CN112129875B - Mass spectrometry method for identifying phosphatidylcholine chain length isomer - Google Patents
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Abstract
The present invention relates to a mass spectrometry method for identifying a phosphatidylcholine chain length isomer, the method comprising the steps of: constructing a phosphatidylcholine chain length isomer and characteristic ion high-resolution mass spectrum simulation database; and (3) establishing a liquid chromatography-high resolution mass spectrometer phosphatidylcholine identification method, carrying out liquid chromatography-high resolution mass spectrometer combined detection on a sample to be detected, and carrying out mass spectrometry analysis according to the accurate mass number mass spectrum characteristics of phosphatidylcholine positive ions and negative ion tertiary fragment ions and the comparison of a simulation database to obtain the identification result of phosphatidylcholine chain length isomers. Can be used for identifying functional phosphatidylcholine and provides technical support for developing novel functional phosphatidylcholine.
Description
Technical Field
The invention belongs to the field of chemical analysis and detection, and particularly relates to a mass spectrometry method for identifying a phosphatidylcholine chain length isomer.
Background
Phosphatidylcholine (PC), a lipid formed by dehydration condensation of glycerol, fatty acid (molecule), phosphoric acid and choline (fig. 1), wherein the fatty acid chain in the molecule has two types of saturated and unsaturated, and is a nonpolar hydrophobic tail; the phosphorylcholine moiety is a dipole ion, a polar hydrophilic head, and is known as a "third nutrient" after proteins and vitamins. It is the material basis of life, the basic material of various membrane structures of cells, and exists in each cell.
Phosphatidylcholine is a major component of cell membranes and can promote the absorption of nutrients by cells and the removal of toxic waste. Can assist the absorption of fat-soluble vitamins A, D, E and K and increase the utilization rate of the vitamins by 100%, and can supplement enough phosphatidylcholine to promote the activity of cells and enhance the immunity and disease resistance of human bodies. Phosphatidylcholine is a powerful emulsifier which breaks down cholesterol and neutral fat in blood into very small particles for absorption and metabolism by tissues, thereby preventing deposition of cholesterol in blood vessels and preventing arteriosclerosis, hypertension and heart diseases caused by cholesterol deposition. Phosphatidylcholine is a relatively typical compatible molecule, and participates in the composition of various lipoproteins, and is an indispensable substance for the catalytic action of various lipid-dependent enzymes. It is involved in the mechanism of action of hormones and plays an important role in the transmission of neuronal excitation.
The phosphatidylcholine-rich material includes: egg yolk, milk, animal brain, bone marrow, heart, lung, liver, kidney, soybean, yeast, etc. Phosphatidylcholine is often bound to proteins in vivo and exists in the form of fatty proteins (lipoproteins). Due to the excellent physiological function of phosphatidylcholine, the U.S. Food and Drug Administration (FDA) specifies: in infant milk powder, lecithin (phosphatidylcholine) must be added.
A large number of researches show that the phosphatidylcholine isomer molecules containing the functional fatty acid in the structure have better performance, not only have the physiological functions of phospholipid and the functional fatty acid, but also have more stable structure, are not easy to generate oxidation reaction, and are more beneficial to the absorption and utilization of human bodies. The functional phosphatidylcholine has a plurality of physiological functions of promoting the development of the brain nervous system, improving memory, reducing blood fat, reducing cholesterol, resisting aging, preventing cancer and the like [1-2]. However, such functional phosphatidylcholine isomer molecules have been difficult to identify.
The high-resolution mass spectrometer has the characteristics of high resolution, high sensitivity and high quality precision, can generate a large amount of mass spectrum information in one scanning, can obtain multi-stage mass spectrum fragments of unknown numbers, and can obtain a large amount of lipid information, thereby realizing detection and identification of complex phosphatidylcholine. In particular, with the development of separation-based "liquid chromatography-mass spectrometry", precise structural analysis of lipids and biological effects caused by precise structures are becoming hot spots for research. Although double bond positional isomers of phosphatidylcholine can be identified by tandem mass spectrometry, functional phosphatidylcholine isomer molecules still need to be mined for a promising way of mass spectrometry feature fragmentation.
Reference is made to: [1] zhang Qin, sun Zhaomin, xu Jie, et al, enzymatic synthesis of phosphatidylserine [ J ] rich in docosahexaenoic acid chinese food journal, 2016, 16 (3): 81-87.
[2] wang Xiang, wei Fang, dong Xuyan, etc. enzymatic synthesis of phospholipids [ J ] rich in arachidonic acid structures chinese oil crop journal, 2015, 37 (6): 889-896.
disclosure of Invention
The invention aims to provide a mass spectrometry method for identifying a Phosphatidylcholine (PC) chain length isomer.
In order to solve the technical problems, the invention adopts the following technical scheme:
a mass spectrometry method for identifying a phosphatidylcholine chain length isomer comprising the steps of:
constructing a phosphatidylcholine chain length isomer and characteristic ion high-resolution mass spectrum simulation database;
and (3) establishing a liquid chromatography-high resolution mass spectrometer phosphatidylcholine identification method, carrying out liquid chromatography-high resolution mass spectrometer combined detection on a sample to be detected, and carrying out mass spectrometry analysis according to the accurate mass number mass spectrum characteristics of phosphatidylcholine positive ions and negative ion tertiary fragment ions and the comparison of a simulation database to obtain the identification result of phosphatidylcholine chain length isomers.
In the scheme, mass spectrum detection conditions in liquid chromatography-high resolution mass spectrum combined detection are as follows: the secondary collision energy CE is 30-40 eV, and multi-stage mass spectrum data of the sample to be detected in the high-resolution mass spectrum are collected.
In the above scheme, the secondary and tertiary fragmentation methods used in mass spectrometry detection are HCD (High-energy C-trap Dissociation).
In the scheme, the liquid chromatography detection conditions in the liquid chromatography-high resolution mass spectrometry combined detection are as follows: the mobile phase A is acetonitrile/water mixed solution with the concentration of 5/5-7/3 and the concentration of v/v, and contains 0.05-0.15% formic acid and 8-12mmol/L ammonium formate; the mobile phase B is an isopropanol/acetonitrile mixed solution with the concentration of 85/15-95/5 and the concentration of v/v, contains 0.05-0.15 percent of formic acid, 8-12mmol/L of ammonium formate, and has the elution gradient of 0min and 10-20 percent of B;4min, 20-40% of B;5min, 45-50% B;22min, 80-85% of B;23-24min, 98-99% B;24.2-30min, 10-20% B.
In the scheme, mass spectrum information is acquired in a positive ion mode and a negative ion mode respectively; the mass spectrum fragment ions combined with MS1 in the positive ion mode obtain unique molecular formula, and are further combined with MS 2 And MS (MS) 3 Identifying the characteristic of phosphatidylcholine which is different from other types of phospholipids, and analyzing whether the phosphatidylcholine is phosphatidylcholine or not; and then, recognizing the chain length isomer of the phosphatidylcholine by combining mass spectrum fragment ions of MS2 in a negative ion mode, and recognizing the fatty acid connected on the phosphoric acid head group of the phosphatidylcholine.
In the scheme, the mobile phase used in the liquid chromatography detection contains formic acid and ammonium formate, the volume percentage of the formic acid is 0.05-0.15%, and the volume percentage of the ammonium formate is 8-12mmol/L.
In the positive ion scan mode, phosphatidylcholine lipids all produce parent ions [ M+H ] in the primary mass spectrum] + Wherein phosphatidylcholine, in the secondary mass spectrum, generates characteristic fragments of choline Head group [ head+OH+H ]] + (m/z 184.0733) production of characteristic fragments [ head+H+OH-N (CH) in a tertiary mass spectrum 3 ) 3 ] + (m/z 124.9998); in the negative ion mode, havePhosphatidylcholine having a choline head group generates a parent ion of [ M+HCOO]-, in a secondary mass spectrum, produce [ R ] 1 COO] - 、[R 2 COO] - 、[M-R 1 CO] - 、[M-R 2 CO] - And structural fragments associated with phosphate head groups. The recognition thought is thus passed through "level-filtering", whereupon recognition analysis of phosphatidylcholine is performed.
In the above scheme, the phosphatidylcholine chain length isomer and characteristic ion high resolution mass spectrum simulation database is characterized in that: according to the known 37 fatty acids (table 1), if two fatty acid acyl chains of phosphatidylcholine are not simultaneously in AB type, if two fatty acid acyl chains of phosphatidylcholine are not simultaneously in AA type, when phosphatidylcholine is in AB type, the phosphate head group type of phosphatidylcholine (fig. 2) and any 2 fatty acids are linked to the glycerol backbone, and all of the AB type phosphatidylcholine (1332 types) can be combined. When the phosphatidylcholine is of AA type, the phosphohead type of the phosphatidylcholine and any 1 fatty acid are connected to a glycerol skeleton, so that 37 types of all AA type phosphatidylcholine can be combined, and a phosphatidylcholine chain length isomer compound database is constructed: comprises 1369 phosphatidylcholine molecular formula, molecular weight, structural formula, and constructing phosphatidylcholine chain length isomer high-resolution mass spectrum database according to three-stage fragment ion mass spectrum characteristics of phosphatidylcholine chain length isomer in high-resolution mass spectrum: comprises a primary characteristic mass spectrum, a secondary characteristic mass spectrum, a tertiary characteristic mass spectrum, positive and negative ion tertiary characteristic fragment ions (10952 total) of phosphatidylcholine chain length isomers and accurate mass numbers.
In the above scheme, the phosphatidylcholine chain length isomer and characteristic ion high resolution mass spectrum simulation database is characterized in that: according to the phosphatidylcholine mass spectrum cleavage law (as shown in fig. 3), for each phosphatidylcholine molecule in the constructed simulation library, the accurate mass numbers of the corresponding secondary mass spectrum and tertiary mass spectrum fragments are generated, and theoretical molecular weights are calculated, so that a simulation database containing lipid molecules, molecular formulas, molecular weights, structural formulas of secondary and tertiary mass spectrum fragments and the accurate mass numbers is constructed.
In the scheme, the method for identifying the phosphatidylcholine by establishing the liquid chromatography-high resolution mass spectrometer comprises the following steps of: by MS1 accurate mass number, mass tolerance (mass tolerance) is set to 1ppm or less, and unique elemental composition is identified.
The invention has the advantages that:
the invention breaks through the accurate mass spectrum characteristics of the three-stage fragment ions of phosphatidylcholine positive ions and negative ions; constructing a phosphatidylcholine chain length isomer and characteristic ion high-resolution mass spectrum simulation database; a liquid chromatography-high resolution mass spectrometer phosphatidylcholine identification method is established. Can be used for identifying functional phosphatidylcholine and provides technical support for developing novel functional phosphatidylcholine.
Drawings
FIG. 1 phosphatidylcholine structural formula
FIG. 2 phosphohead type of phosphatidylcholine of FIG. 1
FIG. 3 phosphatidylcholine positive and negative ion mass spectrum cleavage law
FIG. 4 primary mass spectrum of PC (18:0/18:2) in positive ion mode
FIG. 5 secondary mass spectrum of ion m/z 786.6025 of PC (18:0/18:2) in positive ion mode
FIG. 6 a three-level mass spectrum of the second-level fragment ion m/z184.0733 of PC (18:0/18:2) in positive ion mode
FIG. 7 Primary mass spectrum of PC (18:0/18:2) in negative ion mode
FIG. 8 secondary mass spectrum of ion m/z 830.5888 of PC (18:0/18:2) in negative ion mode
FIG. 9 Primary mass spectrum of PC (18:1/18:1) in positive ion mode
FIG. 10 secondary mass spectrum of ion m/z 786.6025 of PC (18:1/18:1) in positive ion mode
FIG. 11 a three-level mass spectrum of the second-level fragment ion m/z184.0733 of PC (18:1/18:1) in the positive ion mode
FIG. 12 Primary mass spectrum of PC (18:1/18:1) in anion mode
FIG. 13 is a secondary mass spectrum of ion m/z 830.5888 of PC (18:1/18:1) in negative ion mode.
Detailed Description
Example 1
1. Preparation of phosphatidylcholine standard
Phosphatidylcholine PC (18:0/18:2) standard was dissolved with chloroform/methanol mixed solution (2/1, v/v) to prepare a standard stock solution of 1000. Mu.g/mL, and stored in a refrigerator at-30℃in the dark. Before use, the standard solution prepared by 1 mug/mL is diluted by methanol, and the standard solution is prepared on site.
2. Phosphatidylcholine liquid chromatography-mass spectrum acquisition data
A ACQUITY UPLC BEH C column (100 mm. Times.2.1 mm. Times.1.7 μm) was selected on a Dionex high performance liquid chromatography system, the column temperature was set at 40℃and the sample loading was 1. Mu.L. Mobile phase A was acetonitrile/water (6/4, v/v) mixed solution (containing 0.1% formic acid, 10mmol/L ammonium formate); mobile phase B was a mixed solution of isopropanol/acetonitrile (9/1, v/v) (containing 0.1% formic acid, 10mmol/L ammonium formate). Because the particle size of the chromatographic column is small, the column pressure is too high when the flow speed is too high, so the flow speed is set to be 0.2mL/min, and the elution gradient is set to be 0min and 15% B;4min,30% B;5min,48% B;22min,82% B;23-24min,99% B;24.2-30min,15% B.
The mass spectrometer used was a Thermo Orbitrap Fusion (Thermo Fisher Scientific, waltham, MA, USA) mass spectrometer, data acquisition was performed in a Data Dependent MS scan mode in positive and negative ion modes, respectively, and an electrospray ion source (ESI) was selected as the ion source. The ion source region parameters were set as follows: the spray voltage is 3500v (+)/3000 v (-), the capillary temperature is 320 ℃, the evaporation temperature is 320 ℃, and the auxiliary gas (N) 2 ) The gas flow rate was 5arb, and the sheath gas (N) 2 ) The gas flow is 40arb, the scanning range is 150-1200m/z, the Obitrap resolution is 240000, the Automatic Gain Control (AGC) is 1.0e6, and the maximum injection time is 100ms. The secondary and tertiary fragmentation modes are High-energy C-trap Dissociation (HCD), the collision energy is 35+/-5 eV, the resolution of the obitrap is 30000, and the maximum injection time is 60ms.
3. Data analysis
And acquiring primary, secondary and tertiary mass spectrograms under a positive ion mode. When the tolerance mass torlence for lipid recognition was set to 1ppm, m in the primary mass spectrum (FIG. 4)The/z 786.6025 ion is assigned a unique molecular formula C 44 H 84 NO 8 P [ M+H ]] + Ions. Unique molecular formula C 44 H 84 NO 8 P. The 11 possible phosphatidylcholine structures were obtained by matching against the phosphatidylcholine database (see table 2), and the results contained two types of phospholipids, 6 Phosphatidylcholine (PC) and 5 Phosphatidylethanolamine (PE) types (see table 2).
In the positive ion mode, a secondary mass spectrum of m/z 786.6025 was acquired (FIG. 5). M/z184.0733 is matched to the characteristic fragment of choline Head group [ head+OH+H ] in secondary mass spectrometry] + Further, m/z 124.9998 ions in the three-level mass spectrum through m/z184.0733 are matched as [ head+OH+H ]] + (m/z 184.0733) loss [ N (CH) 3 ) 3 ] + Whereas characteristic fragments [ head+H+OH-N (CH) 3 ) 3 ] + (m/z 124.9998). Thus, by the recognition of "grade exclusion", the lipid is excluded from Phosphatidylethanolamine (PE), but 6 possible PC isomers (see table 2).
TABLE 1 37 fatty acid species and names
TABLE 2 PC (18:0/18:2) multistage fragment ions and corresponding identification results in database
In the negative ion mode, when the tolerance for lipid recognition is set to 1ppm, M/z 830.5888 ions in the primary mass spectrum (FIG. 7) are assigned to [ M+HCOO ]] - The unique molecular formula of the ion further proves that the unique molecular formula of the C44H84NO 8P. In the secondary mass spectrum of m/z 830.5888 (FIG. 8), m/z 279.2323 and m/z 283.2635 represent the type information of PC acyl chain by HCD secondary fragmentation, and m/z 279.2323 and m/z 283.2635 are respectively associated with [ C in the database ] 17 H 31 COO] - (C18:2 acyl chain) and [ C ] 17 H 35 COO] - (C18:2 acyl chain) match. Thus, 5 suspected structures were excluded: PC (14:0/22:2), PC (14:1/22:1), (16:0/20:2), PC (16:1/20:1), PC (18:1/18:1), the unique structure of the molecule PC (18:0/18:2) is identified.
Example 2 identification of the Structure of unique PC (18:1/18:1)
1. Preparation of phosphatidylcholine standard
Phosphatidylcholine PC (18:1/18:1) standard was dissolved with chloroform/methanol mixed solution (2/1, v/v) to prepare 1000. Mu.g/mL standard stock solution, which was stored in a-30℃refrigerator in the dark. Before use, the standard solution prepared by 1 mug/mL is diluted by methanol, and the standard solution is prepared on site.
2. Phosphatidylcholine liquid chromatography-mass spectrum acquisition data
A ACQUITY UPLC BEH C column (100 mm. Times.2.1 mm. Times.1.7 μm) was selected on a Dionex high performance liquid chromatography system, the column temperature was set at 40℃and the sample loading was 1. Mu.L. Mobile phase A was acetonitrile/water (6/4, v/v) mixed solution (containing 0.1% formic acid, 10mmol/L ammonium formate); mobile phase B was a mixed solution of isopropanol/acetonitrile (9/1, v/v) (containing 0.1% formic acid, 10mmol/L ammonium formate). Because the particle size of the chromatographic column is small, the column pressure is too high when the flow speed is too high, so the flow speed is set to be 0.2mL/min, and the elution gradient is set to be 0min and 15% B;4min,30% B;5min,48% B;22min,82% B;23-24min,99% B;24.2-30min,15% B.
The mass spectrometer used was a Thermo Orbitrap Fusion (Thermo Fisher Scientific, waltham, MA, USA) mass spectrometer employing Data Dependent MS in positive and negative ion modes, respectively 3 Scanning mode data acquisition and ion source selectionSpray ion source (ESI). The ion source region parameters were set as follows: the spray voltage is 3500v (+)/3000 v (-), the capillary temperature is 320 ℃, the evaporation temperature is 320 ℃, and the auxiliary gas (N) 2 ) The gas flow rate was 5arb, and the sheath gas (N) 2 ) The gas flow is 40arb, the scanning range is 150-1200m/z, the Obitrap resolution is 240000, the Automatic Gain Control (AGC) is 1.0e6, and the maximum injection time is 100ms. The two-stage and three-stage fragmentation modes are HCD, the collision energy is 35+/-5 eV, the Obitrap resolution is 30000, the Automatic Gain Control (AGC) is 5.0e4, and the maximum injection time is 60ms.
3. Data analysis
In the positive ion mode, a primary (fig. 9), a secondary (fig. 10) and a tertiary mass spectrum (fig. 11) are acquired. When the tolerance mass torlence for lipid recognition was set to 1ppm, m/z 786.6025 ions were assigned to unique formula C in the primary mass spectrum (FIG. 9) 44 H 84 NO 8 P [ M+H ]] + Ions. Unique molecular formula C 44 H 84 NO 8 P was aligned with the phosphatidylcholine database and matched to yield 11 possible phosphatidylcholine structures (see Table 3), which contained two types of phospholipids, 6 Phosphatidylcholine (PC) and 5 Phosphatidylethanolamine (PE) types (see Table 3).
In the positive ion mode, a secondary mass spectrum of m/z 786.6025 was acquired (FIG. 10). M/z184.0733 is matched to the characteristic fragment of choline Head group [ head+OH+H ] in secondary mass spectrometry] + Further, m/z 124.9998 ions in the three-level mass spectrum through m/z184.0733 are matched as [ head+OH+H ]] + (m/z 184.0733) loss [ N (CH) 3 ) 3 ]Characteristic fragment [ head+H+OH-N (CH 3) 3, +] + (m/z 124.9998). Thus, by the recognition of "grade exclusion", the lipid was excluded from PE, but 6 possible PC isomers (see table 3).
In the negative ion mode, when the tolerance for lipid recognition is set to 1ppm, M/z 830.5888 ions in the primary mass spectrum (FIG. 11) are assigned to [ M+HCOO ]] - The unique molecular formula of the ion further proves that C 44 H 84 NO 8 Unique molecular formula of P.In the secondary mass spectrum of m/z 830.5888 (FIG. 12), m/z 281.2480 represents the type information of PC acyl chain, m/z 281.2480 and [ C ] in the database by HCD secondary fragmentation 17 H 33 COO] - (C18:1 acyl chain) match. Thus, 5 suspected structures were excluded: PC (14:0/22:2), PC (14:1/22:1), (16:0/20:2), PC (16:1/20:1), PC (18:0/18:2), recognizes the unique structure of the molecule PC (18:1/18:1).
TABLE 3 PC (18:1/18:1) multistage fragment ions and corresponding identification results in database
Example 3
1. Sample pretreatment
The adipocytes were collected and stored at-80℃prior to treatment. mu.L of iced methanol was added to each milligram of frozen cell sample (8. Mu.L/mg). The sample is then vortexed (30 s); after that, 8. Mu.L of iced chloroform was added to each mg of frozen cell sample, and the mixture was vortexed (30 s); then 8. Mu.L of ultra pure water was added per mg of sample, and the sample was vortexed (30 s); the entire operation was performed on wet ice (0 ℃) and then placed on wet ice (0 ℃) for 10 minutes. The sample was then centrifuged (8000 r/min,4 ℃ C., 10 min) and allowed to stand at room temperature for 5 min. The lower liquid (100. Mu.L) was transferred and dried under a stream of nitrogen and then redissolved in 50. Mu.L of methanol/chloroform (2:1, v/v) solution and collected by vortexing to be detected.
2. Chromatographic separation conditions
A ACQUITY UPLC BEH C column (100 mm. Times.2.1 mm. Times.1.7 μm) was selected on a Dionex high performance liquid chromatography system, the column temperature was set at 40℃and the sample loading was 1. Mu.L. Mobile phase A was acetonitrile/water (6/4, v/v) mixed solution (containing 0.1% formic acid, 10mmol/L ammonium formate); mobile phase B was a mixed solution of isopropanol/acetonitrile (9/1, v/v) (containing 0.1% formic acid, 10mmol/L ammonium formate). The flow rate is set to be 0.2mL/min, and the elution gradient is set to be 0min and 15% B;4min,30% B;5min,48% B;22min,82% B;23-24min,99% B;24.2-30min,15% B.
3. Mass spectrometry conditions
4. The mass spectrometer used was Thermo Orbitrap Fusion (Thermo Fisher
Scientific, waltham, mass., USA) mass spectrometry, data Dependent was used in positive and negative ion mode, respectively
MS3 scan mode data acquisition was performed and ion source selection electrospray ion source (ESI). The ion source region parameters were set as follows: the spraying voltage is 3500v (+)/3000 v (-), the capillary temperature is 320 ℃, the evaporation temperature is 320 ℃, and the flow rate of the auxiliary gas (N2) is 5
arb, sheath gas (N2) gas flow rate of 40arb, scan range of 150-1200
m/z, obitrap resolution 240000, automatic Gain Control (AGC) 1.0e6, maximum injection time 100
ms. The secondary and tertiary fragmentation modes are HCD, and the collision energy is 35+/-5
eV, obitrap resolution 30000, automatic Gain Control (AGC) 5.0e4, maximum injection time 60ms. Data results
After the phosphatidylcholine was extracted, isolated and collected from adipocytes, 94 phosphatidylcholine were successfully identified by identifying the phosphatidylcholine chain length isomer using a positive and negative ion simultaneous scanning method (table 4).
TABLE 4 phosphatidylcholine assay results in cancer cells
Claims (7)
1. A mass spectrometry method for identifying a phosphatidylcholine chain length isomer, characterized by: the method comprises the following steps:
constructing a phosphatidylcholine chain length isomer and characteristic ion high-resolution mass spectrum simulation database, wherein the phosphatidylcholine chain length isomer and characteristic ion high-resolution mass spectrum simulation database is characterized in that: generating accurate mass numbers of corresponding secondary mass spectrum and tertiary mass spectrum fragments for each phosphatidylcholine molecule in a phosphatidylcholine chain length isomer compound simulation database according to a phosphatidylcholine mass spectrum splitting rule, and calculating theoretical molecular weights, so as to construct a simulation database containing phosphatidylcholine molecules, molecular formulas, molecular weights, structural formulas of secondary and tertiary mass spectrum fragments and accurate mass numbers;
the method for identifying the phosphatidylcholine by using the liquid chromatography-high resolution mass spectrometer is established, a sample to be detected is subjected to liquid chromatography-high resolution mass spectrometer combination detection, mass spectrometry is carried out according to the accurate mass number mass spectrum characteristics of phosphatidylcholine positive ions and negative ion three-stage fragment ions and the comparison of a simulation database, and the identification result of the phosphatidylcholine chain length isomer is obtained, specifically: collecting mass spectrum information under two scanning modes of a positive ion mode and a negative ion mode respectively, wherein in the positive ion scanning mode, phosphatidylcholine lipid generates parent ions [ M+H ] in primary mass spectrum] + I.e. MS 1 Generating a characteristic fragment of the phosphate Head group of phosphatidylcholine [ head+OH+H ] in a secondary mass spectrum] + I.e. MS 2 Generating characteristic fragments [ head+H+OH-N (CH) in a three-level mass spectrum 3 ) 3 ] + I.e. MS 3 The method comprises the steps of carrying out a first treatment on the surface of the In negative ion mode, phosphatidylcholine with phosphate head group generates parent ion [ M+HCOO ]] - I.e. MS 1 In the secondary mass spectrum, [ R ] is generated 1 COO] - 、[R 2 COO] - 、[M-R 1 CO] - 、[M-R 2 CO] - And structural fragments related to phosphate head group, i.e., MS 2 The method comprises the steps of carrying out a first treatment on the surface of the Binding MS in positive ion mode 1 Mass spectrum fragment ion [ M+H ]] + Obtain unique molecular formula, further combine with MS 2 And MS (MS) 3 Identifying the characteristic of phosphatidylcholine which is different from other types of phospholipids, and analyzing whether the phosphatidylcholine is phosphatidylcholine or not; then combine MS in negative ion mode 1 Further demonstrating the unique molecular formula of phosphatidylcholine, combined with MS 2 Identifying phosphatidylcholine chain length isomer, identifying fatty acid connected with phosphoric acid head group of phosphatidylcholine to obtain identification result of phosphatidylcholine chain length isomer,
wherein: the structural formula of phosphatidylcholine is shown in the following figure:
the phosphate head group of phosphatidylcholine is:
2. the mass spectrometry method of claim 1, wherein: mass spectrum detection conditions in liquid chromatography-high resolution mass spectrum combined detection: the secondary collision energy CE is 30-40 eV, and multi-stage mass spectrum data of the sample to be detected in the high-resolution mass spectrum are collected.
3. The mass spectrometry method of claim 1, wherein: the secondary and tertiary fragmentation modes used in mass spectrometry detection are HCD.
4. The mass spectrometry method of claim 1, wherein: liquid chromatography detection conditions in liquid chromatography-high resolution mass spectrometry combined detection: the mobile phase A is acetonitrile/water mixed solution with the concentration of 5/5-7/3 and the concentration of v/v, and contains 0.05-0.15% formic acid and 8-12mmol/L ammonium formate; the mobile phase B is 85/15-95/5, v/v of isopropanol/acetonitrile mixed solution, 0.05-0.15% of formic acid, 8-12mmol/L of ammonium formate, and the elution gradient is 0min, 10-20% of B;4min, 20-40% of B;5min, 45-50% of B;22min, 80-85% of B;23-24min, 98-99% B;24.2-30min, 10-20% B.
5. The mass spectrometry method of claim 1, wherein: the mobile phase used in the liquid chromatography detection contains formic acid and ammonium formate, the dosage of the formic acid is 0.05-0.15% by volume percent, and the dosage of the ammonium formate is 8-12mmol/L.
6. The mass spectrometry method of claim 1, wherein: in the construction of phosphatidylcholine chain length isomers and characteristic ion high resolution mass spectrum simulation databases: according to the known 37 kinds of fatty acids, if two fatty acid acyl chains of phosphatidylcholine are different and are of AB type, if two fatty acid acyl chains of phosphatidylcholine are identical and of AA type, when phosphatidylcholine is of AB type, the phosphoric acid head group type of phosphatidylcholine and any 2 kinds of fatty acids are connected to a glycerin skeleton, namely 1332 kinds of all AB type phosphatidylcholine can be combined, when phosphatidylcholine is of AA type, the phosphoric acid head group type of phosphatidylcholine and any 1 kind of fatty acids are connected to the glycerin skeleton, namely 37 kinds of AA type phosphatidylcholine can be combined, and a phosphatidylcholine chain length isomer compound simulation database is constructed.
7. The mass spectrometry method of claim 1, wherein: the method for identifying the phosphatidylcholine by using the liquid chromatography-high resolution mass spectrometer comprises the following steps: the mass tolerance is set to be less than or equal to 1ppm through the accurate mass number of MS1, and the unique element composition is identified.
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