A single-cell mass spectrometry data processing method

Abstract

The application discloses a single cell mass spectrum data processing method, and relates to the technical field of data processing. The method comprises the following steps: acquiring a data frame of single cell ionization events, wherein the data frame is used for recording ion data of N ions, and the ion data comprises mass-to-charge ratios and ion signal intensities; performing binning on the N ions according to the ion data of the N ions, thereby obtaining K sub-bins; performing integration on the ions in each of the K sub-bins, thereby obtaining K integrated ions, wherein the K integrated ions comprise integrated ions corresponding to each of the K sub-bins, and the integrated ion data of the integrated ions comprises integrated mass-to-charge ratios and integrated ion signal intensities; and performing integration on the K integrated ions, thereby obtaining a cell superposition data frame corresponding to the data frame, wherein the cell superposition data frame is used for detecting metabolite ion mobility outflow peaks, so that metabolites are annotated through the metabolite ion mobility outflow peaks.

Description

Technical Field

[0001] This application relates to the field of data processing technology, and in particular to a method for processing single-cell mass spectrometry data. Background Technology

[0002] Cells are the basic unit of life. In related technologies, cell flow cytometry can be coupled with electrospray ionization mass spectrometry (ESI-MS) to achieve high throughput and high coverage of metabolite detection.

[0003] However, for metabolite ions in single cells, the above method can only detect the mass-to-charge ratio (m / z) of metabolite ions. If metabolites are annotated only by mass-to-charge ratio, there will be interference from metabolite isomers with the same molecular formula, which will reduce the accuracy of metabolite annotation and thus affect the accurate qualitative and quantitative analysis of subsequent single-cell metabolomics data. Summary of the Invention

[0004] The purpose of this application is to provide a method, apparatus, computer device, and computer-storable medium for processing single-cell mass spectrometry data, which can solve the problem of low accuracy of metabolite annotation in related technologies, affecting the accurate qualitative and quantitative analysis of subsequent single-cell metabolomics data.

[0005] To solve the above-mentioned technical problems, this application is implemented as follows:

[0006] In a first aspect, embodiments of this application provide a method for processing single-cell mass spectrometry data, which may include:

[0007] Acquire a data frame of a single-cell ionization event. The data frame is used to record the ion data of N ions. The ion data includes the mass-to-charge ratio and the ion signal intensity, where N is a positive integer.

[0008] Based on the ion data of N ions, the N ions are divided into boxes to obtain K sub-boxes. The K sub-boxes include at least one of the following types of sub-boxes: a first type of sub-box that meets the preset division conditions and a second type of sub-box that does not meet the preset division conditions. The ions in the first type of sub-box meet at least one of the following conditions: the ions in the first type of sub-box have the same mass-to-charge ratio, or the mass-to-charge ratio of the ions in the first type of sub-box is less than or equal to a first preset threshold. K is a positive integer.

[0009] The ions in each of the K sub-boxes are integrated to obtain K integrated ions. The K integrated ions include the integrated ions corresponding to each of the K sub-boxes. The integrated ion data includes the integrated mass-to-charge ratio and the integrated ion signal intensity. The integrated mass-to-charge ratio is determined by the mass-to-charge ratio and ion signal intensity of each ion in the sub-box, and the integrated ion signal intensity is determined by the ion signal intensity of each ion in the sub-box.

[0010] K integrated ions are integrated to obtain a cell-stacked data frame corresponding to the data frame. The cell-stacked data frame is used to detect the elution peak of metabolite ions, so as to annotate metabolites by the elution peak of metabolite ions.

[0011] In some embodiments provided in this application, the step of "dividing the N ions into K sub-boxes based on the ion data of the N ions" may specifically include:

[0012] In the case where the data frame of a single-cell ionization event is acquired by flow cytometry-mass spectrometry, and the K sub-boxes include a first type of sub-box and a second type of sub-box, the first ion is selected from the N ions based on the ion signal intensity of each ion in the N ions, and the ion signal intensity of the first ion is greater than or equal to a first preset threshold.

[0013] Based on the mass-to-charge ratio of the first ion, the second and third ions are selected from N ions. The mass-to-charge ratio of the second ion is the same as that of the first ion, and the mass-to-charge ratio of the third ion is less than or equal to the second preset threshold.

[0014] The first, second, and third ions are assigned to the first type of sub-box; and the fourth ion, which is not in the first type of sub-box among the N ions, is assigned to the second type of sub-box.

[0015] In some embodiments provided in this application, prior to the step of "dividing the N ions into K sub-boxes based on the ion data of N ions" mentioned above, the single-cell mass spectrometry data processing may further include:

[0016] In the case where the data frame of the single-cell ionization event is acquired by flow cytometry-ion mobility-mass spectrometry, and the ion data also includes ion mobility values, and the K sub-boxes include first-type sub-boxes and second-type sub-boxes, the N ions are divided into M boxes according to the ion mobility value of each ion in the N ions; where each of the M boxes includes P ions, the ion mobility values ​​of the P ions in each box are the same, and the ion mobility values ​​of the ions in different boxes in the M boxes are different, and P and M are integers greater than 1;

[0017] Based on this, the aforementioned step of "dividing the N ions into K sub-boxes based on their ion data" can specifically include:

[0018] Based on the ion signal intensity of each ion in the i-th box out of M boxes, the fifth ion is selected from the P ions in the i-th box. The ion signal intensity of the fifth ion is greater than or equal to the third preset threshold, i∈[1,M].

[0019] Based on the mass-to-charge ratio of the fifth ion, the sixth and seventh ions are selected from the P ions in the i-th box. The mass-to-charge ratio of the sixth ion is the same as that of the fifth ion, and the mass-to-charge ratio of the seventh ion is less than or equal to the fourth preset threshold.

[0020] The fifth, sixth, and seventh ions are assigned to the first type of sub-box in the i-th box; and the eighth ion, which is not in the first type of sub-box in the i-th box, is assigned to the second type of sub-box in the i-th box.

[0021] In some embodiments provided in this application, the integrated ion data of integrated ions also includes integrated ion mobility values, which are the ion mobility values ​​of the sub-box to which the sub-box belongs.

[0022] In some embodiments provided in this application, prior to the step of "dividing the N ions into K sub-boxes based on the ion data of N ions" mentioned above, the single-cell mass spectrometry data processing may further include:

[0023] The mass-to-charge ratio and ion signal intensity of the ions in each sub-box are weighted and summed to obtain a weighted value;

[0024] The integrated mass-to-charge ratio is calculated based on the weighted values ​​and the ion signal intensity of ions in each sub-box.

[0025] Secondly, embodiments of this application provide a method for processing single-cell mass spectrometry data, which may include:

[0026] Extract L test data frames from the set of test data frames, where L is a positive integer;

[0027] Each of the L measured data frames is determined as a data frame of a single-cell ionization event as shown in any of the first aspects, and the steps of the single-cell mass spectrometry data processing method as shown in any of the first aspects are performed to obtain a superimposed data frame of the measured cell, which includes the superimposed data frame of the measured cell for each of the R ions in the measured data frame in the single-cell ionization event.

[0028] Based on the superimposed data frames of the tested cells, the elution peaks of metabolite ion mobility were detected.

[0029] Based on the ion data of metabolite ion mobility elution peaks in single cells, metabolite outcome information of metabolite ion mobility elution peaks in single cells is determined. Metabolite outcome information includes quantitative and qualitative information.

[0030] In some embodiments provided in this application, the step of "extracting L test data frames of single-cell ionization events from the test data frame set" may specifically include:

[0031] When the measured data frame in the measured data frame set is acquired by flow cytometry-ion mobility-mass spectrometry and the user inputs a first preset mass-to-charge ratio and a preset ion mobility value for a single-cell ionization event marker corresponding to a single-cell ionization event, at least two ions matching the first preset mass-to-charge ratio and the preset ion mobility value are extracted from the target measured data frame in the measured data frame set.

[0032] The target ion signal intensity is obtained by summing the ion signal intensities of each of at least two ions;

[0033] The target ion signal intensities of the target measured data frames are sorted according to the chromatographic retention time to obtain the chromatographic elution peaks;

[0034] Based on the first preset peak width, the first peak region is extracted from the chromatographic elution peak, and the chromatographic retention time of the chromatographic peak apex in the first peak region is within the preset chromatographic retention integral range corresponding to the single-cell ionization event marker.

[0035] The data frame corresponding to the first peak region is identified as the measured data frame of the single-cell ionization event being measured.

[0036] In some embodiments provided in this application, the step of "detecting metabolite ion mobility elution peaks based on superimposed data frames of the tested cells" may specifically include:

[0037] The superimposed data frames of the tested cells are arranged according to the ion mobility values ​​of the ions in the superimposed data frames of the tested cells to obtain the first sequence;

[0038] Target ions are selected from the first sequence, and the ion signal intensity of the target ion is greater than or equal to the fifth preset threshold.

[0039] Based on the ion mobility value of the target ion, related ions that match the target ion are screened from the first sequence. The mass-to-charge ratio of the related ion and the mass-to-charge ratio of the target ion satisfy at least one of the following conditions: the mass-to-charge ratio of the related ion is the same as that of the target ion, or the mass-to-charge ratio of the related ion is less than or equal to a sixth preset threshold.

[0040] Based on the target ion and related ions, potential peak targets are constructed;

[0041] If the length of the continuous signal of the potential peak target is less than the seventh preset threshold, the ions in the first sequence are sorted according to the ion signal intensity to obtain the second sequence.

[0042] According to the second preset peak width, extract the second peak region in the second sequence;

[0043] If the ion mobility peak of the target peak in the second peak region is greater than or equal to the eighth preset threshold, the target peak is determined as the peak apex of the metabolite ion mobility effluent peak.

[0044] Based on the mass-to-charge ratio and ion signal intensity of ions in the second peak region, the mass-to-charge ratio of the metabolite ion mobility elution peak is calculated; and the ion mobility value of the target peak is used as the ion mobility value of the metabolite ion mobility elution peak.

[0045] Metabolite ion mobility elution peaks are generated based on the mass-to-charge ratio and ion mobility value of the metabolite ion mobility elution peaks.

[0046] In some embodiments provided in this application, the step of "determining metabolite result information of metabolite ion mobility elution peak in a single cell based on ion data of metabolite ion mobility elution peak in a single cell" may specifically include:

[0047] When the ion data of the metabolite ion mobility effluent peak in a single cell includes the mass-to-charge ratio and ion mobility value of the metabolite ion mobility effluent peak, and the metabolite result information is quantitative, based on the mass-to-charge ratio and ion mobility value of the metabolite ion mobility effluent peak, at least two event ions matching the metabolite ion mobility effluent peak are screened from single-cell ionization events. The mass-to-charge ratio of the event ions satisfies at least one of the following conditions: the mass-to-charge ratio of the event ions is the same as the second preset mass-to-charge ratio; the mass-to-charge ratio of the event ions is less than or equal to the seventh preset threshold; and the ion mobility value of the event ions is less than or equal to the ninth preset threshold.

[0048] The ion signal intensities of each of at least two event ions are summed to obtain quantitative information on the metabolite ion mobility effluent peak in a single cell.

[0049] In some embodiments provided in this application, the step of "determining metabolite result information of metabolite ion mobility elution peak in a single cell based on ion data of metabolite ion mobility elution peak in a single cell" may specifically include:

[0050] When the metabolite result information is qualitative, the metabolite result information of the metabolite ion mobility elution peak in a single cell is determined based on the ion data of the metabolite ion mobility elution peak in a single cell.

[0051] Thirdly, embodiments of this application provide a single-cell mass spectrometry data processing device, which may include:

[0052] The acquisition module is used to acquire data frames of single-cell ionization events. The data frames are used to record ion data of N ions, including mass-to-charge ratio and ion signal intensity, where N is a positive integer.

[0053] The binning module is used to divide the N ions into bins based on the ion data of the N ions to obtain K sub-bins. The K sub-bins include at least one of the following types of sub-bins: a first type of sub-bin that meets the preset division conditions and a second type of sub-bin that does not meet the preset division conditions. The ions in the first type of sub-bin meet at least one of the following conditions: the ions in the first type of sub-bin have the same mass-to-charge ratio, or the mass-to-charge ratio of the ions in the first type of sub-bin is less than or equal to a first preset threshold. K is a positive integer.

[0054] The integration module is used to integrate the ions in each of the K sub-boxes to obtain K integrated ions. The K integrated ions include the integrated ions corresponding to each of the K sub-boxes. The integrated ion data includes the integrated mass-to-charge ratio and the integrated ion signal intensity. The integrated mass-to-charge ratio is determined by the mass-to-charge ratio and ion signal intensity of each ion in the sub-box, and the integrated ion signal intensity is determined by the ion signal intensity of each ion in the sub-box.

[0055] The integration module can also be used to integrate K integrated ions to obtain a cell-overlay data frame corresponding to the data frame. The cell-overlay data frame is used to detect metabolite ion mobility elution peaks so as to annotate metabolites through metabolite ion mobility elution peaks.

[0056] In some embodiments provided in this application, the binning module can be specifically used to, when the data frame of a single-cell ionization event is acquired by flow cytometry-mass spectrometry and the K sub-bins include a first type of sub-bin and a second type of sub-bin, select a first ion from N ions based on the ion signal intensity of each ion in N ions, wherein the ion signal intensity of the first ion is greater than or equal to a first preset threshold.

[0057] Based on the mass-to-charge ratio of the first ion, the second and third ions are selected from N ions. The mass-to-charge ratio of the second ion is the same as that of the first ion, and the mass-to-charge ratio of the third ion is less than or equal to the second preset threshold.

[0058] The first, second, and third ions are assigned to the first type of sub-box; and the fourth ion, which is not in the first type of sub-box among the N ions, is assigned to the second type of sub-box.

[0059] In some embodiments provided in this application, the acquisition module can also be used to, when the data frame of a single-cell ionization event is acquired by flow cytometry-ion mobility-mass spectrometry, and the ion data also includes ion mobility values, and the K sub-boxes include first-type sub-boxes and second-type sub-boxes, divide the N ions into M boxes according to the ion mobility value of each ion in the N ions; wherein each of the M boxes includes P ions, the ion mobility values ​​of the P ions in each box are the same, the ion mobility values ​​of the ions in different boxes in the M boxes are different, and P and M are integers greater than 1;

[0060] Based on this, the binning module may specifically include:

[0061] Based on the ion signal intensity of each ion in the i-th box out of M boxes, the fifth ion is selected from the P ions in the i-th box. The ion signal intensity of the fifth ion is greater than or equal to the third preset threshold, i∈[1,M].

[0062] Based on the mass-to-charge ratio of the fifth ion, the sixth and seventh ions are selected from the P ions in the i-th box. The mass-to-charge ratio of the sixth ion is the same as that of the fifth ion, and the mass-to-charge ratio of the seventh ion is less than or equal to the fourth preset threshold.

[0063] The fifth, sixth, and seventh ions are assigned to the first type of sub-box in the i-th box; and the eighth ion, which is not in the first type of sub-box in the i-th box, is assigned to the second type of sub-box in the i-th box.

[0064] In some embodiments provided in this application, the integrated ion data of integrated ions also includes integrated ion mobility values, which are the ion mobility values ​​of the sub-box to which the sub-box belongs.

[0065] In some embodiments provided in this application, the single-cell mass spectrometry data processing device may further include a computing module; wherein,

[0066] The calculation module is used to perform a weighted summation of the mass-to-charge ratio and ion signal intensity of ions in each sub-box to obtain a weighted value; based on the weighted value and the ion signal intensity of ions in each sub-box, the integrated mass-to-charge ratio is calculated.

[0067] Fourthly, embodiments of this application provide a single-cell mass spectrometry data processing device, which may include:

[0068] The extraction module is used to extract L test data frames of single-cell ionization events from the set of test data frames, where L is a positive integer;

[0069] The processing module is used to determine each of the L measured data frames as a data frame of a single-cell ionization event as shown in any of the first aspects, and to execute the steps of the single-cell mass spectrometry data processing method as shown in any of the first aspects to obtain a superimposed data frame of the measured cell, which includes the superimposed data frame of the measured cell for each of the R ions in the measured data frame in the single-cell ionization event.

[0070] The acquisition module is used to detect the elution peak of metabolite ion mobility based on the superimposed data frames of the tested cells;

[0071] The determination module is used to determine the metabolite result information of the metabolite ion mobility elution peak in a single cell based on the ion data of the metabolite ion mobility elution peak in a single cell. The metabolite result information includes quantitative information and qualitative information.

[0072] In some embodiments provided in this application, the extraction module mentioned above can be used to extract at least two ions that match the first preset mass-charge ratio and preset ion mobility value from the target test data frame in the test data frame set, provided that the test data frame in the test data frame set is acquired by flow cytometry-ion mobility-mass spectrometry and the user inputs the first preset mass-charge ratio and preset ion mobility value of the single-cell ionization event marker corresponding to the single-cell ionization event.

[0073] The target ion signal intensity is obtained by summing the ion signal intensities of each of at least two ions;

[0074] The target ion signal intensities of the target measured data frames are sorted according to the chromatographic retention time to obtain the chromatographic elution peaks;

[0075] Based on the first preset peak width, the first peak region is extracted from the chromatographic elution peak, and the chromatographic retention time of the chromatographic peak apex in the first peak region is within the preset chromatographic retention integral range corresponding to the single-cell ionization event marker.

[0076] The data frame corresponding to the first peak region is identified as the measured data frame of the single-cell ionization event being measured.

[0077] In some embodiments provided in this application, the acquisition module can be specifically used to arrange the superimposed data frames of the tested cells according to the ion mobility values ​​of ions in the superimposed data frames of the tested cells to obtain a first sequence;

[0078] Target ions are selected from the first sequence, and the ion signal intensity of the target ion is greater than or equal to the fifth preset threshold.

[0079] Based on the ion mobility value of the target ion, related ions that match the target ion are screened from the first sequence. The mass-to-charge ratio of the related ion and the mass-to-charge ratio of the target ion satisfy at least one of the following conditions: the mass-to-charge ratio of the related ion is the same as that of the target ion, or the mass-to-charge ratio of the related ion is less than or equal to a sixth preset threshold.

[0080] Based on the target ion and related ions, potential peak targets are constructed;

[0081] If the length of the continuous signal of the potential peak target is less than the seventh preset threshold, the ions in the first sequence are sorted according to the ion signal intensity to obtain the second sequence.

[0082] According to the second preset peak width, extract the second peak region in the second sequence;

[0083] If the ion mobility peak of the target peak in the second peak region is greater than or equal to the eighth preset threshold, the target peak is determined as the peak apex of the metabolite ion mobility effluent peak.

[0084] Based on the mass-to-charge ratio and ion signal intensity of ions in the second peak region, the mass-to-charge ratio of the metabolite ion mobility elution peak is calculated; and the ion mobility value of the target peak is used as the ion mobility value of the metabolite ion mobility elution peak.

[0085] Metabolite ion mobility elution peaks are generated based on the mass-to-charge ratio and ion mobility value of the metabolite ion mobility elution peaks.

[0086] In some embodiments provided in this application, the determining module can specifically be used to, when the ion data of the metabolite ion mobility elution peak in a single cell includes the mass-to-charge ratio and ion mobility value of the metabolite ion mobility elution peak, and the metabolite result information is quantitative information, screen at least two event ions that match the metabolite ion mobility elution peak from single-cell ionization events based on the mass-to-charge ratio and ion mobility value of the metabolite ion mobility elution peak. The mass-to-charge ratio of the event ions satisfies at least one of the following conditions: the mass-to-charge ratio of the event ions is the same as the second preset mass-to-charge ratio; the mass-to-charge ratio of the event ions is less than or equal to the seventh preset threshold; and the ion mobility value of the event ions is less than or equal to the ninth preset threshold.

[0087] The ion signal intensities of each of at least two event ions are summed to obtain quantitative information on the metabolite ion mobility effluent peak in a single cell.

[0088] In some embodiments provided in this application, the determining module can be specifically used to determine the metabolite result information of the metabolite ion mobility elution peak in a single cell based on the ion data of the metabolite ion mobility elution peak in a single cell when the metabolite result information is qualitative information.

[0089] Fifthly, embodiments of this application provide a computer device including a processor, a memory, and a program or instructions stored in the memory and executable on the processor. When the program or instructions are executed by the processor, they implement the steps of the single-cell mass spectrometry data processing method as shown in the first or second aspect.

[0090] In a sixth aspect, embodiments of this application provide a computer-readable storage medium on which a program or instructions are stored, which, when executed by a processor, implement the steps of the single-cell mass spectrometry data processing method as shown in the first or second aspect.

[0091] In a seventh aspect, embodiments of this application provide a chip including a processor and a communication interface, the communication interface being coupled to the processor, the processor being used to run programs or instructions to implement the steps of the single-cell mass spectrometry data processing method as shown in the first or second aspect.

[0092] In summary, the single-cell mass spectrometry data processing method provided in this application can acquire data frames of single-cell ionization events. These data frames record ion data for N ions, including mass-to-charge ratio and ion signal intensity. Then, based on the ion data of the N ions, the N ions are divided into K sub-boxes. Each of the K sub-boxes includes at least one of the following types: a first type of sub-box that meets preset division conditions, and a second type of sub-box that does not meet preset division conditions. The ions in the first type of sub-box meet at least one of the following conditions: the ions in the first type of sub-box have the same mass-to-charge ratio; or the mass-to-charge ratio of the ions in the first type of sub-box is less than or equal to a first preset condition. Threshold; then, the ions in each of the K sub-boxes are integrated to obtain K integrated ions. The K integrated ions include the integrated ions corresponding to each of the K sub-boxes. The integrated ion data includes the integrated mass-to-charge ratio and the integrated ion signal intensity. The integrated mass-to-charge ratio is determined by the mass-to-charge ratio and ion signal intensity of each ion in the sub-box, and the integrated ion signal intensity is determined by the ion signal intensity of each ion in the sub-box. Then, the K integrated ions are integrated to obtain a cell superimposed data frame corresponding to the data frame. The cell superimposed data frame is used to detect the metabolite ion mobility effluent peak, so as to annotate metabolites through the metabolite ion mobility effluent peak. In this way, since the ion mobility elution peak of a metabolite is composed of the signal intensities of ions with a mass-to-charge ratio equal to that metabolite in each ion mobility value bin, ions belonging to that metabolite always appear in a specific number of bins, while noise ion signals appear randomly in different bins. If only the ion mobility elution peak of a certain metabolite in a single-cell ionization event is observed, its signal-to-noise ratio may be very low due to the small amount of metabolite, and it may even be impossible to distinguish between ions belonging to the metabolite and noise ions. Therefore, the single-cell mass spectrometry data processing method provided in this application can greatly increase the signal intensity of the same metabolite ion. Since the noise signal appears randomly, the increase in signal intensity during cell superposition is much smaller than that of the metabolite signal. Therefore, by accumulating the signal intensity of the same ion in all single-cell ionization events, the signal-to-noise ratio of the metabolite ion mobility elution peak and the signal-to-noise ratio of the mass spectrometry peak are improved, thereby promoting the sensitivity and coverage of detecting metabolite ion mobility elution peaks, and thus achieving the accuracy of annotating metabolites through metabolite ion mobility elution peaks. Attached Figure Description

[0093] Figure 1 One of the flowcharts for a single-cell mass spectrometry data processing method provided in this application embodiment;

[0094] Figure 2 A second flowchart illustrating a single-cell mass spectrometry data processing method provided in this application embodiment;

[0095] Figure 3This is one of the structural schematic diagrams of a single-cell mass spectrometry data processing device provided in an embodiment of this application;

[0096] Figure 4 This is a second schematic diagram of the structure of a single-cell mass spectrometry data processing device provided in an embodiment of this application;

[0097] Figure 5 This is a schematic diagram of the structure of a computer device provided in an embodiment of this application. Detailed Implementation

[0098] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0099] The terms "first," "second," etc., used in the specification and claims of this application are used to distinguish similar objects and not to describe a specific order or sequence. It should be understood that such use of data can be interchanged where appropriate so that embodiments of this application can be implemented in orders other than those illustrated or described herein, and the objects distinguished by "first," "second," etc., are generally of the same class and the number of objects is not limited; for example, a first object can be one or more. Furthermore, in the specification and claims, "and / or" indicates at least one of the connected objects, and the character " / " generally indicates that the preceding and following objects are in an "or" relationship.

[0100] Cells are the most basic unit of life, but population analysis easily overlooks the characteristics and heterogeneity of individual cells. The heterogeneity of single-cell metabolism has been widely accepted in the field in recent years and has been preliminarily studied through several methods. Among various techniques, tandem mass spectrometry (MS) has high selectivity, sensitivity, and a wide dynamic range. Coupled with flow cytometry and electrospray ionization mass spectrometry, it can achieve extremely high cell detection throughput and high coverage of metabolite detection, becoming the primary strategy for studying single-cell metabolomics. However, current flow cytometry-mass spectrometry techniques can only detect the mass-to-charge ratio (m / z) of metabolite ions in single cells, lacking another dimension of information for metabolite annotation. The existence of isomers of metabolites with the same molecular formula makes current single-cell metabolite annotation very inaccurate.

[0101] Furthermore, ion mobility-mass spectrometry (IM-MS) is a novel mass spectrometry technique that separates metabolite ions on a millisecond timescale based on their size, shape, and charge in the gas phase, effectively improving resolution and selectivity without increasing analysis time. The collision cross-section (CCS) derived from ion mobility is an important physicochemical property of metabolites and can be used for metabolite annotation. However, for high-throughput, high-dimensional single-cell metabolomics data acquired by flow cytometry-ion mobility-mass spectrometry, there is a lack of highly sensitive, deep-coverage, and automated algorithms to achieve accurate qualitative and quantitative analysis of metabolites. Similarly, there is a lack of highly sensitive data analysis methods for existing single-cell metabolomics or other omics data acquired by mass spectrometry or ion mobility-mass spectrometry.

[0102] Therefore, in order to solve the problems mentioned above, the following will be discussed in conjunction with the appendix. Figures 1-2 The single-cell mass spectrometry data processing provided in this application will be described in detail through specific embodiments.

[0103] First, combined Figure 1 A detailed explanation of the methods for processing single-cell mass spectrometry data is provided.

[0104] Figure 1 This is one of the flowcharts for a single-cell mass spectrometry data processing method provided in the embodiments of this application.

[0105] like Figure 1 As shown, the single-cell mass spectrometry data processing method may include steps 110 to 140.

[0106] Step 110: Acquire a data frame of a single-cell ionization event. The data frame records ion data for N ions, including mass-to-charge ratio and ion signal intensity, where N is a positive integer. Step 120: Based on the ion data of the N ions, divide the N ions into K sub-boxes. Each K sub-box includes at least one of the following types of sub-boxes: a first type of sub-box satisfying a preset division condition, and a second type of sub-box not satisfying the preset division condition. Ions in the first type of sub-box meet at least one of the following conditions: the ions in the first type of sub-box have the same mass-to-charge ratio; the mass-to-charge ratio of ions in the first type of sub-box is less than or equal to a first preset threshold, where K is a positive integer. 130. Integrate the ions in each of the K sub-boxes to obtain K integrated ions. The K integrated ions include the integrated ions corresponding to each of the K sub-boxes. The integrated ion data includes the integrated mass-to-charge ratio and the integrated ion signal intensity. The integrated mass-to-charge ratio is determined by the mass-to-charge ratio and ion signal intensity of each ion in the sub-box, and the integrated ion signal intensity is determined by the ion signal intensity of each ion in the sub-box. Step 140. Integrate the K integrated ions to obtain a cell superimposed data frame corresponding to the data frame. The cell superimposed data frame is used to detect the metabolite ion mobility elution peak to annotate metabolites through the metabolite ion mobility elution peak.

[0107] In this way, since the ion mobility elution peak of a metabolite is composed of the signal intensities of ions with a mass-to-charge ratio equal to that metabolite in each ion mobility bin, the ions belonging to that metabolite always appear in a certain number of bins, while the noise ion signal appears randomly in different bins. If only the ion mobility elution peak of a certain metabolite in a single-cell ionization event is observed, its signal-to-noise ratio may be very low due to the small amount of metabolite, and it may even be impossible to distinguish between ions belonging to the metabolite and noise ions. Therefore, the single-cell mass spectrometry data processing method provided in this application can greatly increase the signal intensity of the same metabolite ion. Since the noise signal appears randomly, the increase in signal intensity during cell superposition is much smaller than that of the metabolite signal. Therefore, by accumulating the signal intensity of the same ion in all single-cell ionization events, the signal-to-noise ratio of the metabolite ion mobility elution peak and the signal-to-noise ratio of the mass spectrometry peak are improved, thereby promoting the sensitivity and coverage of the metabolite ion mobility elution peak.

[0108] It should be noted that the single-cell mass spectrometry data processing method provided in this application embodiment can be applied to other single-cell omics data acquired by mass spectrometry or ion mobility mass spectrometry, such as single-cell proteomics data, and can also be applied to scenarios of single-cell metabolomics data acquired by flow cytometry-ion mobility-mass spectrometry technology.

[0109] The steps mentioned above are explained in detail below.

[0110] First, in step 110, each detected single-cell ionization event is a data frame, and each ion recorded in the data frame has two properties: ion mobility value, mass-to-charge ratio, and ion signal intensity.

[0111] This application provides two methods for acquiring data frames of single-cell ionization events, as detailed below.

[0112] In some embodiments, flow cytometry-mass spectrometry can be used to acquire data frames of single-cell ionization events.

[0113] In other embodiments, data frames of single-cell ionization events can be obtained using flow cytometry-ion mobility-mass spectrometry.

[0114] Secondly, regarding step 120, since the data frames are obtained through the above two techniques in this application embodiment, this application embodiment provides the following two methods to obtain K sub-boxes, the K sub-boxes including a first type of sub-box and a second type of sub-box.

[0115] In some embodiments of this application, the data frames of single-cell ionization events are acquired by flow cytometry-mass spectrometry. Based on this, step 120 may specifically include steps 1201 to 1203.

[0116] Step 1201: Based on the ion signal intensity of each of the N ions, select a first ion from the N ions, wherein the ion signal intensity of the first ion is greater than or equal to a first preset threshold.

[0117] For example, if N is 4, the ion signal intensity of each of the 4 ions can be sorted in descending order, and the ion with the largest ion signal intensity can be determined as the first ion.

[0118] Step 1202: Based on the mass-to-charge ratio of the first ion, select the second and third ions from the N ions. The mass-to-charge ratio of the second ion is the same as that of the first ion, and the mass-to-charge ratio of the third ion is less than or equal to the second preset threshold.

[0119] For example, taking the example shown in step 1201 above, with the first ion as the base point, a second ion with the same mass-to-charge ratio as the first ion is searched from the remaining 3 ions, and a second ion with a mass-to-charge ratio less than the second preset threshold of 20 ppm is searched from the remaining 3 ions.

[0120] Step 1203: The first ion, the second ion, and the third ion are assigned to the first type of sub-box; and the fourth ion, which is not in the first type of sub-box among the N ions, is assigned to the second type of sub-box.

[0121] For example, taking the example shown in step 1202 above, if there is one second ion and one third ion among the remaining three ions, the first ion, the second ion and the third ion can be classified into one category, such as the first sub-box, and the ions other than the first ion, the second ion and the third ion among the four ions can be classified into another category, namely the second sub-box. The second sub-box will only include this ion.

[0122] It should be noted that in some other embodiments of this application, if two of the remaining three ions are second ions and one is a third ion, then the fourth ion will not appear. In this case, the K sub-boxes may only include the first type of sub-boxes. Similarly, if neither the second nor the third ion is among the remaining three ions, then the first ion can be assigned to the first type of sub-box, and the remaining three ions can be assigned to the second type of sub-box. It should also be emphasized that the number of second type sub-boxes can be at least one; that is, if the remaining three ions are assigned to second type sub-boxes, the number of second type sub-boxes can be three. In this case, each of the three ions can be assigned to a separate second type of sub-box.

[0123] In some embodiments of this application, the data frame of the single-cell ionization event is acquired by flow cytometry-ion mobility-mass spectrometry, and the ion data also includes ion mobility values. The K sub-boxes include first-type sub-boxes and second-type sub-boxes. Based on this, before step 120, the single-cell mass spectrometry data processing method may further include step 150:

[0124] Based on the ion mobility value of each of the N ions, the N ions are divided into M boxes; each of the M boxes contains P ions, the ion mobility values ​​of the P ions in each box are the same, and the ion mobility values ​​of the ions in different boxes of the M boxes are different, where P and M are integers greater than 1.

[0125] For example, if N is 4, i.e. ion 1, ion 2, ion 3 and ion 4, then ions with the same ion mobility value among the four ions can be divided into one box. For example, among the four ions, ion 1 and ion 2 have the same ion mobility value, while the ion mobility values ​​of the other ions are different. Then, ion 1 and ion 2 can be divided into the first box, ion 3 into the second box, and ion 4 into the third box. In this case, M is 3.

[0126] Here, in addition to grouping ions 1 and ions 2 with the same ion mobility value into one box, ions with ion mobility values ​​within a preset range can also be grouped into one box. For example, ions with ion mobility values ​​in the range of 1.4518333-1.4497843 can be grouped into one box, and ions with ion mobility values ​​in the range of 0.4541433-0.4520949 can be grouped into another box.

[0127] It should be noted that cross-cell ion binning refers to dividing the range of ion mobility values ​​in each data frame into a fixed number of bins (or scans), with each bin corresponding to a fixed ion mobility value. Furthermore, embodiments of this application can classify all ions from all detected single-cell ionization events according to the binning of ion mobility values ​​described above.

[0128] Based on this, step 120 may specifically include steps 1204 to 1206.

[0129] Step 1204: Based on the ion signal intensity of each ion in the i-th box out of M boxes, select the fifth ion from the P ions in the i-th box. The ion signal intensity of the fifth ion is greater than or equal to the third preset threshold, i∈[1,M].

[0130] For example, taking the example from step 150 above, the following steps are performed on the first of the three boxes: The ion signal intensities of each of the two ions in the first box are sorted in descending order, and the ion with the largest signal intensity is identified as the fifth ion. The same steps are performed on the second box, sorting the ion signal intensities of each of the one ion in the second box in descending order. Since this box contains only one ion, it can be directly identified as the fifth ion. The same steps are performed on the third box as on the second box to obtain the fifth ion in the third box. Thus, the fifth ion in each of the three boxes can be obtained.

[0131] It should be noted that the process of extracting the fifth ion will proceed sequentially according to the signal strength from high to low, and all ions that have not yet been grouped, until all ions in the sub-bin have been grouped.

[0132] Step 1205: Based on the mass-to-charge ratio of the fifth ion, select the sixth and seventh ions from the P ions in the i-th box. The mass-to-charge ratio of the sixth ion is the same as that of the fifth ion, and the mass-to-charge ratio of the seventh ion is less than or equal to the fourth preset threshold.

[0133] For example, taking the example shown in step 1404 above, the sixth ion and the seventh ion are screened from each of the three boxes, with each box as the dimension. For example, taking the first box as an example, the sixth ion with the same mass-to-charge ratio as the fifth ion in the first box is searched, and the seventh ion with a mass-to-charge ratio less than the fourth preset threshold of 20 ppm is searched.

[0134] Here, the seventh ion can also be filtered by the ion mobility value, that is, the seventh ion in the first box whose ion mobility value is less than or equal to the fourth preset threshold of 0.015V*s / cm2 is searched.

[0135] Step 1206: Related ion grouping within the box, that is, assigning the fifth, sixth, and seventh ions to the first type of sub-box in the i-th box; and assigning the eighth ion, which is not in the first type of sub-box in the i-th box, to the second type of sub-box in the i-th box.

[0136] Here, the partitioning principle is similar to step 1203 above, except that step 1206 is a partitioning step that needs to be performed in each box, that is, each box can include at least one of the following sub-boxes: first type sub-box and second type sub-box.

[0137] Furthermore, regarding step 130, the integrated mass-to-charge ratio in this embodiment can be calculated in the following manner, as detailed below.

[0138] In some embodiments of this application, the single-cell mass spectrometry data processing method may further include steps 1601 and 1602 prior to step 130.

[0139] Step 1601: The mass-to-charge ratio and ion signal intensity of the ions in each sub-box are weighted and summed to obtain a weighted value.

[0140] For example, if the first sub-box of K sub-boxes contains 3 ions, namely ion 1, ion 2 and ion 3, then the ion signal intensity of each ion can be used as a weight, and the weighted value can be calculated using the following formula (1):

[0141] Weighted value = Ion signal intensity of ion 1 * mass-to-charge ratio + Ion signal intensity of ion 2 * mass-to-charge ratio + Ion signal intensity of ion 3 * mass-to-charge ratio (1).

[0142] Step 1602: Calculate the integrated mass-to-charge ratio based on the weighted value and the ion signal intensity of ions in each sub-box.

[0143] For example, the mass-to-charge ratio can be calculated using the following formula (2):

[0144] Integrated mass-to-charge ratio = weighted value / (ion signal intensity of ion 1 + ion signal intensity of ion 2 + ion signal intensity of ion 3) (2).

[0145] In some embodiments of this application, the integrated ion signal intensity can be calculated in the following manner, as shown in the following formula (3).

[0146] For example, taking the example shown in steps 1601 and 1602 above, the integrated ion signal of the first sub-box = the ion signal intensity of ion 1 + the ion signal intensity of ion 2 + the ion signal intensity of ion 3.

[0147] In some embodiments of this application, the integrated ion data of integrated ions also includes integrated ion mobility values, which are the ion mobility values ​​of the sub-box to which the sub-box belongs.

[0148] For example, in an embodiment where the data frame of a single-cell ionization event is obtained by flow cytometry-ion mobility-mass spectrometry, N ions are divided into M boxes by ion mobility values, and each of the M boxes corresponds to an ion mobility value. Even if each box is divided into at least one sub-box, the ion mobility value of each sub-box in at least one sub-box is the ion mobility value of that box.

[0149] Therefore, in the embodiments of this application, the metabolite ion signals in each single-cell ionization event can be aligned according to the ion mobility value and mass-to-charge ratio, and the signals of the same ion in different single cells can be accumulated, which can significantly improve the signal-to-noise ratio of the metabolite ion mobility elution peak or mass spectrometry peak, thereby improving the sensitivity and coverage of peak detection.

[0150] Then, regarding step 140, in some embodiments of this application, cell superposition frame generation is performed by arranging the integrated ions obtained in step 130 according to the order of box and mass-to-charge ratio to obtain the cell superposition frame.

[0151] Therefore, in the embodiments of this application, the signal intensity of the same metabolite ion in all single-cell ionization events can be accumulated, while the increase in signal intensity of noise signals during cell superposition is much smaller than that of metabolite signals due to random occurrence. This improves the signal-to-noise ratio of the ion mobility elution peak (EIM) and the mass spectrometry peak (MS peak) of metabolite ions, thereby enhancing the sensitivity and coverage of metabolite ion mobility elution peak detection.

[0152] Based on this, the embodiments of this application establish a single-cell mass spectrometry data processing flow based on cell algorithms, comprising four stages: single-cell ionization event recognition, cell stacking, bottom-up peak assembly, targeted extraction and quantification of metabolites, and multidimensional matching and identification of metabolites, to achieve end-to-end processing of the aforementioned single-cell mass spectrometry data. In this way, it is possible to achieve end-to-end processing of the aforementioned single-cell mass spectrometry data. Figure 1 The content shown enables precise qualitative and quantitative analysis of single-cell metabolomics data.

[0153] Secondly, combining Figure 2 A detailed explanation of the methods for processing single-cell mass spectrometry data is provided.

[0154] Figure 2 This is a second flowchart of a single-cell mass spectrometry data processing method provided in an embodiment of this application.

[0155] like Figure 2 As shown, the single-cell mass spectrometry data processing method may include steps 210 to 280.

[0156] 1) Single-cell ionization event recognition

[0157] Step 210: Extract L test data frames of single-cell ionization events from the set of test data frames, where L is a positive integer.

[0158] Specifically, the measured data frames in the measured data frame set are obtained by flow cytometry-ion mobility-mass spectrometry. Based on this, step 210 may specifically include steps 2101 to 2105.

[0159] Step 2101: Upon receiving the first preset mass-to-charge ratio and preset ion mobility value of the single-cell ionization event marker corresponding to the single-cell ionization event input by the user, extract at least two ions that match the first preset mass-to-charge ratio and preset ion mobility value from the target test data frame in the test data frame set.

[0160] Step 2102: Sum the ion signal intensities of each of at least two ions to obtain the target ion signal intensity of the target measured data frame.

[0161] Step 2103: Sort the target ion signal intensities of the target measured data frames according to the chromatographic retention time to obtain the chromatographic elution peaks.

[0162] Step 2104: Based on the first preset peak width, extract the first peak region on the chromatographic elution peak. The chromatographic retention time of the chromatographic peak apex in the first peak region is within the preset chromatographic retention integral range corresponding to the single-cell ionization event marker.

[0163] Step 2105: The data frame corresponding to the first peak region is determined as the test data frame of the single-cell ionization event being tested.

[0164] For example, using single-cell metabolomics data acquired by ion mobility mass spectrometry (IMS), a data frame is acquired at fixed time intervals using flow cytometry-ion mobility-mass spectrometry (FMS) to identify and extract data frames containing single-cell ionization events. Single cells often contain common, high-abundance metabolites, such as phosphatidylcholine (PC) molecules (34:1) that constitute biological membrane systems. These metabolites can be called single-cell ionization event markers. Thus, based on the user-input preset mass-to-charge ratio of the marker (e.g., an m / z threshold of 20 ppm or 0.004 Da for ions less than 200 Da) and the preset ion mobility value (e.g., a mobility threshold equal to or less than 0.05 V*s / cm²), ions meeting the requirements are extracted from each data frame, and their signal intensities are summed to generate the chromatographic elution peak (EIC) of the marker over time. On this EIC, a local maximum value can be found based on the preset peak width set by the user. This local maximum value must be within the range of the marker response input by the user. Data frames containing the above marker requirements will be considered as single-cell ionization events and extracted from the raw data.

[0165] 2) Cell stacking

[0166] Step 220: Determine each of the L measured data frames as a data frame for a single-cell ionization event, and execute the following... Figure 1 The steps of any of the single-cell mass spectrometry data processing methods shown are used to obtain a superimposed data frame of the tested cell, which includes the superimposed data frame of the tested cell for each of the R ions in the tested data frame during a single-cell ionization event.

[0167] Step 230: Based on the ion data of N ions, divide the N ions into boxes to obtain K sub-boxes. The K sub-boxes include at least one of the following types of sub-boxes: a first type of sub-box that meets the preset division conditions and a second type of sub-box that does not meet the preset division conditions. The ions in the first type of sub-box meet at least one of the following conditions: the ions in the first type of sub-box have the same mass-to-charge ratio, or the mass-to-charge ratio of the ions in the first type of sub-box is less than or equal to a first preset threshold. K is a positive integer.

[0168] Here, please refer to the content in step 120 above, which will not be repeated here.

[0169] Step 240: Integrate the ions in each of the K sub-boxes to obtain K integrated ions. The K integrated ions include the integrated ions corresponding to each of the K sub-boxes. The integrated ion data includes the integrated mass-to-charge ratio and the integrated ion signal intensity. The integrated mass-to-charge ratio is determined by the mass-to-charge ratio and ion signal intensity of each ion in the sub-box, and the integrated ion signal intensity is determined by the ion signal intensity of each ion in the sub-box.

[0170] Here, please refer to the content in step 130 above, which will not be repeated here.

[0171] Step 250: Integrate K integrated ions to obtain a cell-overlay data frame corresponding to the data frame. The cell-overlay data frame is used to detect metabolite ion mobility elution peaks so as to annotate metabolites through metabolite ion mobility elution peaks.

[0172] The purpose of step 250 is to improve the signal-to-noise ratio (SNR) of the ion mobility elution peak of metabolite ions by accumulating the responses of the same ion in all single-cell ionization events, thereby enhancing the sensitivity and coverage of peak detection. All ions from all single-cell ionization events can be collected, and the signal intensities of the same ion belonging to different cells are summed based on m / z and mobility to obtain a cell-overlay data frame. This process consists of four steps: cross-cell ion binning, intra-bin ion grouping, intra-group ion accumulation, and cell-overlay data frame generation. During the accumulation process, the signal intensity of metabolite ions accumulates significantly, while randomly occurring noise signals remain largely unchanged, thus substantially improving the SNR of the metabolite EIM and consequently enhancing the detection of metabolite ion peaks.

[0173] Step 260: The cell superimposed data frame corresponding to the data frame is determined as the cell superimposed data frame to be tested. The cell superimposed data frame to be tested includes the cell superimposed data frame to be tested for each of the R ions in the data frame to be tested in a single cell ionization event.

[0174] 3) Bottom-up assembly

[0175] Step 270: Detect the elution peak of metabolite ion mobility based on the superimposed data frames of the tested cells.

[0176] Specifically, step 270 may include steps 2701 to 2709.

[0177] Step 2701: Arrange the superimposed data frames of the tested cells according to the ion mobility values ​​of the ions in the superimposed data frames of the tested cells to obtain the first sequence.

[0178] Step 2702: Select target ions from the first sequence, wherein the ion signal intensity of the target ion is greater than or equal to the fifth preset threshold.

[0179] Step 2703: Based on the ion mobility value of the target ion, screen related ions that match the target ion from the first sequence. The mass-to-charge ratio of the related ion and the mass-to-charge ratio of the target ion satisfy at least one of the following conditions: the mass-to-charge ratio of the related ion is the same as that of the target ion, or the mass-to-charge ratio of the related ion is less than or equal to a sixth preset threshold.

[0180] Step 2704: Construct potential peak targets based on the target ion and related ions.

[0181] Step 2705: If the length of the continuous signal of the potential peak target is less than a seventh preset threshold, sort the ions in the first sequence according to the ion signal intensity to obtain a second sequence. Conversely, if the length of the continuous signal of the potential peak target is greater than or equal to the seventh preset threshold, remove the potential peak target from the first sequence.

[0182] Step 2706: Extract the second peak region from the second sequence according to the second preset peak width.

[0183] Step 2707: If the ion mobility peak of the target peak in the second peak region is greater than or equal to an eighth preset threshold, the target peak is determined as the peak apex of the metabolite ion mobility effluent peak. The signal-to-noise ratio at the peak apex is less than or equal to a preset signal-to-noise ratio.

[0184] Step 2708: Calculate the mass-to-charge ratio of the metabolite ion mobility effluent peak based on the mass-to-charge ratio of ions in the second peak region and the ion signal intensity; and use the ion mobility value of the target peak as the ion mobility value of the metabolite ion mobility effluent peak.

[0185] Step 2709: Generate metabolite ion mobility elution peaks based on the mass-to-charge ratio of the metabolite ion mobility elution peaks and the ion mobility values ​​of the metabolite ion mobility elution peaks.

[0186] For example, this process may include two steps: assembling a potential peak target and peak detection, detecting metabolite ion mobility effluent peaks in a cell superposition frame. A true metabolite ion mobility effluent peak is a signal that appears continuously in the ion mobility dimension. Therefore, potential peak targets constituting a continuous signal are searched and assembled in the cell superposition frame. Specifically, firstly, all ions in the cell superposition frame are arranged according to their ion mobility values ​​(or binning or scan). Then, the ion with the highest signal intensity is selected as the peak vertex, and the search for its peak target begins. Secondly, in the next ion mobility value bin to the left of this ion, ions with m / z values ​​within a user-defined threshold are searched (e.g., an m / z threshold of 20 ppm or 0.004 Da for ions less than 200 Da). If a suitable ion is found, the search continues to the left of the next ion mobility value bin for other ions, until no suitable ion is found in a certain bin or all bins to the left of the peak vertex have been searched. This search step is then performed sequentially in the ion mobility bins to the right of the peak. The peak and the related ions found on its left and right sides together form the peak target. The length of this peak target must meet the shortest peak target threshold input by the user. If it meets the requirement, the related ions belonging to the peak target are extracted and removed from the cell stacked data frame; if it does not meet the requirement, the related ions are released back into the cell stacked data frame. Furthermore, in descending order of signal intensity, all ions that have not yet been removed and have not been selected as peaks are used as peaks for searching the peak target. After the peak target is assembled, MetCell performs ion mobility peak detection sequentially within the peak target. MetCell determines the local maximum value of the peak width (peakspan) that meets the user-input threshold from the peak target as the peak apex of the EIM, and the signal-to-noise ratio of this peak and the normalized signal-to-noise ratio must meet the user-input requirements. If the above requirements are met, MetCell will take the average of the m / z values ​​of the ions within the user-defined peak width, using the corresponding signal intensity as weights, to obtain the m / z of the EIM, and will take the mobility of the peak apex as the mobility of the EIM. If the peak definition is not met, the m / z and mobility of the EIM will not be output.

[0187] 4) Targeted extraction and quantification of metabolites and multidimensional matching identification of metabolites

[0188] Step 280: Based on the ion data of the metabolite ion mobility elution peak in a single cell, determine the metabolite result information of the metabolite ion mobility elution peak in a single cell. The metabolite result information includes quantitative and qualitative information corresponding to the metabolite ion mobility elution peak.

[0189] Specifically, the ion data of the metabolite ion mobility effluent peak in a single cell includes the mass-to-charge ratio of the metabolite ion mobility effluent peak and the ion mobility value of the metabolite ion mobility effluent peak. The metabolite result information is quantitative information. Based on this, steps 2801 and 2802 are performed.

[0190] Step 2801: Based on the mass-to-charge ratio of the metabolite ion elution peak and the ion mobility value of the metabolite ion elution peak, at least two event ions matching the metabolite ion elution peak are screened from single-cell ionization events. The mass-to-charge ratio of the event ions satisfies at least one of the following conditions: the mass-to-charge ratio of the event ions is the same as the second preset mass-to-charge ratio; the mass-to-charge ratio of the event ions is less than or equal to the seventh preset threshold; and the ion mobility value of the event ions is less than or equal to the ninth preset threshold.

[0191] Step 2802: Sum the ion signal intensities of each event ion in at least two event ions to obtain quantitative information on the metabolite ion mobility effluent peak in a single cell.

[0192] Specifically, metabolites are extracted and quantified. For all ion mobility elution peaks detected by bottom-up peak assembly, the signal intensity of these peaks in a single cell is extracted and used as quantitative information. Specifically, the m / z and mobility corresponding to each detected EIM can be used, according to a user-defined threshold (e.g., an m / z threshold of 20 ppm or 0.004 Da for ions smaller than 200 Da, and a mobility threshold of 0.04 V*s / cm2). Thus, in each single-cell ionization event extracted through the above single-cell ionization event identification steps, ions that meet the requirements are searched, and their signal intensities are summed to obtain the quantitative information of each EIM in each cell.

[0193] Metabolite multidimensional matching identification. MetCell contains a database of 135,638 common metabolites, which includes the mass-to-charge ratio and collision cross-sectional area (CCS) information for each metabolite obtained through experimental acquisition or theoretical calculation. MetCell first converts the mobility of each EIM into CCS, and then searches for and performs multidimensional matching on matching metabolites that meet the requirements based on the user-inputted m / z and CCS threshold, outputting the corresponding matching results.

[0194] It should be noted that the above four stages are used to achieve the entire process of processing ion mobility mass spectrometry single-cell metabolomics data from raw mass spectrometry data to qualitative and quantitative analysis of metabolites.

[0195] It should be noted that flow cytometry-mass spectrometry can also be used to acquire single-cell metabolomics data. In the four modules mentioned above, it is necessary to remove the ion mobility dimension information in modules one, two, four, and five. In module three, it is only necessary to select peaks with a signal-to-noise ratio exceeding a set threshold in the m / z dimension. This technique is also applicable to other single-cell data acquired by mass spectrometry or ion mobility mass spectrometry, such as single-cell proteomics data. In this way, highly sensitive and deep-coverage single-cell metabolomics data analysis can be achieved, detecting more than 10,000 metabolite ion peaks in a single dataset and identifying more than 1,600 metabolites.

[0196] In this way, since the ion mobility elution peak of a metabolite is composed of the signal intensities of ions with a mass-to-charge ratio equal to that metabolite in each ion mobility bin, the ions belonging to that metabolite always appear in a certain number of bins, while the noise ion signal appears randomly in different bins. If only the ion mobility elution peak of a certain metabolite in a single-cell ionization event is observed, its signal-to-noise ratio may be very low due to the small amount of metabolite, and it may even be impossible to distinguish between ions belonging to the metabolite and noise ions. Therefore, the single-cell mass spectrometry data processing method provided in this application can greatly increase the signal intensity of the same metabolite ion. Since the noise signal appears randomly, the increase in signal intensity during cell superposition is much smaller than that of the metabolite signal. Therefore, by accumulating the signal intensity of the same ion in all single-cell ionization events, the signal-to-noise ratio of the metabolite ion mobility elution peak and the signal-to-noise ratio of the mass spectrometry peak are improved, thereby promoting the sensitivity and coverage of the metabolite ion mobility elution peak.

[0197] Based on the same inventive concept, this application provides a single-cell mass spectrometry data processing device, specifically combined with... Figure 3 Please provide a detailed explanation.

[0198] Figure 3 This is one of the structural schematic diagrams of a single-cell mass spectrometry data processing device provided in an embodiment of this application.

[0199] like Figure 3 As shown, the single-cell mass spectrometry data processing device 30 is applied to computer equipment and may specifically include:

[0200] The acquisition module 301 is used to acquire the data frame of a single-cell ionization event. The data frame is used to record the ion data of N ions. The ion data includes the mass-to-charge ratio and the ion signal intensity, where N is a positive integer.

[0201] The binning module 302 is used to divide the N ions into bins based on the ion data of the N ions to obtain K sub-bins. The K sub-bins include at least one of the following types of sub-bins: a first type of sub-bin that meets the preset division conditions and a second type of sub-bin that does not meet the preset division conditions. The ions in the first type of sub-bin meet at least one of the following conditions: the ions in the first type of sub-bin have the same mass-to-charge ratio, or the mass-to-charge ratio of the ions in the first type of sub-bin is less than or equal to a first preset threshold. K is a positive integer.

[0202] Integration module 303 is used to integrate the ions in each of the K sub-boxes to obtain K integrated ions. The K integrated ions include the integrated ions corresponding to each of the K sub-boxes. The integrated ion data includes the integrated mass-to-charge ratio and the integrated ion signal intensity. The integrated mass-to-charge ratio is determined by the mass-to-charge ratio and ion signal intensity of each ion in the sub-box, and the integrated ion signal intensity is determined by the ion signal intensity of each ion in the sub-box.

[0203] The integration module 304 can also be used to integrate K integrated ions to obtain a cell superimposed data frame corresponding to the data frame. The cell superimposed data frame is used to detect the elution peak of metabolite ions so as to annotate metabolites through the elution peak of metabolite ions.

[0204] The single-cell mass spectrometry data processing device 30 provided in the embodiments of this application will be described in detail below.

[0205] In some embodiments provided in this application, the binning module 302 can be specifically used to, when the data frame of a single-cell ionization event is acquired by flow cytometry-mass spectrometry and the K sub-bins include a first type of sub-bin and a second type of sub-bin, select a first ion from N ions based on the ion signal intensity of each ion in N ions, wherein the ion signal intensity of the first ion is greater than or equal to a first preset threshold.

[0206] Based on the mass-to-charge ratio of the first ion, the second and third ions are selected from N ions. The mass-to-charge ratio of the second ion is the same as that of the first ion, and the mass-to-charge ratio of the third ion is less than or equal to the second preset threshold.

[0207] The first, second, and third ions are assigned to the first type of sub-box; and the fourth ion, which is not in the first type of sub-box among the N ions, is assigned to the second type of sub-box.

[0208] In some embodiments provided in this application, the acquisition module 301 can also be used to, when the data frame of a single-cell ionization event is acquired by flow cytometry-ion mobility-mass spectrometry, and the ion data also includes ion mobility values, and the K sub-boxes include first-type sub-boxes and second-type sub-boxes, divide the N ions into M boxes according to the ion mobility values ​​of each ion in the N ions; wherein each of the M boxes includes P ions, the ion mobility values ​​of the P ions in each box are the same, the ion mobility values ​​of the ions in different boxes in the M boxes are different, and P and M are integers greater than 1;

[0209] Based on this, the compartment module 302 may specifically include:

[0210] Based on the ion signal intensity of each ion in the i-th box out of M boxes, the fifth ion is selected from the P ions in the i-th box. The ion signal intensity of the fifth ion is greater than or equal to the third preset threshold, i∈[1,M].

[0211] Based on the mass-to-charge ratio of the fifth ion, the sixth and seventh ions are selected from the P ions in the i-th box. The mass-to-charge ratio of the sixth ion is the same as that of the fifth ion, and the mass-to-charge ratio of the seventh ion is less than or equal to the fourth preset threshold.

[0212] The fifth, sixth, and seventh ions are assigned to the first type of sub-box in the i-th box; and the eighth ion, which is not in the first type of sub-box in the i-th box, is assigned to the second type of sub-box in the i-th box.

[0213] In some embodiments provided in this application, the integrated ion data of integrated ions also includes integrated ion mobility values, which are the ion mobility values ​​of the sub-box to which the sub-box belongs.

[0214] In some embodiments provided in this application, the single-cell mass spectrometry data processing device 30 may further include a computing module; wherein,

[0215] The calculation module is used to perform a weighted summation of the mass-to-charge ratio and ion signal intensity of ions in each sub-box to obtain a weighted value; based on the weighted value and the ion signal intensity of ions in each sub-box, the integrated mass-to-charge ratio is calculated.

[0216] In this way, since the ion mobility elution peak of a metabolite is composed of the signal intensities of ions with a mass-to-charge ratio equal to that metabolite in each ion mobility bin, the ions belonging to that metabolite always appear in a certain number of bins, while the noise ion signal appears randomly in different bins. If only the ion mobility elution peak of a certain metabolite in a single-cell ionization event is observed, its signal-to-noise ratio may be very low due to the small amount of metabolite, and it may even be impossible to distinguish between ions belonging to the metabolite and noise ions. Therefore, the single-cell mass spectrometry data processing method provided in this application can greatly increase the signal intensity of the same metabolite ion. Since the noise signal appears randomly, the increase in signal intensity during cell superposition is much smaller than that of the metabolite signal. Therefore, by accumulating the signal intensity of the same ion in all single-cell ionization events, the signal-to-noise ratio of the metabolite ion mobility elution peak and the signal-to-noise ratio of the mass spectrometry peak are improved, thereby promoting the sensitivity and coverage of the metabolite ion mobility elution peak.

[0217] Based on the same inventive concept, this application provides another single-cell mass spectrometry data processing device, specifically combined with Figure 4 Please provide a detailed explanation.

[0218] Figure 4 This is a second schematic diagram of a single-cell mass spectrometry data processing device provided in an embodiment of this application.

[0219] like Figure 4 As shown, this single-cell mass spectrometry data processing device 40 is applied to computer equipment and may specifically include:

[0220] Extraction module 401 is used to extract L test data frames of single-cell ionization events from the test data frame set, where L is a positive integer;

[0221] Processing module 402 is used to determine each of the L tested data frames as follows: Figure 1 The data frame of the single-cell ionization event shown is executed as follows: Figure 1 The steps of the single-cell mass spectrometry data processing method shown are as follows: to obtain the superimposed data frame of the cell under test, which includes the superimposed data frame of the cell under test for each of the R ions in the single-cell ionization event.

[0222] The acquisition module 403 is used to detect the elution peak of metabolite ion mobility based on the superimposed data frames of the tested cells;

[0223] The determination module 404 is used to determine the metabolite result information of the metabolite ion mobility elution peak in a single cell based on the ion data of the metabolite ion mobility elution peak in a single cell. The metabolite result information includes quantitative information and qualitative information.

[0224] The single-cell mass spectrometry data processing device 40 provided in the embodiments of this application will be described in detail below.

[0225] In some embodiments provided in this application, the extraction module 401 mentioned above can be specifically used to extract at least two ions that match the first preset mass-charge ratio and preset ion mobility value from the target test data frame in the test data frame set, provided that the test data frame in the test data frame set is acquired by flow cytometry-ion mobility-mass spectrometry and the user inputs the first preset mass-charge ratio and preset ion mobility value of the single-cell ionization event marker corresponding to the single-cell ionization event.

[0226] The target ion signal intensity is obtained by summing the ion signal intensities of each of at least two ions;

[0227] The target ion signal intensities of the target measured data frames are sorted according to the chromatographic retention time to obtain the chromatographic elution peaks;

[0228] Based on the first preset peak width, the first peak region is extracted from the chromatographic elution peak, and the chromatographic retention time of the chromatographic peak apex in the first peak region is within the preset chromatographic retention integral range corresponding to the single-cell ionization event marker.

[0229] The data frame corresponding to the first peak region is identified as the measured data frame of the single-cell ionization event being measured.

[0230] In some embodiments provided in this application, the acquisition module 403 may be specifically used to arrange the superimposed data frames of the tested cells according to the ion mobility values ​​of ions in the superimposed data frames of the tested cells to obtain a first sequence;

[0231] Target ions are selected from the first sequence, and the ion signal intensity of the target ion is greater than or equal to the fifth preset threshold.

[0232] Based on the ion mobility value of the target ion, related ions that match the target ion are screened from the first sequence. The mass-to-charge ratio of the related ion and the mass-to-charge ratio of the target ion satisfy at least one of the following conditions: the mass-to-charge ratio of the related ion is the same as that of the target ion, or the mass-to-charge ratio of the related ion is less than or equal to a sixth preset threshold.

[0233] Based on the target ion and related ions, potential peak targets are constructed;

[0234] If the length of the continuous signal of the potential peak target is less than the seventh preset threshold, the ions in the first sequence are sorted according to the ion signal intensity to obtain the second sequence.

[0235] According to the second preset peak width, extract the second peak region in the second sequence;

[0236] If the ion mobility peak of the target peak in the second peak region is greater than or equal to the eighth preset threshold, the target peak is determined as the peak apex of the metabolite ion mobility effluent peak.

[0237] Based on the mass-to-charge ratio and ion signal intensity of ions in the second peak region, the mass-to-charge ratio of the metabolite ion mobility elution peak is calculated; and the ion mobility value of the target peak is used as the ion mobility value of the metabolite ion mobility elution peak.

[0238] Metabolite ion mobility elution peaks are generated based on the mass-to-charge ratio and ion mobility value of the metabolite ion mobility elution peaks.

[0239] In some embodiments provided in this application, the determining module 404 can be specifically used to, when the ion data of the metabolite ion mobility elution peak in a single cell includes the mass-to-charge ratio and ion mobility value of the metabolite ion mobility elution peak, and the metabolite result information is quantitative information, screen at least two event ions that match the metabolite ion mobility elution peak from single-cell ionization events based on the mass-to-charge ratio and ion mobility value of the metabolite ion mobility elution peak. The mass-to-charge ratio of the event ions satisfies at least one of the following conditions: the mass-to-charge ratio of the event ions is the same as the second preset mass-to-charge ratio; the mass-to-charge ratio of the event ions is less than or equal to the seventh preset threshold; and the ion mobility value of the event ions is less than or equal to the ninth preset threshold.

[0240] The ion signal intensities of each of at least two event ions are summed to obtain quantitative information on the metabolite ion mobility effluent peak in a single cell.

[0241] In some embodiments provided in this application, the determining module can be specifically used to determine the metabolite result information of the metabolite ion mobility elution peak in a single cell based on the ion data of the metabolite ion mobility elution peak in a single cell when the metabolite result information is qualitative information.

[0242] The single-cell mass spectrometry data processing device in this application embodiment can be a device, or it can be a component, integrated circuit, or chip in a computer device. The device can be a mobile computer device or a non-mobile computer device.

[0243] For example, mobile computer devices can be mobile phones, tablets, laptops, handheld computers, in-vehicle computer devices, wearable devices, ultra-mobile personal computers (UMPCs), netbooks, or personal digital assistants (PDAs), etc., while non-mobile computer devices can be servers, network attached storage (NAS), personal computers (PCs), televisions (TVs), ATMs, or self-service machines, etc. The embodiments of this application do not impose specific limitations.

[0244] The operating system of the single-cell mass spectrometry data processing device in this application embodiment can be Android, iOS, or other possible operating systems. This application embodiment does not make specific limitations.

[0245] The single-cell mass spectrometry data processing device provided in this application embodiment can achieve... Figures 1 to 2 The various processes implemented in the method implementation examples will not be described again here to avoid repetition.

[0246] In summary, the single-cell mass spectrometry data processing device provided in this application embodiment is characterized by the fact that the ion mobility elution peak of a metabolite is composed of the signal intensities of ions with a mass-to-charge ratio equal to that metabolite in each ion mobility value bin. Ions belonging to the metabolite always appear in a specific number of bins, while noise ion signals appear randomly in different bins. If only the ion mobility elution peak of a certain metabolite in a single-cell ionization event is observed, its signal-to-noise ratio may be very low due to the small amount of metabolite, and it may even be impossible to distinguish between ions belonging to the metabolite and noise ions. Therefore, the single-cell mass spectrometry data processing method provided in this application embodiment can greatly increase the signal intensity of the same metabolite ion. Since the noise signal appears randomly, the increase in signal intensity during cell superposition is much smaller than that of the metabolite signal. Therefore, by accumulating the signal intensity of the same ion in all single-cell ionization events, the signal-to-noise ratio of the metabolite ion mobility elution peak and the signal-to-noise ratio of the mass spectrometry peak are improved, thereby promoting the sensitivity and coverage of the metabolite ion mobility elution peak.

[0247] Optional, such as Figure 5As shown, this application embodiment also provides a computer device 500, including a processor 501, a memory 502, and a program or instructions stored in the memory 502 and executable on the processor 501. When the program or instructions are executed by the processor 501, they implement the various processes of the above-described single-cell mass spectrometry data processing method embodiment and achieve the same technical effect. To avoid repetition, they will not be described again here.

[0248] This application also provides a readable storage medium storing a program or instructions. When the program or instructions are executed by a processor, they implement the various processes of the above-described single-cell mass spectrometry data processing method embodiments and achieve the same technical effects. To avoid repetition, these will not be described again here.

[0249] The processor is the processor in the computer device described in the above embodiments. The readable storage medium includes computer-readable storage media, such as computer read-only memory (ROM), random access memory (RAM), magnetic disk, or optical disk.

[0250] In addition, this application embodiment provides another chip, which includes a processor and a communication interface. The communication interface and the processor are coupled. The processor is used to run programs or instructions to implement the various processes of the above-described single-cell mass spectrometry data processing method embodiment and can achieve the same technical effect. To avoid repetition, it will not be described again here.

[0251] It should be understood that the chip mentioned in the embodiments of this application may also be referred to as a system-on-a-chip, system chip, chip system, or system-on-a-chip, etc.

[0252] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0253] Furthermore, it should be noted that the scope of the methods and apparatus in the embodiments of this application is not limited to performing functions in the order shown or discussed, but may also include performing functions substantially simultaneously or in the reverse order, depending on the functions involved. For example, the described methods may be performed in a different order than described, and various steps may be added, omitted, or combined. In addition, features described with reference to certain examples may be combined in other examples.

[0254] Through the above description of the embodiments, those skilled in the art can clearly understand that the methods of the above embodiments can be implemented by means of software plus necessary general-purpose hardware platforms. Of course, they can also be implemented by hardware, but in many cases the former is a better implementation method. Based on this understanding, the technical solution of this application, in essence, or the part that contributes to the prior art, can be embodied in the form of a software product. This computer software product is stored in a storage medium (such as ROM / RAM, magnetic disk, optical disk) and includes several instructions to cause a terminal (which may be a mobile phone, computer, server, air conditioner, or network device, etc.) to execute the methods of the various embodiments of this application.

[0255] The embodiments of this application have been described above with reference to the accompanying drawings. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A method for processing single-cell mass spectrometry data, characterized in that, include: Acquire a data frame of a single-cell ionization event, wherein the data frame is used to record ion data of N ions, the ion data including mass-to-charge ratio and ion signal intensity, where N is a positive integer; Based on the ion data of the N ions, the N ions are divided into K sub-boxes, wherein the K sub-boxes include at least one of the following types of sub-boxes: a first type of sub-box that meets the preset division conditions, and a second type of sub-box that does not meet the preset division conditions. The ions in the first type of sub-box meet at least one of the following conditions: the ions in the first type of sub-box have the same mass-to-charge ratio, or the mass-to-charge ratio of the ions in the first type of sub-box is less than or equal to a first preset threshold, where K is a positive integer. The ions in each of the K sub-boxes are integrated to obtain K integrated ions. The K integrated ions include the integrated ions corresponding to each of the K sub-boxes. The integrated ion data includes the integrated mass-to-charge ratio and the integrated ion signal intensity. The integrated mass-to-charge ratio is determined by the mass-to-charge ratio and ion signal intensity of each ion in the sub-box, and the integrated ion signal intensity is determined by the ion signal intensity of each ion in the sub-box. The K integrated ions are integrated to obtain a cell-overlay data frame corresponding to the data frame. The cell-overlay data frame is used to detect metabolite ion mobility elution peaks so as to annotate metabolites through the metabolite ion mobility elution peaks.

2. The method according to claim 1, characterized in that, The data frames of the single-cell ionization event are acquired by flow cytometry-mass spectrometry. The K sub-boxes include the first type of sub-box and the second type of sub-box. The process of dividing the N ions into K sub-boxes based on their ion data results in: Based on the ion signal intensity of each of the N ions, a first ion is selected from the N ions, wherein the ion signal intensity of the first ion is greater than or equal to a first preset threshold. Based on the mass-to-charge ratio of the first ion, a second ion and a third ion are selected from the N ions. The mass-to-charge ratio of the second ion is the same as that of the first ion, and the mass-to-charge ratio of the third ion is less than or equal to a second preset threshold. The first ion, the second ion, and the third ion are assigned to the first type of sub-box; and the fourth ion among the N ions, excluding the ions in the first type of sub-box, is assigned to the second type of sub-box.

3. The method according to claim 1, characterized in that, The data frames of the single-cell ionization event are acquired by flow cytometry-ion mobility-mass spectrometry, and the ion data also include ion mobility values. The K sub-boxes include the first type of sub-boxes and the second type of sub-boxes. Before dividing the N ions into K sub-boxes based on their ion data, the method further includes: Based on the ion mobility value of each of the N ions, the N ions are divided into M boxes; wherein each of the M boxes contains P ions, the ion mobility values ​​of the P ions in each box are the same, the ion mobility values ​​of the ions in different boxes of the M boxes are different, and P and M are integers greater than 1. The step of dividing the N ions into K sub-boxes based on their ion data includes: Based on the ion signal intensity of each ion in the i-th box among the M boxes, a fifth ion is selected from the P ions in the i-th box. The ion signal intensity of the fifth ion is greater than or equal to a third preset threshold, i∈[1,M]. Based on the mass-to-charge ratio of the fifth ion, the sixth and seventh ions are selected from the P ions in the i-th box. The mass-to-charge ratio of the sixth ion is the same as that of the fifth ion, and the mass-to-charge ratio of the seventh ion is less than or equal to the fourth preset threshold. The fifth ion, the sixth ion, and the seventh ion are assigned to the first type of sub-box in the i-th box; and the eighth ion, which is not in the first type of sub-box in the i-th box, is assigned to the second type of sub-box in the i-th box.

4. The method according to claim 3, characterized in that, The integrated ion data also includes integrated ion mobility values, which are the ion mobility values ​​of the box to which the sub-box belongs.

5. The method according to claim 1, characterized in that, The method further includes: The mass-to-charge ratio and ion signal intensity of the ions in each sub-box are weighted and summed to obtain a weighted value; The integrated mass-to-charge ratio is calculated based on the weighted value and the ion signal intensity of the ions in each sub-box.

6. A method for processing single-cell mass spectrometry data, characterized in that, include: Extract L test data frames from the set of test data frames, where L is a positive integer; Each of the L measured data frames is determined as a data frame of the single-cell ionization event as described in any one of claims 1-5, and the steps of the single-cell mass spectrometry data processing method as described in any one of claims 1-5 are executed to obtain a superimposed data frame of the measured cells. The superimposed data frame of the measured cells includes the superimposed data frame of the measured cells for each of the R ions in the measured data frame in the single-cell ionization event. Based on the superimposed data frames of the tested cells, the elution peaks of metabolite ion mobility were detected. Based on the ion data of the metabolite ion mobility elution peak in a single cell, the metabolite result information of the metabolite ion mobility elution peak in the single cell is determined. The metabolite result information includes quantitative and qualitative information corresponding to the metabolite ion mobility elution peak.

7. The method according to claim 6, characterized in that, The measured data frames in the set of measured data frames were acquired by flow cytometry-ion mobility-mass spectrometry. The extraction of L test data frames from the set of test data frames to identify single-cell ionization events includes: Upon receiving a user-inputted first preset mass-to-charge ratio and preset ion mobility value for a single-cell ionization event marker corresponding to the single-cell ionization event, at least two ions matching the first preset mass-to-charge ratio and preset ion mobility value are extracted from the target test data frame in the test data frame set. The target ion signal intensity of the target measured data frame is obtained by summing the ion signal intensities of each of the at least two ions. The target ion signal intensities of the target measured data frames are sorted according to the chromatographic retention time to obtain the chromatographic elution peaks; Based on the first preset peak width, a first peak region is extracted from the chromatographic elution peak, and the chromatographic retention time of the chromatographic peak apex in the first peak region is within the preset chromatographic retention integral range corresponding to the single-cell ionization event marker. The data frame corresponding to the first peak region is determined as the test data frame of the single-cell ionization event being tested.

8. The method according to claim 6, characterized in that, The step of detecting metabolite ion mobility effluent peaks based on the superimposed data frames of the tested cells includes: The superimposed data frames of the tested cells are arranged according to the ion mobility values ​​of the ions in the superimposed data frames of the tested cells to obtain a first sequence; Target ions are selected from the first sequence, wherein the ion signal intensity of the target ions is greater than or equal to a fifth preset threshold. Based on the ion mobility value of the target ion, related ions matching the target ion are screened from the first sequence. The mass-to-charge ratio of the related ion and the mass-to-charge ratio of the target ion satisfy at least one of the following conditions: the mass-to-charge ratio of the related ion is the same as that of the target ion, or the mass-to-charge ratio of the related ion is less than or equal to a sixth preset threshold. Based on the target ion and the related ions, potential peak targets are constructed; If the length of the continuous signal of the potential peak target is less than a seventh preset threshold, the ions in the first sequence are sorted according to the ion signal intensity to obtain the second sequence; According to the second preset peak width, extract the second peak region from the second sequence; If the ion mobility peak of the target peak in the second peak region is greater than or equal to the eighth preset threshold, the target peak is determined as the peak apex of the metabolite ion mobility effluent peak. Based on the mass-to-charge ratio and ion signal intensity of the ions in the second peak region, the mass-to-charge ratio of the metabolite ion mobility effluent peak is calculated; and the ion mobility value of the target peak is used as the ion mobility value of the metabolite ion mobility effluent peak. The metabolite ion mobility elution peak is generated based on the mass-to-charge ratio of the metabolite ion mobility elution peak and the ion mobility value of the metabolite ion mobility elution peak.

9. The method according to claim 6, characterized in that, The ion data of the metabolite ion mobility effluent peak in a single cell includes the mass-to-charge ratio of the metabolite ion mobility effluent peak and the ion mobility value of the metabolite ion mobility effluent peak, and the metabolite result information is the quantitative information; The step of determining the metabolite result information of the metabolite ion mobility elution peak in a single cell based on the ion data of the metabolite ion mobility elution peak in a single cell includes: Based on the mass-to-charge ratio of the metabolite ion elution peak and the ion mobility value of the metabolite ion elution peak, at least two event ions matching the metabolite ion elution peak are screened from the single-cell ionization events. The mass-to-charge ratio of the event ions satisfies at least one of the following conditions: the mass-to-charge ratio of the event ions is the same as the second preset mass-to-charge ratio; the mass-to-charge ratio of the event ions is less than or equal to the seventh preset threshold; and the ion mobility value of the event ions is less than or equal to the ninth preset threshold. The ion signal intensity of each of the at least two event ions is summed to obtain quantitative information on the efflux peak of the metabolite ion in the single cell.

10. The method according to claim 6, characterized in that, The metabolite result information is the qualitative information; The step of determining the metabolite result information of the metabolite ion mobility elution peak in a single cell based on the ion data of the metabolite ion mobility elution peak in a single cell includes: Based on the ion mobility value of the metabolite ion mobility effluent peak, the collision cross-sectional area of ​​the metabolite ion mobility effluent peak is generated; Based on the collision cross-sectional area of ​​the elution peak of the metabolite ion mobility and the mass-to-charge ratio of the elution peak of the metabolite ion mobility, the qualitative information is obtained by matching it with the third preset mass-to-charge ratio and preset collision cross-sectional area in the preset metabolite database. The qualitative information includes information on metabolites that match the elution peak of the metabolite ion mobility.

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