Method for ultra-deep detection of red blood cell protein

Abstract

The invention discloses a method for ultra-deep detection of red blood cell protein. The method comprises the following steps: purifying red blood cells, carrying out hypotonic splitting decomposition and precipitation on a purified red blood cell sample, and extracting a cell membrane component of a precipitate and a cytoplasm component of a supernatant; carrying out proteolysis treatment on the cell membrane and cytoplasm component sample to obtain a peptide fragment sample, desalting and washing with a desalting column to obtain an eluent containing peptide fragments, and drying; carrying out trans-liquid chromatography fractionation under an alkaline condition, collecting about 100 peptide fragment fractions, and drying; detecting each fraction by mass spectrometry; and comparing mass spectrum results by using a protein database to identify peptide fragments and proteins to obtain qualitative and quantitative information of the proteins. According to the invention, all proteins in red blood cells are comprehensively and systematically detected to detect more proteins compared with the prior art, the detection sequence coverage of each protein is greatly improved, and more protein and amino acid change information is provided for physiological functions and disease mechanisms of the red blood cells; and more molecular clues are provided for fundamental research and clinical diagnosis and treatment of red blood cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of clinical medicine, and more particularly, to a method for detecting red blood cell proteins. BACKGROUND

[0002] Red blood cells have a unique shape and mainly carry oxygen and carbon dioxide, which is essential for human life. In addition to gas exchange, red blood cells also carry glycosylphosphatidylinositol cross-linked proteins and immune complexes containing complement C3b fragments (16861337), which are involved in cell recognition, adhesion, signal transduction and immune regulation. Recent studies have also shown that red blood cells can bind to DNA through their surface TLR9 receptors (pubmed ID No. 34669439), thereby participating in the infection and pathogenic process of the new coronavirus in the human body. In addition, it is well known that red blood cell membranes also contain different blood group antigens, which are related to the occurrence of certain clinical diseases.

[0003] Clinically, abnormal red blood cell membrane skeleton proteins, metabolic enzymes and specific nuclear proteins can cause spherocytosis, elliptocytosis, stomatocytosis, sickle cell disease, congenital non-spherocytic hemolytic anemia, etc. (29803284, 34889365, 28447420). In addition to red blood cell diseases, red blood cell dysfunction is also involved in cardiovascular diseases, and shows differences in some cancers (such as breast cancer) and neurodegenerative diseases (such as Alzheimer's disease).

[0004] Therefore, the special structure and function of red blood cells, as well as the molecular basis of their own diseases caused by abnormalities, especially at the protein level, need to be systematically studied. The present application aims to provide a method for comprehensively and systematically detecting all proteins in red blood cells to find key proteins that regulate the structure and function of red blood cells, pathogenic molecules related to their diseases, and clinical diagnostic markers.

[0005] The main method for systematic detection of red blood cell proteins (proteomics, i.e., overall non-discriminatory detection of all proteins in a sample) in the field is liquid chromatography-mass spectrometry (LC-MS / MS). In early studies (about 20 years ago), only a few hundred to about 1000 proteins were detected in red blood cells using this technology (pubmed ID numbers 18346024, 17585793, 24976601, 20199679, 14963112, 16861337, 22538302, 26078478). However, according to research analysis, red blood cells contain at least 2000 gene expression products (28263177), and from hematopoietic stem cells to the differentiation of each differentiation stage before mature red blood cells, more than 1200 protein bodies have been directly detected in red blood cells (35084980). Recently, a study reported that nearly 3000 proteins can be detected in red blood cells (31552303, 35858567, 27006477, 28689405).

[0006] Supplementary note: The numbers in parentheses above indicate the ID number of the literature referred to in the Pubmed database; for example, (16861337) in the above text indicates that its ID in the Pubmed database is 16861337. PubMed is a Web-based free biomedical information retrieval system developed by the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (NLM) of the United States, and the ID number is the number assigned by the database to each indexed literature. Each ID number corresponds to a literature. SUMMARY

[0007] 1. Technical problems to be solved:

[0008] To solve the above technical problems, the present application provides a method for ultra-deep detection of red blood cell proteins, which realizes comprehensive and systematic detection of all proteins in red blood cells, can detect more proteins than the prior art, and greatly improves the detection sequence coverage of each protein, thereby providing more protein and amino acid change information for the physiological function and disease mechanism of red blood cells, and providing more molecular clues for the basic research and clinical diagnosis and treatment of red blood cells.

[0009] 2. Technical solutions:

[0010] A method for ultra-deep detection of red blood cell proteins, characterized in that it comprises:

[0011] Step 1: Red blood cell purification; removing white blood cells and platelets from the red blood cell sample to obtain purified red blood cells;

[0012] Step two: separation of the membrane and cytoplasm components; the purified red blood cell sample is low-osmotic lysis and precipitation, and the membrane component of the precipitate and the cytoplasm component of the supernatant are extracted;

[0013] Step three: desalination of the proteolytic peptide; the obtained membrane component and the cytoplasm component of the supernatant sample are subjected to proteolysis treatment, and the obtained peptide sample is subjected to desalination column desalination and washing, and elution is performed to obtain an eluent containing the peptide, and the eluent is dried to obtain the desalted peptide;

[0014] Step four: trans liquid chromatography fractionation under alkaline conditions; 3mg of the desalted peptide obtained in step three is separated by liquid chromatography under alkaline conditions through linear gradient separation, and about 100 peptide fraction is collected and dried;

[0015] Step five: mass spectrometry detection of each peptide fraction by LC-MS / MS; the protein database is used to compare the mass spectrometry detection results to identify the peptide and protein, and the protein qualitative and quantitative information is obtained.

[0016] Further, in step one, after removing the white blood cells from the whole blood sample by filtration, the bottom red blood cell precipitate is obtained by low-speed centrifugation for 3 minutes, and the precipitate is washed with an isotonic buffer without a detergent to obtain the separated and purified red blood cells.

[0017] Further, in step two, the lysis solution is a low-osmotic buffer, and the isotonic salt concentration is, for example, 10-15mM, and the volume of the low-osmotic buffer is more than 10 times the volume of the initial whole blood sample; the supernatant after lysis is the cytoplasm component; after removing the supernatant, the precipitate is washed with the low-osmotic buffer to obtain the membrane component.

[0018] Further, step three specifically includes:

[0019] S31: protein enzymolysis; 4mg of the extracted membrane component and cytoplasm component are extracted, acetonitrile is added to a final concentration of 80%, the proteins in the sample are washed and precipitated, and the sample is washed twice with 80% acetonitrile; then, 0.5mL of 8M urea dissolved in 50mM TEAB at pH 8.5 and 20μg of lysine protease are added, and the sample is shaken at room temperature for about 4 hours until the precipitate is fully dissolved and the solution is clear. Then, dilute the urea to 2M by adding three times the volume of 50mM TEAB at pH 8.5; add 20μg of trypsin to enzymolysis, so that the mass ratio of trypsin in the sample is 100-200:1; after vortexing, place at room temperature, shake for 4-8h; add 1M dithiothreitol DTT to 3mM in the sample, shake at room temperature for 15min, then add 0.1M iodoacetamide IAA to 10mM in the sample, avoid light and shake for 15min; add 1M DTT to 20mM, shake at room temperature for 15min;

[0020] S32: Add 10% formaldehyde FA solution to the two peptide segment samples after enzymatic hydrolysis to a final concentration of about 1%, select 8 desalting columns with a capacity of 1 mg of peptide segment, and desalt the two enzymatic peptide segment samples using four desalting columns, and dry them.

[0021] Further, in the basic condition in step four, C18 chromatographic column is used in trans liquid chromatography; fractionation mobile phase A is 10 mM ammonium formate aqueous solution with pH of 8.0; fractionation mobile phase B is 90% acetonitrile aqueous solution containing 10 mM ammonium formate with pH of 8.0; linear gradient is 5%-5% mobile phase B for 0.01-10 minutes, 5%-40% mobile phase B for 10-110 minutes, 40%-65% mobile phase B for 110-120 minutes, 65%-95% mobile phase B for 120-130 minutes; flow rate is 1 mL / min; 1 mL / tube is collected; about 100 tubes of fractions are collected; drying is performed respectively, and used for mass spectrometry detection in the next step.

[0022] Further, in step five, each fraction is resuspended using 5% formic acid aqueous solution, and then fed into a liquid chromatography mass spectrometry LC-MS / MS for mass spectrometry detection to obtain mass spectrometry raw data, and protein qualitative and quantitative information is obtained by processing the mass spectrometry raw data.

[0023] 3. Beneficial effects:

[0024] (1) The method for detecting blood red cell protein in a high depth disclosed in the application can detect 5264 proteins from red blood cells, including 4777 proteins in a membrane extraction sample, 2350 proteins in a cytoplasm extraction sample, and 1863 proteins detected in both samples. Compared with a human red blood cell protein database and the largest number of red blood cell proteins measured in two databases uniprot and RESPIRE reported, 2348 new proteins are measured, which is the largest number of protein detection so far.

[0025] (2) The method for detecting blood red cell protein in a high depth disclosed in the application has a very high protein sequence coverage due to the realization of high depth detection. Nearly 30% of the detected proteins have a protein sequence of more than 50%. For example, in glucose-6-phosphate dehydrogenase (G6PD) which can cause long-term mild anemia, 65 amino acid sites with natural polymorphism or mutation are detected among 78 amino acid sites. The sequence coverage of other proteins related to red blood cell diseases is more than 50%. The sequence coverage of the detected red blood cell blood type related proteins also reaches more than 60% in part, which provides key molecular clues for the analysis of clinical disease mechanisms at the protein level.

[0026] In conclusion, the method for detecting hemoglobin protein provided by the scheme not only realizes the detection of more hemoglobin protein types in terms of breadth, but also greatly improves the sequence coverage of related proteins in terms of depth, which has a profound significance for clinical treatment. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The complete process of separating and purifying red blood cells and the verification of separation effect in the scheme;

[0028] Figure 2 The separation effect of red blood cell membranes and cytoplasm is verified by detecting their protein components under SDS-PAGE and conventional mass spectrometry;

[0029] Figure 3 The approximate experimental process of the ultra-deep detection in the scheme, the large fractionation of sample peptides under alkaline conditions, and the detection results of peptides and proteins;

[0030] Figure 4 The number of proteins identified in the scheme and the distribution in red blood cells and cytoplasm;

[0031] Figure 5 Comparison of the proteins detected by the ultra-deep mass spectrometry method with the existing related reports and databases;

[0032] Figure 6 The protein amino acid sequence coverage detected by the ultra-deep mass spectrometry method and examples. DETAILED DESCRIPTION

[0033] The application will be specifically described below with reference to the drawings.

[0034] Example 1:

[0035] A method for ultra-deep detection of hemoglobin protein, characterized in that it comprises:

[0036] Step 1: Red blood cell purification; removing white blood cells and platelets from the red blood cell sample to obtain purified red blood cells;

[0037] Step 2: Separation of red blood cell membrane and cytoplasmic components; low-osmotic lysis and precipitation of the purified red blood cell sample, and extraction of the cell membrane component of the precipitate and the cytoplasmic component of the supernatant;

[0038] Step 3: Proteinase digestion peptide desalting; the obtained cell membrane component and supernatant cytoplasmic component samples are subjected to proteinase digestion treatment, and the peptide sample is subjected to desalting column desalting and washing to obtain an eluate containing peptides, and the eluate is dried to obtain desalted peptides;

[0039] Step four: trans-liquid chromatography fractionation under alkaline conditions; 3 mg of the peptide segment obtained by desalting in step three was separated by liquid chromatography under alkaline conditions by linear gradient, and about 100 peptide segment fractions were collected and dried;

[0040] Step five: mass spectrometry detection of each peptide segment fraction by LC-MS / MS; using a protein database to compare the mass spectrometry results to identify the peptide segment and the protein, and obtain protein qualitative and quantitative information.

[0041] Further, after removing white blood cells from the whole blood sample in step one by filtration, the bottom red blood cell precipitate was obtained by low-speed centrifugation for 3 minutes, and washed with isotonic buffer without detergent to obtain separated and purified red blood cells.

[0042] Further, the lysis solution in step two is a low-osmotic buffer with an isotonic salt concentration of, for example, 10-15 mM, and the volume of the low-osmotic buffer is more than 10 times the volume of the initial whole blood sample; the supernatant after lysis is the cytoplasmic component; after removing the supernatant, the membrane component is obtained by washing the precipitate with low-osmotic buffer.

[0043] Example 2:

[0044] Step three specifically includes:

[0045] S31: protein enzymolysis; 4 mg of protein was extracted from the cell membrane component and the cytoplasmic component, acetonitrile was added to a final concentration of 80%, and the protein in the sample was washed and precipitated, and then washed twice with 80% acetonitrile; then, 0.5 mL of 8M urea dissolved in 50mM TEAB at pH 8.5 and 20 μg of lysine protease were added, and the sample was shaken at room temperature for about 4 hours until the precipitate was fully dissolved and the solution was clear. Then, dilute the urea to 2M by adding three times the volume of 50mM TEAB at pH 8.5; add 20 μg of trypsin to enzymolysis, so that the mass ratio of trypsin in the sample is 100-200:1; after vortexing, place at room temperature, shake for 4-8h; add 1M dithiothreitol DTT to 3mM in the sample, shake at room temperature for 15min, then add 0.1M iodoacetamide IAA to 10mM in the sample, avoid light and shake for 15min; add 1M DTT to 20mM, shake at room temperature for 15min;

[0046] S32: add 10% formaldehyde FA solution to a final concentration of about 1% in each of the two enzymolysis peptide segment samples, select 8 desalting columns with a peptide segment capacity of 1 mg, desalt the two enzymolysis peptide segment samples with four desalting columns, and dry.

[0047] Further, in step four, during the reverse liquid chromatography under alkaline conditions, a C18 column was used; the fractionated mobile phase A was a 10 mM ammonium formate aqueous solution at pH 8.0; the fractionated mobile phase B was a 90% acetonitrile aqueous solution containing 10 mM ammonium formate at pH 8.0; the linear gradient was: 5%–5% mobile phase B for 0.01–10 min, 5–40% mobile phase B for 10–110 min, 40–65% mobile phase B for 110–120 min, and 65–95% mobile phase B for 120–130 min; the flow rate was 1 mL / min; 1 mL was collected per tube; approximately 100 tubes of fraction were collected; each fraction was dried for subsequent mass spectrometry detection.

[0048] Furthermore, in step five, each fraction is first resuspended in a 5% formic acid aqueous solution, and then injected into a liquid chromatography-mass spectrometry (LC-MS / MS) instrument to obtain raw mass spectrometry data. The raw mass spectrometry data is then processed to obtain qualitative and quantitative information on proteins.

[0049] Example 3:

[0050] This embodiment is used to illustrate the purification of red blood cells from whole blood and the separation effect of their cell membranes from the cytoplasm in this application.

[0051] 1. Take 1 mL of human peripheral blood (EDTA anticoagulated), filter it through a white blood cell (WBC) filter to remove white blood cells, then centrifuge at low speed (800g) for 3 minutes. Collect the red blood cell pellet near the bottom of the tube to avoid residual white blood cells and platelets, and wash it three times with phosphate-buffered saline (PBS) to purify the red blood cells. The red blood cells are processed according to the procedure of this invention (…). Figure 1 (A and 1B), whether under a microscope or under automated blood cell counting instruments ( Figure 1 C) all showed that white blood cells and platelets were effectively removed. Figure 1 D).

[0052] To avoid interference from hemoglobin, which accounts for over 98% of total protein in the erythrocyte cytoplasm, on the detection of other proteins by mass spectrometry, this protocol separates the membrane proteins of erythrocytes from the cytoplasmic fraction. A 10-fold diluted phosphate-buffered saline (0.1×PBS) solution, at a volume 10 times the initial whole blood volume (if the volume is insufficient, the salt ions released during erythrocyte lysis will increase the osmotic pressure of the solution, preventing the remaining erythrocytes from being completely lysed), is added to the purified erythrocyte pellet to induce lysis through hypotonic lysis, which is then confirmed under a microscope. After centrifugation (20,000g, 10 minutes), the upper half of the supernatant is collected as the cytoplasmic fraction. After removing the remaining supernatant, the pellet is washed three times with 0.1×PBS until no red residue remains, and the pellet near the bottom of the tube is collected as the membrane fraction.

[0053] Figure 1The complete process of separation and purification of red blood cells and the verification of the separation effect in this scheme. (A) Purification of red blood cells in whole blood and separation of membrane and cytoplasm. (B) Purified red blood cells at different stages in the process of lysis and separation of membrane and cytoplasm. (C) Staining and microscopic observation of the separation effect of red blood cells, showing that white blood cells and platelets are effectively removed after separation. (D) Automatic blood cell instrument counting, showing efficient removal of white blood cells and platelets.

[0054] 2. Protein electrophoresis and mass spectrometry identification after separation of red blood cell membrane and cytoplasm. Take 20 μg of all proteins of purified red blood cells, i.e. the same amount of red blood cell membrane and cytoplasm separation samples from the starting whole blood, and perform common SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). After Coomassie blue staining, the protein components of cytoplasm and cytoplasm on the gel are almost completely different (the most concentrated band near the bottom is hemoglobin, almost completely in cytoplasm) Figure 2 The separation effect of red blood cell membrane and cytoplasm in this scheme is verified by their protein components in SDS-PAGE and routine mass spectrometry detection; the membrane proteins and cytoplasmic proteins of the whole red blood cells are effectively separated Figure 2 A).

[0055] The separated membrane proteins and cytoplasmic proteins are treated and mass spectrometry detected Figure 2 B, see Example 2 for detailed method steps), in addition to the 455 proteins detected in both component samples, 1058 proteins are detected in the membrane protein and 146 proteins are detected in the cytoplasm, the two components have a large difference in the types of proteins identified Figure 2 C); although part of the proteins are detected in both components, the content distribution is quite different. From the relative level of all proteins in the membrane and cytoplasm, most of the differences are obvious Figure 2 D). In addition, the membrane protein accounts for 41.8% in the membrane component, while the membrane protein accounts for only 26.2% in the cytoplasmic sample, with a significant difference Figure 2 E). Almost all the commonly recognized red blood cell membrane specific proteins are almost completely present in the separated membrane component Figure 2 F).

[0056] These indicate that red blood cells are effectively purified, and their membrane and cytoplasmic components are also effectively separated.

[0057] Figure 2The separation effect of the red blood cell membrane and cytoplasm in the present application is verified by the protein components in the SDS-PAGE and conventional mass spectrum detection. (A) SDS-PAGE shows the protein components of red blood cells and their separated membranes and cytoplasm, which indicates that they are effectively separated; (B) The liquid chromatogram of the protein samples of the separated red blood cell membrane and cytoplasm in the mass spectrum detection process shows the distribution of different peak types, which indicates the different peptide segments in the sample and the protein composition in the original sample; (C) The number of proteins detected in the red blood cell membrane and cytoplasm components; (D) The level distribution of the detected proteins in the red blood cell membrane and cytoplasm components; (E) The relative content difference of the membrane proteins and cytoplasmic proteins in the membrane and cytoplasm extracts; (F) The highest content of the proteins in the red blood cell membrane and cytoplasm reported in the literature is detected in the red blood cell membrane and cytoplasm extracts in the present application. PSM: peptide-spectru-match, peptide-spectrum-match, i.e. the number of times a peptide is detected.

[0058] Example 4:

[0059] This example is used to illustrate the specific process of ultra-depth detection of the present application. The general process of experimental operation is summarized in Appendix Figure 3 A.

[0060] 1. Preparation of clinical red blood cell samples. The red blood cell samples were from 100 clinical individuals, of which four different blood types (A, B, AB, and O) accounted for 25 cases respectively; in each group of blood type individuals, different genders, ages, and clinical disease states were included. Such samples provide individual selection to maximize the differences in red blood cell protein components among clinical individuals, providing the most comprehensive red blood cell proteomic information for the clinic. The red blood cell samples collected from each individual every day were respectively subjected to red blood cell purification and membrane plasma separation, then part of each was taken, and the membrane components and cytoplasmic components of all samples were mixed and-80°C frozen with the remaining part of each sample. After all 100 samples were collected and processed and frozen, at the time of detection, all mixed parts were taken out and mixed to form two independent samples (membrane components and cytoplasmic components) of red blood cell components from 100 individual sources, and then subjected to ultra-depth mass spectrometry analysis.

[0061] 2、Membrane and cytosol protein samples were digested into peptides. 4 mg of protein from each of the membrane and cytosol fractions were extracted and washed and precipitated with 80% acetonitrile. The samples were washed twice with 80% acetonitrile. Then, 0.5 mL of 8 M urea (dissolved in 50 mM TEAB, pH 8.5) and 20 μg of LysC enzyme were added, and the samples were shaken at room temperature for about 4 hours until the precipitate was completely dissolved and the solution was clear. Then, the urea was diluted to 2 M by adding three times the volume of 50 mM TEAB, pH 8.5; 20 μg of trypsin was added to digest the samples, and the mass ratio of trypsin to sample was 100-200: 1; the samples were vortexed and shaken at room temperature for 4-8 hours; 1 M DTT (dithiothreitol) was added to the samples to a concentration of 3 mM, and the samples were shaken and incubated at room temperature for 15 minutes; 0.1 M IAA (iodoacetamide) was added to the samples to a concentration of 10 mM, and the samples were shaken and incubated in the dark for 15 minutes; and 1 M DTT was added to the samples to a concentration of 20 mM, and the samples were shaken and incubated at room temperature for 15 minutes.

[0062] 3、The peptides after enzymatic digestion and reductive alkylation were desalted and purified. 10% formic acid (FA) was added to the peptide samples to a concentration of 1%, and the samples were shaken and mixed. Eight desalting columns with a capacity of 1 mg of peptides were selected, and four desalting columns were used to desalt each of the two samples of digested peptides. 3 mL of 100% acetonitrile was passed through the columns, and then 3 mL of 0.1% FA was passed through the columns. The digested peptide samples were added to the columns at a rate of 1 mg per column, and the samples were filtered. 3 mL of 0.1% FA was passed through the columns to wash away the salts and impurities that did not bind to the columns. Finally, 1 mL of 40% acetonitrile in 0.1% FA was added to the columns, and the purified peptides were eluted and collected, and the samples were dried.

[0063] 4、The membrane and cytosol protein peptides after enzymatic digestion and desalting were subjected to transverse liquid chromatography under alkaline conditions. The system included a liquid chromatography system (Shimadzu) and a C18 column (XBridge C18 column 3.5 um, 4.6 mm x 250 mm, Waters), and a liquid chromatography system (fractionation mobile phase A: 10 mM ammonium formate aqueous solution (NH4COOH), pH 9.0; fractionation mobile phase B: 90% acetonitrile aqueous solution containing 10 mM ammonium formate, pH 9.0).

[0064] Resuspend 4 mg of desalted peptides in each sample with 0.5 mL of mobile phase A; prepare the liquid chromatography system, and wash the system with at least 50 mL of fractionated mobile phase B, then equilibrate the system with at least 50 mL of fractionated mobile phase A; load the 0.5 mL of mobile phase A to resuspend 4 mg of desalted peptides in the protein sample; fractionate the sample using a gradient mobile phase, and the specific gradient is: 0.01-10 min, 5%-5% B; 10-110 min, 5-40% B; 110-120 min, 40-65% B; 108-110 min, 65-95% B; 110-120 min, 95-95% B. The liquid flow rate is 1 mL / min. The automatic collector collects at a rate of 1 tube / min. About 100 tubes are collected in total; and the collected fraction tubes are dried.

[0065] 5. After fractionation, each peptide sample is subjected to mass spectrometry detection. Resuspend about 1 μg of the desalted and dried peptide sample with 25 μL of 5% FA formic acid to make a solution, centrifuge (20000g, 3 min), and take 20 μL of the resuspended solution for liquid chromatography tandem mass spectrometry (LC-MS / MS) detection. Liquid chromatography instrument: nanoliter liquid chromatography system (Thermo EASY nLC 1200). Chromatography column: self-made in the laboratory, Reprosil pure C18 AQ, inner diameter 75 μm, length 25 cm, filled with C18 microspheres (particle size 1.9 μm); column temperature: 50°C. Mobile phase A: containing 0.2% formic acid, 3% DMSO, and the rest is deionized water; mobile phase B: 80% acetonitrile, 0.2% formic acid, 3% DMSO, and the rest is deionized water; gradient elution: continuously adjusted according to different fractions. Elution time is 120 min; injection flow rate is 0.3 μL / min. Mass spectrometer (Thermo Q Exactive HF X) scanning conditions: DDA mode to collect mass spectrometry data, spray voltage 2.1 kV, capillary temperature (320°C); primary full scan resolution (60,000, 200 m / z), AGC target (3e6), Maximum ion time (50 ms), primary mass scan range (350-1500), Top N (20), secondary full scan: resolution (15,000, 200 m / z), AGC target (1e5), Maximum ion time (105 ms); secondary scan range (Fixed first mass 100 m / z), fragmentation mode (HCD), fragmentation normalized collision energy (NCE) (29), dynamic exclusion time (30.0 s).

[0066] 6、Mass spectrometry raw data peptide and protein identification and quantitative analysis. Mass spectrometry raw data analysis is processed by ThermoProteome Discoverer 2.5 software search database. The reviewed human protein sequence in Swiss Prot protein database is the database. The main search parameters are as follows: the urea methylation on cysteine (Carbamidomethyl) is set as a fixed modification parameter; the oxidation modification on methionine and the acetylation modification (Acetyl) on the N terminal of protein are set as variable modification parameters. Specific enzyme cutting selects trypsin, and the maximum number of allowed polypeptide cutting is 2.

[0067] 7、Red blood cell membrane and cytoplasmic protein detection and result analysis. The final fraction samples of the peptides for machine after fractionation are 82 and 94 respectively, and the number of peptides detected in each fraction sample is about 12000 and 5000 respectively, and the number of proteins detected is about 2500 and 1000 respectively (Appendix Figure 3 B). The total number of proteins detected in red blood cell membrane and cytoplasmic samples is 4777 and 2350 respectively (Appendix Figure 4 A), and after combining the two, a total of 5264 proteins are detected, including 2914 proteins specifically detected in the membrane component, 487 proteins specifically detected in the cytoplasmic component, and 1863 proteins detected in both components (Appendix Figure 4 B). The content distribution of all 5264 proteins detected in the membrane and cytoplasm has obvious difference (Appendix Figure 4 C), which is consistent with the general biological knowledge of protein distribution in red blood cells and the experimental effect of separating and extracting red blood cell membrane and cytoplasm, indicating the reliability of the mass spectrometry detection and quantitative results. The 100 proteins with the highest content detected in the cell membrane and cytoplasmic components are listed in the table (Appendix 1 and Appendix 2).

[0068] Table 1 100 proteins with the highest content on red blood cell membrane detected by this ultra-deep mass spectrometry method

[0069]

[0070]

[0071]

[0072]

[0073] Table 2 100 unique proteins with the highest abundance in cytoplasmic extract

[0074]

[0075]

[0076]

[0077]

[0078] Figure 3 The general experimental procedure of the ultra-deep detection of the present solution, and the extensive fractionation of the sample peptide segments under alkaline conditions and the detection results of the peptide segments and proteins. (A) The general procedure of the ultra-deep detection of red blood cells. (B) The extensive fractionation of the sample peptide segments under alkaline conditions in the process of the ultra-deep detection, and the number of peptide segments and proteins detected in each fraction finally detected by mass spectrometry.

[0079] Figure 4 The number of proteins identified in the present solution and other information, and the distribution in red blood cells and cytoplasm. (A) The number of proteins detected by mass spectrometry ultra-deep detection of the red blood cell membrane and cytoplasm extract of the present solution and other information. "#" is the number; FDR: false discovery rate, the lower the better, generally less than 0.05 (5%). PSM: peptide-spectrum-match, the number of times a peptide segment is detected. (B) The number of proteins detected by mass spectrometry ultra-deep detection of the red blood cell membrane and cytoplasm extract. (C) The protein level distribution of the mass spectrometry ultra-deep detection of the red blood cell membrane and cytoplasm extract.

[0080] 8、Comparison of the present solution with existing reports on red blood cell protein identification and databases. The database data set constructed by the ultra-deep detection of the present research was compared with two most influential reports on red blood cell (RBC) proteomics (https: / / pubmed.ncbi.nlm.nih.gov / 28689405 / , https: / / pubmed.ncbi.nlm.nih.gov / 31552303 / ) and two protein databases

Uniprot database (https: / / www.uniprot.org / ) and RESPIRE database (https: / / www.dsimb.inserm.fr / respire)

[0081] Table 3. The 100 most abundant proteins in red blood cells detected by the ultra-deep mass spectrometry method

[0082]

[0083]

[0084]

[0085]

[0086]

[0087] The present application lists the 100 most abundant proteins in red blood cells detected, such as Table 3, which can be seen that the new proteins include some less expressed hemoglobin subunit epsilon (HBE1) in adult red blood cells, non-red blood cell expressed actin (ACTA1, a-skeletal muscle actin) and tubulin (TUBB4B, TUBB2A, TUBB1, etc.). All these proteins have their specific peptides, which indicate their true existence, and also reflect the superiority of ultra-deep detection.

[0088] Figure 5 Comparison of proteins detected by the ultra-deep mass spectrometry method of the present application with existing related reports and databases. (A) Comparison of the number of proteins in the existing related reports and red blood cell protein data. Two reports are from the research group (https: / / pubmed.ncbi.nlm.nih.gov / 28689405 / ) and the Weekes research group (https: / / pubmed.ncbi.nlm.nih.gov / 31552303 / ). (B) Comparison of the levels of proteins detected by the present application and the report of the research group of the present application, which illustrates the reliability of the protein detection of the present application.

[0089] 9、Red blood cell protein sequencing coverage of the present application. Due to the ultra-deep detection of the present application, the sequence coverage of the proteins detected has also been greatly improved, which is conducive to the detection of mutant amino acids corresponding to gene mutations that cause red blood cell diseases. Nearly 1% of the proteins of the present application have almost 100% of the amino acid sequences detected, and nearly 30% of the proteins have nearly 50% of the sequences detected and covered (Appendix 2). Figure 6 ​A) For example, glucose-6-phosphate-1-dehydrogenase (G6PD) is a protein associated with hemolytic anemia. Congenital deficiency or reduced function caused by genetic mutation is the most common cause of red blood cell metabolic disorders, affecting more than 400 million people worldwide. In the database constructed by the ultra-deep detection method of the present application, the sequence coverage of G6PD reaches 76%, covering 65 of the 78 natural amino acid variation sites (Appendix Figure 6 B), greatly improving the possibility of discovering amino acid variations at the protein level, and providing molecular clues for revealing the underlying mechanisms of clinical red blood cell diseases.

[0090] Figure 6 The protein amino acid sequence coverage detected by the ultra-deep mass spectrometry method of the present application and examples. (A) Protein sequence coverage detected by the present application. (B) Protein sequence of glucose-6-phosphate dehydrogenase (G6PD) detected in the present application. G6PD deficiency or mutation is a common cause of red blood cell disease. Its reduced or lost function can induce hemolytic anemia under certain triggers. The red underlined part in the figure is the detected peptide segment. The red labeled amino acid is the site that can be varied.

[0091] The protein corresponding to the common mutant gene causing red blood cell disease and the sequence coverage of the protein determining the red blood cell blood group antigen are summarized in Tables 4 and 5, respectively, providing a reference for the molecular analysis of related proteins in clinical diseases.

[0092] Table 4. Sequence detection coverage of mutant proteins related to red blood cell disease detected by the ultra-deep mass spectrometry method of the present application

[0093]

[0094]

[0095] Table 5. Sequence detection coverage of red blood cell blood group related proteins detected by the ultra-deep mass spectrometry method of the present application

[0096]

[0097]

[0098] Although the present application has been disclosed with the above preferred embodiments, they are not intended to limit the present application, and any person skilled in the art can make various changes or modifications without departing from the spirit and scope of the present application. Therefore, the scope of protection of the present application should be determined by the scope of protection defined by the claims of the present application.

Claims

1. A method for ultra-deep detection of hemoglobin protein, characterized in that: Comprising: Step one: red blood cell purification; removing white blood cells and platelets from the red blood cell sample to obtain purified red blood cells; Step two: separation of red blood cell membrane and cytoplasm components; low-osmotic lysis of the purified red blood cell sample, precipitation, extraction of the cell membrane component of the precipitate and the cytoplasm component of the supernatant; Step three: desalting of the proteinase digestion peptide segment; The obtained cell membrane component and supernatant cytoplasm component samples are subjected to proteinase digestion treatment to obtain peptide segment samples, which are then subjected to desalting column desalting and washing, elution to obtain eluate containing peptide segments, and drying to obtain desalted peptide segments; Step four: trans liquid chromatography fractionation under alkaline conditions; 3 mg of the desalted peptide segments obtained in step three are separated by liquid chromatography under alkaline conditions through linear gradient separation, and about 100 peptide segment fractions are collected and dried; Step five: mass spectrometry detection of each peptide segment fraction by LC-MS / MS; using a protein database to compare the mass spectrometry detection results to identify peptide segments and proteins, and obtain protein qualitative and quantitative information.

2. The method of claim 1, wherein the method is for ultra-deep detection of hemoglobin protein. In step one, after removing white blood cells from the whole blood sample by filtration, the red blood cells at the bottom are obtained by low-speed centrifugation for 3 minutes, and the isosmotic buffer without detergent is used for washing to obtain the separated and purified red blood cells.

3. The method of claim 1, wherein the method is for ultra-deep detection of hemoglobin protein. In step two, the lysis solution is a low-osmotic buffer with an isotonic salt concentration of, for example, 10-15 mM, and the volume of the low-osmotic buffer is more than 10 times the volume of the initial whole blood sample; the supernatant after lysis is the cytoplasm component; after removing the supernatant, the precipitate is washed with the low-osmotic buffer to obtain the membrane component.

4. The method of claim 1, wherein the method is for ultra-deep detection of hemoglobin protein. Step three specifically includes: S31: proteinase digestion; 4 mg of protein is extracted from each of the extracted cell membrane component and cytoplasm component, acetonitrile is added to a final concentration of 80%, and the protein in the sample is washed and precipitated, and then washed twice with 80% acetonitrile; then, 0.5 mL of 8 M urea dissolved in 50 mM TEAB at pH 8.5 and 20 μg of lysine protease are added, and the precipitate is dissolved and the solution is clarified at room temperature for about 4 hours; then, dilute the urea to 2 M by adding three times the volume of 50 mM TEAB at pH 8.5; add 20 μg of trypsin for digestion, so that the mass ratio of trypsin in the sample is 100-200:1; after vortexing, place at room temperature, shake for 4-8 h; add 1 M DTT to 3 mM in the sample, shake at room temperature for 15 min, then add 0.1 M IAA to 10 mM in the sample, avoid light, shake for 15 min; add 1 M DTT to 20 mM, shake at room temperature for 15 min; S32: 10% formaldehyde (FA) solution is added to each of the two peptide segment samples after digestion to a final concentration of about 1%, eight desalting columns with a peptide segment capacity of 1 mg are selected, and the two digestion peptide segment samples are desalted using four desalting columns, and dried.

5. The method of claim 1, wherein the method is for ultra-deep detection of hemoglobin protein. In step four, the C18 column was used for trans liquid chromatography under basic conditions; the fractionation mobile phase A was 10 mM ammonium formate aqueous solution with pH 8.0; the fractionation mobile phase B was 90% acetonitrile aqueous solution containing 10 mM ammonium formate with pH 8.0; the linear gradient was 5%~5% mobile phase B for 0.01~10 min, 5~40% mobile phase B for 10~110 min, 40~65% mobile phase B for 110~120 min, 65~95% mobile phase B for 120~130 min; the flow rate was 1 mL / min; 1 mL / tube was collected; about 100 tubes of fractions were collected; drying was performed respectively for the next mass spectrometry detection.

6. The method of claim 1, wherein the method is for ultra-deep detection of hemoglobin protein. In step five, each fraction was resuspended with 5% formic acid aqueous solution, and then was injected into the liquid chromatography mass spectrometry LC-MS / MS for mass spectrometry detection to obtain mass spectrometry raw data, and the mass spectrometry raw data was processed to obtain protein qualitative and quantitative information.

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