A method for evaluating the extraction efficiency of vesicles in a sample

Abstract

The application discloses a method for evaluating extraction efficiency of vesicles in a sample, which comprises adding a liposome standard sample containing standard substance to a sample to be detected, extracting and detecting the sample, analyzing the content of the standard substance, and evaluating the extraction efficiency of the vesicle component in the sample extraction process. The method is simple and reliable, can effectively evaluate the purification efficiency of the vesicles, and improves the stability of the vesicle proteomics.

Description

Technical Field

[0001] This invention belongs to the field of vesicle proteomics technology, specifically, it relates to a method for evaluating the vesicle extraction efficiency in a sample. Background Technology

[0002] 1. Composition of liposomes

[0003] Some amphiphilic molecules, such as many natural and synthetic surfactants, spontaneously form ordered molecular assemblages with closed bilayer structures when dispersed in water; these are also called vesicles or liposomes. A vesicle is a spherical bilayer composed of one or more lipids. The type, number, and ratio of lipids can vary, and they can be derived from natural sources or synthetically. At least one (or some) of the lipids is amphiphilic, defined as having a hydrophilic portion and a hydrophobic portion (typically a hydrophilic head and a hydrophobic tail). The hydrophobic portion typically extends towards the hydrophobic phase (inside the bilayer), while the hydrophilic portion typically faces the aqueous phase (outside the bilayer, possibly between adjacent adjacent bilayer surfaces). The hydrophilic portion may contain polar or charged groups, such as carbohydrates, phosphates, carboxyl groups, sulfate groups, amino groups, thiol groups, nitro groups, hydroxyl groups, and other similar groups. The hydrophobic portion may contain nonpolar groups, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbon groups and one or more aromatic groups, alicyclic groups, or groups substituted with alicyclic groups. Examples of amphiphilic compounds include, but are not limited to, phospholipids, amino esters, sphingolipids, and phosphate ethanol amino esters.

[0004] Lipids typically refer to phospholipids, but they can also be anionic and neutral (including zwitterionic and polar) lipids, including anionic and neutral phospholipids. At a given pH, neutral lipids exist as uncharged or neutral zwitterionic forms. At physiological pH, such lipids include, for example, dioleoylphosphatidylglycerol (DOPG), diphosphatidylcholine, diphosphatidylethanolamine, sphingomyelin, cephalin, cholesterol, cerebrosides, and dioleoylglycerols. Examples of zwitterionic lipids include, but are not limited to, dioleoylphosphatidylcholine (DOP), dimylazolipidylcholine (DMPC), and dioleoylphosphatidylserine (DOPS). Anionic lipids are negatively charged lipids at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diphosphatidylserine, diphosphatidic acid, N-dodecylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-pentylphosphatidylethanolamine, lysine phosphatidylglycerol, palm oil phosphatidylglycerol (POPG), and other anionic modifying groups bound to neutral lipids.

[0005] Anionic lipids and neutral lipids are collectively referred to as noncationic lipids in this paper. Such lipids may contain phosphorus, but are not limited to this. Examples of noncationic lipids include lecithin, lysophosphatidylethanolamine, phosphatidylethanolamine, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyroxyphosphatidylethanolamine (DMPE), distearate phosphatidylethanolamine (DSPE), oleoylphosphatidylethanolamine (POPE), palmitoylphosphatidylcholine (POPC), methionine phosphatidylcholine (EPC), distearate phosphatidylcholine (DSPC), and dioleoylphosphatidylcholine (DOP). C) Dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palm oleoylphosphatidylglycerol (POPG), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, palm oleophobic phosphatidylethanolamine (POPE), 1-sclero-2-oleo-phosphatidylethanolamine (SOPE), phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetylphosphatide, and cholesterol. Other non-phospholipases include stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, ricinoleic acid glyceryl acetate, hexadecyl stearate, myristate isopropionyl, amphoteric acrylic polymers, triethanolamine dodecyl sulfate, alkylaryl sulfate polyethoxylated fatty amides, bis(octadecyl)dimethylammonium bromide, dimethylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, cephalin, and cerebrosides. In some cases, lipids such as lysophosphatidylcholine and lysophosphatidylethanolamine may be used. Non-cationic lipids also include polyethylene glycol-based polymers such as PEG 2000, PEG 5000, and polyethylene glycol conjugated with phospholipids or ceramides (called PEG-Cer).

[0006] 2. Biological significance of liposomes

[0007] Vesicles are common membrane structures in eukaryotic cells, possessing a phospholipid bilayer. They are important biologically derived liposomes, serving as essential functional components of the intracellular membrane system and carriers for the directed transport of intracellular substances. Currently, omics research on these fluid-borne vesicles, including but not limited to exosome microscopy and platelet microscopy, is receiving increasing attention due to the growing sophistication of extraction and analytical techniques.

[0008] Platelets and intracellular organelles, such as the endoplasmic reticulum and Golgi apparatus, can secrete vesicles, which are themselves relatively large vesicles. Human body fluids, including blood, urine, cerebrospinal fluid, pleural fluid, and saliva, all contain vesicles released by cells, primarily exosomes. These vesicles contain substances released from various tissues and organs of the organism, thus holding a wealth of potentially usable physiological or pathological information. Exosomes are membranous vesicles widely distributed in various body fluids and secreted by multiple cells. They have a vesicle structure formed by a lipid bilayer, with a diameter generally between 30 and 120 nm. They contain cell-specific proteins, lipids, and nucleic acids. Besides carrying and transmitting important signaling molecules, forming a novel intercellular communication system that alters the function of other cells, they play a crucial role in many physiological and pathological processes. Many types of cells in the human body can secrete exosomes. Exosomes are also found in human body fluids (blood, urine, saliva, and milk), and exosomes secreted by cells are present in the conditioned culture medium of cells in vitro.

[0009] Vesicle extraction is crucial for studying the components within vesicles, and its efficiency directly impacts the qualitative and quantitative identification of the target substances. However, currently only a limited number of methods exist for the qualitative and quantitative analysis of pure vesicles, and there is no effective means to assess vesicle purification efficiency. For vesicle proteomics, the purification efficiency directly determines the amount of proteome extracted, which in turn affects the quality of subsequent proteomic identification data. Summary of the Invention

[0010] Purpose of the invention: To address the shortcomings of current methods for evaluating the extraction efficiency of vesicle structures in various extraction schemes, this invention provides a method that utilizes liposomes to encapsulate protein or peptide standards to construct liposome standards. These liposome standards are added to the target extract during the extraction process, and the content of the corresponding standards is detected and analyzed using appropriate methods to evaluate the extraction efficiency of vesicle structures in proteomics.

[0011] Technical solution: To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0012] A method for evaluating the vesicle extraction efficiency in a sample includes the following steps:

[0013] 1) Prepare liposome standards containing protein or peptide compound standards;

[0014] 2) Take two equal amounts of samples, one as the target sample and the other as the control sample. Add liposome standard to the target sample and add an equal amount of diluent to the control sample. Extract vesicles from both samples simultaneously.

[0015] 3) Take the sample after treatment in step 2), add liposome standard to the control sample, the amount added is the same as the amount added to the target sample in step 2), add an equal amount of diluent to the target sample, and perform proteomic detection pretreatment on both samples at the same time.

[0016] 4) Detect the sample after step 3) and analyze the content of protein or peptide compound standard substances. The ratio of the content in the target sample to the content in the control sample is the vesicle structure extraction efficiency.

[0017] Furthermore, the preferred or specific implementation scheme is as follows:

[0018] 1. Preparation of liposome standards:

[0019] a. Liposome standards consist of a double-layered outer shell of liposomes and standard substances contained within them;

[0020] b. The liposome bilayer shell has an amphiphilic polymeric perimembrane, and the amphiphilic liposomes have a size ranging from 10 nm to 10 μm.

[0021] The size is based on the target liposome profiling type, such as exosomes: 30-150 nm, platelets: 1-8 μm; c. Lipids in liposomes include anionic lipids and neutral lipids, collectively referred to as non-cationic lipids in this paper.

[0022] Lipids can contain phosphorus, but they are not limited to this. Examples of non-cationic lipids include lecithin, lysophosphatidylethanolamine, lysophosphatidylethanolamine, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearate phosphatidylethanolamine (DSPE), oleoylphosphatidylethanolamine (POPE), palmitoylphosphatidylcholine (POPC), methionine phosphatidylcholine (EPC), distearate phosphatidylcholine (DSPC), and dioleoylphosphatidylcholine (DOP). C) Dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palm oleoylphosphatidylglycerol (POPG), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, palm oleophobic phosphatidylethanolamine (POPE), 1-sclero-2-oleo-phosphatidylethanolamine (SOPE), phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetylphosphatide, and cholesterol. Other non-phosphorus lipids include stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glyceryl ricinoleate, hexadecyl stearate, isopropionyl myristate, amphoteric acrylic polymers, triethanolamine dodecyl sulfate, alkylaryl sulfate polyethoxylated fatty amides, bis(octadecyl)dimethylammonium bromide, dimethylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, cephalin, and cerebrosides. In some cases, lipids such as lysophosphatidylcholine and lysophosphatidylethanolamine can be used. Non-cationic lipids also include polyethylene glycol-based polymers such as PEG 2000.

[0023] PEG5000 and polyethylene glycol conjugated with phospholipids or neuroamines (referred to as PEG-Cer). Preferably, the lipids are lipids that closely resemble the components of real biological cell membranes, such as lecithin, sphingomyelin, cephalin, etc.

[0024] d. The standard substance contained is an amino acid chain, which can be a protein or peptide; the standard substance is preferably a substance not present in the target sample, including substances with different amino acid sequences, different isotope labels, different site modifications, different tag modifications, etc.

[0025] e. Liposome synthesis methods include, but are not limited to, injection method, thin film dispersion method, ultrasonic dispersion method, and high pressure homogenization method, with microfluidic injection method and ultrasonic dispersion method being preferred;

[0026] 2. Add the liposome standard to the target sample, along with the extraction process:

[0027] a. The target sample is a natural sample containing vesicle structures, including body fluid samples such as plasma, urine, cerebrospinal fluid, pleural fluid, saliva, etc., as well as tissue or cell lysates, cell culture media, microbial cell lysates, microbial culture media samples, etc.

[0028] b. The vesicle extraction process includes steps such as adsorption of vesicles and cleaning to remove impurities, such as high-speed centrifugation, filtration, chromatography, material adsorption, and antibody binding.

[0029] 3. Sample pretreatment for subsequent proteomics analysis:

[0030] The detection of standard substances in liposome standards includes the detection of proteins and peptides;

[0031] The preferred method for detecting proteins and peptides is high-performance liquid chromatography-mass spectrometry (HPLC-MS), which can also be applied to related qualitative and quantitative protein experiments, such as Western blot colorimetry, ELISA quantification, and other experiments based on the binding of corresponding antibodies, including immunoluminescence, chemiluminescence, and fluorescence colorimetry.

[0032] The pretreatment steps for proteomics detection include reductive alkylation, protein purification, enzymatic digestion, desalting, and peptide purification, which serve as pretreatment for subsequent liquid chromatography-mass spectrometry methods.

[0033] The relevant qualitative and quantitative experiments for proteins involve direct or simple processing (such as resuspension, boiling, elution, etc.) of the vesicle samples after adsorption and washing, followed by experiments based on the binding of corresponding antibodies, such as Western blot color development, ELISA quantification, immunoluminescence, chemiluminescence, and fluorescence color development.

[0034] 4. The processed samples are tested, and the content of protein or peptide compound standard substances is analyzed. The ratio of the content in the standard sample to the content in the control sample is the vesicle structure extraction efficiency.

[0035] a. The preferred method for proteomic sample detection is a non-targeted method based on liquid chromatography-mass spectrometry, including label-free quantification, data-independent acquisition mass spectrometry (DIA-MS), tandem mass spectrometry labeling (TMT), in vitro isotope labeling (iTRAQ), and matrix-assisted laser desorption / ionization (MALDI); targeted methods, including parallel reaction monitoring (PRM) and multiple reaction detection mass spectrometry (MRM-MS), can also be used.

[0036] b. For non-targeted methods based on liquid chromatography-mass spectrometry, the method itself detects all information in the sample, extracts data from it based on the information of protein or peptide standards, compares the ratios of the obtained quantitative data, and obtains the vesicle structure extraction efficiency.

[0037] c. For targeted methods based on liquid chromatography-mass spectrometry, parameters need to be set according to the information of protein or peptide standards. While obtaining the information of the target substance in the sample, the quantitative information of the protein or peptide standards is detected. The ratio of the obtained quantitative data is compared to obtain the vesicle structure extraction efficiency.

[0038] d. Related qualitative and quantitative protein experiments, including Western blot colorimetry, ELISA quantification, and other experiments based on antibody binding such as immunoluminescence, chemiluminescence, and fluorescence colorimetry, all operate on the principle of antibody-specific binding to protein or peptide markers. The luminescent groups on the antibody produce color. Due to differences in the proportion of recovered liposomes, the content of protein or peptide markers varies, resulting in differences in color development. Quantitative comparisons are performed by analyzing the color intensity.

[0039] The extraction efficiency of vesicle structures was obtained;

[0040] Beneficial effects: Compared with the prior art, the present invention extracts and detects samples by adding liposomes containing standard substances to the sample to be tested, and analyzes the content of the standard substances, thereby evaluating the extraction efficiency of vesicle components during the sample extraction process. This method is simple and reliable, can effectively evaluate the purification efficiency of vesicles, and improve the stability of vesicle proteomics. Attached Figure Description

[0041] Figure 1 This is a flowchart illustrating the method for evaluating the vesicle extraction efficiency in samples according to the present invention.

[0042] Figure 2 The content of bovine serum albumin in each component was determined by Western blot. Detailed Implementation

[0043] The following provides a comprehensive description of the present invention. The embodiments described are the most preferred embodiments of the present invention, but the present invention is not limited to the following embodiments.

[0044] Example 1

[0045] 1) Preparation of liposome standards: Bovine serum albumin was used in this study to evaluate human samples.

[0046] 1. Add lecithin to chloroform, remove the chloroform using rotary evaporation, and distribute the lecithin evenly in the rotary evaporation flask;

[0047] 2. Add 1 mg / mL bovine serum albumin solution to the rotary evaporator flask and shake thoroughly to obtain multilayer liposomes (MLV); 3. Centrifuge the multilayer liposomes (MLV) at 12,000 rpm for 10 min, discard the supernatant, and you can obtain liposome standard 1 with an average particle size of 1.5 μm.

[0048] 4. Transfer the multilayer liposomes (MLV) to centrifuge tubes and place them in a 0°C water bath;

[0049] 5. Immerse the probe of the ultrasonic cell disruptor into the sample, adjust the ultrasonic power to about 100W, set the timer to 10-30min, and the effective ultrasonic time to 50%. The dispersion must change from milky white to opalescent – ​​so that the window frame can be seen through the sample when facing sunlight.

[0050] 6. Centrifuge at 12,000 rpm for 10 min to obtain liposomes with a diameter of about 100 nm, thus obtaining liposome standard 2; 7. Detect liposome standard 1 and liposome standard 2 using the BCA detection method. Dilute both liposomes to 0.01 mg / mL according to the measured concentration.

[0051] 2) Use liposome markers to evaluate the recovery efficiency of liposomes of different sizes.

[0052] 1. Take three 40μL human plasma samples, add 10μL of water to plasma A, add 10μL of liposome evaluation sample 1 to plasma B, and add 10μL of liposome evaluation sample 2 to plasma C;

[0053] 2. Three plasma samples were processed using a commercially available plasma protein adsorption kit, which involved adding nano-adsorption magnetic beads and buffer solution, incubating thoroughly, magnetically separating the supernatant, and washing three times with washing buffer.

[0054] 3. Add 10 μL of liposome evaluation sample 1 or liposome evaluation sample 2 to sample A after plasma adsorption, and add 10 μL of water to samples B and C;

[0055] 4. Perform the same pre-proteomic processing on the three samples from step 3, including reductive alkylation, enzymatic digestion, and desalting, to obtain proteomic samples, and then perform liquid chromatography-mass spectrometry detection.

[0056] 5. The mass spectrometry data obtained from the detection were extracted and analyzed to obtain the quantitative data of bovine serum albumin in three samples A, B and C, as shown in Table 1;

[0057] Table 1. Quantitative data of bovine serum albumin

[0058]

[0059]

[0060] 6. As shown in Table 1, the kit used in this study achieved a recovery rate of 76.23% for large-sized (1 μm level) liposome vesicle structures, which is lower than the recovery rate of 92.43% for small-sized (100 nm level) liposome vesicle structures. This indicates that the kit is more suitable for the recovery of small-sized (100 nm level) liposome vesicle structures.

[0061] 3) Monitor the purification process of the exosome purification kit using liposome standard 2.

[0062] 1. Collect 50 mL of culture medium supernatant from HeLa cells and concentrate it using a 100 kDa ultrafiltration centrifuge tube to 2-3 mL. Transfer the concentrated sample from the ultrafiltration tube to a new centrifuge tube.

[0063] 2. Add 10 μL of liposome standard 2 to the concentrated sample;

[0064] 3. Take a commercially available exosome purification column (exclusion method), add 2 column volumes of PBS to replace the blocking buffer for activation;

[0065] 4. Add 1 mL of concentrated sample into the activated exosome purification column;

[0066] 5. Once all samples have entered the purification column, PBS is continuously added for elution. Each half column volume of elution buffer is collected as one fraction sample.

[0067] 6. Samples from each component were taken for electrophoresis, and Western blot was used to detect bovine serum albumin concentration to identify the component containing the exosomes, such as... Figure 2 ;

[0068] 7. It was confirmed that the bands of components 3, 4, and 5 were clear, indicating that their bovine serum albumin content was high. In other words, liposome standard 1 had a high content of these three components, which indicates that it has a high concentration of exosomes. These three components were combined and concentrated to the expected volume using a 100kDa ultrafiltration centrifuge tube, which is the purification of exosomes.

Claims

1. A method for evaluating the vesicle extraction efficiency in a sample, characterized in that, Includes the following steps: 1) Prepare liposome standards encapsulating protein or peptide compounds; 2) Take two equal amounts of samples, one as the target sample and the other as the control sample. Add liposome standard to the target sample and add an equal amount of diluent to the control sample. Extract vesicles from both samples simultaneously. 3) Take the sample processed in step 2), add liposome standard to the control sample, the amount added is the same as the amount added to the target sample in step 2), add an equal amount of diluent to the target sample, and perform proteomic detection pretreatment on both samples at the same time; the pretreatment includes reductive alkylation, protein purification, enzymatic digestion, desalting, and peptide purification steps to obtain proteomic samples. 4) Perform liquid chromatography-mass spectrometry on the sample after step 3) and analyze the content of protein or peptide compound standard substances. The ratio of the content in the target sample to the content in the control sample is the vesicle structure extraction efficiency.

2. The method for evaluating the vesicle extraction efficiency in a sample according to claim 1, characterized in that, In step 1), the lipids in the liposome standard are selected from anionic lipids or neutral lipids; the protein or peptide compound standard is selected from substances not present in the target sample.

3. The method for evaluating the vesicle extraction efficiency in a sample according to claim 1, characterized in that, In step 1), the particle size range of the liposome standard is 10 nm-10 μm, and its size is based on the target liposome omics type, such as exosomes: 30-150 nm, platelets: 1-8 μm.

4. The method for evaluating the vesicle extraction efficiency in a sample according to claim 1, characterized in that, In step 1), the preparation method of the liposome standard includes injection method, thin film dispersion method, ultrasonic dispersion method or high pressure homogenization method.

5. The method for evaluating the vesicle extraction efficiency in a sample according to claim 1, characterized in that, In step 2), the sample is selected from body fluid samples containing vesicle structures, tissue or cell lysates, cell culture media, microbial cell lysates, or microbial culture media; the diluent is selected from phosphoric acid, citric acid, carbonic acid, acetic acid, barbituric acid, Tris buffer, or physiological saline, or from water.

6. The method for evaluating the vesicle extraction efficiency in a sample according to claim 1, characterized in that, In step 2), the extraction of the vesicles includes the steps of adsorbing vesicles and washing to remove impurities.

7. The method for evaluating the vesicle extraction efficiency in a sample according to claim 1, characterized in that, In step 3), the diluent is selected from phosphoric acid, citric acid, carbonic acid, acetic acid, barbituric acid, Tris buffer or physiological saline, or water.

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