Enrichment and extraction method for soluble protein in tissue sample

Abstract

The invention discloses a method for enriching and extracting soluble protein in a tissue sample, which comprises the following steps: adding the tissue sample into a low-salt buffer solution, homogenizing, centrifuging, collecting supernatant precipitate, resuspending the precipitate by using Triton, and obtaining the supernatant to obtain an extracting solution; adding anhydrous acetonitrile to precipitate protein and collecting precipitate; carrying out proteolysis reductive alkylation and quenching reaction treatment on the precipitate to obtain a peptide fragment sample; desalting by using a peptide fragment sample desalting cleaning solution; after washing, adding a desalted eluent for eluting, collecting an eluent containing the peptide fragment, and drying to obtain a desalted peptide fragment; performing reversed-phase fractionation chromatography under an alkaline condition; and finally, LC-MS / MS detection is carried out. Under the condition that a strong denaturing agent is not introduced, secretory protein, cytoplasm protein and membrane protein can be selectively enriched, meanwhile, most of cell nucleus protein and tissue interstitial fibrin are effectively removed, and therefore the detection capacity of follow-up mass spectrometric detection on extremely-low-abundance circulating markers is remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of soluble protein extraction, specifically to the enrichment, extraction, and detection of soluble proteins in tissue samples, thereby increasing the possibility of discovering disease biomarkers in peripheral blood from proteomics analysis of clinical tissue samples. Background Technology

[0002] Extracting all proteins from diseased tissue samples and comprehensively analyzing them using mass spectrometry to identify differentially expressed proteins and further verify their levels in peripheral blood has long been a common and primary method for discovering serum protein biomarkers in clinical testing. This method typically involves lysing the entire tissue using reagents such as urea to extract all proteins, which are then analyzed. However, serum proteins are mostly soluble proteins derived from secreted, cytoplasmic, or membrane proteins within tissue cells. The proteins extracted from the entire tissue sample include poorly soluble proteins such as nuclear proteins and interstitial proteins, which are generally difficult to secrete into peripheral blood, making them less likely to become peripheral blood biomarkers. Furthermore, their high concentrations in the sample can interfere with mass spectrometry detection of highly soluble proteins with potential peripheral blood biomarker value, such as secreted, cytoplasmic, or membrane proteins. Therefore, how to extract and enrich proteins from tissue samples that may potentially appear in peripheral blood is an urgent technical problem to be solved. Summary of the Invention

[0003] 1. The technical problem to be solved: To address the aforementioned technical problems, this invention provides a method for enriching and extracting readily soluble proteins from tissue samples. Utilizing a low-salt approach combined with Triton X-100, the method gently extracts tissue samples, resulting in significant enrichment of readily soluble proteins. This method leverages the fact that readily soluble proteins are more likely to be secreted into peripheral blood than nucleoproteins and intercellular matrix proteins, thus increasing the likelihood of discovering protein molecules that could serve as disease biomarkers in peripheral blood, providing original findings for clinical laboratory-aided diagnosis of diseases.

[0004] 2. Technical Solution: A method for enriching and extracting readily soluble proteins from tissue samples, characterized by comprising: Step 1: Enrichment and extraction of soluble proteins; add tissue samples to low-salt buffer, homogenize and centrifuge to collect supernatant and precipitate, resuspend the precipitate with Triton and obtain supernatant to obtain the extract; Step 2: Protein precipitation; Add anhydrous acetonitrile to the extract to precipitate the protein, and collect the precipitate; Step 3: Proteolytic digestion and reductive alkylation: The precipitate is subjected to proteolytic digestion, protein reductive treatment, protein alkylation treatment, and quenching reaction treatment in sequence to obtain peptide samples; Step 4: Peptide desalting: Load the peptide sample into the desalting column and desalt it using desalting washing solution; wash, then add desalting eluent again for elution, collect the eluent containing peptides, and dry it to obtain desalted peptides; Step 5: Reverse-phase fractionation chromatography under alkaline conditions; the desalted peptides are fractionated by reverse liquid chromatography, and the collected peptide fractions are dried; Step 6: LC-MS / MS detection: The peptide sample collected by fractionation is injected into a liquid chromatography-mass spectrometry (LC-MS) instrument for mass spectrometry detection; the raw mass spectrometry data are analyzed using a protein database.

[0005] Further, in step one, the tissue sample is human tissue or mouse tissue; the low-salt buffer is a Tris-HCl aqueous solution with a molar concentration of 25 mM and a pH of 7.4, or a Hepes aqueous solution with a molar concentration of 25 mM and a pH of 7.5; in step one, the low-salt buffer contains a protease inhibitor mixture PIC; the volume ratio of 100 times working concentration PIC aqueous solution to low-salt buffer is 1:100.

[0006] Further, step one specifically includes: taking tissue samples, adding low-salt buffer at a ratio of 1 g tissue sample to 10 mL low-salt buffer, wherein PIC aqueous solution has been added to the low-salt buffer; homogenizing in a Dounce homogenizer, centrifuging at 4 ℃ and 20000 g for 5 min, collecting the supernatant LS fraction, and retaining the precipitate; resuspending the retained precipitate in a 1% Triton X-100 aqueous solution, the volume of which is the same as that of the supernatant LS fraction; centrifuging again to obtain the supernatant Triton; mixing the LS fraction and the supernatant Triton to form a Mix sample, which is used for subsequent detection.

[0007] Furthermore, in step two, the volume ratio of the extract to anhydrous acetonitrile is 1:4. The protein precipitate is retained by centrifugation and washed three times with 80% acetonitrile.

[0008] Further, step three specifically involves: adding the precipitate obtained in step two to an 8 M urea solution, the solvent of which is a 25 mM TEAB aqueous solution with a pH of 8.5; simultaneously adding one working concentration of PIC aqueous solution to the urea solution to prevent protein degradation; rotating and mixing at room temperature for at least 4 hours or overnight at 4°C until the precipitate is fully dissolved or the solution becomes clear; then, according to the protein concentration and total amount, adding Lys-C enzyme at a mass ratio of enzyme to substrate of 1:100 and performing enzymatic hydrolysis at room temperature; after 4 hours, diluting the sample after Lys-C hydrolysis with three times the volume of 25 mM TEAB to a 2 M urea solution, then adding trypsin for digestion, and incubating at 37°C for at least 4 hours; subsequently, adding 1 mM dithiothreitol for reduction for 30 min, 3 mM iodoacetamide for alkylation in the dark for 15 min, and finally terminating the reaction with 1.5 mM dithiothreitol.

[0009] Furthermore, in step four, a C18 desalting column is used; the desalting washing solution is a 0.1 wt% formic acid aqueous solution; and the desalting eluent is a 60-65 wt% acetonitrile aqueous solution containing 0.1 wt% formic acid.

[0010] Further, 4 mg of dried peptide fragments were redissolved in an aqueous solution of NH4HCO2 with a molar concentration of 10 mM and a pH of 8.0; the dissolved peptide fragments were separated using an XBridge C18 column with a mobile phase of 15% to 45%, and approximately 100 fractions were collected and dried.

[0011] Further, in step six, each fraction obtained in step five is resuspended in 5% formic acid, and 1 µg is injected into the DDA mode of the electrostatic field orbital trap ultra-high resolution mass spectrometer; the raw mass spectrometry data are analyzed using a protein database.

[0012] 3. Beneficial effects: (1) This invention provides a method for enriching and extracting readily soluble proteins from tissue samples. It utilizes a mild lysis method combining a low-salt buffer with the neutral detergent Triton X-100 to enrich readily soluble proteins in tissues while inhibiting poorly soluble proteins such as nucleoproteins in the precipitate. When this method was used to extract brain tissue, membrane proteins and cytoplasmic proteins were significantly extracted, while nucleoproteins were not. Mass spectrometry analysis of all protein components in the extracted brain tissue samples revealed that readily soluble proteins such as membrane proteins, cytoplasmic proteins, and secretory proteins were more abundant in the readily soluble samples extracted with low-salt and Triton X-100 compared to the whole tissue samples, while the poorly soluble samples after readily soluble extraction contained more nucleoproteins. Furthermore, the relative levels of readily soluble proteins in the low-salt and Triton X-100 samples were significantly higher than those in the poorly soluble precipitate samples after readily soluble extraction.

[0013] (2) Using this method, 15 tissue samples from mice—heart, lung, esophagus, small intestine, large intestine, liver, gallbladder, spleen, kidney, pancreas, prostate, ovary, bladder, muscle, and blood vessels—were extracted. It was found that membrane proteins and cytoplasmic proteins were almost all effectively extracted, while nucleoproteins were retained. Mass spectrometry analysis showed that the number of membrane proteins, cytoplasmic proteins, and secretory proteins in the readily soluble extracted samples was higher than that in the whole tissue samples and the precipitate samples after readily soluble extraction, while the precipitate samples after readily soluble extraction contained a higher number of nucleoproteins. Overall protein analysis of the whole tissue samples, readily soluble extracted samples, and the poorly soluble precipitate samples after extraction revealed that readily soluble proteins were enriched in all these tissues.

[0014] (3) The method of using ultra-deep mass spectrometry proteomics analysis to detect soluble extracts of brain tissue was compared with similar methods reported for detecting whole brain tissue samples. It was found that this method detected a greater number of cytoplasmic proteins, membrane proteins, and secretory proteins, and had an enrichment effect on these proteins. The concentration of most protein markers in cerebrospinal fluid extracted by this method was 10-100 times higher than that in the reported methods for direct analysis of whole tissues, which greatly improved the detection sensitivity.

[0015] In summary, this mild tissue sample extraction method, utilizing low salt and Triton X-100, demonstrates significant enrichment of readily soluble proteins. Because these proteins are more likely to be secreted into peripheral blood than nucleoproteins and intercellular matrix proteins, this method is more likely to discover protein molecules that can serve as disease biomarkers in peripheral blood, providing original findings for clinical laboratory-aided diagnosis of diseases. Attached Figure Description

[0016] Figure 1 This is a schematic diagram illustrating the optimal concentration and number of repeated extractions for extracting mouse cerebral cortex tissue using Triton X-100. Figure 2 The experimental procedure (A) and sample (B) for the gentle extraction of mouse brain tissue using this method are shown in the figure. Figure 3 To verify the SDS-PAGE and Western blot images of the whole brain tissue lysate and extracted samples from the mouse in the example; Figure 4 To verify the heatmap analysis of the LC-MS / MS mass spectra and analysis results of the four protein samples in the example; Figure 5 To validate the results, SDS-PAGE and Western blot were used to demonstrate the mild extraction results of 16 mouse tissues. Figure 6To verify the mass spectrometry results of the mild extraction of 16 mouse tissues in the example; Figure 7 This outlines the workflow for mild extraction and deep proteomics analysis using this method. Detailed Implementation

[0017] The present invention will now be described in detail with reference to specific embodiments.

[0018] A method for enriching and extracting readily soluble proteins from tissue samples, characterized by comprising: Step 1: Enrichment and extraction of soluble proteins; add tissue samples to low-salt buffer, homogenize and centrifuge to collect supernatant and precipitate, resuspend the precipitate with Triton and obtain supernatant to obtain the extract; Step 2: Protein precipitation; Add anhydrous acetonitrile to the extract to precipitate the protein, and collect the precipitate; Step 3: Proteolytic digestion and reductive alkylation: The precipitate is subjected to proteolytic digestion, protein reductive treatment, protein alkylation treatment, and quenching reaction treatment in sequence to obtain peptide samples; Step 4: Peptide desalting: Load the peptide sample into the desalting column and desalt it using desalting washing solution; wash, then add desalting eluent again for elution, collect the eluent containing peptides, and dry it to obtain desalted peptides; Step 5: Reverse-phase fractionation chromatography under alkaline conditions; the desalted peptides are fractionated by reverse liquid chromatography, and the collected peptide fractions are dried; Step 6: LC-MS / MS detection: The peptide sample collected by fractionation is injected into a liquid chromatography-mass spectrometry (LC-MS) instrument for mass spectrometry detection; the raw mass spectrometry data are analyzed using a protein database.

[0019] Further, in step one, the tissue sample is human tissue or mouse tissue; the low-salt buffer is a Tris-HCl aqueous solution with a molar concentration of 25 mM and a pH of 7.4, or a Hepes aqueous solution with a molar concentration of 25 mM and a pH of 7.5; in step one, the low-salt buffer contains a protease inhibitor mixture PIC; the volume ratio of 100 times working concentration PIC aqueous solution to low-salt buffer is 1:100.

[0020] Further, step one specifically includes: taking tissue samples, adding low-salt buffer at a ratio of 1 g tissue sample to 10 mL low-salt buffer, wherein PIC aqueous solution has been added to the low-salt buffer; homogenizing in a Dounce homogenizer, centrifuging at 4 ℃ and 20000 g for 5 min, collecting the supernatant LS fraction, and retaining the precipitate; resuspending the retained precipitate in a 1% Triton X-100 aqueous solution, the volume of which is the same as that of the supernatant LS fraction; centrifuging again to obtain the supernatant Triton; mixing the LS fraction and the supernatant Triton to form a Mix sample, which is used for subsequent detection.

[0021] Furthermore, in step two, the volume ratio of the extract to anhydrous acetonitrile is 1:4. The protein precipitate is retained by centrifugation and washed three times with 80% acetonitrile.

[0022] Further, step three specifically involves: adding the precipitate obtained in step two to an 8 M urea solution, the solvent of which is a 25 mM TEAB aqueous solution with a pH of 8.5; simultaneously adding one working concentration of PIC aqueous solution to the urea solution to prevent protein degradation; rotating and mixing at room temperature for at least 4 hours or overnight at 4°C until the precipitate is fully dissolved or the solution becomes clear; then, according to the protein concentration and total amount, adding Lys-C enzyme at a mass ratio of enzyme to substrate of 1:100, and performing enzymatic hydrolysis at room temperature; after 4 hours, diluting the 8 M urea to a 2 M urea solution with three times the volume of 25 mM TEAB of the sample after Lys-C enzymatic hydrolysis, and then adding trypsin for digestion, and incubating at 37°C for at least 4 hours; subsequently, adding 1 mM dithiothreitol for reduction for 30 min, 3 mM iodoacetamide for alkylation in the dark for 15 min, and finally terminating the reaction with 1.5 mM dithiothreitol.

[0023] Furthermore, in step four, a C18 desalting column is used; the desalting washing solution is a 0.1 wt% formic acid aqueous solution; and the desalting eluent is a 60-65 wt% acetonitrile aqueous solution containing 0.1 wt% formic acid.

[0024] Further, 4 mg of dried peptide fragments were redissolved in an aqueous solution of NH4HCO2 with a molar concentration of 10 mM and a pH of 8.0; the dissolved peptide fragments were separated using an XBridge C18 column with a mobile phase of 15% to 45%, and approximately 100 fractions were collected and dried.

[0025] Further, in step six, each fraction obtained in step five is resuspended in 5% formic acid, and 1 µg is injected into the DDA mode of the electrostatic field orbital trap ultra-high resolution mass spectrometer; the raw mass spectrometry data are analyzed using a protein database.

[0026] Example 1: This embodiment used two 9-month-old mice for tissue protein extraction, and explored the appropriate concentration of Triton X-100 using mouse brain tissue. It was found that a 1% concentration of Triton X-100 added to low-salt buffer (LS) could fully extract readily soluble proteins from tissue samples (see attached). Figure 1 ).

[0027] In the figure, WTL represents whole tissue lysis buffer, i.e., tissue is ground in low-salt buffer with 1 working concentration of protease inhibitor mixture PIC added; LS represents low-salt buffer, i.e., 25 mM Tris-HCl, pH 7.4; Pellet represents the precipitate that is undissolved in both low-salt buffer and 1% Triton X-100 buffer; Triton represents the supernatant extracted after resuspending the precipitate in Triton. This example includes the following steps: 1. Extraction of mouse tissue proteins The mice were dissected, and the brain and other tissues were removed and immediately frozen at -80ºC. After dissection, the mouse brain tissue was separated into left and right hemispheres along the sagittal midline and collected for subsequent protein extraction and analysis. The left brain of the mouse was weighed and showed 0.208g. The brain was placed in a Dounce homogenizer, (1) 2ml LS buffer (10ml low salt buffer (25mM Tris-HCl, pH 7.4) per g of tissue) and 20ul 100×PIC (PIC is a mixture of protease inhibitors, and 100× indicates that the concentration is 100 times the working concentration) was added, and the brain tissue was homogenized several times on ice. The homogenate was carefully aspirated from the homogenizer and dispensed into EP tubes at 400ul each and immediately frozen at -80°C. One tube was reserved for use, and then centrifuged at 20,000g for five minutes to collect the supernatant of the separation liquid, i.e., the supernatant LS fraction, as thoroughly as possible. In this example, 320ul of the supernatant LS fraction was actually taken out. The collected precipitate (represented as precipitate 1 in the figure) was thoroughly resuspended in 320 μL of 1.0% Triton X 100 low-salt buffer and 3.2 μL of 100×PIC. The volume of 1.0% Triton X 100 low-salt buffer added was equal to the volume of the aspirated LS fraction, in order to restore the initial solution volume. After centrifugation at 20,000 g for five minutes, the supernatant Triton buffer was collected; in this example, 300 μL was obtained. All the above steps were performed on ice. The flowchart of the extraction process and sample images are attached. Figure 2 As shown in A and B.

[0028] 2. Protein precipitation Retain precipitate ( Figure 2The sample, designated as precipitate 2 (Pellet), was washed twice with 80% acetonitrile (ACN). The supernatant was discarded, leaving only the precipitate. The precipitate was left exposed for approximately 5 minutes to allow 80% of the ACN to evaporate. The precipitate (Pellet fraction) was then dissolved in 200 μL of 8M urea and 2 μL of 100×PIC solution, and incubated overnight at 4°C using a shaker to obtain the Pellet protein sample. The LS fraction and the supernatant Triton fraction were combined (as the extract, mixed sample) for proteomic analysis.

[0029] 3. The protein concentration of the Mix component was determined using a protein quantification BCA kit, and the sample concentration was 9.86 ug / ul.

[0030] 4. SDS-PAGE and Western Blot analysis were performed on the extracted components. Heating each extracted sample, pre-treated with LDS loading buffer, to 75ºC for 10 minutes under 10 mM DTT conditions. Then loading onto 4-12% gradient Bis-Tris gels (M00654, SurePAGE, Genscript) for electrophoresis: 4%-12% pre-cast gels and equilibration plates were added to the electrophoresis tank. ddH2O was added to the inner tank to check for leaks. After standing for three minutes without any change in the liquid level, freshly prepared MOPS electrophoresis buffer was added. The 15-well comb was carefully and slowly removed, ensuring the wells remained intact. Using a 10 μL pipette, 1.5 μL of pre-stained protein marker and each protein (each sample was mixed with 4xLDS to a 1xLDS sample) were added sequentially to the comb (WTL component, LS component, Triton component, Pellet component: 2, 2, 2, 1 μL respectively). When aspirating the sample, aspirate slowly to avoid air bubbles. Position the pipette directly over the well and slowly and vertically load the sample, being careful not to let it overflow due to excessive speed. Add recyclable electrophoresis buffer to the outer tank. Set the electrophoresis parameters to 150V for 40 minutes. Carefully remove the gel from the clamp, keeping it moist with water to prevent it from drying out and cracking. Gently rinse the surface of the gel with ddH2O to remove the electrophoresis buffer. Then, stain with Coomassie blue, covering it with plastic wrap to prevent evaporation. Shake on a shaker at room temperature for 1 hour, observing for protein bands. Adjust the staining time as needed based on the staining progress. Afterward, recover the staining solution, destain the gel in water, and observe the stained protein bands. The SDS-PAGE results are attached. Figure 3 A, Appendix Figure 5 As shown, this method can effectively separate protein bands.

[0031] Take another gel and perform electrophoresis in the same manner. Then perform Western blotting on the gel block. Activate the nitrocellulose membrane (0.2 μm) by placing it in transfer buffer containing methanol for 5 minutes. Use a transfer sandwich clamp to clamp and fix it on the transfer tank in the following order: black gel, white membrane, sponge pad, three layers of filter paper, gel block, nitrocellulose membrane, three layers of filter paper, sponge pad. Pour in the transfer buffer (50 ml of rapid transfer buffer (20X) + 850 ml of ddH2O + 100 ml of methanol + 1 ml of 10% LDS). Apply a constant current of 400 mA for 40 min. The protein is transferred to the nitrocellulose membrane. The membrane was removed and rinsed off the transfer buffer under a slow stream of water. It was then blocked with 5% skim milk in TBST buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.05% Tween-20) for approximately 15 minutes. The membrane was washed with TBST buffer, front and back sides, for 3 minutes each. It was then incubated with primary antibody in 3% bovine serum albumin in 1×TBST at room temperature for 1 hour. The primary antibody was recovered, and the membrane was washed with TBST buffer, front and back sides, for 3 minutes each. Next, horseradish peroxidase-conjugated secondary antibody in 2.5% milk powder was added, and the membrane was incubated at room temperature for 1 hour. After TSBT washing and cleaning, the signal was visualized using a chemical substrate (SuperSignal WestPico, 34087, Thermo Scientific) for imaging. Primary antibodies included: anti-sodium / potassium transporter (ab300507, Abcam), anti-amyloid precursor protein (APP) (ab32136, Abcam), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (60004-1, Proteintech), anti-α-tubulin (#2144, Cell Signaling Technology), anti-Lamin B1 (ab133741, Abcam), and anti-histone H4 (ab177840, Abcam). Western blotting results are attached. Figure 3 B, Appendix Figure 5 As shown, membrane proteins (ATP1A and APP) and cytoplasmic proteins (Tubulin and GAPDH) are mainly distributed in the mildly extracted LS and Triton fractions, while nuclear proteins (Lamin B1 and Histone4) are mainly retained in the final precipitate.

[0032] 5. Enzymatic digestion of extracted components and analysis by LC-MS / MS Take 500 μL of protein samples from each extraction fraction. Based on the protein concentration quantification result of the Mix fraction (9.86 μg / μL), the Mix fraction contains approximately 493 μg. Add 1500 μL of 100% ACN to the extracted Mix sample to form 80% ACN for protein precipitation. After centrifugation, discard the supernatant. Wash twice repeatedly with 1.5 mL of 80% CAN, discarding the supernatant and retaining the precipitate. Dissolve the precipitate in 8M urea (25 mM TEAB, pH 8.5). Add 5 μg of LysC at a 1 / 100 enzyme / substrate ratio and react at room temperature for 4 hours or longer until the solution becomes clear. Then, dilute the urea to 2 M with 3 volumes of 25 mM TEAB (pH 8.5) and add trypsin (1 / 100 enzyme / substrate ratio) to continue digestion in solution. After reacting at room temperature for 4-8 hours, add DTT for reduction, followed by IAA basicization and DTT termination. The samples were acidified with 1% formic acid, desalted by passing them through a C18 column, and dried for LC MS / MS analysis.

[0033] Desalting steps for protein samples: 1. 100% methanol, 200 μL, column chromatography (activation) 2. Wash with 200ul of elution salt solution (60% ACN + 0.1% FA) 3. Equilibrate twice with 200ul of 0.1% FA. 4. Add sample and collect filtrate (the filtrate will be filtered a total of 3 times, starting from step one). 5. 200ul 0.1% FA twice 6. Rinse twice with 200 μL of 5% ACN + 0.5% FA. 7. Rinse once with 200ul of elution salt solution. 8. Collect all the elution salt solution, dry it, and prepare it for use in the machine. 9. Finally, rinse the column with 100% methanol for next use. In mass spectrometry analysis, WTL, Mix (LS+Triton), and Triton and Pellet components of the tissue sample were detected (see attached). Figure 6 A), and statistically analyzed the number of cytoplasmic proteins, membrane proteins, and secretory proteins detected. It was found that the number of these proteins in the mixed sample extracts of most tissue samples was higher than that in the WTL (Appendix). Figure 6 B). Comparing the levels of these proteins in the Mix and Pellet samples, and then taking the logarithm of Log2, reveals that the protein count tends to skew towards a positive value. This indicates that the levels of these proteins in the Mix sample are higher than those in the Pellet sample, meaning that these proteins are relatively enriched in the Mix sample. (See appendix) Figure 6 C).

[0034] Example 2 This method was used to extract readily soluble proteins (Mix component) from the brain tissue of Alzheimer's disease (AD) model mice and to perform ultra-deep protein detection (see attached). Figure 7 A), namely, RPLC fractionation of the desalted peptides under alkaline pH conditions: A liquid chromatography system (Shimadzu) and a C18 column (XBridge C18 column 3.5 μm, 4.6 mm x 250 mm, Waters) were used. In the liquid chromatography system, the fractionated mobile phase A was 10 mM ammonium formate aqueous solution (NH4COOH), pH 9.0; the fractionated mobile phase B was 90% acetonitrile aqueous solution containing 10 mM ammonium formate, pH 9.0.

[0035] ① Dissolve and resuspend 4 mg of desalted peptide (based on protein) from each sample in 0.5 mL of mobile phase A. ② Prepare the liquid chromatography system, and after washing the system with at least 50 mL of fractionated mobile phase B, equilibrate the system with at least 50 mL of fractionated mobile phase A; ③ Dissolve 4 mg of desalted peptides (based on protein content) in 0.5 mL of mobile phase A and resuspend the protein sample; ④ Fractionate the sample using gradient flow, with the specific gradient design as follows: 0.01-15 minutes, 5%-5% B; 15-105 minutes, 5-40% B; 105-108 minutes, 40-65% B; 108-110 minutes, 65-95% B; 110-120 minutes, 95-95% B.

[0036] Liquid flow rate: 1 mL / min. An automatic collector was used to collect 1 tube per minute. Approximately 120 tubes were collected in total.

[0037] ⑤ Drain the collected fractionation tubes.

[0038] (6) Mass spectrometry detection and protein identification of peptides in each fractionation tube after drying: The specific experimental procedure is described above, or as shown below: The collected fraction samples were dissolved in 5% formic acid to a concentration of approximately 1 μg / µl and then resuspended. The resuspended samples were then loaded onto a mass spectrometer (Thermo Q Exactive HF-X) for detection. This mass spectrometer was connected in series with a nano-liquid chromatography system (Thermo EASY-nLC 1200).

[0039] Liquid chromatography conditions: Laboratory-made chromatographic column: Reprosil pure 120Å C18-AQ, inner diameter 75 µm, length 25 cm, packed with C18 microspheres, C18 microsphere particle size 1.9 µm; column temperature: 50℃; mobile phase A: containing 0.2% formic acid, 3% DMSO, balance deionized water; mobile phase B: 80% acetonitrile, 0.2% formic acid, 3% DMSO, balance deionized water; gradient elution: approximately 1 μg peptide was loaded into the mass spectrometer (Q Exactive HF-X, Thermo) and subjected to a linear gradient elution with acetonitrile in data-dependent acquisition (DDA) mode for approximately two hours.

[0040] Mass spectrometry chromatographic conditions: Mass spectrometry data were acquired using the DDA mode of the Thermo Q Exactive_HF-X with the following parameters: spray voltage 2.1 kV; capillary temperature 320 °C; first-stage full scan resolution 60,000 (at 200 m / z), AGC target: 3e6, maximum ion time: 50 ms, first-stage mass scan range 350-1500; Top N: 20; second-stage full scan resolution 15,000 (at 200 m / z), AGC target: 1e5, maximum ion time: 105 ms, second-stage fixed-first mass 100 m / z; fragmentation mode HCD, normalized collision energy (NCE) 29, dynamic exclusion time 30.0 s.

[0041] Peptide retrieval: Raw mass spectrometry data analysis was performed using Thermo Proteome Discoverer 2.5 software for database searching. The Swiss-Prot protein database contained reviewed human protein sequences. The main search parameters were as follows: cysteine ​​methylation (Carbamidomethyl) was set as a fixed modification parameter; methionine oxidation and N-terminal acetylation were set as variable modification parameters. Trypsin was selected for specific enzyme digestion, with a maximum allowed number of missed peptide cleavages of 2.

[0042] Ultra-depth mass spectrometry (UTM) analysis of brain tissue detected 2066 cytoplasmic proteins, 1349 membrane proteins, 445 membrane and cytoplasmic proteins, and 662 secretory proteins. This significantly higher result than a study using the same mouse model to detect whole-brain WTL (with appendix). Figure 7B). Analysis of the readily soluble proteins described in this invention and the research report using the above methods also showed that the readily soluble proteins extracted by the method of this invention were enriched (see appendix). Figure 7 C). The proteins detected in this experiment were sorted from highest to lowest according to the human plasma proteins and concentrations recorded in the Human Protein Atlas (HPA) database. It was found that the proteins extracted using this method could cover up to the lowest recorded protein level (approximately pg / mL) (see appendix). Figure 7 D). A review of reported cerebrospinal fluid protein markers revealed that almost all of them were enriched using this method's extraction and mass spectrometry analysis (see appendix). Figure 7 E), indicating that the present invention has a stronger ability to detect these proteins that can be released into cerebrospinal fluid and even peripheral blood.

[0043] Example 3 For the mice used in Example 1, one 9-month-old C57BL / 6J mouse and one 5×FAD17 mouse were selected. Immediately after perfusion, tissues such as brain, heart, liver, spleen, lung, kidney, muscle, large intestine, small intestine, prostate, ovary, bladder, and gallbladder were dissected and flash-frozen at -80℃, with all operations performed on ice. Mixed samples of different tissues obtained using this method were purified and enzymatically digested to obtain samples of different tissues.

[0044] When using mass spectrometry to detect the components of these different tissue samples separately (see appendix) Figure 4 A), the protein components identified in each group again showed differences from one another (see appendix). Figure 4 B). Cellular localization analysis of the proteins in the identification results revealed that the total number of cytoplasmic proteins, membrane proteins, and secretory proteins in WTL, LS, Triton, and Pellet were 1422, 1372, 1199, and 1080, respectively. When the number of these proteins in the LS and Triton components was combined, a total of 1504 proteins were found, exceeding the number detected when the tissue sample was analyzed as a whole (WTL). (See Appendix) Figure 4 C).

[0045] Based on the analysis results of the above embodiments, this invention addresses the problem of the lack of efficient and selective extraction methods for potential peripheral blood biomarker proteins from tissue samples in the prior art. It provides a tissue protein extraction scheme based on a mild, low-salt, nonionic surfactant. This method can selectively enrich secretory proteins, cytoplasmic proteins, and membrane proteins without introducing strong denaturants, while effectively removing most nuclear proteins and interstitial fibrin proteins, thereby significantly improving the detection capability of subsequent mass spectrometry for extremely low abundance circulating biomarkers. This method is applicable to various types of animal or human tissue samples, and is simple, low-cost, and highly reproducible. It can be widely used in proteomics-based disease biomarker discovery, clinical auxiliary diagnosis, and related mechanism research, and has significant promotion and application value.

[0046] Although the present invention has been disclosed above with reference to preferred embodiments, these are not intended to limit the invention. Any person skilled in the art can make various changes or modifications without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be defined by the scope of the claims of this application.

Claims

1. A method for enriching and extracting readily soluble proteins from tissue samples, characterized in that: include: Step 1: Enrichment and extraction of readily soluble proteins; add tissue samples to low-salt buffer, homogenize and centrifuge to collect the supernatant and precipitate, resuspend the precipitate with Triton, and then obtain the supernatant to obtain the extract; Step 2: Protein precipitation; Add anhydrous acetonitrile to the extract to precipitate the protein, and collect the precipitate; Step 3: Proteolytic digestion and reductive alkylation: The precipitate is subjected to proteolytic digestion, protein reduction, protein alkylation, and quenching reaction in sequence to obtain peptide samples; Step 4: Peptide desalting: Load the peptide sample into the desalting column and desalt it using desalting washing solution; Wash, then add desalting eluent for elution, collect the eluent containing peptides, and dry to obtain desalted peptides; Step 5: Reverse-phase fractionation chromatography under alkaline conditions; the desalted peptides are fractionated by reverse liquid chromatography, and the collected peptide fractions are dried; Step 6: LC-MS / MS detection: The peptide sample collected by fractionation is injected into a liquid chromatography-mass spectrometry (LC-MS) instrument for mass spectrometry detection; the raw mass spectrometry data are analyzed using a protein database.

2. The method for enriching and extracting readily soluble proteins from tissue samples according to claim 1, characterized in that: In step one, the tissue sample is human tissue or mouse tissue; the low-salt buffer is a Tris-HCl aqueous solution with a molar concentration of 25 mM and a pH of 7.4 or a Hepes aqueous solution with a molar concentration of 25 mM and a pH of 7.5; in step one, the low-salt buffer contains a protease inhibitor mixture PIC; the volume ratio of 100 times working concentration PIC aqueous solution to low-salt buffer is 1:

100.

3. The method for enriching and extracting readily soluble proteins from tissue samples according to claim 2, characterized in that: Step one specifically includes: taking tissue samples and adding low-salt buffer at a ratio of 1 g tissue sample to 10 mL low-salt buffer, wherein PIC aqueous solution has been added to the low-salt buffer; homogenizing the sample in a Dounce homogenizer and centrifuging at 4 ℃ and 20000 g for 5 min, collecting the supernatant LS fraction and retaining the precipitate; resuspending the retained precipitate in a 1% Triton X-100 aqueous solution, the volume of which is the same as that of the supernatant LS fraction; centrifuging again to obtain the supernatant Triton; mixing the LS fraction and the supernatant Triton to form a Mix sample, which is used for subsequent detection.

4. The method for enriching and extracting readily soluble proteins from tissue samples according to claim 3, characterized in that: In step two, the volume ratio of the extract to anhydrous acetonitrile is 1:

4. After centrifugation, the protein precipitate is retained and washed three times with 80% acetonitrile.

5. The method for enriching and extracting readily soluble proteins from tissue samples according to claim 3, characterized in that: Step 3 is as follows: Add the precipitate obtained in Step 2 to an 8 M urea solution. The solvent for the 8 M urea solution is a 25 mM TEAB aqueous solution with a pH of 8.

5. At the same time, add 1 working concentration of PIC aqueous solution to the 8 M urea solution to prevent protein degradation. Rotate and mix at room temperature for more than 4 hours or at 4°C overnight until the precipitate is fully dissolved or the solution becomes clear. Then, according to the protein concentration and total amount, add Lys-C enzyme at a mass ratio of 1:100 to substrate and perform enzymatic hydrolysis at room temperature. After 4 hours, dilute the 8 M urea to a 2 M urea solution with three times the volume of 25 mM TEAB of the sample after Lys-C enzymatic hydrolysis, and then add trypsin for digestion. Digest at 37°C for at least 4 hours. Subsequently, add 1 mM dithiothreitol for reduction for 30 min, 3 mM iodoacetamide for alkylation in the dark for 15 min, and finally terminate the reaction with 1.5 mM dithiothreitol.

6. The method for enriching and extracting readily soluble proteins from tissue samples according to claim 1, characterized in that: In step four, a C18 desalting column is used; the desalting washing solution is a 0.1 wt% formic acid aqueous solution; the desalting eluent is a 60-65 wt% acetonitrile aqueous solution containing 0.1 wt% formic acid.

7. The method for enriching and extracting readily soluble proteins from tissue samples according to claim 1, characterized in that: In step five, 4 mg of dried peptide fragments were redissolved in an aqueous solution of NH4HCO2 with a molar concentration of 10 mM and a pH of 8.

0. The dissolved peptide fragments were then separated using an XBridge C18 column with a mobile phase of 15% to 45%, and approximately 100 fractions were collected and dried.

8. The method for enriching and extracting readily soluble proteins from tissue samples according to claim 1, characterized in that: In step six, each fraction obtained in step five is resuspended in 5% formic acid, and 1 µg is injected into the DDA mode of the electrostatic field orbital trap ultra-high resolution mass spectrometer; the raw mass spectrometry data are analyzed using a protein database.

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