Method for automatically identifying positions of unsaturated fatty acid and double bonds thereof

Abstract

The invention discloses a method for automatically identifying unsaturated fatty acid and double bond positions thereof. According to the invention, the principle of regular fragmentation of unsaturated fatty acid in a collision dissociation mode of a liquid chromatography-tandem mass spectrometer after the unsaturated fatty acid is subjected to a Pattern-Buchi (PB) reaction is utilized; the invention develops an automatic data processing method for unsaturated fatty acid mass spectrum data acquired in two modes of a data dependent acquisition mode and a data non-dependent acquisition mode in liquid chromatography-tandem mass spectrometry, and can automatically identify unsaturated fatty acid and double bond positions thereof from huge DDA data of liquid chromatography-tandem mass spectrometry. And the quantitative analysis of the unsaturated fatty acid is realized by utilizing the strength information of the characteristic fragment ions in the DIA data, so that the accuracy and the efficiency of the qualitative and quantitative analysis of the unsaturated fatty acid are remarkably improved.

Description

Technical Field

[0001] The invention relates to the technical field of biological mass spectrometry analysis, and in particular to a method for automatically identifying unsaturated fatty acids and double bond positions thereof. Background Art

[0002] Liquid chromatography tandem mass spectrometry (LC-MS / MS) is an important technology widely used for qualitative analysis of various organic molecules. Among them, liquid chromatography can separate complex samples, and the separated components enter the tandem mass spectrometer in sequence according to polarity. The primary mass spectrometer source is an electrospray ionization source (ESI for short), which can obtain the molecular ion peak of the component; the secondary mass spectrometer uses collision induced dissociation (CID for short) or high energy collision dissociation (HCD for short) technology for fragmentation, which can obtain the characteristic peaks of molecular fragmentation.

[0003] Fatty acid (FA) is the most basic component of the lipidome in the human body and has a great influence on the composition and content of various substances in the lipidome. Especially for unsaturated fatty acids, changes in the position structure of their carbon-carbon double bonds may cause or indicate the occurrence of diseases. However, HPLC-MS / MS cannot fragment them at the carbon-carbon double bond position to produce secondary fragments, so it is necessary to perform a derivatization reaction on the carbon-carbon double bond position. Using the principle of photocyclization addition reaction (such as Paternò-Büchi reaction, referred to as PB reaction), the carbonyl group can react with the carbon-carbon double bond in fatty acids to generate a four-membered ring structure. Since the four-membered ring generated after the PB reaction has a large tension, it can be fragmented in the tandem mass spectrometry, thereby obtaining characteristic fragments.

[0004] Due to the huge amount of data in LC-MS / MS, there may be tens of thousands of secondary mass spectrometry data in one LC-MS / MS, so the data needs to be specifically screened. The reaction products will produce complex fragments under the fragmentation of the secondary mass spectrometry, and the characteristic fragments produced by double bonds at different positions will overlap in the secondary mass spectrometry, so the fragments in the secondary mass spectrometry need to be paired and calculated. However, there are still certain algorithmic and technical gaps in the detection of positional isomerism of fatty acids using LC-MS / MS and PB reactions. Summary of the invention

[0005] In order to solve the above technical problems, the present application provides a method for automatically identifying unsaturated fatty acids and the positions of their double bonds.

[0006] The present invention provides a method for automatically identifying unsaturated fatty acids and double bond positions thereof, comprising the following steps:

[0007] (1) mixing a sample to be tested with a reaction reagent and performing a photocycloaddition reaction to obtain a reaction product, detecting the reaction product using a data-dependent acquisition mode in a liquid chromatography tandem mass spectrometry to obtain first detection data, and detecting the reaction product using a data-independent acquisition mode in the liquid chromatography tandem mass spectrometry to obtain second detection data, wherein the sample to be tested includes at least one unsaturated fatty acid, and the reaction reagent is a compound containing a carbonyl group;

[0008] (2) performing a qualitative analysis based on the first detection data to determine the primary ions corresponding to the reaction products and the target secondary fragments corresponding to each of the primary ions, thereby obtaining a qualitative analysis result;

[0009] (3) Based on the qualitative analysis result, a quantitative analysis is performed on the second detection data to obtain a quantitative analysis result.

[0010] In some embodiments, in step (2), a qualitative analysis is performed based on the first detection data to determine the primary ions corresponding to the reaction products and the target secondary fragments corresponding to each of the primary ions to obtain a qualitative analysis result, including:

[0011] Obtain the primary ion and theoretical fragment ion set corresponding to the unsaturated fatty acids in the lipid metabolite database;

[0012] Performing data extraction on the first detection data to obtain primary mass spectrum data and secondary mass spectrum data;

[0013] Determining the primary ion corresponding to the reaction product based on the theoretical reaction product mass in the primary mass spectrum data;

[0014] Screening out theoretical fragments corresponding to each of the primary ions from the theoretical fragment ion set;

[0015] Based on the theoretical fragments, the target secondary fragments within the retention time of the primary ions are matched from the secondary mass spectrometry data to obtain qualitative analysis results of the types of unsaturated fatty acids and double bond positions.

[0016] In some embodiments, the method of obtaining primary ions and theoretical fragment ion sets corresponding to unsaturated fatty acids in a lipid metabolite database includes: screening unsaturated fatty acids from the lipid metabolite database to obtain a double bond position reference data set; based on the fragmentation principle of the reaction product under secondary mass spectrometry, performing mass simulation on the fragment molecular formulas corresponding to the unsaturated fatty acids in the double bond position reference data set to obtain theoretical fragments corresponding to each unsaturated fatty acid, wherein the theoretical fragment ion set includes theoretical fragments corresponding to each of the unsaturated fatty acids.

[0017] In some embodiments, the data extraction of the first detection data to obtain primary mass spectrum data and secondary mass spectrum data includes: format conversion of the original data of the first detection data to obtain first detection data; and data extraction of the converted first detection data to obtain the primary mass spectrum data and the secondary mass spectrum data.

[0018] In some embodiments, determining the primary ion corresponding to the reaction product based on the theoretical reaction product mass in the primary mass spectrometry data includes: obtaining the theoretical retention time of the unsaturated fatty acids in the double bond position reference data set in the primary mass spectrometry data, and correction parameters of the unsaturated fatty acids for the retention time before and after the reaction; determining the primary ion corresponding to the reaction product based on the retention time, the theoretical retention time and the correction parameters.

[0019] In some embodiments, the secondary mass spectrometry data includes multiple secondary fragments, and based on the theoretical fragments, the target secondary fragments corresponding to the primary ions are determined from the secondary mass spectrometry data to obtain qualitative analysis results, including: obtaining the theoretical fragment mass corresponding to the theoretical fragments, and extracting the actual fragment mass corresponding to each of the secondary fragments from the secondary mass spectrometry data; comparing the theoretical fragment mass with the actual fragment mass, and determining the target secondary fragments corresponding to the primary ions and the fragment detection rate of the target secondary fragments based on the comparison result, and the qualitative analysis results include the fragment detection rate.

[0020] In some embodiments, the theoretical fragment mass and the actual fragment mass are compared, and the target secondary fragments corresponding to the primary ions and the fragment detection rate of the target secondary fragments are determined based on the comparison results, including: obtaining a preset mass error threshold; calculating the mass difference between the theoretical fragment mass and the actual fragment mass, and taking the secondary fragments corresponding to the mass difference less than the preset mass difference as the target secondary fragment; and determining the fragment detection rate of the target secondary fragment based on the number of the target secondary fragments and the theoretical fragments.

[0021] In some embodiments, after determining the target secondary fragments corresponding to the primary ions and the fragment detection rate of the target secondary fragments based on the comparison results, it also includes: establishing an extracted ion flow corresponding to the secondary fragments in each of the primary ions based on the secondary mass spectrometry data, the extracted ion flow including the target secondary fragments corresponding to the primary ions; determining the unsaturation degree of the unsaturated fatty acid corresponding to the primary ion; if the unsaturated fatty acid is a monounsaturated fatty acid, determining the target retention time and target signal intensity corresponding to the target secondary fragments, and the qualitative analysis results also include the target retention time and target signal intensity corresponding to the target secondary fragments.

[0022] In some embodiments, after determining the unsaturation degree of the unsaturated fatty acid corresponding to the primary ion, it also includes: if the unsaturated fatty acid is a polyunsaturated fatty acid, screening out the retention time corresponding to the target secondary fragment with the highest signal intensity in the extracted ion flow as the target retention time, and screening out the secondary mass spectrometry signal intensity corresponding to the target secondary fragment with the highest signal intensity in the extracted ion flow as the target signal intensity, and the qualitative analysis result also includes the target retention time and the target signal intensity.

[0023] In some embodiments, in step (3), based on the qualitative analysis result, the second detection data is quantitatively analyzed to obtain a quantitative analysis result, including: searching the second detection data for the primary ions corresponding to the reaction products and the target secondary fragments corresponding to each of the primary ions; determining the retention time of each of the target secondary fragments and matching it with the target retention time in the qualitative analysis result; based on the retention time, searching the second detection data for secondary fragment peaks that are within a preset range of the target retention time, and performing quantitative analysis on the secondary fragment peaks to obtain the quantitative analysis result.

[0024] It can be seen from the above technical scheme that the present specification provides a method for automatically identifying unsaturated fatty acids and the positions of their double bonds, using a carbonyl-containing compound as a PB reaction reagent, utilizing the principle of regular fragmentation of unsaturated fatty acids in collision dissociation after a PB reaction, and combining the data-dependent acquisition mode (Data Dependent Acquisition, abbreviated as DDA) and the data-independent acquisition mode (Data Independent Acquisition, abbreviated as DIA) in liquid chromatography tandem mass spectrometry to identify unsaturated fatty acids from the raw data of a large liquid tandem mass spectrometry. On the one hand, based on the DDA data, unsaturated fatty acids in non-targeted analysis can be quickly and accurately screened out and the number and position of their double bonds can be determined; on the other hand, based on the DIA data, quantitative analysis of unsaturated fatty acids can be performed. The combination of the two improves the accuracy and efficiency of qualitative and quantitative analysis of unsaturated fatty acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0026] Figure 1 A schematic flow chart of a method for automatically identifying unsaturated fatty acids and double bond positions provided in an embodiment of the present application;

[0027] Figure 2 A diagram showing the fragmentation law of the photocycloaddition reaction product under collision fragmentation involved in the embodiments of the present application;

[0028] Figure 3A The bar graphs are the signal intensities of FAx and FAy of each double bond in the monounsaturated fatty acid FA18:1 (Δ6, Δ9, Δ11) and the polyunsaturated fatty acid FA22:6 (Δ4, 7, 10, 13, 16, 19) under the DDA mode and the average of FAx and FAy under three parallel data acquisitions;

[0029] Figure 3B The standard curve of the signal intensity of FAx, FAy and the average of FAx and FAy of the secondary mass spectrometry fragments of FA18:1(Δ9) in DDA mode;

[0030] Figure 4AThe bar graphs are the peak areas of FAx and FAy of each double bond in the monounsaturated fatty acid FA18:1 (Δ6, Δ9, Δ11) and the polyunsaturated fatty acid FA22:6 (Δ4, 7, 10, 13, 16, 19) under three parallel data acquisitions in the DIA mode;

[0031] Figure 4B The standard curve of the peak areas of FAx, FAy and the average of FAx and FAy of the secondary mass spectrometry fragments of FA18:1(Δ9) in DIA mode;

[0032] Figure 5 A schematic diagram of a process for performing data analysis on a sample to be tested provided in an embodiment of the present application;

[0033] Figure 6 A schematic diagram of the qualitative analysis process provided in the embodiments of the present application. DETAILED DESCRIPTION

[0034] In order to facilitate the understanding of this specification, the specification will be described more fully below with reference to the relevant drawings. The preferred embodiments of this specification are given in the drawings. However, this specification can be implemented in many different forms without departing from the core spirit of this specification and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of this specification more thorough and comprehensive.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this specification belongs. The terms used herein in this specification are only for the purpose of describing specific embodiments and are not intended to limit this specification. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0036] The method for automatically identifying unsaturated fatty acids and their double bond positions provided in the embodiment of the present application mainly includes three steps: liquid chromatography-tandem mass spectrometry dual-mode data acquisition, DDA qualitative analysis and DIA quantitative analysis. Among them, the method of qualitative analysis removes redundancy with fatty acid information in an existing database, and obtains information such as the carbon number, degree of unsaturation and double bond position of unsaturated fatty acids from the database. At the position where PB reacts to form a four-membered ring, the four-membered ring will be broken in the secondary mass spectrum to produce a pair of characteristic fragments that conform to the fragmentation law, and all its theoretical secondary fragments are calculated. Traverse all secondary spectra, and the precursor ions that may be fatty acid PB products are used as screening objects to calculate the detection rate, time and intensity of theoretical fragments. The qualitative results obtained using DDA are quantified against DIA, so that isomers are identified and quantified.

[0037] Specifically, Figure 1The schematic diagram of the process flow of the method for automatically identifying unsaturated fatty acids and their double bond positions provided in the embodiment of the present application is shown. Figure 1 As shown, the embodiment of the present application provides a method for automatically identifying unsaturated fatty acids and double bond positions thereof, comprising the following steps:

[0038] S10. Mixing the sample to be tested with the reaction reagent and performing a photocycloaddition reaction to obtain a reaction product, detecting the reaction product using a data-dependent acquisition mode in a liquid chromatography tandem mass spectrometry to obtain first detection data, and detecting the reaction product using a data-independent acquisition mode in a liquid chromatography tandem mass spectrometry to obtain second detection data, wherein the sample to be tested includes at least one unsaturated fatty acid, and the reaction reagent is a compound containing a carbonyl group.

[0039] In the present application, the sample to be tested may be pre-treated. For example, if the sample to be tested contains protein, the sample to be tested may be subjected to protein precipitation. If fatty acids need to be extracted, the solvent system needs to be compatible with LC-MS / MS.

[0040] Figure 2 FIG. 2 shows the fragmentation law of the photocycloaddition reaction product under collision fragmentation involved in the embodiment of the present application. Figure 2 As shown, in the embodiment of the present application, the unsaturated fatty acids in the sample to be tested can react with the PB reaction reagent to form a four-membered ring. Due to the large tension of the four-membered ring, it will break in the high-energy collision, thereby generating characteristic fragment ions of the PB reagent, and the nitrogen-containing end will produce a high-intensity mass spectrometry signal. The double bond of a fatty acid will produce both positive and negative addition products, and two characteristic fragments will be generated in the secondary mass spectrum, among which the product of the carboxyl end break can be expressed as FAx, and the product of the carbon end break can be expressed as FAy.

[0041] There are two acquisition modes in LC-MS / MS, namely Data Dependent Acquisition (DDA) and Data Independent Acquisition (DIA). Most of the current data analysis focuses on DDA, which has the advantage of a small secondary acquisition window and can accurately obtain fragments of substances with higher signals. Most of the fragments are believed to come from the same parent ion. When DIA collects secondary fragments, the window is larger and more substances can be covered. The secondary mass spectra of almost all substances can be obtained, and the fragment peaks are continuous during the secondary mass spectrometry acquisition. Compared with DDA, it has a good peak shape and is easy to obtain the peak area. At present, the qualitative analysis of fatty acids using PB reaction still relies on DDA detection, and the intensity of the secondary mass spectrometry fragments is directly used as the quantitative result, but this quantitative result has a large error.

[0042] Figure 3A The bar graph shows the signal intensity of FAx and FAy of each double bond in the monounsaturated fatty acid FA18:1 (Δ6, Δ9, Δ11) and the polyunsaturated fatty acid FA22:6 (Δ4, 7, 10, 13, 16, 19) under three parallel data acquisitions in DDA mode, as well as the average value of FAx and FAy. Figure 3B The standard curve of the signal intensities of FAx, FAy and the average of FAx and FAy of the secondary mass spectrometry fragments of A18:1(Δ9) in DDA mode is shown. Figure 4A The bar graphs show the peak areas of FAx and FAy of each double bond in the monounsaturated fatty acid FA18:1 (Δ6, Δ9, Δ11) and the polyunsaturated fatty acid FA22:6 (Δ4, 7, 10, 13, 16, 19) under three parallel data acquisitions in the DIA mode, as well as the average values ​​of FAx and FAy. Figure 4B The standard curve of the peak areas of FAx, FAy and the average of FAx and FAy of the secondary mass spectrum fragments of FA18:1(Δ9) in DIA mode is shown.

[0043] Depend on Figure 3A , Figure 3B , Figure 4A and Figure 4B It can be seen that the use of DIA's secondary fragment peak area to quantify the secondary fragments can make up for the lack of DDA quantification.

[0044] Taking the above factors into consideration, the embodiments of the present application respectively adopt the data-dependent acquisition mode and the data-independent acquisition mode in liquid chromatography tandem mass spectrometry to detect the reaction products, wherein the data-dependent acquisition mode is adopted to detect the reaction products to obtain the first detection data, and the data-independent acquisition mode in liquid chromatography tandem mass spectrometry is adopted to detect the reaction products to obtain the second detection data, and then the first detection data and the second detection data are combined to qualitatively and quantitatively analyze the unsaturated fatty acids.

[0045] The liquid chromatography tandem mass spectrometer used in the embodiments of the present application is a high-resolution liquid chromatography-mass spectrometry instrument, wherein the mass error of the primary mass spectrometer should be less than 5 ppm, and the mass error of the secondary mass spectrometer should be less than 20 ppm.

[0046] Figure 5 The schematic diagram of the process of data analysis of the sample to be tested provided in the embodiment of the present application is shown. Figure 5As shown, since the first detection data is mass spectrometry data measured in a data dependent acquisition mode (DDA), that is, in the embodiment of the present application, the unsaturated fatty acids in the sample to be tested are qualitatively analyzed based on the DDA data. After obtaining the qualitative analysis results, the unsaturated fatty acids in the sample to be tested are quantitatively analyzed based on the DIA data (i.e., the second detection data) and the qualitative analysis results.

[0047] S20, performing a qualitative analysis based on the first detection data, determining the primary ions corresponding to the reaction products and the target secondary fragments corresponding to each primary ion, and obtaining a qualitative analysis result.

[0048] in, Figure 6 The schematic diagram of the qualitative analysis process provided by the embodiment of the present application is shown. Figure 6 As shown, step S20 may include:

[0049] Step S201, obtaining a set of primary ions and theoretical fragment ions corresponding to unsaturated fatty acids in a lipid metabolite database.

[0050] The lipid metabolism database can be LIPID MAPS. LIPID MAPS (Lipid Metabolites and Pathways Strategy) is a comprehensive lipid metabolite database designed to systematically study lipid molecules, their metabolic pathways and functions. LIPID MAPS provides detailed information on more than 40,000 lipids, including chemical structure, physical properties, distribution and biological functions. In addition, this database also includes lipid data from a variety of organisms (such as humans, mice and rats).

[0051] Among them, obtaining the theoretical fragment ion set corresponding to the unsaturated fatty acids in the lipid metabolite database can include: screening unsaturated fatty acids from the lipid metabolite database to obtain a double bond position reference data set; based on the fragmentation principle of the reaction product under the secondary mass spectrometry, performing mass simulation on the fragment molecular formulas corresponding to the unsaturated fatty acids in the double bond position reference data set to obtain the theoretical fragments corresponding to each unsaturated fatty acid, and the theoretical fragment ion set includes the theoretical fragments corresponding to each unsaturated fatty acid.

[0052] Since the database of fatty acids in LipidMaps contains the structure and position information of fatty acids, the embodiment of the present application can use the data in the database to retain the data of all unsaturated fatty acids and extract the position information of each unsaturated fatty acid. Specifically, all theoretical fragments under each unsaturated fatty acid can be simulated according to the aforementioned formula (1) and formula (2), and a database of fatty acids, double bond positions and secondary theoretical fragments corresponding to each other can be established, that is, a theoretical fragment ion set.

[0053] Step S202: extract the first detection data to obtain primary mass spectrum data and secondary mass spectrum data. The primary mass spectrum data and secondary mass spectrum data in this step are corresponding mass spectrum data in the DDA mode.

[0054] Among them, performing data extraction on the first detection data to obtain primary mass spectrum data and secondary mass spectrum data can include: converting the data format of the first detection data to obtain converted first detection data; performing data extraction on the converted first detection data to obtain primary mass spectrum data and secondary mass spectrum data.

[0055] In an embodiment of the present application, before the first detection data is processed, the data format of the first detection data can be converted into a format recognizable in the python library, for example, the first detection data can be converted into a .mzXML format. After the data format conversion of the first detection data, the converted first detection data can be obtained, and accordingly, the data format of the converted first detection data can be in a .mzXML format. Primary mass spectrometry data and secondary mass spectrometry data can be extracted from the converted first detection data.

[0056] Step S203: Determine the primary ion corresponding to the reaction product based on the theoretical reaction product mass in the primary mass spectrum data. For ease of explanation, the primary ion corresponding to the reaction product is represented as PB FA.

[0057] Among them, based on the theoretical reaction product mass in the primary mass spectrometry data, determining the primary ion corresponding to the reaction product can include: obtaining the retention time of unsaturated fatty acids in the primary mass spectrometry data set in the double bond position reference data set, and correction parameters for the retention time of unsaturated fatty acids before and after the reaction; based on the retention time, the theoretical retention time and the correction parameters, determining the retention time of the primary ion corresponding to the reaction product.

[0058] In the embodiment of the present application, in the extraction of the primary peak, a specific mass of extracted ion flow can be established first, and then the peak position can be calibrated using the function for finding the signal peak in Python (i.e., find_peaks function). The fatty acid peaks before the reaction are narrow, and many single peaks show Gaussian distribution. PB FA peaks often do not show a Gaussian distribution. PB When calibrating the peak position of FA, its peak width needs to be considered. For substances with greater unsaturation, a larger peak search distance is given. When looking for the end points at both ends of the peak, search from the cap to the left and right respectively. Since the peak often rises, falls, and then rises again, a larger stable window can be given at the end position to avoid premature termination.

[0059] In complex biological samples, not only fatty acids are contained, but also other lipids such as phospholipids and triglycerides with fatty acyl chains as the basic structure. Before the tandem mass spectrometry fragmentation, source cleavage will occur, resulting in the appearance of source cleavage products with the same mass as the fatty acid PB reaction product. The parents of these source cleavages are all substances with smaller polarity and have a longer retention time in the reverse column. In the embodiment of the present application, this feature can be used to eliminate the false positive results of non-free fatty acids. For example, the peak time of the fatty acid before the reaction can be calculated first, and then used to calibrate the fatty acid product after the reaction.

[0060] First, calculate the fatty acids contained before the reaction and the retention time (RT) of each fatty acid. For easy distinction, the retention time corresponding to the fatty acid before the reaction is called the theoretical retention time. Since the peak time of homologues is linear, the peak time of all fatty acids can be predicted by this rule. The four monounsaturated fatty acids FA 16:1, FA 18:1, FA 20:1, and FA 22:1 are abundant in biological samples and have good mass spectrometry response. They can be used to establish a standard curve of carbon number and RT when the degree of unsaturation is 1.

[0061] When the degree of unsaturation is higher, the slope of the standard curve is equivalent to that when the degree of unsaturation is 1. With each increase in unsaturation, the theoretical retention time in the reversed phase column gradually decreases and the difference is equivalent. Therefore, the RT difference between FA 18:2 and FA18:1 can be selected as the correction parameter to predict the theoretical RT of all detected fatty acids, and the reasonable actual peak time of fatty acids can be retained when the mass error and time error allow. PB The peak interval of FA is due to the addition of a reaction reagent. PB Compared with fatty acids, FA has a higher polarity and its peak time will be earlier. Similarly, FA 18:1 can be used before reaction. PB The maximum retention time difference of FA was used as the calibration parameter, and the peak time of fatty acids was used to calibrate the reasonable PB Retention time of FA.

[0062] Step S204, screening out theoretical fragments corresponding to each primary ion from the theoretical fragment ion set. After the primary ion corresponding to the reaction product is determined, the theoretical fragments corresponding to each primary ion can be screened out from the theoretical fragment ion set.

[0063] Step S205: Based on the theoretical fragments, the target secondary fragments within the retention time of the primary ion are matched from the secondary mass spectrometry data to obtain the qualitative analysis results of the unsaturated fatty acid types and double bond positions. After determining the theoretical fragments corresponding to each primary ion, it is necessary to compare the actual peak results of the secondary mass spectrometry data with the theoretical peak results of the theoretical fragments, and then determine the target secondary fragments of the primary ion based on the comparison results, and determine the fragment detection rate based on the difference between the target secondary fragments and the theoretical fragments.

[0064] The secondary mass spectrum data may include multiple secondary fragments.

[0065] On this basis, step S205, based on the theoretical fragments, matches the target secondary fragments within the retention time of the primary ion from the secondary mass spectrometry data to obtain the qualitative analysis results of the unsaturated fatty acid types and double bond positions, which may include: obtaining the theoretical fragment mass corresponding to the theoretical fragments, and extracting the actual fragment mass corresponding to each secondary fragment from the secondary mass spectrometry data; comparing the theoretical fragment mass with the actual fragment mass, and determining the target secondary fragments corresponding to the primary ion and the fragment detection rate of the target secondary fragments based on the comparison results. The qualitative analysis results include the fragment detection rate.

[0066] In the present embodiment, the precursor ions of all secondary mass spectrometry data can be PB The data of FA are extracted and matched to its secondary mass spectrometry data. Without considering the isomerism of the double bond position, the secondary mass spectrometry data of the same FA are merged. Match the theoretical fragment mass in the LipidMaps database, extract the extracted ion flow in the secondary mass spectrometry that meets the fragment mass within the mass error, and take the time at the highest point of the signal intensity as the retention time of the fragment. Due to the poor secondary response of low-concentration polyunsaturated fatty acids, some secondary mass spectrometry fragments are missing, so the secondary mass spectrometry fragment detection rate of some fatty acids cannot reach 1.0.

[0067] Among them, comparing the theoretical fragment mass with the actual fragment mass, and determining the target secondary fragments corresponding to the primary ions and the fragment detection rate of the target secondary fragments based on the comparison results, can include: obtaining a preset mass error threshold; calculating the mass difference between the theoretical fragment mass and the actual fragment mass, and taking the secondary fragments corresponding to the mass difference less than the preset mass difference as the target secondary fragments; based on the number of target secondary fragments and theoretical fragments, determining the fragment detection rate of the target secondary fragments. The preset mass error threshold can be set according to actual conditions, and the comparison in this application is not limited.

[0068] After determining the target secondary fragments corresponding to the primary ions and the fragment detection rate of the target secondary fragments based on the comparison results, the method can also include: establishing an extracted ion flow corresponding to the secondary fragments in each primary ion based on the secondary mass spectrometry data, the extracted ion flow including the target secondary fragments within the retention time of the primary ion; determining the unsaturation degree of the unsaturated fatty acid corresponding to the primary ion; if the unsaturated fatty acid is a monounsaturated fatty acid, determining the target retention time and target signal intensity corresponding to the target secondary fragments, and the qualitative analysis results also include the target retention time and target signal intensity corresponding to the target secondary fragments.

[0069] like Figure 3A and Figure 4A As shown, for polyunsaturated fatty acids, the mass signals of FAx and FAy far from the double bond position at the carboxyl end are stronger, and the signals of FAx and FAy at the position of UN=6, i.e., Δ19, in FA 22:6. The double bond at the maximum position of each polyunsaturated fatty acid is selected as the result, and the mass, retention time, signal intensity, and fragment detection rate of FAx and FAy are output. In this regard, after determining the unsaturation degree of the unsaturated fatty acid corresponding to the primary ion, it can also include: if the unsaturated fatty acid is a polyunsaturated fatty acid, the retention time corresponding to the target secondary fragment with the highest signal intensity is screened out in the extracted ion flow as the target retention time, and the secondary mass spectrometry signal intensity corresponding to the target secondary fragment with the highest signal intensity is screened out in the extracted ion flow as the target signal intensity, and the qualitative analysis results also include the target retention time and the target signal intensity.

[0070] In the embodiment of the present application, considering the randomness of the DDA data (first detection data) during collection, the data can be collected twice or more (e.g., three times, four times, etc.) in parallel during qualitative analysis, and a comprehensive judgment is made based on the results of each parallel experiment to determine the qualitative analysis results of the sample to be tested, so as to reduce the experimental error caused by the randomness of the DDA mode. After determining the qualitative analysis results, the DDA data is used to find the peak value in the DIA data (second detection data) of each sample to be tested for statistical analysis to obtain the quantitative analysis results.

[0071] S30, based on the qualitative analysis result, the second detection data is quantitatively analyzed to obtain a quantitative analysis result. In the embodiment of the present application, after obtaining the qualitative analysis result in the DDA mode, the primary ions in the DIA mode data can be searched, and according to the retention time of the theoretical fragments recorded in the qualitative analysis result, the peak position of the theoretical fragments at a similar retention time under the corresponding primary ions is searched and the peak area is calculated, so as to obtain the quantitative analysis result.

[0072] Among them, step S30 may include: searching for the primary ions corresponding to the reaction products and the target secondary fragments corresponding to each primary ion in the second detection data; determining the retention time of each target secondary fragment, and matching it with the target retention time in the qualitative analysis result; based on the retention time, searching for the secondary fragment peaks within a preset range with the target retention time from the second detection data, performing quantitative analysis on the secondary fragment peaks, and obtaining quantitative analysis results.

[0073] When determining the peak area of ​​the secondary mass spectrum fragments by the DIA mode, the method in the aforementioned step S20 can be used to extract the peak area corresponding to RT in the qualitative analysis result using the secondary mass spectrum data in the second detection data. The peak shape of the extracted ion chromatogram under the secondary mass spectrum of FAx and FAy is usually Gaussian distribution relative to its parent ion, and the error in calculating the area will also be smaller.

[0074] According to the above analysis, the embodiment of the present application obtains the theoretical mass-to-charge ratio of paired ions according to the addition and fragmentation characteristics to match all secondary fragments to screen unsaturated fatty acids in the sample to be tested and determine the double bond position of the unsaturated fatty acids. Since both DDA and DIA are methods for collecting secondary mass spectrometry data, excluding the contingency of DDA, the peak time of DIA is theoretically the same as that of DDA. Therefore, the embodiment of the present application uses the retention time and intensity of the theoretical fragments of the secondary mass spectrometry of DDA with higher sensitivity as a reference, and searches for the corresponding peaks in the DIA data for integral quantification, which can improve the accuracy and efficiency of qualitative and quantitative analysis of unsaturated fatty acids.

[0075] The following are specific preparation examples involved in the above content of the present disclosure. It should be clear that the following examples are only for illustrating the method disclosed above for screening the double bond position of unsaturated fatty acids using liquid chromatography tandem mass spectrometry, and the specific implementation methods and parameters used therein are only one or several methods in accordance with the numerous processes and methods described above. Those skilled in the art can use other parameters according to the content introduced in this specification to screen the double bond position of unsaturated fatty acids according to the above method, and perform qualitative and quantitative analysis of unsaturated fatty acids without deviating from the core spirit disclosed in the application.

[0076] Example 1

[0077] This embodiment provides a method for screening the double bond position of unsaturated fatty acids using liquid chromatography tandem mass spectrometry, using a mixed standard as a sample to be tested and 2-acetylpyridine (2-AP) as a PB reaction reagent. Among them, the mixed standard includes 20 unsaturated fatty acids. The nomenclature of each unsaturated fatty acid refers to LipidMaps. The abbreviations of the 18 fatty acids are FA14:1(9Z), FA 15:1(10Z), FA16:1(9Z), FA 17:1(10Z), FA 18:1(6Z), FA18:1(9Z), FA18:1(11Z), FA18:2(9Z,12Z), FA 18:3(9Z,12Z,15Z), FA 18:3(6Z,9Z,12Z), FA 20:1(11Z), FA 20:2(11Z,14Z), FA 20:3(11Z,14Z,17Z), FA 20:3(8Z,11Z,14Z), FA20:4(5Z,8Z,11Z,14Z), FA 20:5(5Z,8Z,11Z,14Z,17Z), FA 22:1(13Z), FA 22:2(13Z,16Z), FA 22:6(4Z,7Z,10Z,13Z,16Z,19Z), FA 24:1(15Z).

[0078] S1. Detection

[0079] The mixed standard was dissolved in isooctane to obtain a mixed standard solution with a concentration of 10 μg / mL. The mixed standard solution was mixed evenly with the PB reaction reagent 2-AP, 20 μL of the solution was injected into a high-transmittance quartz tube, and the solution was irradiated with a 254 nm wavelength ultraviolet lamp for 2 minutes. The reaction products were detected using the data-dependent acquisition mode (DDA) and the data-independent acquisition mode (DIA) in the liquid chromatography-tandem mass spectrometry. Among them, the reactant was detected using the data-dependent acquisition mode (DDA) in the liquid chromatography-tandem mass spectrometry to obtain the first detection data; the reactant was detected using the data-independent acquisition mode (DIA) in the liquid chromatography-tandem mass spectrometry to obtain the second detection data.

[0080] S2. Qualitative analysis

[0081] After the file format of the first detection data is converted to .mzXML, the algorithm is used to calculate it. The fatty acid database downloaded from Lipid Maps is used for de-redundancy, and unsaturated fatty acids with non-repeated positions are retained. The carbon number, unsaturation and double bond position of the unsaturated fatty acids are obtained from the information in the library. At the position where PB reacts to form a four-membered ring, the HCD secondary mass spectrometer will break the four-membered ring to produce a pair of characteristic fragments that conform to the fragmentation law, and calculate the theoretical secondary fragments of all unsaturated fatty acids. Among them, the product of the carboxyl end rupture can be expressed as FAx, and the product of the carbon end rupture can be expressed as FAy.

[0082] FAx = C x H (2x-2a) O2 C7H8N (Formula 1)

[0083] FAy=C y H (2y-2FA_UN+2a) C7H8N (Formula 2)

[0084] Among them, x is its position under the Δ nomenclature, a is its ath double bond in the Δ direction, y is its position under the ω nomenclature, and FA_UN is the degree of unsaturation of the fatty acid. According to these two structural formulas, the molecular formulas of FAx and FAy can be calculated from fatty acids with double bond position information, and then the molecular weight can be simulated.

[0085] Based on the above principles, primary data extraction is performed first in qualitative analysis. In the process of extracting primary data, false positive results of non-free fatty acids can be eliminated. The peak time of fatty acids before the reaction is calculated first, and then used to calibrate the fatty acid products after the reaction.

[0086] Specifically, the fatty acids contained before the reaction and the retention time (RT) of each fatty acid are first calculated. Since the peak time of homologues is linear, the peak time of all fatty acids can be predicted by this rule. The monounsaturated fatty acids FA 16:1, FA 18:1, FA 20:1, and FA 22:1 are abundant in biological samples and have good mass spectrometry response. They can be used to establish a standard curve of carbon number and RT when the degree of unsaturation is 1.

[0087] When the degree of unsaturation is higher, the slope of the standard curve is equivalent to that when the degree of unsaturation is 1. With each increase in unsaturation, the RT in the reverse phase column gradually decreases and the difference is equivalent. Therefore, the RT difference between FA 18:2 and FA 18:1 can be selected as the correction parameter to predict the theoretical RT of all detected fatty acids, and the reasonable actual peak time of fatty acids can be retained when the mass error and time error allow. The peak interval of all PBFA after extraction is PBCompared with fatty acids, FA has a higher polarity and its peak time will be earlier. PB The maximum retention time difference of FA was used as the calibration parameter, and the peak time of fatty acids was used to calibrate the reasonable PB Retention time of FA.

[0088] After the primary data is extracted, the secondary data can be extracted and identified. All secondary spectra are traversed and those within the mass error of 20ppm are considered as screening objects. The theoretical fragment detection rate, intensity and retention time of the theoretical fragments in the library are determined. Specifically, all the precursor ions of the secondary mass spectrometry data are PB The data of FA are extracted and matched to its secondary mass spectrometry data. Without considering the isomerism of the double bond position, the secondary mass spectrometry data of the same FA are merged. Match the theoretical fragment mass in the LipidMaps database, extract the extracted ion flow in the secondary mass spectrometry that meets the fragment mass within the mass error, and take the time at the highest point of the signal intensity as the retention time of the fragment. Due to the poor secondary response of low-concentration polyunsaturated fatty acids, some secondary mass spectrometry fragments are missing, so the secondary mass spectrometry fragment detection rate of some fatty acids cannot reach 1.0.

[0089] For monounsaturated fatty acids, the final output will be the corresponding results of mass, retention time, intensity, and detection rate of FAx and FAy.

[0090] For polyunsaturated fatty acids, since the mass signals of FAx and FAy far away from the double bond position at the carboxyl end are stronger, the double bond at the maximum position of each polyunsaturated fatty acid is selected as the result, and the mass, retention time, intensity and detection rate of FAx and FAy are output.

[0091] S3. Quantitative analysis

[0092] In the second detection data, find the extracted ion flow diagram of the fragment ion corresponding to the DDA theoretical fragment peak time to calculate the peak area for quantitative analysis. When taking the peak area of ​​the secondary mass spectrometry fragment of DIA, it is necessary to find the precursor ion of the corresponding data collection like DDA. The precursor ion is calculated using the peak of the parent ion in the primary secondary mass spectrometry data. After selecting the precursor ion, extract the extracted ion flow corresponding to FAx and FAy under the precursor ion. Referring to the peak area corresponding to RT in the DDA result, the extracted ion flow under the secondary mass spectrometry of FAx and FAy usually presents a Gaussian distribution relative to its parent ion, and the error when calculating the area will also be smaller.

[0093] Table 1 shows the data calculation results of Example 1 after LC-MS / MS analysis using the mixed standard as the sample to be tested.

[0094] Table 1. Data analysis results of Example 1

[0095]

[0096]

[0097] Example 2

[0098] This example adopts the same analytical method as Example 1, except that: this example uses serum as the sample to be tested. The serum extraction is a methyl tert-butyl ether (MTBE) extraction method, taking a certain volume of serum, adding methanol to precipitate protein and vortexing, adding MTBE and vortexing, shaking extraction, adding ultrapure water and centrifuging, taking the upper layer of MTBE extracted lipids and vacuum drying the solvent. Then, it is re-dissolved with a solvent of acetonitrile / isopropanol / water = 65 / 30 / 5 for standby use.

[0099] Table 2 shows the data calculation results of Example 2 after LC-MS / MS analysis using serum as the test sample.

[0100] Table 2. Data analysis results of Example 2

[0101]

[0102]

[0103]

[0104]

[0105] As shown in Table 2, all components with high content in serum can be detected by secondary mass spectrometry and their positions can be calculated. However, due to insufficient secondary collection of some components, the theoretical fragment detection of some polyunsaturated fats cannot reach 100%, but the results are still quite referenceable.

[0106] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0107] The above-described embodiments only express several embodiments of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. A method for automatically identifying unsaturated fatty acids and their double bond positions, It is characterized in that The following steps are involved: (1) mixing a sample to be tested with a reaction reagent and performing a photocycloaddition reaction to obtain a reaction product, detecting the reaction product using a data-dependent acquisition mode in a liquid chromatography tandem mass spectrometer to obtain first detection data, and detecting the reaction product using a data-independent acquisition mode in the liquid chromatography tandem mass spectrometer to obtain second detection data, wherein the sample to be tested includes at least one unsaturated fatty acid, and the reaction reagent is a compound containing a carbonyl group; (2) performing a qualitative analysis based on the first detection data to determine the primary ions corresponding to the reaction products and the target secondary fragments corresponding to each of the primary ions, thereby obtaining a qualitative analysis result; (3) Based on the qualitative analysis result, a quantitative analysis is performed on the second detection data to obtain a quantitative analysis result.

2. The method for automatically identifying unsaturated fatty acids and double bond positions thereof as claimed in claim 1, It is characterized in that In the step (2), a qualitative analysis is performed based on the first detection data to determine the primary ions corresponding to the reaction products and the target secondary fragments corresponding to each of the primary ions to obtain a qualitative analysis result, including: Obtain the primary ion and theoretical fragment ion set corresponding to the unsaturated fatty acids in the lipid metabolite database; Performing data extraction on the first detection data to obtain primary mass spectrum data and secondary mass spectrum data; Determining the primary ion corresponding to the reaction product based on the theoretical reaction product mass in the primary mass spectrum data; Screening out theoretical fragments corresponding to each of the primary ions from the theoretical fragment ion set; Based on the theoretical fragments, the target secondary fragments within the retention time of the primary ions are matched from the secondary mass spectrometry data to obtain qualitative analysis results of the types of unsaturated fatty acids and double bond positions.

3. The method for automatically identifying unsaturated fatty acids and double bond positions thereof as claimed in claim 2, It is characterized in that The method of obtaining the primary ion and theoretical fragment ion set corresponding to the unsaturated fatty acid in the lipid metabolite database includes: Screening unsaturated fatty acids from the lipid metabolite database to obtain a double bond position reference data set; Based on the fragmentation principle of the reaction product under secondary mass spectrometry, mass simulation is performed on the fragment molecular formulas corresponding to the unsaturated fatty acids in the double bond position reference data set to obtain theoretical fragments corresponding to each unsaturated fatty acid, and the theoretical fragment ion set includes the theoretical fragments corresponding to each unsaturated fatty acid.

4. The method for automatically identifying unsaturated fatty acids and double bond positions thereof as claimed in claim 2, It is characterized in that The step of extracting the first detection data to obtain primary mass spectrum data and secondary mass spectrum data includes: Converting the format of the original data of the first detection data to obtain first detection data; Data extraction is performed on the converted first detection data to obtain the primary mass spectrum data and the secondary mass spectrum data.

5. The method for automatically identifying unsaturated fatty acids and double bond positions thereof as claimed in claim 2, It is characterized in that The determining of the primary ion corresponding to the reaction product comprises: Obtaining the retention time of the unsaturated fatty acid product in the double bond position reference data set in the primary mass spectrum data, and the correction parameters of the unsaturated fatty acid before and after the reaction for the retention time; Based on the retention time, the theoretical retention time and the calibration parameter, the primary ion corresponding to the reaction product is determined.

6. The method for automatically identifying unsaturated fatty acids and double bond positions thereof as claimed in claim 2, It is characterized in that The secondary mass spectrum data includes a plurality of secondary fragments, The step of determining the target secondary fragments corresponding to the primary ions from the secondary mass spectrometry data based on the theoretical fragments to obtain a qualitative analysis result includes: Obtaining theoretical fragment masses corresponding to the theoretical fragments, and extracting actual fragment masses corresponding to each of the secondary fragments from the secondary mass spectrum data; The theoretical fragment mass and the actual fragment mass are compared, and based on the comparison result, the target secondary fragment corresponding to the primary ion and the fragment detection rate of the target secondary fragment are determined, and the qualitative analysis result includes the fragment detection rate.

7. The method for automatically identifying unsaturated fatty acids and double bond positions thereof as claimed in claim 6, It is characterized in that The comparing the theoretical fragment mass with the actual fragment mass, and determining the target secondary fragment corresponding to the primary ion and the fragment detection rate of the target secondary fragment based on the comparison result, comprises: Obtaining a preset quality error threshold; Calculating the mass difference between the theoretical fragment mass and the actual fragment mass, and taking the secondary fragment corresponding to the mass difference being smaller than the preset mass difference as the target secondary fragment; Based on the number of the target secondary fragments and the theoretical fragments, a fragment detection rate of the target secondary fragments is determined.

8. The method for automatically identifying unsaturated fatty acids and double bond positions thereof as claimed in claim 6, It is characterized in that After determining the target secondary fragments corresponding to the primary ions and the fragment detection rate of the target secondary fragments based on the comparison result, the method further includes: Based on the secondary mass spectrometry data, establishing an extracted ion flow corresponding to each secondary fragment in the primary ion, wherein the extracted ion flow includes a target secondary fragment corresponding to the primary ion; Determining the degree of unsaturation of the unsaturated fatty acid corresponding to the primary ion; If the unsaturated fatty acid is a monounsaturated fatty acid, the target retention time and the target signal intensity corresponding to the target secondary fragment are determined, and the qualitative analysis result also includes the target retention time and the target signal intensity corresponding to the target secondary fragment.

9. The method for automatically identifying unsaturated fatty acids and double bond positions thereof as claimed in claim 8, It is characterized in that After determining the unsaturation degree of the unsaturated fatty acid corresponding to the primary ion, the method further comprises: If the unsaturated fatty acid is a polyunsaturated fatty acid, the retention time corresponding to the target secondary fragment with the highest signal intensity is screened out in the extracted ion flow as the target retention time, and the secondary mass spectrometry signal intensity corresponding to the target secondary fragment with the highest signal intensity is screened out in the extracted ion flow as the target signal intensity, and the qualitative analysis result also includes the target retention time and the target signal intensity.

10. The method for automatically identifying unsaturated fatty acids and double bond positions thereof as claimed in claim 1, It is characterized in that In the step (3), based on the qualitative analysis result, the second detection data is quantitatively analyzed to obtain a quantitative analysis result, including: Searching the second detection data for the primary ions corresponding to the reaction products and the target secondary fragments corresponding to each of the primary ions; Determining the retention time of each of the target secondary fragments and matching it with the target retention time in the qualitative analysis result; Based on the retention time, secondary fragment peaks having a preset range with respect to the target retention time are searched from the second detection data, and quantitative analysis is performed on the secondary fragment peaks to obtain the quantitative analysis result.

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