WO2024022312A1

WO2024022312A1 - Capillary electrophoresis-based lipidome analysis system as well as use thereof, and lipidome analysis method

Info

Publication number: WO2024022312A1
Application number: PCT/CN2023/109024
Authority: WO
Inventors: 欧阳证; 吴俊函; 张文鹏
Original assignee: 清华大学
Priority date: 2022-07-25
Filing date: 2023-07-25
Publication date: 2024-02-01
Also published as: CN115236170A

Abstract

Provided are a capillary electrophoresis-based lipidome analysis system as well as the use thereof, and a lipidome analysis method. The analysis system comprises a voltage apparatus, a capillary tube, a photochemical reactor, an ionization interface and a mass spectrometer, the voltage apparatus being electrically connected to the capillary tube, the capillary tube being connected to the mass spectrometer by means of the ionization interface, and the photochemical reactor being arranged on the capillary tube.

Description

Lipidome analysis system based on capillary electrophoresis and its application and lipidome analysis method

priority information

This disclosure requests the priority and rights of the patent application with patent application number 202210884394.6 filed with the State Intellectual Property Office of China on July 25, 2022, and the full text of which is incorporated herein by reference.

Technical field

The present disclosure belongs to the technical field of mass spectrometry, and specifically relates to a capillary electrophoresis-based lipidome analysis system and its application and a lipidome analysis method.

Background technique

Lipids play an important role in organisms. They are not only the main components of the cytoskeleton, but also participate in cell metabolism, material transport, and information transmission. Lipidomics analyzes and studies overall lipids, compares changes in lipid metabolism under different physiological states, identifies lipid biomarkers, and reveals the important role of lipids in life activities. With the development of "soft ionization" technologies such as electrospray ionization, mass spectrometry has become the most commonly used and effective tool for lipidomics analysis. Usually, liquid chromatography-high resolution mass spectrometry and liquid chromatography-tandem mass spectrometry are used to identify lipids. However, conventional tandem mass spectrometry analysis can only identify information on lipid subclasses and fatty acid chain lengths, and it is still difficult to characterize the complete structure of lipids. The structure of lipids is extremely complex, and lipids can be divided into different types according to the different head groups of their molecules. In addition, the structural characteristics of lipids such as fatty acid chains, fatty acid chain positions, carbon-carbon double bond positions, and double bond geometric configurations can all change, resulting in a large number of lipid isomers.

Among them, the ratio of lipid carbon-carbon double bond isomers has been confirmed to change in the tissues and body fluids of various tumor patients, and is a potential disease biomarker. Methods commonly used to identify the position of carbon-carbon double bonds in lipids include: ultraviolet photodissociation (UVPD), ozone-induced dissociation (OzID), photochemical (Paternò-Büchi, PB) derivatization reaction, etc. Among them, the combination of PB reaction and tandem mass spectrometry is currently one of the main strategies for identifying lipid double bonds. Among them, liquid chromatography-mass spectrometry (LC-MS) technology combined with photochemical derivatization reactions provides a feasible solution for high-throughput photochemically derived lipid structure analysis.

Structural lipidomics technology based on liquid chromatography-mass spectrometry has some limitations: first, the online derivatization reaction is limited by column pressure and chromatographic separation conditions; in addition, liquid chromatography has a large injection volume and cannot analyze small volumes. Analyze the lipid profile of a sample such as a small number of cells. These technical problems limit the application of lipidomics in microbiological lipid analysis and medicine. Developments in lipid biomarker screening for disease.

Therefore, improvements in lipidome analysis systems are necessary.

public content

The present disclosure aims to improve at least one of the above technical problems, at least to a certain extent.

In order to improve the above technical problems, the present disclosure provides a lipid group analysis system based on capillary electrophoresis. The analysis system includes a voltage device, a capillary tube, a photochemical reactor, an ionization interface and a mass spectrometer; the voltage device and the capillary electrophoresis connection, the capillary tube is connected to the mass spectrometer through the ionization interface; the photochemical reactor is provided on the capillary tube. Therefore, by arranging a photochemical reactor on the capillary tube, online photochemical reaction on the capillary tube can be realized. That is, the disclosed system can undergo a chemical derivatization reaction during electrophoretic separation, and realize the analysis of the fine structure of lipids through mass spectrometer analysis. ; The disclosed system realizes the analysis of trace samples and direct complex samples while retaining the characteristics of high-throughput photochemical derivatization; in addition, the disclosed system also has the advantage of high analytical sensitivity.

According to an embodiment of the present disclosure, the photochemical reactor includes a photochemical reaction window and a light source; the photochemical reaction window is located on the capillary tube; the orthographic projection of the light source on the capillary tube covers the photochemical reaction window, for example, The light source may be located on one side of the capillary tube (eg, upper side, lower side), and the light emitted by the light source may at least cover the photochemical reaction window. As a result, photochemical reactions can be carried out online during capillary electrophoresis separation.

According to an embodiment of the present disclosure, the capillary tube includes a base layer and a polyimide coating layered in a stack, and the polyimide coating layer is located outside the capillary tube; the capillary tube at the photochemical reaction window includes a base layer . This can prevent the quartz capillary from breaking, increase the transmittance at the photochemical reaction window, ensure that the photochemical reaction proceeds fully, and further improve the accuracy and precision of the analysis system.

According to an embodiment of the present disclosure, the number of the photochemical reactors is greater than or equal to 1.

According to an embodiment of the present disclosure, when the number of the photochemical reactors is greater than 1, there is no overlapping area between the wavebands emitted by the light sources of different photochemical reactors. As a result, different types of lipids in the lipid group can fully undergo photochemical reactions, further improving the accuracy of the analysis system.

According to an embodiment of the present disclosure, the mass spectrometer is a triple quadrupole mass spectrometer or a mass spectrometer capable of collision-induced fragmentation.

The present disclosure also provides the application of the previously described capillary electrophoresis-based lipidome analysis system in biological lipid extract analysis and/or lipid biomarker screening.

The present disclosure also provides a method for analyzing lipid groups using the capillary electrophoresis-based lipid group analysis system described above. The method includes: adding buffer and lipid groups to the capillary, turning on the voltage device, and A voltage is applied to both ends of the capillary, and the lipid group is electrophoretically separated in the capillary; a photochemical reaction occurs when the lipid group flows through the chemical reactor; a mass spectrometer signal is obtained through a mass spectrometer, and the lipid group is analyzed . Therefore, this method has all the features and advantages of the lipidome analysis system based on capillary electrophoresis mentioned above, which will not be described again here.

According to an embodiment of the present disclosure, the buffer includes a derivatization reagent, and the derivatization reagent includes at least one of acetone, benzophenone, diacetylpyridine, acetophenone derivatives, benzoylpyridine, and phenylglyoxylate. species, the above derivatization reagents are compatible with mass spectrometry.

According to an embodiment of the present disclosure, the buffer further includes a buffer electrolyte, and the buffer electrolyte includes at least one of formic acid, ammonium acetate, and ammonium bicarbonate; optionally, when the buffer electrolyte is solid, the buffer electrolyte The electrolyte is added in the form of a buffer electrolyte aqueous solution; the buffer also includes a solvent, and the solvent includes acetonitrile. Both the buffer electrolyte and the solvent are compatible with mass spectrometry.

According to embodiments of the present disclosure, when the buffer electrolyte is solid, the concentration of the buffer electrolyte in the added buffer electrolyte aqueous solution is 10-50 mmol/L, and the pH of the buffer electrolyte aqueous solution is 8-10.

According to an embodiment of the present disclosure, the volume ratio of the derivatization reagent to the solvent and the buffer electrolyte is (1-5):(2-6):(1-5).

According to an embodiment of the present disclosure, when the derivatization reagent and the buffer electrolyte are solid, in the solution formed by the derivatization reagent, the solvent and the buffer electrolyte aqueous solution, the concentration of the derivatization reagent is 2-20mmol/L, the volume ratio of the solvent to water is (5-9): (1-5).

According to an embodiment of the present disclosure, before the buffer and lipid group are added to the capillary, the method further includes: adding an inorganic alkali solution to the capillary for activation, and then flushing the capillary with water and buffer. As a result, the activated capillary can generate electroosmotic flow under high voltage, which can achieve better separation of different components with different charges in the lipid group.

Description of drawings

Figure 1 is a schematic diagram of a lipid profile analysis system based on capillary electrophoresis in one embodiment of the present disclosure.

Figure 2 is a flow chart of a method for analyzing lipid groups in one embodiment of the present disclosure.

Figure 3 shows a lipid profile analysis system based on capillary electrophoresis that implements lipid carbon-carbon double bond light diffraction in one embodiment of the present disclosure. Flowchart of biochemical analysis.

Figure 4 is a capillary electrophoresis separation chromatogram of 10 nanoliters of bovine liver polar lipid extract in an acetone buffer system containing a derivatization reagent in an embodiment of the present disclosure.

Figure 5 shows an example of the present disclosure, after the phosphatidylcholine (PC) component of 10 nanoliters of bovine liver polar lipid extract was separated by capillary electrophoresis in an acetone buffer system containing a derivatization reagent. First level mass spectrum.

Figure 6 shows an unreacted fraction of the phosphatidylethanolamine (PE) component of 10 nanoliters of bovine liver polar lipid extract separated by capillary electrophoresis in an acetone buffer system containing a derivatization reagent in an embodiment of the present disclosure. level mass spectrum.

Figure 7 shows a spectrum of tandem mass spectrometry analysis of PC 16:0_18:1 in 10 nanoliters of bovine liver polar lipid extract after photoderivatization in an acetone buffer system containing derivatization reagent in an embodiment of the present disclosure. .

Figure 8 is an example of the present disclosure. PE 17:0_22:4 in 10 nanoliters of bovine liver polar lipid extract was analyzed by cascade mass spectrometry after photoderivatization in an acetone buffer system containing photoderivatization reagent. Spectrum.

Figure 9 is a capillary electrophoresis separation chromatogram of 10 nanoliters of bovine liver polar lipid extract in a buffer system containing photoderivatization reagent benzophenone in one embodiment of the present disclosure.

Figure 10 is an example of the present disclosure, cascade mass spectrometry analysis of PC 16:0_18:1 in 10 nanoliters of bovine liver polar lipid extract after photoderivatization in a buffer system containing the derivatization reagent benzophenone. spectrum.

Figure 11 is a capillary electrophoresis separation chromatogram of 10 nanoliters of bovine liver polar lipid extract in an ammonium bicarbonate buffer system containing acetone in one embodiment of the present disclosure.

Figure 12 shows a negative mode tandem mass spectrum of PC 16:0_18:1 in 10 nanoliters of bovine liver polar lipid extract in an ammonium bicarbonate buffer system containing acetone in one embodiment of the present disclosure.

Figure 13 is a chromatogram of capillary electrophoresis separation of 10 nanoliters of lipid extract from 50 cells in an acetone buffer system containing derivatization reagent in one embodiment of the present disclosure.

Detailed ways

Embodiments of the present disclosure are described in detail below. The embodiments described below are illustrative and are only used to explain the present disclosure and are not to be construed as limitations of the present disclosure. If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field or product instructions will be followed. If the manufacturer of the reagents used is not indicated, they are all conventional products that can be purchased commercially.

The present disclosure provides a lipid group analysis system based on capillary electrophoresis. Refer to Figure 1. The analysis system includes a voltage device, a capillary tube, a photochemical reactor, an ionization interface and a mass spectrometer; the voltage device is electrically connected to the capillary tube, The capillary tube is connected to the mass spectrometer through the ionization interface; the photochemical reactor is provided on the capillary tube. Therefore, the analysis system of the present disclosure can electrophoretically separate lipid groups in a capillary tube, and use a photochemical reactor to perform online derivatization of the separated unsaturated lipids. The products after the photochemical reaction enter the mass spectrometer through the ionization interface. Capillary electrophoresis-mass spectrometry analysis can be used to analyze the fine structure of lipids. Moreover, the analysis system of the present disclosure can analyze trace samples and has the advantage of high analysis sensitivity.

The disclosed mass spectrometry analysis process can achieve a variety of different reaction purposes, including evaluating the reaction efficiency of chemical derivatization reactions, conducting in-depth lipid subclasses, fatty acid chain lengths, carbon-carbon double bond positions, and sn isomer analysis. Quality fine structure analysis, etc. Specifically, using the system of the present disclosure and using a multi-mode tandem mass spectrometry method, the positional information of complex lipid C=C can be identified, the positional isomers of lipid C=C can be relatively quantified, and stem cells and organoids can also be realized. Simultaneous analysis of multiple lipid isomers in trace amounts of rare biological samples, the analytical sensitivity can reach the single cell level.

It should be noted that the term "lipidome" encompasses different classes of lipids.

For ease of understanding, the working principle of the capillary electrophoresis-based lipidome analysis system in this disclosure is briefly explained below:

A tiny amount (about 10nL) of the lipid sample enters the capillary. Driven by the high voltage of the voltage device, the lipid sample undergoes electrophoresis in the capillary containing the buffer containing the derivatization reagent. When the lipid containing unsaturated double bonds When the mass flow passes through the photochemical reaction window, it is illuminated by ultraviolet light, and corresponding photochemical reactions occur to produce corresponding photochemical derivatives. The entire photochemical process and the electrophoretic separation process occur simultaneously without adding any unnecessary analysis time; moreover, the disclosed system inherits the advantages of capillary electrophoresis mass spectrometry in the application scenario of micro-sample analysis, and can analyze micro-samples, allowing photochemical derivatization of lipids. Structural analysis methods are applied to more scenarios.

According to an embodiment of the present disclosure, the photochemical reactor includes a photochemical reaction window and a light source; the photochemical reaction window is located on the capillary tube; the orthographic projection of the light source on the capillary tube covers the photochemical reaction window, that is, the light source The emitted light may cover at least the photochemical reaction window. By setting up a photochemical reactor, photochemical reactions can be carried out online during capillary electrophoresis separation.

Those skilled in the art can adjust the position of the photochemical reactor on the capillary as needed. Specifically, those skilled in the art can adjust the position of the photochemical reaction window on the capillary, the length of the photochemical reaction window, and the emitted light from the light source as needed. band.

In some embodiments of the present disclosure, the light source is used to emit ultraviolet light. For example, the light source may emit ultraviolet light at 254 nm. Further, the light source can be an ultraviolet lamp.

In some embodiments of the present disclosure, the length of the photochemical reaction window is 2 cm, and the distance between the light source and the photochemical reaction window is 1 cm.

According to an embodiment of the present disclosure, the capillary tube includes a base layer and a polyimide coating layered in a stack, and the polyimide coating layer The imine coating is located on the outside of the capillary; the capillary at the photochemical reaction window includes a base layer. In some embodiments of the present disclosure, the capillary tube is a quartz capillary tube, and the base layer is made of fused quartz. Quartz has the disadvantage of being extremely brittle and easily broken. By arranging polyimide on the surface of the base layer near the outer side of the tube, The coating can increase the flexibility of the base layer and prevent the quartz capillary from breaking. Since polyimide is opaque, in order to allow the photochemical reaction at the photochemical reaction window to fully proceed, the polyimide coating at the photochemical reaction window needs to be removed, thereby increasing the light transmittance at the photochemical reaction window. The matrix layer made of fused quartz has good light transmittance. Almost all the light emitted by the light source can pass through the matrix layer, allowing the photochemical reaction to fully proceed and further improving the accuracy and precision of the analysis system.

According to an embodiment of the present disclosure, the number of photochemical reactors on the capillary tube is greater than or equal to 1. As a result, different types of lipids in the lipid group can fully undergo photochemical reactions, further improving the precision and accuracy of the analysis system.

According to some embodiments of the present disclosure, when the number of the photochemical reactors is greater than 1, there is no overlapping area between the wavebands emitted by the light sources of different photochemical reactors. Since the lipid group contains different types of lipids, and the properties of different types of lipids are different, the photochemical reaction bands corresponding to different types of lipids are different. By setting up multiple photochemical reactors, the light sources of the multiple photochemical reactors are There is no overlapping area between the emitted wavelength bands, which allows different types of lipids in the lipid group to fully carry out photochemical reactions, further improving the precision and accuracy of the analysis system.

The ionization interface in the present disclosure can ionize the components flowing out of the capillary electrophoresis, and the ionized components enter the mass spectrometer. The ionization interface includes, but is not limited to, a nanoelectrospray interface.

According to an embodiment of the present disclosure, the mass spectrometer is a triple quadrupole mass spectrometer or a mass spectrometer capable of collision-induced dissociation (CID).

The present disclosure also provides the application of the previously described capillary electrophoresis-based lipidome analysis system in biological lipid extract analysis and/or lipid biomarker screening. Therefore, the lipidome analysis system proposed in this disclosure can achieve high-throughput, highly sensitive, and fine structure analysis of lipids in trace amounts of complex samples.

According to embodiments of the present disclosure, the lipid biomarkers may specifically be lipid biomarkers of medical disease samples. The samples include, but are not limited to, clinical samples such as tissues, body fluids, and dried blood spots, or rare and small amounts of biological samples such as exosomes, stem cells, and even single cells.

The lipid biomarker has a C=C position isomer or a sn position isomer of the fatty acid chain.

The inventor applied the lipidome analysis system to the analysis of micro-cells (50 levels). Through this structure analysis process, 34 unsaturated lipid molecules were identified in 50 cell-level lipid extracts, among which Includes 15 pairs of double bond isomers. In addition, by directly analyzing dried blood spots, the inventor found that the ratio of the C=C double bond isomer content in the dried blood spots of normal and type 2 diabetic patients has a significant difference, which provides a basis for clinical diagnosis. The search for biomarkers provides new directions.

The present disclosure also provides a method for analyzing lipid groups using the capillary electrophoresis-based lipid group analysis system described above. Referring to Figure 2, the method includes:

S100. Add the buffer and lipid group to the capillary tube, turn on the voltage device, apply voltage at both ends of the capillary tube, and the lipid group is electrophoretically separated in the capillary tube; the lipid group flows through the chemical reactor photochemical reaction occurs;

According to an embodiment of the present disclosure, the buffer includes a derivatization reagent, and the derivatization reagent includes at least one of acetone, benzophenone, diacetylpyridine, acetophenone derivatives, benzoylpyridine, and phenylglyoxylate. species; the above derivatization reagents are compatible with mass spectrometry.

According to an embodiment of the present disclosure, the buffer further includes a buffer electrolyte, the buffer electrolyte includes at least one of formic acid, ammonium acetate, and ammonium bicarbonate, and the buffer electrolyte may be compatible with mass spectrometry.

In some embodiments of the present disclosure, when the buffer electrolyte is solid, the buffer electrolyte is added in the form of an aqueous buffer electrolyte solution.

According to an embodiment of the present disclosure, the buffer further includes a solvent, and the solvent includes acetonitrile.

After a large number of exploratory experiments, the inventor found that the above-mentioned derivatized reagents, buffer electrolytes, and solvents functionally support and cooperate with each other. The buffer composed of them can produce stable electrical connections during electrophoresis and form a stable Electroosmotic flow achieves electrophoretic separation.

According to embodiments of the present disclosure, when the buffer electrolyte is solid, the concentration of the buffer electrolyte in the added buffer electrolyte aqueous solution is 10-50mmol/L, for example, it can be 10mmol/L, 12mmol/L, 15mmol/L, 20mmol /L, 25mmol/L, 30mmol/L, 35mmol/L, 40mmol/L, 45mmol/L, 50mmol/L, the pH of the buffer electrolyte aqueous solution is 8-10, for example, it can be 8, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.

According to an embodiment of the present disclosure, the volume ratio of the derivatization reagent to the solvent and the buffer electrolyte is (1-5):(2-6):(1-5), for example, it can be 1:2:1 , 1:3:1, 1:6:1, 1:6:5, 2:4:3, 2:5:5, 3:4:3, 3:4:4, 3:5:3, 3 :6:3, 4:4:3, 4:5:3, 4:6:3, 5:2:1, 5:4:1, 5:6:5.

According to an embodiment of the present disclosure, when the derivatization reagent and the buffer electrolyte are solid, in the solution formed by the derivatization reagent, the solvent and the buffer electrolyte aqueous solution, the concentration of the derivatization reagent is 2-20mmol/L, such as 2mmol/L, 3mmol/L, 4mmol/L, 5mmol/L, 6mmol/L, 7mmol/L, 8mmol/L, 10mmol/L, 15mmol/L, 20mmol/L, the solvent The volume ratio to water is (5-9):(1-5), such as 5:1, 6:1, 7:1, 7:3, 7:4, 7:5, 8:1, 8:3 , 8:5, 9:1, 9:2, 9:5.

According to an embodiment of the present disclosure, before the buffer and lipid group are added to the capillary, the method further includes: adding an inorganic alkali solution to the capillary for activation, and then flushing the capillary with water and buffer. In some embodiments of the present disclosure, the capillary is a quartz capillary. Adding an inorganic base can activate the hydroxyl groups of the capillary. Through activation, the silicon-oxygen bonds in the quartz can be opened to generate silicon hydroxyl groups. Under high voltage, the buffer solution The silanols in it can generate an electric double layer. The driving force of capillary electrophoresis is the generation of electroosmotic flow, which allows for better separation of different lipid components with differences in charge within the lipidome.

According to embodiments of the present disclosure, inorganic bases include, but are not limited to, sodium hydroxide.

It should be understood that capillaries can also be chemically modified. The present disclosure does not limit the specific method of chemical modification, as long as the chemically modified capillary can generate electroosmotic flow under high voltage.

In the present disclosure, the photochemical reactor is arranged on the capillary tube. Specifically, the photochemical reaction window is located on the capillary tube; thus, the photochemical process and the electrophoretic separation process can occur simultaneously without adding any unnecessary analysis time.

In some embodiments of the present disclosure, the photochemical reactor can be disposed on one side close to the end of the capillary (the side close to the mass spectrometer). At this time, most of the different types of lipids in the lipid group flow through the photochemical reactor. Electrophoretic separation is achieved through capillaries, and photochemical reactions occur as they flow through chemical reactors, thereby increasing the accuracy and precision of the analysis.

According to some embodiments of the present disclosure, the distance between the photochemical reactor and the ionization interface is less than or equal to 15 cm.

S200. Obtain mass spectrum signals through a mass spectrometer, and analyze the lipid group.

Through tandem mass spectrometry analysis, the fine structure of lipids can be analyzed.

The present disclosure will be described below with reference to specific embodiments. It should be noted that these embodiments are only illustrative and do not limit the disclosure in any way.

Example 1: Capillary electrophoresis using acetone as derivatization reagent, online PB photochemical derivatization and mass spectrometry system analysis of phospholipid C=C double bond position isomers in bovine liver extract

In a specific embodiment, the flow chart of the specific implementation analysis of the capillary electrophoresis online PB photochemical derivatization and mass spectrometry system is shown in Figure 3. The specific implementation steps are:

(1) Prepare a buffer solution containing acetone. The specific composition of the buffer solution is acetone: acetonitrile: 12mmol/L ammonium acetate aqueous solution with a volume ratio of 3:4:3, pH=9.6;

Prepare a methanol:isopropanol:10mmol/L ammonium acetate aqueous solution with a volume ratio of 3.5:3.5:3, and then add 0.1% acetic acid as the sheath flow fluid.

The main function of the buffer in (1) above is to make electrophoresis produce a stable electrical connection and form a stable electroosmotic flow to achieve electrophoretic separation; the function of the sheath flow liquid is to provide a stable electrical connection for mass spectrometry electrospray at the capillary electrophoresis-mass spectrometry interface and Assists in ionization of samples.

(2) Use 1 mole per liter of sodium hydroxide aqueous solution to activate a 50 cm long quartz capillary for 30 minutes, and rinse it with water and buffer for 30 minutes.

(3) Use ablation to remove the polyimide coating outside the 2 cm capillary at 15 cm from the end of the quartz capillary to set it as a photochemical reaction window.

(4) Load the prepared buffer containing acetone into the electrophoresis capillary through pressure, and add sheath fluid to the capillary electrophoresis-mass spectrometry interface so that the entire electrophoresis forms a stable circuit closed loop, and the electrophoresis effluent is discharged through the capillary electrophoresis mass spectrometry interface. ionization.

The capillary electrophoresis mass spectrometry interface in (4) above adopts the nanosheath flow electrospray capillary electrophoresis mass spectrometry interface.

(5) Load 10 nanoliters of bovine liver polar lipid extract into the electrophoresis capillary through pressure injection.

(6) Apply a high voltage of 20kV to both ends of the capillary to generate an electroosmotic flow in the capillary and conduct electrophoresis to separate different components; and apply a high voltage of 20kV at the capillary electrophoresis mass spectrometry interface to generate electrospray to assist in ionization of the sample.

(7) Open the light-emitting unit module near the photochemical reaction window. Specifically, the light-emitting unit emits 254nm ultraviolet light, and the distance between the light-emitting unit and the photochemical reaction window on the capillary is 1 cm.

(8) The PB photochemical reaction occurs when each component of the sample flows through the photochemical reaction window.

(9) Obtain the corresponding mass spectrum signal through the tandem mass spectrometry platform, record the relationship between the total ion current of the mass spectrum and time, and obtain the chromatogram of capillary electrophoresis; in order to further obtain structural information, the mass spectrometer will be analyzed in tandem mass spectrometry mode.

The tandem mass spectrometry platform in (9) above will separately collect sample data before the reaction to obtain information such as lipid type and chain length, and then collect sample data after the reaction to obtain lipid carbon-carbon double bond position information.

As shown in Figure 4, through the above steps, good separation can be achieved for multiple types of phospholipids in complex lipid analytes, and the number of theoretical plates can reach about 20,000. The primary mass spectrometry information of different components can be well obtained through chromatographic separation. For example, Figure 5 and Figure 6 respectively show the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) components without reaction. level mass spectrum. At the same time, after the PB reaction, the carbon-carbon double bond position of the unsaturated phospholipid can be identified. As shown in Figure 7, cascade mass spectrometry analysis of the PB derivatization product of PC 16:0_18:1 can accurately identify its location. There are 9- and 11-position double bond position isomers on the unsaturated fatty acid chain; in addition to monounsaturated phospholipids, polyunsaturated phospholipids can still be accurately identified, as shown in Figure 8, with PE 17:0_22:4 For example, cascade analysis of its PB derivatives can accurately identify the positions of its unsaturated double bonds at positions 7, 10, 13 and 16 respectively.

The capillary electrophoresis-based lipidome analysis system of the present disclosure, using acetone as a derivatization reagent, can be used to identify unsaturated lipids in bovine liver extracts. The unsaturated lipid analysis method proposed in this disclosure can achieve large-scale fine structure analysis of lipids in a small amount of complex samples of 10 nanoliters. The inventor applied this unsaturated lipid analysis method to the analysis of bovine liver extract, and through this structure analysis process, 163 unsaturated lipid molecules were identified in the bovine liver polar lipid extract. It provides a feasible lipid analysis solution for future research on biological clinical trace samples.

Example 2: Capillary electrophoresis using benzophenone as derivatization reagent, online PB photochemical derivatization and mass spectrometry analysis of lipid C=C double bond position isomers in bovine liver extract

Buffers containing benzophenone derivatized reagents get rid of the limitations of acetone and promote the improvement of mass spectrometry ionization efficiency. effect; in addition, during the PB reaction process, compared with the +58Da derivatization product peak produced when acetone is used as a derivatization reagent, benzophenone will produce a +182Da derivatization product peak, which will provide insights into the lipid identification of complex samples. help.

In this example, benzophenone is a non-volatile substance. A large amount of benzophenone entering the mass spectrometer will pollute the mass spectrometer, so it is not suitable for use in liquid chromatography with a large flow rate. However, because of the flow rate of capillary electrophoresis used in this system, Extremely low, there is no need to worry about problems such as the reagents used contaminating the mass spectrometer.

The bovine liver polar lipid extract was analyzed with reference to a method similar to Example 1. The difference is that the buffer is formed by mixing benzophenone, acetonitrile, and ammonium acetate aqueous solution. In the ammonium acetate aqueous solution, the ammonium acetate The concentration is 12mmol/L, and the pH of the ammonium acetate aqueous solution is 9.6. In the buffer solution formed by mixing benzophenone, acetonitrile, and ammonium acetate aqueous solution, the concentration of benzophenone is 5 mmol/L, and the volume ratio of acetonitrile to water is 7:3. Figure 9 is an electrophoresis chromatogram of bovine liver lipid extract in a buffer system containing benzophenone derivatization reagent, which still shows good separation of different lipid components. Figure 10 shows the identification of the unsaturated phospholipid PC 16:0_18:1 using benzophenone as the PB reaction reagent, and information on the presence of carbon-carbon double bond isomers at positions 9 and 11 can be obtained.

Example 3: Analysis of lipid sn positional isomers in bovine liver extract using capillary electrophoresis mass spectrometry in a buffer system prepared with ammonium bicarbonate

In addition to the derivatization identification of double bond position isomers, the analysis of sn isomers is also an important level in lipid structure identification. Tandem mass spectrometry analysis of bicarbonate adducts of phosphatidylcholine is an important solution for the analysis of sn isomers of phosphatidylcholine phospholipids.

Mass spectrometry analysis of bovine liver polar lipid extract was performed with reference to a method similar to Example 1. The difference is that the electrolyte of the buffer is changed from ammonium acetate to ammonium bicarbonate. The composition of the buffer in Example 3 is a volume ratio of 3 : 4:3 acetone: acetonitrile: 12 mmol/L ammonium bicarbonate aqueous solution with pH = 9.6; in addition, the electrolyte of the sheath flow liquid in Example 3 also changes accordingly, and the specific ratio of the sheath flow liquid in Example 3 is volume The ratio of methanol:isopropyl alcohol:10mmol/L ammonium bicarbonate is 2.5:2.5:5. In addition, the electrophoresis voltage was adjusted from 20kV to 16kV, the electrospray voltage applied at the interface was also changed from 2kV to minus 2.8kV, and the mass spectrometer was adjusted to negative mode analysis.

Figure 11 shows the capillary electrophoresis separation chromatogram of 10 nanoliters of bovine liver polar lipid extract in an acetone-containing ammonium bicarbonate buffer system. It can be clearly found that the phosphatidylcholine (PC) components are effectively separated. Figure 12 shows the negative mode tandem mass spectrum of PC 16:0_18:1 in 10 nanoliters of bovine liver polar lipid extract in an ammonium bicarbonate buffer system containing acetone. The corresponding sn-1 can be clearly seen. and sn-2 diagnostic ions m/z 419 and m/z 445. At the same time, the corresponding two fatty acid chain information m/z 255 and m/z 281 can be seen. In addition, with the help of acetone as a photoderivatization reagent, the double-bond lipid structure identification as in Example 1 can still be achieved.

Example 4: Analysis of lipid isomers in a small amount of cells using capillary electrophoresis mass spectrometry

The capillary electrophoresis mass spectrometry analysis system described in the present disclosure has the characteristics of low sample consumption and high sensitivity, and can Analyze minute amounts of samples. In biological clinical research, the analysis of rare and trace samples, such as cancer stem cells, hematopoietic stem cells, exosomes, etc., is of great research significance for diseases and biological development.

Analyze the sample extract of about 50 HeLa cells using a method similar to Example 1, except that the sample is 2 μL of cell extract obtained by liquid-liquid microextraction. Through capillary electrophoresis separation, the phosphatidylcholine (PC) component and phosphatidylethanolamine (PE) component can be clearly seen in Figure 13. 34 lipid isomers were accurately identified through photochemical PB reaction and tandem mass spectrometry analysis. . It is worth noting that the actual injection volume during the analysis was only 10 nL each time, and the actual analyzed sample volume was much lower than the single-cell level, indicating that the disclosed system has the capability of single-cell level sample analysis.

In the description of this specification, reference to the description of the terms "one embodiment," "another embodiment," "some embodiments," "examples," "specific examples," or "some examples" or the like is intended to be in conjunction with the implementation. An example or example describes a specific feature, structure, material, or characteristic that is included in at least one embodiment or example of the present disclosure. In this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification unless they are inconsistent with each other.

Although the embodiments of the present disclosure have been shown and described above, it can be understood that the above-mentioned embodiments are illustrative and should not be construed as limitations of the present disclosure. Those of ordinary skill in the art can make modifications to the above-mentioned embodiments within the scope of the present disclosure. The embodiments are subject to changes, modifications, substitutions and variations.

Claims

A lipidome analysis system based on capillary electrophoresis, wherein the analysis system includes a voltage device, a capillary tube, a photochemical reactor, an ionization interface and a mass spectrometer;

The voltage device is electrically connected to the capillary tube, and the capillary tube is connected to the mass spectrometer through the ionization interface;

The photochemical reactor is located on the capillary tube.
The lipid group analysis system based on capillary electrophoresis according to claim 1, wherein the photochemical reactor includes a photochemical reaction window and a light source;

The photochemical reaction window is located on the capillary;

The orthographic projection of the light source on the capillary tube covers the photochemical reaction window.
The lipid group analysis system based on capillary electrophoresis according to claim 1 or 2, wherein the capillary tube includes a matrix layer and a polyimide coating layer arranged in a stack, and the polyimide coating layer is located on the capillary tube. the outside of;

The capillary tube at the photochemical reaction window includes a matrix layer.
The lipid profile analysis system based on capillary electrophoresis according to any one of claims 1 to 3, wherein the number of the photochemical reactors is greater than or equal to one.
The lipid profile analysis system based on capillary electrophoresis according to any one of claims 1 to 4, wherein when the number of the photochemical reactors is greater than 1, the light sources of different photochemical reactors emit There is no overlapping area between the bands.
The lipid profile analysis system based on capillary electrophoresis according to any one of claims 1 to 5, wherein the mass spectrometer is a triple quadrupole mass spectrometer or a mass spectrometer capable of collision-induced fragmentation.
Application of the lipid profile analysis system based on capillary electrophoresis according to any one of claims 1 to 6 in biological lipid extract analysis and/or lipid biomarker screening.
A method for analyzing lipid groups using the capillary electrophoresis-based lipid group analysis system according to any one of claims 1 to 6, wherein the method includes:

Add the buffer solution and lipid group to the capillary tube, turn on the voltage device, apply voltage to both ends of the capillary tube, and the lipid group is electrophoretically separated in the capillary tube; when the lipid group flows through the chemical reactor, photochemical reactions;

Mass spectrometry signals are acquired by a mass spectrometer, and the lipid panel is analyzed.
The method of claim 8, wherein the buffer includes a derivatization reagent, the derivatization reagent includes acetone, benzophenone, diacetylpyridine, acetophenone derivatives, benzoylpyridine, phenylglyoxylic acid At least one ester.
The method according to claim 9, wherein the buffer solution further includes a buffer electrolyte, and the buffer electrolyte includes at least one of formic acid, ammonium acetate, and ammonium bicarbonate.
The method according to claim 10, wherein when the buffer electrolyte is solid, the buffer electrolyte is added in the form of an aqueous buffer electrolyte solution;

The buffer also includes a solvent including acetonitrile.
The method according to claim 11, wherein when the buffer electrolyte is solid, the concentration of the buffer electrolyte in the added buffer electrolyte aqueous solution is 10-50 mmol/L, and the pH of the buffer electrolyte aqueous solution is 8-10.
The method according to claim 12, wherein the volume ratio of the derivatization reagent to the solvent and the buffer electrolyte is (1-5):(2-6):(1-5).
The method according to claim 11 or 12, wherein when the derivatization reagent and the buffer electrolyte are solid, in the solution formed by the derivatization reagent, the solvent and the buffer electrolyte aqueous solution, the The concentration of the derivatization reagent is 2-20 mmol/L, and the volume ratio of the solvent to water is (5-9): (1-5).
The method according to any one of claims 8 to 14, wherein before the buffer and lipid group are added to the capillary, the method further includes: adding an inorganic alkali solution to the capillary for activation, and then using water and buffer. Liquid flushes the capillary tube.