrometry analysis, the method comprising the following steps: introducing precursor ions into an ion mobility spectrometer (IMS), sequentially releasing the precursor ions from the IMS according to their ion mobility, introducing the released precursor ions into a mass filter, fragmenting the precursor ions transmitted through the mass filter to produce fragment ions, and performing mass spectrometry measurement on the fragment ions, controlling the IMS and the mass filter in a synchronized manner to perform multiple IM scans, during which the precursor ions are sequentially released from the IMS and during which the mass window of the mass filter is continuously or stepwise moved toward lower or higher m / z values, and in the IM scans, adjacent mass windows associated with continuous mass spectrometry measurements of the fragment ions overlap, so that the precursor ions transmitted through the mass filter during the IM scan are located in at least one continuous scanning region in the m / z‑IM plane, and the at least one continuous scanning region extends in a substantially diagonal direction in the m / z‑IM plane.

Description

Technical Field

[0001] The present invention belongs to the field of mass spectrometry. More specifically, the present invention relates to a method for data independent combined ion mobility and mass spectrometry analysis. Background Art

[0002] Proteomics based on mass spectrometry has become a powerful technology for identifying and quantifying thousands of proteins. However, due to the limited speed, sensitivity and resolution of current mass spectrometers, the coverage of complete proteomes is very challenging. In bottom-up proteomics, peptides are usually separated by liquid chromatography with a peak width of several seconds, and mass spectra are obtained with a time-of-flight (TOF) instrument in about 100 μs. This allows the increase of ion mobility as the third dimension of separation therebetween. In Meier et al.'s Online parallel accumulation–serial fragmentation (PASEF) with a novel trapped ion mobility mass spectrometer, http: / / dx.doi.org / 10.1101 / 336743, a scanning mode called synchronous accumulation continuous fragmentation (PASEF) has been described, which uses the timsTOF Pro instrument of BrukerDaltonik, which combines a trapped ion mobility mass spectrometer with a quadrupole mass filter and a time-of-flight mass analyzer. It has been found that this scanning mode allows high sequencing speeds under good sensitivity.

[0003] However, the PASEF scan mode in its original form is itself a so-called data-dependent acquisition mode, in which specific precursors are selected sequentially. Especially for screening or clinical applications, a so-called data-independent acquisition (DIA) scheme is preferred, in which ion groups are recursively isolated by mass filters and fragmented simultaneously, resulting in a convolved fragment ion spectrum consisting of fragments from many different precursors. A data-independent variant of the PASEF method (called diaPASEF) is described in Florian Meier et al. Parallel accumulation-serialfragmentation combined with data-independent acquisition (diaPASEF): bottom-up proteomics with near optimal ion usage, BioarXiv doi: https: / / doi.org / 10.1101 / 656207. In this work, a deconvolution scheme for mass spectra is also described. Deconvolution consists in associating the fragments found in the mass spectrum with the corresponding precursors. However, deconvolution in the diaPASEF scheme remains challenging. For example, using a mass window width of 25 Da for the mass window of a quadrupole mass filter, in a proteomic analysis of a tryptic digest of the proteome of a human cell line, up to several thousand precursors are found in the ion mobility range where typical precursors typically occur, making deconvolution a very difficult task. Summary of the invention

[0004] The problem of the present invention is to provide a method and an apparatus for data-independent combined ion mobility and mass spectrometry analysis, which allows an improved association of detected fragments with the corresponding precursor ions. This problem is achieved by the method and the apparatus according to the independent claims. Preferred embodiments are defined in the dependent claims.

[0005] According to one aspect of the invention, the problem is solved by a method comprising the following steps:

[0006] introducing the precursor ions into an ion mobility separator (IMS), also known in the art as an ion mobility spectrometer,

[0007] releasing precursor ions from the IMS sequentially according to their ion mobility,

[0008] introducing the released precursor ions into a mass filter that selectively transmits precursor ions having m / z values ​​falling within a controllable mass selection window,

[0009] fragmenting precursor ions transmitted through the mass filter to produce fragment ions,

[0010] performing mass spectrometry on the fragment ions, wherein each fragment ion is associated with a mass window and an ion mobility (IM) range, and

[0011] Correlate the detected fragments with their corresponding precursor ions.

[0012] Note that the "IM range" associated with the fragment ions is typically defined by the time interval during the sequential release of the precursor ions from the IMS, where all precursor ions released in that time interval will be subjected to the same mass spectral measurement, such as associated with the same TOF push in a TOF mass analyzer. Although reference is made to an IM range here, the range is typically very narrow, so that one can also speak of an "IM value".

[0013] In the present invention, the IMS and the mass filter are controlled in a synchronized manner so as to perform a plurality of IM scans, during which precursor ions that increase or decrease IM are sequentially released from the IMS, and during which the mass window of the mass filter is moved continuously or stepwise toward a lower or higher m / z value, respectively. In the IM scan, adjacent mass windows associated with consecutive mass spectrometry measurements of target ions overlap each other, for example, overlapping at least 10%, preferably at least 30%, more preferably at least 60% and most preferably at least 90% of their width, so that the precursor ions transmitted by the mass filter during the IM scan are located in at least one continuous scan region in the m / z-IM plane, which extends in a substantially diagonal direction in the m / z-IM plane. For example, if a TOF mass analyzer is used, the mass window can be moved after each TOF push or after a given number of TOF pushes. Adjacent scan regions associated with different IM scans overlap in the m / z direction, for example, overlapping at least 33%, preferably at least 50% and most preferably at least 66% of their width in the m / z direction. Furthermore, the step of associating the detected fragments with their corresponding precursor ions is based on determining or utilizing corresponding mass windows and IM ranges associated with various occurrences of the fragments in the mass spectrometry measurement.

[0014] Note that the "occurrence of a fragment" can be binary information in the simplest case, i.e., whether the fragment is found to be present in a given mass window and IM range. However, in a practical implementation, the "occurrence" can have a quantitative component and, for example, can represent the intensity of the fragment in a mass spectrometry measurement. Therefore, in a preferred embodiment, the occurrence of the fragment corresponds to the relative intensity or absolute intensity of the fragment in a mass spectrometry measurement. Reference to the "occurrence of a fragment" anywhere below should be understood to refer to either binary information (the presence of the fragment was found / not found) and quantitative information about the intensity of the fragment without further mention.

[0015] According to the invention, the scanning area, i.e. the area covered in the m / z-IM plane in one IM scan, is arranged approximately diagonally. This approximately diagonal scanning area will also be referred to herein as a "maximum information data independent acquisition (MIDIA)-frame". The term "approximately diagonal" should be interpreted in a broad manner and in particular does not necessarily require that the MIDIA extends along a straight line. Instead, the term "approximately diagonal" mainly reflects the fact that in consecutive scanning areas both the m / z range and the IM range vary simultaneously.

[0016] The motivation for the roughly diagonal direction is that in many applications, the mass and ion mobility of the precursors are related to each other. This means that for a given charge value z, the precursors are usually roughly distributed along a diagonal (possibly curved) line in the m / z-IM plane. Therefore, arranging the MIDIA frames roughly along this diagonal direction allows for optimal use of the available precursors in the scan.

[0017] Although focusing on this diagonal region in the m / z-IM plane has been considered in the diaPASEF method mentioned in the background of the present invention, the region is still scanned using multiple separate "vertical" scans, referred to herein as "PASEF frames", in which a certain mass window of the mass filter is kept fixed and the IM values ​​are scanned. Once the IM scan has covered the target area, the mass window is jumped by a multiple of its width to a new position, and the vertical scan is continued at the new position until the entire precursor ion mobility range is covered. In the next PASEF frame, a different m / z range is targeted that does not overlap with the previous frame or subsequent frames. Although the MIDIA acquisition method described herein is a special type of diaPASEF, the terms "diaPASEF" and "PASEF frame" as used herein generally refer to the way the method is performed in the prior art, and the new acquisition method is referred to herein as "MIDIA".

[0018] The reason for using vertical frames in the prior art is that this is the mode of operation in which the equipment actually used to perform the prior art diaPASEF procedure was designed. Firstly, a person skilled in the art would not see any advantage in using diagonal scanning, since vertical diaPASEF scanning also allows coverage of the entire target area. Therefore, according to common sense, there would not be any reason to make an effort to restructure the available equipment accordingly.

[0019] However, the inventors have noticed that using diagonal MIDIA frames, in fact more information about the precursors is encoded into the collective mass spectrometric data acquired using MIDIA frames as defined above. One reason for this increase in information must be related to the overlap of MIDIA frames in the m / z direction. Depending on the size of the overlap, each precursor may be reflected in more than one MIDIA frame. For example, in a preferred embodiment, adjacent MIDIA frames may overlap by 2 / 3 in the m / z direction, meaning that each precursor with a given m / z value will be presented in three different MIDIA frames. By identifying these MIDIA frames, the possible m / z range is then reduced to 1 / 3 the size of the mass window of the respective MIDIA frame. Therefore, the size of the mass window can be chosen to be relatively large, and typically 40% to 60% larger than diaPASEF, thereby allowing better sensitivity while allowing resolution of m / z values ​​at a sub-mass window scale.

[0020] Another reason for the increase in information is that even within a single MIDIA frame, the mass windows are moving relative to each other. It can be seen that the precursors exhibit a certain extension or average extension ΔIM in the m / z plane in the IM direction. 前体 , which means that the same precursor will be covered by different adjacent IM ranges within the same MIDIA frame, or in other words, the same precursor will be present in its adjacent "subframes". Since these subframes will generally be shifted relative to each other in the m / z direction, the presence of a precursor in a selected subframe encodes additional information about its m / z value. This additional information is not captured in data obtained using prior art diaPASEF frames, where each precursor is present in only one and only one diaPASEF frame, and where the presence in multiple subframes in any given PASEF frame due to spreading in the IM direction does not provide any additional information about its m / z value.

[0021] In a preferred embodiment, the step of associating the detected fragments with their corresponding precursor ions is based on evaluating the consistency of the experimental precursors with the mass windows and IM ranges associated with the various occurrences of the fragments in the mass spectrometry. As previously described, each fragment ion subjected to mass spectroscopy is associated with a corresponding mass window and IM range associated with the mass filter and IMS, or in other words, with a corresponding subframe of the MIDIA frame. Then, if a specific fragment is found to occur in the mass spectral data, and the occurrence of the fragment can be associated with a set of MIDIA subframes, these MIDIA subframes can be checked for consistency with the experimental precursors.

[0022] In a preferred embodiment, the step of associating the detected fragments with their corresponding precursor ions comprises determining the intersection of mass windows associated with the occurrence of the fragments in the mass spectrometry, and identifying the corresponding precursor ions based at least in part on the intersection. In this way, the mass or m / z value of the precursor ion can be determined with high specificity and on a scale that is lower than the width of the mass window of the mass filter used. Note that unlike the diaPASEF scheme, all mass windows in which a particular fragment has ever appeared - provided that it is indeed produced by only one precursor - will be identical, and determining the intersection will not add any information. When determining the intersection of mass windows, the range of corresponding IM values ​​associated with the fragments can also be considered. The IM values ​​of fragments belonging to the same precursor should be within a specific IM distribution width, which is typically found for a single precursor.

[0023] In a preferred embodiment, the method further comprises the step of performing mass spectrometry in an operating mode in which the mass filter and fragmentation are disabled so that all precursor ions sequentially released from the IMS are subjected to mass spectrometry without fragmentation, thereby obtaining an m / z spectrum as a function of the ion mobility of the precursor ions. This operating mode is generally referred to in the art as MS1 mode. In MS1 ​​mode, high-resolution m / z spectra can be obtained, and the spread or average spread ΔIM of the precursor in the m / z-IM plane can also be discerned. 前体 (i.e., the aforementioned “IM distribution width”) information.

[0024] In a related embodiment, based on the m / z spectrum of the precursor ion, a "fingerprint" of at least one precursor ion is established, the fingerprint comprising a set of mass windows and associated IM ranges in the mass windows and associated IM ranges covered by the IM scan, which is consistent with the appearance of the at least one precursor ion in the m / z spectrum, or in other words, consistent with the position of the precursor in the m / z-IM plane. Here, the step of associating the detected fragments with their corresponding precursors preferably comprises the step of establishing a set of mass windows and corresponding IM ranges of the mass filter and IMS associated with the occurrence of the detected fragments, and comparing the set with the fingerprint of one or more precursors.

[0025] Here, the "quality window and corresponding IM range" may correspond, for example, to the aforementioned MIDIA subframe. However, MS1 data is usually not collected in a scanning procedure using overlapping MIDIA frames as used in the MS2 measurement of the present invention, so that the "fingerprint" based on the MIDIA subframe cannot be directly distinguished from the MS1 data.

[0026] Therefore, in a preferred embodiment, the method further comprises the step of performing a "pseudo MS2" measurement of the precursor ion, wherein the IMS and the mass filter are controlled in a synchronized manner so as to perform overlapping IM scans of the same type as in the mass spectrometry measurement of the fragment ions, but without fragmentation of the precursor ion. Since the m / z of the precursor is retained in the pseudo MS2 measurement, the pseudo MS2 measurement allows the MS1 measurement data and the "real" MS2 measurement data (including the fragments) to be correlated with each other.

[0027] In a preferred embodiment, the step of associating the detected fragments with their corresponding precursors is based on a comparison, correlation or matching of the pseudo-MS2 data with real MS2 data obtained in the mass spectrometry measurement performed on the fragment ions. Graphically, pseudo-MS2 includes the same information as MS1 data, but in the "format" or data space of an MS2 measurement, which makes it particularly easy to compare, correlate or match precursor information with fragment information.

[0028] In an alternative embodiment, the method further comprises a step of predicting or calculating the expected occurrence (particularly intensity distribution) of the corresponding fragments with respect to at least the IM range and the number of IM scans in the plurality of IM scans based on the performance of the precursors in the MS1 mass spectrometry, and the step of associating the detected fragments with their corresponding precursors is based on a comparison, correlation or match of the expected occurrence (particularly intensity distribution) with the actual MS2 data obtained in the mass spectrometry performed on the fragment ions. Thus, in this embodiment, the MS1 mass spectrometry data is converted into a format for acquiring MS2 data, such as MIDIA subframes, thereby allowing comparison, correlation or matching.

[0029] In an alternative embodiment, the MS2 data may be converted into a format for use in an MS1 ​​measurement. For this purpose, the method comprises a step of predicting or calculating the expected intensity distribution of possible precursors as a function of at least IM and m / z based on the "real" MS2 data obtained in the mass spectrometry measurement (i.e., using MIDIA mode) of a given fragment, and the step of associating the detected fragments with their corresponding precursors is based on a comparison, correlation or matching of this expected intensity distribution of possible precursors with the data obtained in the MS1 mass spectrometry measurement.

[0030] In both embodiments, the predicting or calculating step may be performed using a model or algorithm based at least in part on one or more matrix-based methods, neural networks, random forests, support vector machines or other machine learning methods. Here, the model or algorithm is preferably derived or trained using at least in part the results of the pseudo MS2 measurements as ground truth data.

[0031] As mentioned above, the precursors generally have an elongated form in the m / z-IM plane, and an average extension ΔIM in the IM direction 前体 Furthermore, the movement of the edges of adjacent scan areas of the IM scan relative to each other in the m / z direction is accompanied by an edge offset ΔIM in the IM direction between the edges of adjacent scan areas. 帧 In a preferred embodiment, the shift of adjacent scanning areas is adapted to the average precursor extension ΔIM 前体 , so that ΔIM 前体 ≥ΔIM 帧 , Optimize ΔIM 前体 ≥1.2·ΔIM 帧 And more preferably ΔIM 前体 ≥1.5·ΔIM 帧。 In this way, it can be ensured that each precursor is covered by at least two IM scans, and possibly at least three IM scans, resulting in a more characteristic "fingerprint" of the corresponding precursor and fragment.

[0032] In a preferred embodiment, the mass filter is a quadrupole RF device.

[0033] In a preferred embodiment, the IMS is a capture IMS (TIMS) device. Here, the TIMS device preferably includes a first TIMS and a second TIMS, wherein the first TIMS is configured to constantly receive precursor ions and transfer the received precursor ions to the second TIMS in a time-controlled manner, and the second TIMS is configured to perform the IM scan, wherein the precursor ions are released from the second TIMS in sequence according to their ion mobility, and wherein preferably, in each IM scan, the precursor ions of lower ion mobility are released before the precursor ions of higher ion mobility. In other embodiments, the IMS can be a traveling wave IMS device or a drift tube IMS, which will require an elevated m / z window due to different separation principles. The IMS device preferably includes an upstream trap for receiving ions, while a downstream IMS device performs the IMS scan.

[0034] In a preferred embodiment, the method further comprises the step of providing the precursor ion by separating the precursor molecule from the sample and ionizing the precursor molecule. Preferential separation of the precursor molecule from the sample is preferably carried out by chromatography, and more preferably by liquid chromatography. In a preferred embodiment, the sample preferably comprises a biological material, particularly a peptide, lipid, metabolite or other small molecule of mass 50 to 5000Da. Other examples of samples may include proteins, sugars, pesticides or drugs.

[0035] The precursor ions can be generated using one of spray ionization (e.g., electrospray (ESI) or thermal spray), desorption ionization (e.g., matrix-assisted laser / desorption ionization (MALDI) or secondary ionization), chemical ionization (CI), photoionization (PI), electron impact ionization (EI), or gas discharge ionization.

[0036] The precursor ions can be fragmented by one of collision induced dissociation (CID), surface induced dissociation (SID), photodissociation (PD), electron capture dissociation (ECD), electron transfer dissociation (ETD), collision activation followed by electron transfer dissociation (ETCD), activation simultaneous with electron transfer dissociation (AI-ETD), and fragmentation by reaction with highly excited or free radical neutral particles.

[0037] The fragment ions and / or precursor ions can be measured by one of a time-of-flight analyzer (eg, using orthogonal ion injection), an electrostatic ion trap, an RF ion trap, and an ion cyclotron frequency ion trap.

[0038] According to another aspect of the present invention, a device for data-independent combined ion mobility and mass spectrometry is provided. The device comprises: an ion mobility separator (IMS) for receiving precursor ions and sequentially releasing precursor ions from the IMS according to their ion mobility; a mass filter arranged to receive the released precursor ions and selectively transmit precursor ions whose m / z values ​​fall within a controllable mass window; a fragmentation device for fragmenting the precursor ions transmitted through the mass filter to produce fragment ions; a device for performing mass spectrometry on the fragment ions, wherein each fragment ion is associated with a mass window and an ion mobility (IM) range; and a control system, the control system being configured to control the IMS and the mass filter in a synchronized manner so as to perform multiple IM scans, during which the precursor ions of the IM are increased or decreased. The bulk ions are released from the IMS sequentially and during this time the mass window of the mass filter is moved continuously or stepwise towards lower or higher m / z values, respectively; wherein the control system (32) is configured to control the IMS (16) and the mass filter (26) in a synchronized manner so that in the IM scan, adjacent mass windows associated with consecutive mass spectrometry measurements of fragment ions overlap, for example by at least 10%, preferably at least 30%, more preferably at least 60% and most preferably at least 90% of their width, so that the precursor ions transmitted through the mass filter during the IM scan are located in at least one consecutive scan region in the m / z-IM plane, which extends in a substantially diagonal direction in the m / z-IM plane. Here, adjacent scan regions associated with different IM scans overlap in the m / z direction, for example by at least 33%, preferably at least 50% and most preferably at least 66% of their width in the m / z direction.

[0039] The apparatus may also include an ion source.

[0040] In a preferred embodiment, the control system is further configured to associate detected fragments with their corresponding precursor ions based on evaluating the consistency of experimental precursor pairs with the mass window and IM range associated with various occurrences of the fragments in the mass spectrometry measurement.

[0041] In a preferred embodiment, the control system is further configured to associate the detected fragments with their corresponding precursor ions, wherein the association preferably includes determining an intersection of mass windows associated with the occurrence of the fragments observed in the mass spectrometry measurement, and identifying the corresponding precursor ions based at least in part on the intersection.

[0042] In a preferred embodiment, the control system is also configured to control the device to perform MS1 mass spectrometry in an operating mode, wherein the mass filter and the fragmentation device are disabled so that all precursor ions sequentially released from the IMS are subjected to mass spectrometry without fragmentation, thereby obtaining an m / z spectrum as a function of the ion mobility of the precursor ions.

[0043] In a preferred embodiment, the control system is configured to establish a fingerprint of at least one precursor ion based on the m / z spectrum of the precursor ion, the fingerprint comprising a set of mass windows and associated IM ranges in the mass window and associated IM range covered by the IM scan, which is consistent with the representation of the at least one precursor ion (34) in the m / z spectrum.

[0044] Here, the control system is preferably configured for associating the detected fragments with their corresponding precursors, wherein the association includes establishing a set of mass windows and corresponding IM ranges of the mass filter and IMS associated with the occurrence of the detected fragments, and comparing the set with the fingerprints of one or more precursors.

[0045] In a preferred embodiment of the device, the control system is configured to control the step of controlling the device to perform a pseudo-MS2 measurement on precursor ions, wherein the IMS and the mass filter are controlled in a synchronized manner so as to perform the same type of overlapping IM scans as in the mass spectrometry measurement performed on the fragment ions, but without fragmenting the precursor ions.

[0046] In a preferred embodiment of the apparatus, the control system is configured to associate detected fragments with their corresponding precursors based on comparison, correlation or matching of the pseudo MS2 data with real MS2 data obtained in the mass spectrometry measurements performed on the fragment ions.

[0047] In a preferred embodiment of the device, the control system is configured to predict or calculate the expected occurrence, in particular the intensity distribution, of the corresponding fragments with respect to at least the IM range and the IM scan number in the multiple IM scans based on the performance of the precursor in the MS1 mass spectrometry measurement, and the control system is also configured to associate the detected fragments with their corresponding precursors based on comparison, correlation or matching of the expected occurrence, in particular the intensity distribution, with the real MS2 data obtained in the mass spectrometry measurement performed on the fragment ions.

[0048] In a preferred embodiment of the device, the control system is configured to predict or calculate the expected intensity distribution of possible precursors as a function of at least IM and m / z based on the actual MS2 data obtained in the mass spectrometry measurement of a given fragment, and the control system is also configured to associate the detected fragments with their corresponding precursors based on a comparison, correlation or match of this expected intensity distribution of possible precursors with the data obtained in the MS1 mass spectrometry measurement.

[0049] In a preferred embodiment of the device, the control system is configured to perform the prediction or calculation using a model or algorithm based at least in part on one or more matrix-based methods, neural networks, random forests, support vector machines or other machine learning methods.

[0050] In a preferred embodiment, the mass filter is a quadrupole RF device.

[0051] In a preferred embodiment, the IMS is one of a trapping IMS (TIMS) device, a travelling wave IMS device and a drift tube IMS. The mass spectrometer may be one of a time-of-flight analyser (e.g. using orthogonal ion injection), an electrostatic ion trap, an RF ion trap and an ion cyclotron frequency ion trap.

[0052] In a preferred embodiment, the TIMS device comprises a first TIMS and a second TIMS, wherein the first TIMS is configured to constantly receive precursor ions and transfer the received precursor ions to the second TIMS in a time-controlled manner, and the second TIMS is configured to perform the IM scan, wherein the precursor ions are sequentially released from the second TIMS according to their ion mobility. Here, in each IM scan, precursor ions with lower ion mobility are released before precursor ions with higher ion mobility.

[0053] In a preferred embodiment, the apparatus further comprises a separator and an ionization device for providing the precursor ions by separating the precursor molecules from the sample and ionizing the precursor molecules. The separator is preferably a chromatographic device, and more preferably a liquid chromatography device (12). The sample preferably comprises biological material, in particular one of a peptide, lipid, metabolite or other small molecule having a mass of 50 to 5000 Da.

[0054] In a preferred embodiment of the apparatus, the control system (32) is configured to control the IMS (16) and the mass filter (26) in a synchronized manner such that

[0055] - the precursor has an average extension ΔIM in the m / z-IM plane 前体 ,

[0056] - the movement of the edges of adjacent scan areas of the IM scan relative to each other in the m / z direction is accompanied by an edge offset ΔIM in the IM direction between the edges of adjacent scan areas 帧 ,as well as

[0057] - the shifting of adjacent scanning areas is adapted to the average precursor extension ΔIM 前体 , so that ΔIM 前体 ≥ΔIM 帧 , Optimize ΔIM 前体 ≥1.2·ΔIM 帧 And more preferably ΔIM 前体 ≥1.5·ΔIM 帧 . BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 is a schematic illustration of a system according to an embodiment of the present invention.

[0059] Figure 2 The distribution of precursor ions in the m / z-IM plane is shown, as well as the position of the PASEF frame and an arrow indicating the procedure of the third IM scan in the DiPASEF method.

[0060] Figure 3 Shown with Figure 2 Distribution of precursor ions in the same m / z-IM plane and PASEF frame in , but the arrow indicates the procedure of the fourth IM scan in the diaPASEF method.

[0061] Figure 4 The general location of the diagonal MIDIA frames in the m / z-IM plane is shown.

[0062] Figure 5is a close-up view of the diaPASEF and MIDIA frames in the m / z-IM plane, both divided into subframes according to the IM value.

[0063] Figure 6 An overlay of three diagonal MIDIA frames in the m / z-IM plane is shown.

[0064] Figure 7 Illustrated is the overlay of several MIDIA frames for a certain IM value and how the mass of the precursor can be determined at sub-frame m / z resolution by its appearance in each MIDIA frame.

[0065] Figure 8 The positions of the first diaPASEF frame and the first MIDIA frame in the m / z-IM plane relative to the target precursor are shown.

[0066] Fig. 9 The positions of the first and second diaPASEF frames and MIDIA frames relative to the target precursor in the m / z-IM plane are shown.

[0067] Fig.10 The positions of the first to third diaPASEF frames and MIDIA frames relative to the target precursor in the m / z-IM plane are shown.

[0068] Fig.11 is a table summarizing the position of the MIDIA subframes of 10 different MIDIA frames in the m / z-IM plane and the identity with a given precursor ion.

[0069] Fig.12 _ is a table illustrating the specificity of a subframe set with the same m / z value.

[0070] Figures 13 to 16 Shown are the MIDIA frame timings and the corresponding intensities as a function of the IMI covered by the fragments of the three precursors.

[0071] Fig.17 is a schematic diagram illustrating the steps of associating fragments with their corresponding precursors based on correlation of intensity-IMI relationships of multiple overlapping MIDIA frames.

[0072] Fig.18 is a schematic diagram illustrating the steps of associating fragments with their corresponding precursors based on comparison of conversion coefficients of intensity-IMI relationships of multiple overlapping MIDIA frames.

[0073] Fig.19is a schematic diagram illustrating the steps of associating fragments with their corresponding precursors based on calculated correlations between data associated with a plurality of MIDIA cycles, each MIDIA cycle comprising a plurality of MIDIA scans, wherein the plurality of MIDIA cycles cover a corresponding retention time range associated with a liquid chromatography peak.

[0074] Fig. 20 Shown are the intensities as a function of the ion mobility index (IMI) of known precursors obtained in an MS1 ​​measurement and in exemplary eighth and ninth MIDIA frames, respectively.

[0075] Fig.21 is a table summarizing the locations of the edges of the mass window as a function of IMI for the eighth MIDIA frame and the corresponding transmission coefficients for selected m / z values.

[0076] Fig. 22 is a table summarizing the locations of the edges of the mass window as a function of IMI for the ninth MIDIA frame and the corresponding transmission coefficients for selected m / z values.

[0077] Fig.23 is shown in the MS1 measurement and in each of the 20 consecutive MIDIA frames Fig. 20 3D plot of the intensities obtained for known precursors.

[0078] Fig.24 Shown with Fig.23 The same data, but presented as a heatmap. DETAILED DESCRIPTION

[0079] It should be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and do not limit the methods and apparatus described herein. In this application, the use of the singular may include the plural, unless otherwise specifically stated. In addition, the use of "or" means "and / or", when applicable or unless otherwise stated. Those of ordinary skill in the art will recognize that the following description is illustrative only and is not intended to be limiting in any way. Such technicians will readily come up with other embodiments that benefit from the present disclosure. Various examples of exemplary embodiments will now be referenced in detail, as shown in the accompanying drawings.

[0080] exist Figure 1 In the embodiment shown, a system 10 for data independent combined ion mobility and mass spectrometry is shown. The system 10 includes a sample separation device 12 for separating a sample. The sample is typically a biological sample, such as a peptide. In the embodiment shown, the sample separation device 12 is a liquid chromatography device, but other separation devices, such as a gas chromatography device, a capillary electrophoresis separation device, etc., may also be used.

[0081] The sample of separation, or more precisely the material of the sample, is introduced into an ion source 14, where they are ionized, for example, by electrospray ionization (ESI), matrix-assisted laser desorption / ionization (MALDI) or electron impact ionization (EI), to form precursor ions. The precursor ions are introduced into an ion mobility spectrometer or separator 16, which sequentially releases the precursor ions according to their ion mobility. In the illustrated embodiment, IMS16 is a so-called capture IMS (TIMS) device, in which ions are preferably captured by the opposing force of the gas flow and the reaction DC electric field along the axial direction. The ions are radially limited by the electric RF field. The TIMS device 16 shown includes an inlet funnel 18, a first TIMS20, a second TIMS22 and an outlet funnel 24. Here, the first TIMS20 is configured to constantly receive precursor ions and transfer the received precursor ions to the second TIMS22 in a time-controlled manner. The second TIMS (22) is configured to perform an IM scan, wherein the precursor ions are released sequentially from the second TIMS 22 according to the ion mobility of the precursor ions. In the illustrated embodiment, in each IM scan, the precursor ions with lower ion mobility are released before the precursor ions with higher ion mobility. In other embodiments, the IMS may be a traveling wave IMS device (TWIMS) or a drift tube IMS (DT-IMS). Due to different separation principles, these IMS release precursor ions with higher ion mobility before precursor ions with lower ion mobility.

[0082] Downstream of the IMS 16, a mass filter is provided. The mass filter 26 selectively transmits precursor ions having m / z values ​​falling within a controlled mass selection window. In the embodiment shown, the mass filter is a quadrupole RF device 26.

[0083] Precursor ions falling within the current mass window of the quadrupole RF device 26 are transmitted to a fragmentation device 28, where the precursor ions are fragmented into fragment ions. In the illustrated embodiment, the fragmentation device is a collision cell 28. However, other types of fragmentation devices are also feasible, such as surface induced dissociation fragmentation devices, electron transfer dissociation (ETD) devices, electron capture dissociation (ECD) devices, ultraviolet light induced dissociation (UVPD) devices, etc.

[0084] The system 10 also includes a mass spectrometer 30, which performs mass spectrometric measurements on the fragment ions. In the illustrated embodiment, the mass spectrometer is a time-of-flight (TOF) analyzer.

[0085] Finally, the system 10 includes a control system 32, which is configured to control each component of the system 10 and also perform data analysis of the mass spectra obtained by the TOF analyzer 30. The control system 32 includes one or more microprocessors and a memory for storing suitable computer codes for performing system control and data analysis functions. The control system 32 can be a single unit, or can be a distributed system including different control units with separate processors and / or dedicated control circuits, ASICs, etc. that are in data communication with each other.

[0086] Figure 1 The system 10 shown in can be operated in two operating modes, which are generally referred to as MS1 and MS2 (tandem MS or MS / MS) modes, respectively. If operated in MS1 ​​mode, the mass filter 26 and the collision cell 28 are disabled so that all precursor ions sequentially released from the second TIMS 22 are transmitted to the TOF analyzer 30 without mass selection or fragmentation, thereby obtaining a high-resolution m / z spectrum as a function of the mobility of the precursor ions. The precursors are also associated with the corresponding retention times of the chromatographic device 12. In Figure 2 The m / z mobility plots of observed / measured precursor ion intensities obtained in MS1 ​​mode are shown in FIG. Figure 2 In the graph of , the horizontal axis defines the m / z value of the precursor, while the vertical axis defines the counter ion mobility (1 / K0), such that larger values ​​on the vertical axis correspond to lower mobilities. Figure 2 In the diagram of FIG. 1 , individual precursors are represented by vertically elongated points, i.e., with a certain extension in the mobility direction, because the ions are accumulated in the TIMS device 16 with a certain spatial distribution, resulting in a certain variation in the mobility measured by the TIMS device 16 for the same precursor. That is, the precursor ion species released from the TIMS device 16 have a temporal pulse width that can be in the order of milliseconds. The flight time in the TOF analyzer is shorter than the TIMS pulse width, so that multiple TOF spectra are measured for each released TIMS pulse.

[0087] from Figure 2 From the m / z-mobility diagram, it can be seen that there is a correlation between the ion mobility of the precursor and the m / z value. Generally, the ion mobility decreases with the increase of the m / z value, so that the precursor is usually located in the area extending along the two diagonals in the m / z mobility diagram, where Figure 2 The two regions visible in the graph correspond to different charge states (z values) of the ions.

[0088] This correlation between the mass and mobility of precursors has been exploited by the data-independent simultaneous accumulation-serial fragmentation (diaPASEF) method, which is described in Florian Meier et al. Parallel accumulation-serial fragmentation combined with data-independent acquisition (diaPASEF): bottom-up proteomics with near optimal ion usage, BioarXiv doi: https: / / doi.org / 10.1101 / 656207. Figure 2 and Figure 3 The method comprises a plurality of IM scans during which precursor ions are sequentially released from the TIMS device 16 according to their mobility, starting with the lower mobility ( Figure 2 The vertical axis of the graph shows a large anti-mobility value) and moves toward higher mobilities (i.e., Figure 2 During each IM scan, a plurality (usually two or four, as shown in the figure) of different mass windows of the quadrupole mass filter 26 are applied in sequence, so as to cover only the relevant part of the m / z-mobility plane, i.e., the region where precursors are to be found.

[0089] For example, Figure 2 The third IM scan in a series of such IM scans is shown in FIG: This scan starts with the highest anti-mobility value, while the mass window of the quadrupole mass filter 26 remains fixed at a relatively high value. The width of the mass window is chosen to be 25 Da. All precursors released from the TIMS device 16 and located in this mass window will reach the collision cell 28, where they will be fragmented. The fragments are subjected to TOF mass spectrometry using a TOF analyzer 30. The TOF analyzer 30 records the mass spectra of the available fragments one by one, for example at a repetition rate of 10 kHz, wherein each mass spectrum is associated with a corresponding TOF push. During the time course, the mass spectra of the fragments are recorded by the TOF analyzer 30. Figure 2 Precursors with increasingly lower back-mobility values, indicated by the rightmost vertical downward pointing arrows in FIG. 1 , are released from the TIMS device 16 and - provided they meet the fixed mass window of the quadrupole mass filter 26 - reach the collision cell 28 and subsequently the TOF analyzer 30. Thus, a different ion mobility range or value is associated with each TOF push of the TOF analyzer 30.

[0090] Once the inverse mobility value drops below the target region, i.e., away from the region of the majority of the expected precursor ions, the mass window is Figure 2Then, the IM scan is continued in a different, shifted mass window. Figure 2 This process was continued for four different mass windows, each 25 Da wide, as shown in . Figure 2 Each set of rectangles acquired during a single TIMS separation / IM scan shown in is referred to herein as a "diaPASEF frame," where each rectangle covers a 25 Da mass window and a corresponding IM range.

[0091] Figure 3 The fourth subsequent IM scan / diaPASEF frame is illustrated. The fourth IM scan begins with a mass window that is directly adjacent to the starting mass window of the third IM scan and has a slightly higher back mobility so as to cover the relevant portion in the m / z-IM plane. The fourth IM scan is also distributed over four non-intersecting mass windows. The process is continued until the entire target range is covered. The results of this process are encoded into a four-dimensional data space that includes the time parameters of the separation device, such as the retention time, m / z mass window, ion mobility and intensity of the liquid chromatography device 12. Then, target data extraction can be performed according to the four dimensions described in the article by Meier et al. above. The diaPASEF method is "data independent" in that it does not include the preparation or separation of any specific precursor. Instead, all precursors provided by the liquid chromatography device 12 are subjected to the PASEF method, and since the mass window of the mass filter 26 is selected to match the current ion mobility range, the utilization of the available precursor ions is quite high. Since this mode of operation of the device includes two mass selection steps, namely preselection of a mass window by quadrupole mass filter 26 and mass spectrometric measurement of fragment ions generated by precursors in collision cell 28 by TOF analyzer 30, the process represents the above-mentioned MS / MS mode of operation.

[0092] In the current best application of the diaPASEF procedure, 16 PASEF frames are used to cover the entire precursor ion space with a mass window width of 25 Da and typically 150-500 TOF pushes per PASEF frame. For a 100 ms TIMS accumulation time, the total cycle time for covering the MS1 frame and all 16 diaPASEF frames is about 1.7 seconds. Since the diaPASEF frames do not overlap in the m / z direction, each precursor is scanned during only one diaPASEF frame, resulting in a 5.88% duty cycle for the corresponding fragment ions.

[0093] Next, refer to Figure 4 , explaining a method according to an embodiment of the present invention. Figure 4Again, diaPASEF frames corresponding to the first and second IM scans in the m / z ion mobility plane are shown. A type of diagonal frame used in embodiments of the present invention is also shown. The method of the present invention will be referred to herein as "Maximum Information Data Independent Acquisition (MIDIA)" and the corresponding diagonal frames are referred to as "MIDIA-frames". Each MIDIA frame is generated during the course of an IM scan, during which, as before, precursor ions with increasing mobility (shown in the figure as decreasing reverse mobility) are released from the TIMS device 16 in sequence. For this purpose, the terms "MIDIA frame" and "MIDIA scan" are used herein synonymously. However, the mass window of the mass filter 26 is continuously or stepwise moved toward lower m / z values ​​so that adjacent mass windows in the IM scan associated with continuous mass spectrometry measurements of the target ions overlap each other and so that all precursor ions transmitted from the mass filter during the IM scan are located in a continuous scan area (MIDIA frame / scan) in the m / z-IM plane. This is in contrast to the case where the mass window of the mass filter 26 is also moved in steps toward lower m / z values ​​so that adjacent mass windows in the IM scan associated with continuous mass spectrometry measurements of the target ions overlap each other and so that all precursor ions transmitted from the mass filter during the IM scan are located in a continuous scan area (MIDIA frame / scan) in the m / z-IM plane. Figure 4 The diaPASEF frame shown in is different in that each IM scan is distributed over several non-intersecting mass windows within the diaPASEF frame, the m / z values ​​of these non-intersecting mass windows are typically separated by a distance that is a multiple of the mass window width itself.

[0094] As described above, in the present invention, the mass window of the quadrupole mass filter 16 can be moved continuously or stepwise. However, since the mass spectrometry measurements of the fragments are performed as discrete events, even in the case where the mass window is continuously moved, two consecutive mass spectrometry measurements will be associated with different discrete mass window ranges, which correspond to the progress made by continuously moving the mass window between two consecutive mass spectrometry measurements (e.g., between two consecutive TOF pushes). This situation is indeed similar to that of the IM value, because the precursor ions are also continuously released from the TIMS device 16, and the IM value or IM range is only defined by the time relative to the sequential TOF pushes continuously released from the TIMS device 16.

[0095] Figure 5 A close-up view of the diaPASEF frame on the left and the MIDIA frame on the right are shown, both in the m / z-IM plane. Figure 1 Precursors obtained in the MS1 mode of operation of the device 10 are again shown as vertically elongated dots. A diaPASEF frame is a portion of a diaPASEF scan during which a mass window of 25 Dalton width is held at a fixed position. The diaPASEF frame is divided vertically into 20 fields or "subframes" of ion mobility, wherein each of these fields or subframes corresponds to a TOF push. Within the diaPASEF frame, a specific sample precursor having an m / z value of 500 is shown at reference numeral 34.

[0096] The MIDIA frame on the right corresponds to a full scan of ion mobility. The MIDIA frame is likewise divided into fields or subframes, each having a width corresponding to the mass window of the quadrupole mass separator 26 in the m / z dimension, and each corresponding to one TOF push. The IM value or range of the subframe is associated with the time of the TOF push, or more generally, with the time of the associated mass spectrometry measurement performed on the fragments of the precursor. The scan direction is again from higher to lower (i.e., downward in the figure) of the reverse mobility, but unlike the diaPASEF frame on the left, the mass window of the quadrupole mass filter 26 moves between each TOF push. It does not matter whether this movement is performed in steps or continuously, what is important is that each mass spectrometry measurement associated with a subsequent TOF push is associated with a different position of the mass window. The mass window used for the exemplary MIDIA scan is 36 Da wide, and is therefore wider than those used in diaPASEF (25 Da). In addition, it can be seen that the mass windows associated with consecutive TOF pushes (or more generally: consecutive mass spectrometry measurements) substantially overlap each other to a large extent. Due to the variance of the IM values ​​associated with most precursors, or in other words, the elongation of the points representing the precursors in the m / z-IM diagram exceeds the IM range covered by each subframe, the same precursor can be covered by different shifted subframes with different m / z windows. This fact allows to evaluate information about the m / z value of the precursor at the sub-mass window resolution, as will be explained below.

[0097] Although this is not envisaged in the preferred embodiment, in the method of the invention it is also conceivable to perform a small amount of TOF pushes without moving the mass window in between, and only then to move the mass window. However, even in this case, the mass windows associated with the TOF pushes just before and just after the move will overlap each other. This is again different from diaPASEF frames, where each time a mass window is moved, it is moved to a position that does not overlap with the previous position. Furthermore, the mass windows of the individual diaPASEF frames are selected so that they do not overlap significantly, so that typically each precursor will only be present in one diaPASEF frame.

[0098] Figure 6 and Figure 4 Similar, but showing the positions of the next two MIDIA scans (Scan 2 and Scan 3). Note that the numbering of the scans refers to their position in the m / z-IM plane, not (or not necessarily) the order in which they were recorded. Figure 6As can be seen in Figure 1, adjacent scan regions associated with individual scans overlap each other by approximately two-thirds of their area. That is, for a given IM value, the mass windows in adjacent MIDIA scans shift by 12 Da, or 1 / 3 of the mass window width of 36 Da. This overlap indicates that each precursor will appear in three MIDIA frames within the corresponding IM range associated with the precursor. To understand this, one can focus on Figure 6 A horizontal region 36 is represented by a horizontal rectangle in FIG. 3 which represents a region associated with a certain IM value traversed by different MIDIA frames.

[0099] In this horizontal region 36, adjacent MIDIA frames are shifted by 12Da relative to each other. Figure 7 , where each MIDIA frame overlaps with the horizontal region 36 shown. Each rectangle corresponds to the intersection of a MIDIA frame and the horizontal region 36. Figure 6 In the m / z-IM map, these regions will be located on top of each other, but in Figure 6 In the image, they are moved vertically to better distinguish them from each other.

[0100] exist Figure 7 Also shown in , is an exemplary table summarizing the number of each MIDIA frame, and the lower and upper boundaries ("Start" and "End") with respect to its m / z value when intersecting region 36. Twelve MIDIA frames are represented in the table, covering the m / z range of 400 to 580 Da within horizontal region 36. Precursor 34 with an M / z value of 500 Da will appear in three MIDIA frames, namely MIDIA frames 6, 7 and 8. Therefore, it can be inferred from the precursor appearing in these three MIDIA frames within the narrow IM range defined by horizontal region 36 that the mass must be from the start of the eighth subframe (496 Da) to the end of the sixth subframe (508 Da), i.e. within the m / z range of 12 Da. In contrast, in diaPASEF, each precursor will appear in only one diaPASEF frame, and although it may appear in different subframes (according to different IM values), no additional information about the m / z value can be discerned therefrom. It can thus be seen that, although the mass window used in the MIDIA frame (36 Da) is wider than the mass window used in diaPASEF (25 Da), the data acquired using the MIDIA acquisition scheme encodes more specific information about the m / z values ​​of the precursors.

[0101] However, from the following Figures 8 to 12As will become apparent from the discussion of , due to the unique overlap of the MIDIA frames, the combination of the IM values ​​and the m / z values ​​associated with a single subframe shifted in the m / z direction (or, in other words, the diagonal direction of the MIDIA frames), the MIDIA data actually encodes information about the mass of the precursor ions at a much higher resolution than just one-third of a single mass window, and in many cases only up to the order of a single Dalton. This is particularly true if additional information about the precise position of the precursor ions in the m / z-IM plane and their spread in ion mobility is utilized, which can be readily obtained from MS1 measurements using the apparatus 10, which can be performed simultaneously with MS / MS measurements.

[0102] For comparison, Figure 8 Also shown is a diaPASEF frame covering a first scan of an exemplary precursor 34 at m / z equal to 500 on the left, and a first MIDIA scan on the right, where it can be seen that the first MIDIA scan covers at least the upper edge of the corresponding intensity peak or "spot" in the m / z-IM plane. Furthermore, since the intensity peak or "spot" is elongated in the vertical (ion mobility) direction, it overlaps with different subframes of the mutually shifted subframes of the first MIDIA scan, so that even this single MIDIA scan already contains m / z information with a resolution lower than the width of the individual mass windows, which is due to the above reference. Figure 7 Similar reasons as explained.

[0103] Fig. 9 Again, the dashed line Figure 8 , and the position of the adjacent "second" scan indicated by a solid line. Likewise, the terms "first" and "second" do not, or at least do not necessarily, refer to the order in which the scans are performed, but rather to their position relative to each other in the m / z-IM plane. Fig. 9 As shown on the left side of , the diaPASEF frame of the second scan covers a completely new m / z range and therefore cannot give additional information about the m / z value of the target precursor 34. However, the second MIDIA scan covers most of the target precursor peaks and therefore contains additional information about the m / z values ​​and ion mobilities of the peaks. Finally, Fig.10 The first and second scans are shown in dashed lines, and the third scan is shown in solid lines. Again, it can be seen that the third MIDIA scan covers most of the precursor peaks, and therefore contains additional information about their m / z values ​​and ion mobilities.

[0104] A typical task is to associate the fragments identified in the mass spectrometry measurements made by the TOF analyzer 30 with the corresponding precursors. Each measurement using the TOF analyzer 30 is associated with a corresponding subframe of the MIDIA scan or frame. Figures 8 to 10As will be apparent from the discussion of the present invention, a particular precursor (e.g., precursor 34) will be present in multiple subframes of multiple MIDIA scans or frames. If a particular fragmentation occurrence is detected in a given mass spectrometry set, the corresponding subframes of the multiple MIDIA scans can be determined. The subframe set defines a "fingerprint" for precursor 34, from which its m / z value can be determined with very high accuracy.

[0105] Fig.11 is a table summarizing the positions of the subframes within the 10 MIDIA scans. The first column of the table lists the number of TOF pushes, which can be considered as an "ion mobility index" (IMI). Each integer represents a certain ion mobility value or a (small) ion mobility range.

[0106] For each IM index, the second and third columns specify the lower and upper boundaries of the total quality window covered by the entire MIDIA frame ("Start / End").

[0107] The fourth column again lists the IM Index, but further indicates in bold and shaded letters the possible IM Index values ​​for a given precursor 34. That is, as in e.g. Figure 8 As can be seen in FIG. 1 , the precursor 34 is represented by an elongated point extending in the vertical direction in the m / z-IM plane, which means that for the same precursor 34, a range of IM values ​​can be measured using the TIMS device 16. For example, Fig.11 As can be seen in Figure 5, the distribution of IM values ​​is centered around IM index 59, but the precursor can also be found in subframes corresponding to IM indices from 53 to 65. The variance of this distribution of IM values ​​can be obtained from the data recorded in the MS1 operating mode.

[0108] MIDIA frames 0 to 9 are represented in columns 5 to 14. Each number in these columns indicates the center of the mass window of the corresponding subframe for a given MIDIA frame of IM index. For example, in the fifth column corresponding to the first MIDIA frame (MIDIA frame number 0), the number "445.7" indicates that the center of the mass window of the subframe in the first MIDIA frame corresponding to IM index 54 is centered at m / z value 445.7, i.e., 427.7 to 463.7, because the mass window is 36 Da wide in this case.

[0109] In the columns representing MIDIA frames, the m / z values ​​of these subframes are printed in boldface, which may include the precursor 34 with an m / z value of 500 Da. For example, in the column corresponding to MIDIA frame number 5, at an IM index of 63, the m / z value 517.0 is printed in boldface, because with a mass window 36 Da wide centered around this value, the precursor with an m / z value of 500 will still be barely transmitted by the quadrupole mass filter 26. However, the subframe of the same MIDIA frame number 5 corresponding to an IM index value of 64 (mass window centered around 518.3) will no longer allow the precursor 34 with m / z equal to 500 to reach the fragmentation unit 28, which is why this value is printed in normal font.

[0110] In the columns corresponding to MIDIA frames, all subframes are highlighted and shaded by the corresponding frame, which is consistent with the expected IM index range of 53 to 65 and the center of the quality window being compatible with precursor 34, which is located in the interval between 482 and 518. It can be seen that, as expected, precursor 34 will be found primarily in three MIDIA frames, namely MIDIA frame numbers three, four and five, and possibly also in two subframes of MIDIA frames 2 and 6.

[0111] The set of subframes in which the precursor 34 will be allowed to reach the fragmentation unit 28 can be considered to be a "fingerprint" of the precursor 34 in the MIDIA data. For example, suppose that a particular fragment appears in multiple mass spectrometry measurements, and it is desired to check whether the fragment can be associated with the precursor 34. Since each mass spectrometry measurement corresponds to a subframe of a MIDIA scan, it is possible to check whether the occurrence of the fragment in each subframe is consistent with the "fingerprint". Here, the criterion of "consistent with the fingerprint" can be formulated as a quantitative criterion using a statistical scoring scheme to produce a likelihood value or probability value of consistency as a function of the deviation of the fingerprint and the set of subframes in which the fragment is actually found, thereby allowing for the handling of data noise that will inevitably occur. The statistical scoring scheme may include associating certain weights with certain deviations from the fingerprint. For example, if the fragment is not present in the mass spectrometry measurement associated with the subframe of the fifth MIDIA frame of IM index 59 (having the center of the mass window that exactly matches the m / z value of 500 for the precursor 34, and having an IM index in the middle of the ion mobility distribution of the precursor 34), this will make it very unlikely that the fragment is indeed associated with the precursor 34. On the other hand, if a fragment should be lost in a subframe associated with, for example, an IM index at the border of the distribution (eg, 53 or 65), this will not significantly reduce the probability of a match.

[0112] There are many ways to use "fingerprints" to associate fragments with their precursors, and the present invention is not limited to any particular one of them. This matching can even be done using a machine learning algorithm, such as a deep learning algorithm like a convolutional neural network, which can be trained on real measurement data or simulated measurement data, for example by turning off the collision energy, thereby keeping the complete precursor signal in MIDIA frames, which can be used as a ground truth dataset for algorithm training, as will be explained in more detail below.

[0113] However, as can become apparent from the above discussion, the association of detected fragments with their corresponding precursor ions is based on the information contained in the occurrences of the fragments in multiple MIDIA subframes, or in more general language, based on "determining or utilizing the corresponding mass windows and IM ranges associated with the various occurrences of said fragments in the mass spectrometry measurement", keeping in mind that each MIDIA subframe corresponds to a specific IM range (which can be parameterized by the ion mobility index (IMI)) and the corresponding mass window associated with the IMI in a given MIDIA scan. The occurrences of fragments in multiple MIDIA subframes are highly specific to the corresponding precursors and can therefore be used as a "fingerprint" to associate the fragments with their precursors.

[0114] Note that for a simpler explanation, in the above explanation the expression "occurrence" is used in the sense of binary information about whether a certain fragment is present in a MIDIA subframe. However, in a practical implementation, the "occurrence" may have a quantitative component and may, for example, represent the intensity of the fragment in a TOF measurement associated with a MIDIA subframe. Such a "quantitative presence" or intensity represents additional information of a "fingerprint" that may be used to associate a fragment with its precursor.

[0115] Furthermore, again for simplicity of illustration, in the above description, the retention time associated with the liquid chromatography device 12 is not considered, i.e., the explanation focuses only on the case of a given retention time. In general, the retention time defines another dimension to be considered in data analysis, as will become more apparent from the following discussion.

[0116] Regardless of the precise manner in which the "fingerprint" is used to associate fragments with precursors, the fundamental question, of course, is how specific the fingerprint actually is with respect to the m / z values ​​that determine the likely precursor. This can be seen from Fig.12, where all subframes in the relevant m / z range appearing in any MIDIA scan are listed in order of increasing center of the mass window. Each subframe can be represented by the corresponding number and corresponding IM index of the MIDIA scan to which it belongs, listed in the first and second columns, respectively. The third column represents the center of the mass window, while the fourth and fifth columns represent the lower and upper boundaries, respectively. The remaining columns list the m / z values ​​of possible potential precursors in steps of 0.2 Da. If precursor 34 (or other potential precursors with a given mass and IM index) falls into the subframe, it is represented by "1", if not, it is represented by "0". In addition, in the table, some m / z ranges consistent with the same selection of subframes are surrounded by printed boxes in the figure. This means that the precursors within these m / z ranges exhibit the same "fingerprint", and the width of the m / z range corresponding to the same fingerprint defines the potential specificity of the method with respect to determining m / z values.

[0117] according to Fig.12 As can be seen from the table in , the typical m / z range that will result in the same fingerprint in this example is up to 0.8 Da, and can be as low as 0.4 Da. This shows that fingerprints have the potential to distinguish precursors, or determine the m / z value of precursors with an accuracy of about 1 Da, and theoretically even lower.

[0118] This also demonstrates that the particular data acquisition in MIDIA frames as described herein does significantly increase the information encoded therein compared to the information encoded in non-overlapping vertical prior art PASEF frames, which is why the name "maximum information data-independent acquisition" was chosen. Exactly how the information is then derived from the data depends, among other things, on the problem to be solved, and is not limiting of the invention. In some cases, a particular fragment will be observed in many measurements, and it will simply be desirable to know the possible m / z values ​​for a possible precursor. In other embodiments, the possible precursors may be known, for example from MS1 measurements performed intermittently with MS / MS measurements, as well as the variance of their ion mobilities, and it may then be desirable to determine the likelihood that a certain fragment is associated with that particular precursor, for example to generate a weighted precursor-fragment ion relationship that can be used as input to a database search engine or to query a spectral library to identify target analytes (e.g., peptides, lipids, metabolites, or other small molecules with a mass of 50 to 5000 Da).

[0119] Thus, those skilled in the art will appreciate that there are many possible methods to exploit the information contained in the IM-related TOF measurements obtained by overlapping MIDIA frames for the purpose of associating fragments with their precursors, and that the present invention is not limited to any particular method therein. Indeed, a variety of data evaluation methods will yield superior results, as they all benefit from the fact that a given random occurrence (as a binary event or intensity) in the various MIDIA subframes associated with different MIDIA scans encodes highly specific information about the corresponding precursor ion. Thus, all of these variances rely on "determining or exploiting the corresponding mass windows and IM ranges associated with the various occurrences of a given fragment in the mass spectrometry measurement".

[0120] It should therefore be understood that the present invention is not limited to any particular data evaluation process, and a few examples are outlined below.

[0121] In a preferred embodiment, IMS / MS is used to measure MS1 data, i.e., the data of unfragmented precursor ions. The measurement data can be organized into four-dimensional tuples (RT, IMI, m / z, intensity), wherein RT is retention time, and IMI is ion mobility index. Intensity can be absolute intensity or relative intensity. Tuples are referred to as MS1-(4D-tuples) below. Therefore, these MS1-(4D-tuples) define the intensity as a function of RT and IMI for each precursor characterized by its m / z value.

[0122] MS2 data of fragment ions are collected using overlapping MIDIA frames. This generates a 5-dimensional tuple (RT, m / z, MIDIA scan #, IMI, intensity), which is referred to herein as a MIDIA-(5D-tuple), where "MIDIA scan #" represents the number of MIDIA scans in a given MIDIA cycle. Note that the corresponding mass window of the mass filter is defined by the MIDIA scan # and the IMI together with the measurement protocol, which defines the synchronization of the IMS device and the mass filter (a quadrupole in the illustrated embodiment) for each individual MIDIA scan.

[0123] Additionally or alternatively, the exact position of the mass window of the mass filter as a function of the MIDIA scan # and the IMI can be obtained by a "pseudo-MS2" measurement, which is performed in the same manner as a true MS2 measurement, but the precursor ions are not fragmented. These pseudo-MS2 measurements can be performed separately or in parallel with the true MS2 measurements. For example, between every two MIDIA scans or MIDIA cycles of a true MS2 measurement, a pseudo-MS2 MIDIA scan or cycle can be interspersed, which would be an example of parallel acquisition of pseudo-MS2 data. From these pseudo-MS2 data, the exact position of the mass window can be reconstructed.

[0124] In addition, since pseudo-MS2 data preserves the m / z value of the precursor, the same ions in the MS1 data set and the pseudo-MS2 data set are directly associated. Therefore, pseudo-MS2 data can be used as ground truth data for any type of algorithm or model, and the algorithm or model predicts or calculates the intensity distribution of IMI and MIDIA scan # expected in the corresponding fragment ions in the MIDIA-(5D-tuple) of the MS2 data based on the performance of the precursor in the MS1-(4D-tuple) space. For the prediction or calculation of the expected distribution, various algorithms and models can be used, such as matrix-based methods, neural networks, random forests, support vector machines or other machine learning methods. In this way, once such an algorithm or model is established, the MS1 data present in the four-dimensional MS1-(4D-tuple) space can be converted into the corresponding five-dimensional data in the MIDIA-(5D-tuple) space, which can be compared, matched or related to the MS2 data of the fragment, so that a given fragment is associated with its precursor without having to continuously perform pseudo-MS2 measurements. Obviously, this conversion has nothing to do with the m / z values ​​contained in the MIDIA-(5D-tuple), since the m / z of the fragments cannot be derived from the MS1 data.

[0125] In an alternative, pseudo-MS2 data can also be used to derive algorithms and models that allow the calculation or prediction of the intensity distribution as a function of RT, IMI and (possibly) m / z of the corresponding precursor ion based on the MIDIA-(5D-tuple) associated with a given fragment (i.e., RT, m / z, MIDIA scan #, IMI, intensity). These algorithms or models can also be based on matrix-based methods, neural networks, random forests, support vector machines or other machine learning methods. In this way, once such an algorithm or model is established, the MS2 data present in the five-dimensional MIDIA-(5D-tuple) space can be converted into corresponding four-dimensional data in the MS1-(4D-tuple) space and can be compared, matched or correlated with the MS1 data of the experimental precursor, thereby associating a given fragment with its precursor without having to continuously perform pseudo-MS2 measurements.

[0126] More specifically, in one embodiment, the matching or association of precursor ions (MS1) and fragment ions (true MS2) can be performed, for example, by a scoring function based on:

[0127] (i) predicted or simulated MIDIA-(5D-tuple) fragment intensity distributions derived from the MS1-(4D-tuple) intensity distributions of the experimental precursor ions using one of the above algorithms and models, and

[0128] (ii) MIDIA-(5D-tuple) intensity distribution of a given fragment ion obtained in MS2 measurement.

[0129] As mentioned before, in this scoring, the m / z values ​​of measured and predicted MIDIA-(5D-tuple) are not taken into account, since the predicted MIDIA-(5D-tuple) derived from the MS1 measurement does not contain m / z information about possible fragments. Instead, the starting point for the match will be a given fragment with a specific m / z value, and the match focuses on the similarity or consistency of the other four dimensions of the predicted or simulated MIDIA-(5D-tuple) with the corresponding measured MIDIA-(5D-tuple) of the fragment.

[0130] Note that the MIDIA-scan #, IMI and intensity values ​​for all MIDIA-(5D-tuples) where the m / z values ​​correspond to the m / z of a given fragment represent what is more generally referred to as the above "mass window and IM range associated with various occurrences of said fragment in the mass spectrometry measurement". In the current specific embodiment, the "mass window" is parameterized by the MIDIA-scan # and IMI, the "IM range" is represented by the IMI, and the "various occurrences of the fragment" correspond to a subset of the MIDIA-(5D-tuples) whose m / z values ​​match the m / z value of the fragment, where the corresponding intensity values ​​add a quantitative measure for the "occurrence". In addition, selecting the MIDIA-(5D-tuple) intensity distribution for a given fragment ion, for example by selecting all MIDIA-(5D-tuples) with the fragment m / z value, can be considered as an example of "determining the corresponding mass window and IM range associated with various occurrences of the fragment in the MS2 measurement".

[0131] In another embodiment, the matching or association of the precursor ion (MS1) and the fragment ion (MS2) can be performed by a scoring function based on:

[0132] (i) MIDIA-(5D-tuple) intensity distributions of the experimental precursor ions obtained by pseudo-MS2 data collected in parallel with the real MS2 data, and

[0133] (ii) MIDIA-(5D-tuple) intensity distribution of a given fragment obtained in a real MS2 measurement.

[0134] Again in this context, taking into account in the scoring the selection of the MIDIA-(5D-tuple) intensity distribution for a particular fragment is a specific example of the aforementioned "determining the respective mass windows and IM ranges associated with the various occurrences of said fragment".

[0135] In yet another embodiment, the matching or association of the precursor ions (MS1) and the fragment ions (MS2) can be performed, for example, by a scoring function based on:

[0136] (i) RT, IMI and intensity distribution of the corresponding precursor ions predicted from the MIDIA-(5D-tuple) intensity distribution of the corresponding fragments,

[0137] (ii) the m / z range of the precursor ions predicted from the MIDIA-(5D-tuple) intensity distribution of the corresponding fragments, and

[0138] (iii) Measured MS1-(4D-tuple) intensity distribution.

[0139] The scoring function adopted in the above examples can be relevant, or can be carried out using matrix-based prediction models, neural networks, random forests, support vector machines or other machine learning methods. The above embodiments are based on the available situation of MS1 data, so all possible precursor ions are all known. However, this is not always necessary, because the appropriate algorithm of the above type can be used to reconstruct the IMI, RT and m / z of the MIDIA-(5D-tuple) intensity distribution, possible precursor ions from each independent fragment. For this purpose, preferably in the first step, all fragments related to the same precursor (speculation) are identified. Note that the fragments derived from the same precursor can be identified by their highly correlated distribution in the MIDIA-(5D-tuple) space in all dimensions except the m / z value. Then, based on the MIDIA-(5D-tuple) intensity distribution of each fragment, the IMI, RT and m / z of the possible precursor ions can be determined, which can be used for database search or compared with the spectral library.

[0140] Next, refer to Figures 13 to 19 , another exemplary embodiment will be described that further illustrates how and why the use of MIDIA scans and frames allows very precise association of fragments with their precursors. Fig.13 The three precursors P1, P2 and P3 located in the m / z-IM plane are shown, together with the position of the first MIDIA scan in a MIDIA cycle of a real MS2 measurement. The precursors P1, P2 and P3 are also considered to have an elongated shape in the IM direction, which means that each precursor is associated with a range of IMI that is associated with the inverse rate of the TIMS pulse and has a Fig.13 In the expression ΔIM 前体 As mentioned above, this expansion or “stretching” of the precursor in the IM direction ΔIM 前体 The reason for this is that each IMI is associated with a TOF push, and the frequency of the TOF push is higher than the rate of the TIMS pulses when the precursor ions are released from the TIMS. Furthermore, it can be seen that the positions of the IM ranges associated with precursors P2 and P3 are identical, which makes the correct attribution of the individual fragments particularly difficult using prior art methods.

[0141] In an MS2 measurement, five dimensions of data are acquired (RT, m / z, MIDIA scan #, IMI, intensity), which are organized into the above-mentioned MIDIA-(5D-tuple). For illustrative purposes, three fragments F1, F2 and F3 originating from precursors P1, P2 and P3, respectively, should be considered. In practice, the task is then to identify for each fragment Fn (where n is an integer) its corresponding precursor. For this purpose, one can look in a first step at the intensity distribution of a given fragment Fn for a given first MIDIA scan (i.e. MIDIA scan #=1), defined by its m / z value, as a function of the IMI. In Fig.13 Three fragments F1, F2 and F3 are shown in the figure. Although IMI is actually a discrete variable, it is shown as a continuous variable in the figure for simplicity. As can be seen from the figure, during the lower IMI value range, the first fragment F1 first has an intensity peak, while F2 and F3 have intensity peaks at the same IMI value range at the same time. The simultaneous occurrence of intensity of F2 and F3 in the same IMI value range indicates that it may be difficult to correctly specify their respective precursor ions. However, it can be seen that the lower end of the precursor P2 in the m / z-IM diagram is not covered by the first MIDIA frame, so that the intensity-IMI diagrams of F2 and F3 are not exactly the same at all. On the contrary, for fragment F2, the intensity of the highest IMI value is "cut off" because for these highest IMI values, the corresponding precursor P2 falls outside the mass window of the mass filter (quadrupole). The figure of F2 shows the actual intensity-IMI diagram obtained with a solid line, and the dotted line indicates how the curve will look when the mass window is not cut off. In the absence of "truncation", the intensity-IMI diagrams of fragments F2 and F3 are actually indistinguishable from each other.

[0142] Fig.14 The same situation is shown for the second MIDIA scan (i.e., MIDIA scan #=2). Fig.14 As can be seen in Figure 1, the second MIDIA frame corresponds to a shifted copy of the first MIDIA frame, which is shifted by a distance Δ in the m / z direction. m , the distance Δ m Corresponding to 1 / 3 of the frame width. m , for the same m / z, there is a vertical offset ΔIM between the edges of adjacent MIDIA frames 帧 , which is also in Fig.14 Shown in.

[0143] Fig.14 The newly added intensity-IMI maps of the fragments F1, F2 and F3 of the second MIDIA frame are also shown in . It can be seen that in this case, the second MIDIA frame cuts off the upper end of the precursor P1, which is reflected in the corresponding intensity-IMI map of the fragment F1.

[0144] Fig.15 The same situation is shown for the third MIDIA scan (i.e., MIDIA scan #=3). Fig.15 As can be seen in , the third MIDIA frame corresponds to a shifted copy of the first MIDIA frame, which is shifted by 2 / 3 of its width towards higher m / z values. Fig.15 The cumulative intensity-IMI map of the fragments F1, F2 and F3 up to the third MIDIA frame is also shown in FIG. It can be seen that in this case, the third MIDIA frame cuts off the upper part of each of the precursors P1, P2 and P3, but the sizes of these cut-off parts are different from each other, resulting in Fig.15 Characteristic intensity-IMI diagrams of the corresponding fragments F1 to F3 are shown in FIG.

[0145] final, Fig.16 The same situation is shown for the fourth MIDIA scan (i.e., MIDIA scan #=4). Here, the fourth MIDIA frame is shifted by one full frame width relative to the first MIDIA frame. Precursors P1 and P3 are no longer covered by the fourth MIDIA frame, but the lower end of precursor P2 is still captured, which is reflected in the intensity-IMI map of the corresponding fragment F2.

[0146] The "cutoff points" in the fragment intensity-IMI plot are very characteristic and encode information that allows each of the fragments Fn to be correctly associated with its corresponding precursor Pn, for example by determining the correlation, as will be described next. In the illustrated embodiment, the ΔIM of the IM range covered by each precursor ion Pn is 前体 The size is greater than the vertical offset ΔIM 帧 , which guarantees that for each precursor, there will be at least one such cutoff point. Figures 13 to 16 In the example schematically illustrated in , precursors P1 and P2 do have two such truncation points, whereas P3 has only one.

[0147] Fig.17 Schematically illustrated is how a fragment Fx is associated with a corresponding precursor among the possible precursors, wherein in the exemplary example, for simplicity, only three precursors P1 to P3 are available and only four MIDIA frames are considered. The corresponding precursor is identified by calculating the correlation between the intensity-IMI diagrams of all four MIDIA frames of a given fragment Fx and the corresponding intensity-IMI diagrams of all available precursors (in this simple case precursors P1, P2 and P3). As mentioned above, these intensity-IMI diagrams can be obtained directly by pseudo-MS2 measurements, organizing the data generated by pseudo-MS2 measurements into MIDIA-(5D-tuples) of the same type as real MS2. This allows the correlations to be calculated in a direct way.

[0148] In an alternative, an intensity-IMI map of a "virtual MIDIA scan" of precursor ions P1 to P3 can be artificially generated based on the MS1 data alone, for example using one of the algorithms described above for this purpose. Here, the term "virtual MIDIA scan" means that in this case, the MS1 data are not generated using an actual MIDIA scan, but rather pseudo MS2 data are derived if they have been obtained, for example in order to predict how the data will look.

[0149] Regardless of how exactly one obtains the intensity-IMI distribution or waveform of the precursors for all four (real or virtual) MIDIA frames, from Fig.17 It can be directly seen that the data of Fx has the highest correlation with the data of P1, so that fragment Fx can be identified as fragment F1.

[0150] Instead of calculating correlations, the matching between fragments and precursor data can of course be done in various other ways. Fig.18 As shown in , the measured intensity-IMI distribution of the set of MIDIA frames associated with the fragment Fx can be transformed, such as a wavelet transform, to produce a set of transform coefficients Ci(Fx). A similar transformation can be applied to each precursor Pn, producing a corresponding set of transform coefficients Ci(Pn) for n=1, ..., 3. The set of transform coefficients Ci(Fx) can then be compared with the set of transform coefficients Ci(Pn) of the corresponding precursor Pn, and the precursor with the best match in coefficients (in this case, P1) is associated with the fragment Fx.

[0151] Note that creating an intensity-IMI plot for various MIDIA scans from MS2 data is also an example of "determining corresponding mass windows and IM ranges associated with various occurrences of the fragments in the mass spectrometry" since each non-vanishing intensity represents an "occurrence" and the MIDIA scan # and IMI together define the mass window associated with the mass filter.

[0152] exist Figures 13 to 18 In the discussion of , in order to keep the discussion simple and focused, only a single MIDIA cycle consisting of four MIDIA frames is discussed, and the RT dimension has been ignored so far. Of course, in practical applications, the RT dimension will also be considered. For example, Fig.19 As shown in , correlations between data associated with a plurality of MIDIA cycles can be calculated, each MIDIA cycle comprising a plurality of MIDIA scans or frames, wherein the plurality of MIDIA cycles cover one LC peak, ie, RT range.

[0153] The inventors' current studies have found that MIDIA acquisition not only allows for a much higher specificity regarding precursor quality, but is also advantageous in terms of sensitivity and speed compared to diaPASEF currently known in the art. In the best mode of currently known diaPASEF, the total cycle time is currently 1.7 seconds.

[0154] In contrast, in one embodiment of the present invention, 20 MIDIA scans can be used, each performed in 50 ms and consisting of a 36-Da wide mass window, and overlapping the adjacent MIDIA frames by 2 / 3 in the m / z direction. In this embodiment, the cycle time is thus reduced to 1.1 seconds (including MS1 scans and internal instrument "overhead"), and the sensitivity is found to be increased by about 50%. In addition, in Figure 7 Based on the basic principle, the specificity is increased by at least 100%, that is, to 12 Da instead of 25 Da, but using the above reference Fig.11 and Fig.12 The specificity of the interpreted fingerprint can be increased much higher than this.

[0155] In another embodiment, a high-resolution measurement can be performed, which does not improve the speed of diaPASEF, but allows further increase in specificity. In this embodiment, the mass window is chosen to be only 24 Da wide. In this embodiment, 32 MIDIA frames are used, again overlapping by 2 / 3 in the m / z direction, and again using 50 ms dedicated to each MIDIA scan. In this case, the specificity reaches at least 8 Da, even without using the mass window as described above. Fig.11 and Fig.12 Additional information encoded in the interpreted fingerprint.

[0156] If high speed is an issue, in yet another embodiment, the individual MIDIA scans are performed in only 25 ms each, with a mass window size of 36 Da and an overlap of 2 / 3. The sensitivity is again about 50% higher than in the case of diaPASEF, while the cycle time is reduced to 0.6 seconds, and thus to about 1 / 3 of that of diaPASEF.

[0157] Fig. 20 Exemplary measurements according to an embodiment of the present invention are shown. Fig. 20 In FIG. 1 , the diamonds connected by dashed lines represent the intensity of the precursor ion with an m / z value of 550.3 obtained in the MS1 measurement as a function of ion mobility. It can be seen that for this precursor ion, the intensity is distributed over a range of IMI values ​​according to a bell-shaped distribution, as described above.

[0158] This precursor ion has been subjected to a MIDIA procedure, wherein in each MIDIA frame the mass window of the mass filter is adjusted as a function of the IMI, but without subsequent fragmentation of the precursor ion. The triangle symbols connected by dotted lines show the intensity of the precursor after passing through the mass filter in the eighth MIDIA frame. The purpose of this measurement is also to confirm the high accuracy of determining the m / z value of the precursor using the information contained in the measurement from the MIDIA frames.

[0159] Fig.21 A table summarizing how the position of the quality window in the eighth MIDIA frame varies as a function of the IMI is shown. More precisely, Fig.21 The second and third columns of the table show the left and right edges of the mass window for a given IMI value. The fourth to fourteenth columns summarize the transmission coefficients for m / z values ​​of 548.5 to 553.5 m / z. In an ideally simplified view, one would expect the transmission coefficient to switch between 0% and 100% at the left and right edges of the mass window, but in practice, the transmission efficiency of the quadruple across multiple IMI values ​​is of course gradually changing. For example, for an IMI value of 277, where the left edge of the mass window is 548.5, it can be seen that the transmission coefficient for the precursor with an m / z of 548.5 is 60%, gradually increases for larger m / z values, and only reaches a maximum at 551.5 m / z. In addition, the maximum transmission coefficient in this case is 120%, not 100% as expected, because the percentage is expressed as a comparison with the transmission rate obtained for the precursor when the mass selectivity of the mass filter is turned off. As the skilled person will appreciate, when mass selection is "off", the transmission rate is slightly lower than when selection is on and the precursor falls exactly in the middle of the mass window. Fig. 20 The reason why the maximum intensity associated with the eighth MIDIA frame (i.e., the triangle) exceeds the intensity of the quality-selected precursor without the quality filter (i.e., the diamond).

[0160] from Fig.21 As can be seen from the table, for all cases where IMI is below 275, the transmission coefficient of the precursor ion with m / z 550.3 should be 0, which is exactly Fig. 20 At an IMI value of 276, according to Fig.21 The transmission coefficient of the table should be somewhere between 14% and 32%, which is consistent with Fig. 20 The transmission coefficient for m / z 550 to 550.5 reaches a maximum of 120% at an IMI of 278, which is also Fig. 20 Shown in.

[0161] The full transmission factor of 120% is maintained until an IMI of 289, at which point the transmission factor begins to decline and reaches zero above an IMI of 293, which is also Fig. 20 Shown in.

[0162] From the IMI-dependent transmission coefficients associated with each MIDIA frame and the measured intensities, the masses of the precursor ions can be determined with very high accuracy. In fact, by substituting the intensity obtained in the eighth MIDIA frame ( Fig. 20 The triangle in Fig.21 The transmission coefficients match those of the ions, so we can predict that the m / z must be between 550 and 550.5.

[0163] Additional information can be obtained from other MIDIA frames. For example, in Fig. 20 In FIG, the circles connected by dotted lines represent the intensities obtained in the ninth MIDIA frame. Fig. 22 The table summarizes the transmission coefficients of the quality filter as a function of the IMI for the ninth MIDIA frame. Fig. 20 It can be seen with the naked eye that the transmission coefficient remains at or near zero at IMI 280 and rises to 120% during consecutive IMI values ​​281 and 282, with the transmission coefficient being just under 60% at the intermediate IMI value 281. Therefore, it can be discerned again that the m / z of the precursor ion must be 550 to 550.5.

[0164] Since each fragment ion can be detected in multiple MIDIA frames (in the embodiment shown, at least three MIDIA frames, but typically quite a few more, depending on the spread of the precursor ions in the IMI direction), the accumulated information from the transmission patterns associated with the various MIDIA frames allows a very precise estimation of the precursor m / z, typically with an accuracy of + / -1 m / z.

[0165] Although in Fig. 20 In Figure 2, only two intensity-IMI curves are shown, but Fig.23 is a 3D plot showing the intensity as a function of IMI for all 20 MIDIA frames. It can be seen that the precursor can indeed be seen in multiple MIDIA frames, and the appearance (in this case: intensity) of the precursor in each of these frames, especially the step-like rise in intensity when the precursor first falls into the mass window, contains very precise information about the m / z of the precursor that can be exploited in the method of the present invention.

[0166] Fig.23 Shown with Fig. 22 The same data, but shown as a heat map where darker colors represent higher intensities.

[0167] Although the present invention has been described in terms of specific embodiments, it should be understood that changes and modifications thereto that will occur in the art should be considered aspects of the present invention. Therefore, the present invention should only be limited to such limitations as appear in the claims.

Claims

1. A data independent combined ion mobility and mass spectrometry method comprising the following steps: introducing the precursor ions (34) into an ion mobility spectrometer (IMS) (16), sequentially releasing precursor ions (34) from the IMS (16) according to their ion mobility, introducing the released precursor ions (34) into a mass filter (26) which selectively transmits precursor ions (34) having m / z values ​​falling within a controllable mass window, fragmenting the precursor ions (34) transmitted through the mass filter (26) to produce fragment ions, performing mass spectrometry on the fragment ions, wherein each fragment ion is associated with a mass window and an ion mobility (IM) range, and Correlating the detected fragments with their corresponding precursor ions (34), wherein the IMS (16) and the mass filter (26) are controlled in a synchronized manner to perform a plurality of IM scans, during which precursor ions (34) that increase or decrease IM are sequentially released from the IMS (16) and during which the mass window of the mass filter (26) is moved continuously or stepwise toward lower or higher m / z values, respectively, wherein in the IM scans, adjacent mass windows associated with successive mass spectrometric measurements of fragment ions overlap, such that the precursor ions (34) transmitted through the mass filter (26) during one IM scan are located in at least one continuous scan region in the m / z-IM plane, the at least one continuous scan region extending in a substantially diagonal direction in the m / z-IM plane, wherein adjacent scan regions associated with different IM scans overlap in the m / z direction, And wherein the step of associating the detected fragments with their corresponding precursor ions (34) is based on determining or utilizing corresponding mass windows and IM ranges associated with various occurrences of the fragments in the mass spectrometry measurement.

2. A method as claimed in claim 1, wherein in the IM scan, adjacent mass windows associated with continuous mass spectrometry measurements of fragment ions overlap by at least 10% of their width, and / or wherein adjacent scan areas associated with different IM scans overlap by at least 33% of their width in the m / z direction.

3. A method as claimed in claim 1, wherein in the IM scan, adjacent mass windows associated with continuous mass spectrometry measurements of fragment ions overlap by at least 30% of their width, and / or wherein adjacent scan areas associated with different IM scans overlap by at least 50% of their width in the m / z direction.

4. A method as claimed in claim 1, wherein in the IM scan, adjacent mass windows associated with continuous mass spectrometry measurements of fragment ions overlap by at least 60% of their width, and / or wherein adjacent scan areas associated with different IM scans overlap by at least 66% of their width in the m / z direction.

5. The method of claim 1 or 2, wherein the occurrence of the fragments corresponds to the relative intensity or absolute intensity of the fragments in the mass spectrometry measurement.

6. A method as claimed in claim 1 or 2, wherein the step of associating the detected fragments with their corresponding precursor ions (34) is based on evaluating the consistency of the mass windows and IM ranges associated with various occurrences of the fragments in the mass spectrometry for experimental precursor pairs, or wherein the step of associating the detected fragments with their corresponding precursor ions (34) includes determining the intersection of mass windows associated with the occurrences of the fragments in the mass spectrometry, and identifying the corresponding precursor ions (34) at least in part based on the intersection.

7. A method as claimed in claim 1 or 2, wherein the method further comprises the step of performing MS1 mass spectrometry in an operation mode in which the mass filter (26) and the fragmentation are disabled so that all precursor ions (34) sequentially released from the IMS (16) are subjected to mass spectrometry without fragmentation, thereby obtaining an m / z spectrum as a function of the ion mobility of the precursor ions (34).

8. A method as claimed in claim 7, wherein based on the m / z spectrum of the precursor ion (34), a fingerprint of at least one precursor ion (34) is established, and the fingerprint includes a set of mass windows and related IM ranges in the mass window and related IM range covered by the IM scan, which is consistent with the performance of the at least one precursor ion (34) in the m / z spectrum.

9. A method as claimed in claim 8, wherein the step of associating the detected fragments with their corresponding precursors includes establishing a set of mass windows and corresponding IM ranges of the mass filter (26) and IMS (16) associated with the occurrence of the detected fragments, and comparing the set with the fingerprint of one or more precursor ions (34).

10. A method as claimed in claim 1 or 2, wherein the method also includes the step of performing a pseudo MS2 measurement on the precursor ions, wherein the IMS (16) and the mass filter (26) are controlled in a synchronized manner so as to perform the same type of overlapping IM scans as in the mass spectrometry measurement performed on the fragment ions, but without fragmenting the precursor ions.

11. A method as claimed in claim 10, wherein the step of associating the detected fragments with their corresponding precursors is based on comparison, correlation or matching of pseudo MS2 data obtained in the pseudo MS2 measurement performed on the precursor ions and real MS2 data obtained in the mass spectrometry measurement performed on the fragment ions.

12. A method as claimed in claim 7, wherein the method also includes a step of predicting or calculating the expected occurrence, in particular the intensity distribution, of the corresponding fragments with respect to at least the IM range and the IM scan number in the multiple IM scans based on the performance of the precursors in the MS1 mass spectrometry, and wherein the step of associating the detected fragments with their corresponding precursors is based on comparing, correlating or matching the expected occurrence, in particular the intensity distribution with the real MS2 data obtained in the mass spectrometry measurement performed on the fragment ions.

13. A method as claimed in claim 7, wherein the method further comprises a step of predicting or calculating an expected intensity distribution of possible precursors as a function of at least IM and m / z based on actual MS2 data obtained in the mass spectrometry of a given fragment, and wherein the step of associating the detected fragments with their corresponding precursors is based on a comparison, correlation or matching of the expected intensity distribution of the possible precursors with the data obtained in the MS1 mass spectrometry.

14. A method as claimed in claim 12 or 13, wherein the predicting or calculating step is performed using a model or algorithm based at least in part on one or more matrix-based methods, neural networks, random forests, support vector machines or other machine learning methods.

15. A method as claimed in claim 14, wherein the method further comprises the step of performing a pseudo MS2 measurement on the precursor ions, wherein the IMS (16) and the mass filter (26) are controlled in a synchronized manner so as to perform overlapping IM scans of the same type as in the mass spectrometry measurement performed on the fragment ions, but without fragmenting the precursor ions, wherein the results of the pseudo MS2 measurement are at least partially used as ground truth data to derive or train the model or algorithm.

16. The method of claim 1 or 2, wherein in the m / z-IM plane, the precursor has an average extension ΔIM 前体 , wherein the edges of adjacent scan areas of the IM scan move relative to each other in the m / z direction with an offset ΔIM between the edges of the adjacent scan areas in the IM direction 帧 , and wherein the shifting of adjacent scanning areas is adapted to the average precursor extension ΔIM 前体 , so that ΔIM 前体 ≥ΔIM 帧 .

17. The method of claim 16, wherein the shifting of adjacent scanning areas is adapted to the average precursor extension ΔIM 前体 , so that ΔIM 前体 ≥1.2·ΔIM 帧 .

18. The method of claim 16, wherein the shifting of adjacent scanning areas is adapted to the average precursor extension ΔIM 前体 , so that ΔIM 前体 ≥1.5·ΔIM 帧 .

19. The method of claim 1 or 2, wherein the mass filter (26) is a quadrupole RF device (26), and / or wherein the mass spectrometry is performed using a TOF mass analyzer (30).

20. The method of claim 1 or 2, wherein the IMS (16) is one of a Trapping IMS (TIMS) device, a Drift IMS device, and a Traveling Wave IMS device.

21. A method as claimed in claim 20, wherein the TIMS device (16) includes a first TIMS (20) and a second TIMS (22), wherein the first TIMS (20) is configured to constantly receive precursor ions (34) and transfer the received precursor ions (34) to the second TIMS (22) in a time-controlled manner, and the second TIMS (22) is configured to perform the IM scan, wherein the precursor ions (34) are released from the second TIMS (22) in sequence according to their ion mobility.

22. The method of claim 21, wherein in each IM scan, precursor ions (34) of lower ion mobility are released before precursor ions (34) of higher ion mobility.

23. The method of claim 1 or 2, wherein the method further comprises the step of providing the precursor ions (34) by separating precursor molecules from a sample and ionizing the precursor molecules.

24. The method of claim 23, wherein separation of precursor molecules from the sample is performed by chromatography.

25. The method of claim 23, wherein the sample comprises one of a peptide, a lipid, a metabolite or other small molecule having a mass of 50 to 5000 Da.

26. An apparatus (10) for data independent combined ion mobility and mass spectrometry analysis, the apparatus comprising: an ion mobility separator (IMS) (16) for receiving precursor ions (34) and sequentially releasing the precursor ions (34) from the IMS (16) according to their ion mobility, a mass filter (26) arranged to receive the released precursor ions (34) and selectively transmit precursor ions (34) having m / z values ​​falling within a controllable mass window, a fragmentation device (28) for fragmenting the precursor ions (34) transmitted through the mass filter (26) to generate fragment ions, means for performing mass spectrometry on the fragment ions, wherein each fragment ion is associated with a mass window and an ion mobility (IM) range, and Control system (32), wherein the control system (32) is configured to control the IMS (16) and the mass filter (26) in a synchronized manner so as to perform a plurality of IM scans, during which precursor ions (34) that increase or decrease IM are sequentially released from the IMS (16), and during which the mass window of the mass filter (26) is continuously or stepwise moved towards lower or higher m / z values, respectively, wherein the control system (32) is configured to control the IMS (16) and the mass filter (26) in a synchronized manner such that in the IM scan, adjacent mass windows associated with successive mass spectrometric measurements of fragment ions overlap, such that the precursor ions (34) transmitted through the mass filter (26) during the IM scan are located in at least one continuous scan region in the m / z-IM plane, the at least one continuous scan region extending in a substantially diagonal direction in the m / z-IM plane, Wherein the control system (32) is configured to control the IMS (16) and the mass filter (26) in a synchronized manner such that adjacent scan regions associated with different IM scans overlap in the m / z direction.

27. The apparatus (10) of claim 26, wherein in the IM scan, adjacent mass windows associated with consecutive mass spectrometric measurements of fragment ions overlap by at least 10% of their width, And / or wherein the control system (32) is configured to control the IMS (16) and the mass filter (26) in a synchronized manner such that adjacent scan areas associated with different IM scans overlap in the m / z direction by at least 33% of their width in the m / z direction.

28. The apparatus (10) of claim 26, wherein in the IM scan, adjacent mass windows associated with consecutive mass spectrometry measurements of fragment ions overlap by at least 30% of their width, And / or wherein the control system (32) is configured to control the IMS (16) and the mass filter (26) in a synchronized manner such that adjacent scan areas associated with different IM scans overlap in the m / z direction by at least 50% of their width in the m / z direction.

29. The apparatus (10) of claim 26, wherein in the IM scan, adjacent mass windows associated with consecutive mass spectrometric measurements of fragment ions overlap by at least 60% of their width, And / or wherein the control system (32) is configured to control the IMS (16) and the mass filter (26) in a synchronized manner such that adjacent scan areas associated with different IM scans overlap in the m / z direction by at least 66% of their width in the m / z direction.

30. An apparatus (10) as described in claim 26 or 27, wherein the control system is also configured to associate the detected fragments with their corresponding precursor ions (34) based on evaluating the consistency of the mass window and IM range associated with various occurrences of the fragments in the mass spectrometry measurement for experimental precursor pairs, or wherein the control system is also configured to associate the detected fragments with their corresponding precursor ions.

31. The apparatus (10) of claim 30, wherein the correlating comprises determining an intersection of mass windows associated with occurrences of the fragments in the mass spectrometry measurements, and identifying corresponding precursor ions (34) based at least in part on the intersection.

32. An apparatus (10) as described in claim 26 or 27, wherein the control system is further configured to control the apparatus to perform MS1 mass spectrometry in an operating mode, wherein the mass filter (26) and the fragmentation device are disabled so that all precursor ions (34) sequentially released from the IMS (16) are subjected to mass spectrometry without fragmentation, thereby obtaining an m / z spectrum as a function of the ion mobility of the precursor ions (34).

33. An apparatus (10) as described in claim 32, wherein the control system is configured to establish a fingerprint of at least one precursor ion based on the m / z spectrum of the precursor ion (34), the fingerprint comprising a set of mass windows and related IM ranges in the mass window and related IM range covered by the IM scan, which is consistent with the performance of the at least one precursor ion (34) in the m / z spectrum.

34. An apparatus (10) as claimed in claim 33, wherein the control system is configured to associate the detected fragments with their corresponding precursors, wherein the association includes establishing a set of mass windows and corresponding IM ranges of the mass filter (26) and IMS (16) associated with the occurrence of the detected fragments, and comparing the set with the fingerprint of one or more precursor ions.

35. The apparatus (10) of claim 26 or 27, wherein the mass filter (26) is a quadrupole RF device.

36. An apparatus (10) as described in claim 26 or 27, wherein the control system (32) is configured to control the apparatus to perform a pseudo-MS2 measurement on precursor ions, wherein the IMS (16) and the mass filter (26) are controlled in a synchronized manner to perform the same type of overlapping IM scans as in the mass spectrometry measurement performed on the fragment ions, but without fragmenting the precursor ions.

37. An apparatus (10) as described in claim 36, wherein the control system (32) is configured to associate the detected fragments with their corresponding precursors based on comparison, correlation or matching of pseudo MS2 data obtained in the pseudo MS2 measurement performed on the precursor ions and real MS2 data obtained in the mass spectrometry measurement performed on the fragment ions.

38. An apparatus (10) as described in claim 32, wherein the control system (32) is configured to predict or calculate the expected occurrence, in particular, intensity distribution, of corresponding fragments with respect to at least the IM range and the IM scan number in the multiple IM scans based on the performance of the precursor in the MS1 mass spectrometry, and the control system (32) is also configured to associate the detected fragments with their corresponding precursors based on comparison, correlation or matching of the expected occurrence, in particular, intensity distribution with the actual MS2 data obtained in the mass spectrometry measurement performed on the fragment ions.

39. An apparatus (10) as described in claim 32, wherein the control system (32) is configured to predict or calculate the expected intensity distribution of possible precursors as a function of at least IM and m / z based on the actual MS2 data obtained in the mass spectrometry measurement of a given fragment, and the control system (32) is also configured to associate the detected fragments with their corresponding precursors based on a comparison, correlation or matching of the expected intensity distribution of the possible precursors with the data obtained in the MS1 mass spectrometry measurement.

40. An apparatus (10) as described in claim 38, wherein the control system (32) is configured to perform the prediction or calculation using a model or algorithm based at least in part on one or more matrix-based methods, neural networks, random forests, support vector machines or other machine learning methods.

41. An apparatus as claimed in claim 26 or 27, wherein the IMS (16) is one of a capture IMS (TIMS) device (16), a drift-type IMS device and a traveling wave IMS device, and / or wherein the mass spectrometry device is a TOF mass analyser (30).

42. An apparatus (10) as described in claim 41, wherein the TIMS device (16) includes a first TIMS (20) and a second TIMS (22), wherein the first TIMS (20) is configured to constantly receive precursor ions (34) and transfer the received precursor ions (34) to the second TIMS (22) in a time-controlled manner, and the second TIMS (22) is configured to perform the IM scan, wherein the precursor ions (34) are released from the second TIMS (22) in sequence according to their ion mobility.

43. The apparatus (10) of claim 42, wherein in each IM scan, precursor ions (34) of lower ion mobility are released before precursor ions (34) of higher ion mobility.

44. The apparatus (10) of claim 26 or 27, wherein the apparatus (10) further comprises a separator (12) and an ionization device for providing the precursor ions (34) by separating precursor molecules from a sample and ionizing the precursor molecules.

45. The apparatus (10) of claim 44, wherein the separator is a liquid chromatography device (12), and wherein the sample comprises one of a peptide, a lipid, a metabolite or other small molecule having a mass of 50 to 5000 Da.

46. ​​The apparatus of claim 26 or 27, wherein the control system (32) is configured to control the IMS (16) and the mass filter (26) in a synchronized manner such that - the precursor has an average extension ΔIM in the m / z-IM plane 前体 , - the movement of the edges of adjacent scan areas of the IM scan relative to each other in the m / z direction is accompanied by a displacement of the edges in the IM direction ΔIM between the edges of the adjacent scan areas 帧 ,as well as - the shifting of adjacent scanning areas is adapted to the average precursor extension ΔIM 前体 , so that ΔIM 前体 ≥ΔIM 帧 .

47. The apparatus of claim 46, wherein the shifting of adjacent scanning areas is adapted to the average precursor spread ΔIM 前体 , so that ΔIM 前体 ≥1.2·ΔIM 帧 .

48. The apparatus of claim 46, wherein the shifting of adjacent scanning areas is adapted to the average precursor spread ΔIM 前体 , so that ΔIM 前体 ≥1.5·ΔIM 帧 .

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