Synthetic peptide assisted single-cell proteomics detection method

Abstract

The invention discloses a synthetic peptide assisted single-cell proteomics detection method. The polypeptide containing the amino acid sequence as shown in SEQ ID NO: 1 is adopted, protein can be adsorbed, the loss of a sample in the protein extraction process is effectively reduced, and the mass spectrometry detection result is not influenced. In addition, the method is simple, rapid and high in throughput, does not need special equipment or consumables, and is suitable for the technical field of proteomics detection.

Description

Technical Field

[0001] The present invention relates to proteomics detection technology, and in particular to a method for single-cell proteomics detection assisted by synthetic peptides. Background Art

[0002] Single-cell proteomics studies are expected to provide more precise information about cell function and fill the gap between genotype and phenotype. Considering that a single mammalian somatic cell contains only 200-300 picograms of protein and that proteins are not amplifiable, researchers have proposed a series of solutions to reduce sample loss, including reducing the number of steps required for handling, reducing the processing volume, and using signal-enhanced samples, nanodroplets chips, oil-air droplets chips, or carrier proteins. However, these previously described techniques have various shortcomings, such as limitations in experimental design, complex sample preparation, or impact on data collection quality. These limitations make single-cell proteome detection still extremely challenging. Summary of the invention

[0003] The present invention aims to provide a method for single-cell proteomics detection assisted by synthetic peptides, that is, a simple, rapid and high-throughput single-cell proteomics method.

[0004] In order to achieve the above object, according to one aspect of the present invention, a polypeptide is provided, wherein the polypeptide comprises the amino acid sequence shown in SEQ ID NO:1.

[0005] According to a second aspect of the present invention, there is provided an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the above polypeptide.

[0006] According to the third aspect of the present invention, an expression vector is provided, wherein the expression vector comprises the above-mentioned nucleic acid molecule.

[0007] According to a fourth aspect of the present invention, a host cell is provided, wherein the host cell contains at least one copy of the above expression vector; or the above nucleic acid molecule is integrated into the genome of the host cell.

[0008] According to a fifth aspect of the present invention, a protein extraction method is provided, comprising lysing a tissue sample or a cell sample in a container coated with the above polypeptide to obtain a sample protein extract.

[0009] Furthermore, the container comprises a centrifuge tube.

[0010] Furthermore, the lysis includes ultrasonic lysis.

[0011] According to a sixth aspect of the present invention, there is provided a protein extraction kit, comprising a container coated with the above polypeptide and a lysis buffer reagent.

[0012] According to a seventh aspect of the present invention, there is provided a protein group detection method, comprising the following steps:

[0013] The protein in the sample to be tested is extracted by the above protein extraction method to obtain a protein extract of the sample to be tested;

[0014] digesting the protein extract of the sample to be tested in the container to obtain a protein digest of the sample to be tested; and

[0015] The protein digest of the sample to be tested is subjected to liquid chromatography-mass spectrometry analysis to obtain a proteomics test result of the sample to be tested.

[0016] Furthermore, the digestion includes digestion with pancreatic enzymes.

[0017] By applying the technical solution of the present invention, a polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 1 is used to adsorb proteins, effectively reduce sample loss during protein extraction, and does not affect mass spectrometry detection results. In addition, the method is simple, rapid and high-throughput, does not require special dedicated equipment or consumables, and can complete high-throughput preparation of 96-well samples within 3 to 4 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0019] Figure 1 The liquid phase gradient of the LC-MS machine for micro-samples in the proteomic detection operation process of the present application is shown;

[0020] Figure 2 shows a single cell proteome map according to Example 1;

[0021] Figure 3 A single cell protein localization result diagram according to Example 1 is shown;

[0022] Figure 4 A diagram showing the clustering results of healthy human single cells according to Example 1 is shown;

[0023] Figure 5 A diagram showing the patient single cell clustering result according to Example 1 is shown;

[0024] Figure 6 The figure shows the drug target analysis result according to Example 1. DETAILED DESCRIPTION

[0025] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0026] The proteomic detection operation process is as follows:

[0027] 1. Synthetic peptide coated PCR tubes

[0028] 1) The ordered synthetic peptide (sequence FFWIK VFFIK VFFVK IFFVK IFFVK IFFVK (SEQ ID NO: 1), purity 99.99%) was dissolved in water to 0.2 μg / μL and stored at -20°C for 6 months.

[0029] 2) Add 80 μL of the synthetic peptide solution prepared in step 1) to a 200 μL low-adsorption PCR tube and incubate at room temperature for 8 h.

[0030] 3) Completely remove the synthetic peptide solution from the tube and air-dry the tube. The tube can be stored at -20°C.

[0031] 2. Micro-sample protein extraction and enzymatic hydrolysis

[0032] 1) Collect the cells in the 200 μL low-adsorption PCR tube coated in step 1, store in less than 5 μL PBS solution, and immediately perform the following operations or store at -20°C. It is recommended that the cell samples should not be repeatedly frozen and thawed.

[0033] Note: The operation of opening the sample tube is performed in the clean bench. For each batch of samples, a sample of 1000 Hela cells or 100 Hela cells is made as a quality control, which is loaded on the machine and analyzed together with the samples. For samples with more than 100 cells, 1000 Hela cells are used as quality control, and for samples with 100 cells or less, 100 Hela cells are used as quality control.

[0034] 2) Add 50mM ammonium bicarbonate solution to the sample until the total volume of the sample is 5μL (100 cells) or 10μL (1000 cells). If the original sample solution exceeds 10μL, do not add ammonium bicarbonate solution. Add DTT to the sample to a final concentration of 5mM (the required volume of DTT is small, so the DTT stock solution can be diluted in advance before use).

[0035] 3) When adding liquid to the tube, add the liquid in mid-air or with the tip of the gun against the wall of the tube. Do not touch the liquid. After adding, centrifuge in a desktop centrifuge for 5 seconds to allow all the liquid to gather at the bottom of the tube.

[0036] 4) Place the PCR tube on a hollow yellow 96-well plate, float the plate on the water surface of a water bath, immerse the sample portion of the tube in water, cover the portion of the yellow plate and tube that is exposed to the water surface with parafilm, and perform water bath ultrasound for 15 minutes at 37°C.

[0037] 5) For trace animal tissues or large amounts of cells that cannot be lysed by water bath sonication, use an ice bath probe sonication for 5 minutes (2s / 3s) (power 40%-60%, adjusted according to the volume and hardness of the sample). Before probe sonication, the volume needs to be made up and DTT added, and after sonication, place in a 37°C water bath for 15 minutes.

[0038] 6) Remove the sample from the water bath and centrifuge it in a desktop centrifuge for 5 seconds to allow all the liquid to accumulate at the bottom of the tube. After the sample returns to room temperature, add IAM at a final concentration of 10 mM and let it stand in a dark room for 30 minutes.

[0039] 7) Add 1 μL of 1 μg / μL Promega Trypsin enzyme and perform enzymolysis at 37°C for 2 h.

[0040] 8) Visually assess the sample volume (compared with the volume of 8 μL of control liquid added to an empty tube). If the volume is more than 8 μL, concentrate it with a vacuum freeze dryer for 5-10 minutes; do not completely drain the sample, and do not remove the sample to measure the volume to avoid sample loss. Then, depending on the situation, add mass spectrometer A liquid to the total volume to 7-8 μL for the next step of mass spectrometer detection.

[0041] 3. Protein extraction of library samples

[0042] For the library construction samples corresponding to trace samples, the conventional protein extraction and enzymatic hydrolysis methods of each laboratory can be used, and more than 50 μg of protein needs to be prepared.

[0043] 4. Methods for LC-MS (liquid chromatography-mass spectrometry platform) of trace samples

[0044] 1) Confirm that the instrument is in qualified condition; check the trace samples. There should be no cracks on the wall of the ep tube. The sample volume in the tube should be 7-8μL. If the volume is too much or too little, it can be concentrated and supplemented with organic A liquid using a vacuum freeze concentrator. It cannot be completely drained. The volume of the same batch of samples should be consistent.

[0045] 2) Centrifuge the trace sample at a speed below 13,000g for 30 seconds to allow the liquid to accumulate at the bottom of the tube. Do not increase the speed to avoid breaking the sample tube.

[0046] 3) Transfer all trace samples to the sample liner tube with a sample volume of 6-7 μL. For the same project, wash the samples of biological and technical replicates once every 3-5 times, and wash the samples between different groups.

[0047] 4) Before loading the trace sample, replace the new injection transfer fluid, submit 3 1-hour sequences to clean the chromatographic column, and search the last cleaning result with protein search library software, using the same library as the trace sample. The sample can be loaded only if the number of residual protein identifications is less than 60, otherwise continue cleaning;

[0048] 5) Use a conventional C18 proteomics column for UPLC liquid phase analysis. The liquid phase parameters are shown in Figure 1 .

[0049] 6) The trace samples for library construction were loaded onto the Thermo Lumos mass spectrometer with the following parameters: MS: resolution 240,000, AGC target 4e5, maximum IT 100ms, scan range 350-1500; MS / MS: resolution 30,000, AGC target 5e4, maximum IT 100ms, Loop count / top N 2, isolation window 1.6m / z, first mass 100.

[0050] Thermo HF / HFX mass spectrometry parameters: MS: resolution 240,000, AGC target 3e6, maximum IT100ms, scan range 350-1500; MS / MS: resolution 30,000, AGC target 1e5, maximum IT150ms, Loop count / top N 2, isolation window 1.6m / z, first mass 100, NCE 26%.

[0051] 7) Thermo Lumos mass spectrometer parameters for trace samples without library construction were: MS: resolution 120,000, AGC target 4e5, maximum IT 100ms, scan range 350-1500; MS / MS: resolution 30,000, AGC target 5e4, maximum IT 100ms, Loop count / top N 10, isolation window 1.6m / z.

[0052] Thermo HF / HFX mass spectrometry parameters MS: resolution 120,000, AGC target 3e6, maximum IT100ms, scan range 350-1500; MS / MS: resolution 30,000, AGC target 1e5, maximum IT100ms, Loop count / top N 10, isolation window 1.6m / z, first mass 100, NCE 26%.

[0053] 5. LC-MS method for library sample construction

[0054] 1) For library construction, it is recommended to take 20-50 μg of sample and divide it into 20 groups using the High PH RP method. The peptide amount of each group should be no less than 1 μg. If the sample amount is less than 20 μg, the number of groups can be reduced.

[0055] 2) When the library samples are put on the LC-MS machine, the same instrument should be used as the trace samples, and the liquid phase gradient should be consistent, and the same chromatographic column should be used. The mass spectrometry parameters can use the conventional proteomics machine parameters of each laboratory. Thermo Lumos mass spectrometer parameter setting reference: MS: resolution 120,000, AGC target 1e5, maximum IT 50ms, scan range 350-1500; MS / MS: resolution 30,000, AGC target 2e4, maximum IT 50ms, Loopcount / top N 20, isolation window 1.6m / z.

[0056] Note: There is no mandatory requirement for the order in which library construction samples and trace samples are loaded onto the machine.

[0057] 6. Off-line data analysis software and parameter setting reference

[0058] 1) Use MaxQuant software and set “No fraction” in the Raw files module.

[0059] 2) Group-specific parameter settings: select "Trypsin / P" in "Enzyme"; select "Oxidation (M), Acetyl (Protein N-term), Carbamidomethyl (C), Deamidation (NQ), Gln->pyro-Glu" in "Variable modification"; select LFQ in "Label-free quantification", and then select 1 in "LFQ min.ratio count" (if it is an identification analysis, keep this item at the default setting: None).

[0060] 3) Global parameters setting: Sequences setting includes databases (downloaded by yourself or derived from genome and transcriptome sequencing); Include contaminants. When the search database species is "human", do not select "Include contaminants"; Adv.identification setting checks the Match between runs option; Labelfree quantification setting checks iBAQ.

[0061] 4) For parameters not specified in 1)-3), the default parameters of MaxQuant software were used.

[0062] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0063] Example 1: Using single-cell proteomics methods to describe the CD34+ single-cell proteomics map

[0064] We applied this single-cell proteomics technology to obtain single-cell protein profiles of scarce / trace clinical samples. We collected peripheral blood from healthy subjects and patients with chronic total occlusion (CTO) of the coronary artery, isolated circulating CD34+ cells, and used this technology to describe the protein profiles of 240 circulating CD34+ single cells.

[0065] CTO is an important branch of coronary artery disease, characterized by complete atherosclerotic occlusion of the coronary artery. Circulating CD34+ progenitor cells derived from bone marrow play an important role in endothelial cell repair after vascular injury or atherosclerosis development, but their exact function in CTO is unclear due to the extremely small number of cells.

[0066] We used flow cytometry (FACS) to isolate CD34+ cells from peripheral blood mononuclear cells (PBMCs) and applied this technology to the proteomic analysis of CD34+ single cells. Proteomic data analysis was performed on 40 single CD34+ cells collected from peripheral blood samples of 3 CTO patients and 3 healthy donors, for a total of 240 single cell samples. The average number of CD34+ single cell proteins in healthy donors was 1057±253, 931±150, and 1058±287, respectively; the average number of CD34+ single cell proteins in CTO patients was 867±209, 844±211, and 870±187, respectively (n=40, Figure 2 ). Similar to HeLa cells, most proteins are subcellularly localized in the cytoplasm, nucleus and cell membrane ( Figure 3 ).

[0067] Drawing on the analysis method of single-cell transcriptome data, we analyzed the cellular heterogeneity and cell clustering diversity of this batch of data. We used the UMAP dimensionality reduction algorithm (Uniform Manifold Approximation and Projection) to analyze the single-cell proteomic data. 120 single cells from CTO patients and 120 single cells from healthy donors were clustered into three subgroups, Con-0 / 1 / 2 (healthy people, Figure 4 )CTO-0 / 1 / 2(patients, Figure 5 ). IngenuityPathway Analysis (IPA), a software for functional / pathway analysis based on large-scale omics data, clearly showed that: 1) Con-0 and CTO-0 subpopulation cells share molecular functions involved in arterial occlusion, atherosclerosis, coronary artery disease, and other CTO-related occlusions; 2) Con-1 subpopulations express proteins involved in arterial development under normal conditions, in contrast, CTO-2 expresses proteins involved in vascular lesions when CTOs occur. 3) Con-2 and CTO-1 subpopulations uniquely express some proteins involved in angiogenesis and vasculogenesis, which are important for the repair and alternative resolution of vascular lesions. Proteins involved in angiogenesis and vasculogenesis were significantly regulated in the CTO-1 subpopulation, showing poor vascular repair ability under disease conditions. This finding was only present at the single-cell level, but not in the control 2000-cell / tube samples, where CD34+ cells were only found to be involved in CTO-related occlusions. This means that single-cell level analysis is necessary to determine subtle changes in protein expression and reveal cell-specific functions.

[0068] In addition, we compared the differentially expressed proteins between CTO and control groups in single cells with drug targets approved by the U.S. Food and Drug Administration (FDA) to find potential drug targets, of which 25 proteins were annotated as "drug targets". A key drug target, aldehyde dehydrogenase 2 (ALDH2), is only lowly expressed in the CTO-1 subpopulation that plays angiogenesis and angiogenesis functions ( Figure 6 ), suggesting the potential role of ALDH2 agonists in the treatment of CTO.

[0069] In conclusion, functional analysis of proteins expressed in single cells showed that CD34+ subpopulations have different functions, and subpopulations in CTOs show stronger ability to promote arterial occlusion, participate in vascular lesions, and inhibit angiogenesis and vasculogenesis. The results clearly show that single-cell proteomic studies provide precise cellular functional information and provide conclusive evidence from proteomic expression changes and pathway enrichment.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A polypeptide, characterized in that The polypeptide comprises the amino acid sequence shown in SEQ ID NO:

1.

2. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleic acid sequence encoding the polypeptide of claim 1.

3. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule of claim 2.

4. A host cell, characterized in that The host cell contains at least one copy of the expression vector according to claim 3; or the nucleic acid molecule according to claim 2 is integrated into the genome of the host cell.

5. A protein extraction method, characterized in that: The method comprises lysing a tissue sample or a cell sample in a container coated with the polypeptide according to claim 1 to obtain a sample protein extract.

6. The protein extraction method according to claim 5, characterized in that The container comprises a centrifuge tube.

7. The protein extraction method according to claim 5 or 6, characterized in that: The lysis includes ultrasonic lysis.

8. A protein extraction kit, characterized in that: The method comprises a container coated with the polypeptide according to claim 1 and a lysis buffer reagent.

9. A protein group detection method, characterized in that: The steps include: Extracting the protein in the sample to be tested by the protein extraction method according to any one of claims 5 to 7 to obtain a protein extract of the sample to be tested; Digesting the protein extract of the sample to be tested in the container to obtain a protein digest of the sample to be tested; and The protein digest of the sample to be tested is subjected to liquid chromatography-mass spectrometry analysis to obtain a proteomics test result of the sample to be tested.

10. The protein group detection method according to claim 9, characterized in that: The digestion includes digestion with trypsin.

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