A method for detecting SARS-CoV-2 S1 protein

Abstract

The present invention relates to a detection method for SARS-CoV-2S1 protein. In this method, the SARS-CoV-2S1 protein is pre-treated by solution enzymatic hydrolysis to obtain S1 peptides, a C18 chromatographic column is used to separate the samples, and an orbitrap high-resolution mass spectrometer is used for data acquisition. First, data acquisition of S1 peptide samples is performed under the data-dependent mode (DDA) of the mass spectrometer, 6-10 high-intensity specific peptides with a large number of peptide matching spectra (PSM) are selected, and a peptide library of parallel reaction monitoring mode (PRM) is constructed; finally, the SARS-CoV-2S1 protein is identified according to the constructed PRM peptide library. This technology can realize high-sensitivity and high-precision detection of SARS-CoV-2S1 protein, and is expected to become an important supplementary method for nucleic acid detection in human trace virus detection and cargo surface trace virus detection, and play an important role in curbing the spread of SARS COV-2.

Description

Technical Field

[0001] The invention relates to a method for detecting SARS-CoV-2 S1 protein, and belongs to the technical field of biological analysis. Background Art

[0002] SARS-CoV-2 is a single-stranded RNA virus. The spike glycoprotein (S protein) on the surface of the virus particle is a key protein for coronavirus to infect host cells. It binds to the angiotensin-converting enzyme II (ACE2) of the host cell. The type 2 transmembrane serine protease in the host cell cleaves the S protein into two parts, S1 and S2. S1 has a receptor binding domain (RBD) that binds to the host cell and has large variability, with obvious differences between different viruses or different strains of the same virus. Therefore, it is of great significance to achieve highly sensitive and specific detection of SARS-CoV-2 S1 protein.

[0003] For the rapid and accurate detection of the new coronavirus, the most commonly used detection technologies currently include nucleic acid testing and serological testing. SARS-CoV-2 nucleic acid detection is based on the use of metagenomic next-generation sequencing technology to determine the nucleic acid sequence, mainly using three conservative sequences in the viral genome (open reading frame 1ab, nucleocapsid protein N, and envelope protein E) as detection targets, and using real-time reverse transcription polymerase chain reaction (RT-PCR) to detect SARS-CoV-2 nucleic acid. Nucleic acid testing has the advantages of simple operation, high sensitivity, and strong specificity, and can detect patients in the window period; however, problems such as sampling time and location, experimental operation, diagnostic reagent quality, virus mutation, and viral load differences can lead to "false negative" results.

[0004] Serological testing for SARS-CoV-2 antibodies targets immunoglobulins M and G (IgM and IgG). IgM appears about one week after the early stage of viral infection and is a diagnostic indicator for acute infection, but its concentration is relatively low and its duration is short. IgG appears about three weeks after viral infection and remains in the body for a long time, and the antibody titer continues to increase. Serological testing is simple and quick to operate, and can be used as an effective supplement to nucleic acid testing. However, since a window period is required from the virus entering the body to the production of specific antibodies, the test may also result in "false negatives", and antibody serological tests based on N or S proteins are easily affected by endogenous or exogenous interferences and may result in "false positives".

[0005] Mass spectrometry-based omics analysis technology has the advantages of high throughput, high sensitivity and simple operation. It can accurately characterize nucleic acids, proteins and metabolites qualitatively and quantitatively, and has been widely used in infectious disease diagnosis, biomarker discovery, pathogen identification and pathogenic mechanism research.

[0006] In this patent, in response to the current problems of "false positive" and "false negative" in the detection of the new coronavirus, a highly sensitive detection method for SARS-CoV-2 S1 protein based on targeted mass spectrometry parallel reaction monitoring mode has been developed. Separation technology is used to reduce the impact of interferences, and high-sensitivity mass spectrometry detection technology is combined to achieve sensitivity, specificity and reliability of SARS-CoV-2 S1 protein detection. Summary of the invention

[0007] The purpose of the present invention is to provide a method for detecting SARS-CoV-2 S1 protein, which solves the problems of "false positive" and "false negative" in the detection of the new coronavirus.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] A method for detecting SARS-CoV-2 S1 protein, comprising the following steps:

[0010] (1) Extraction and purification of SARS-CoV-2 S1 protein: SARS-CoV-2 virus particles were centrifuged at 60,000-80,000 g for 1-3 h to break the virus particles; the precipitate was then suspended by oscillation with 70% formic acid, and then an equal volume of acetonitrile was added and oscillated; then centrifuged at 14,000-16,000 g for 5-20 min, and the supernatant, i.e., the extracted protein solution, was removed; finally, the SARS-CoV-2 S1 protein was purified by size exclusion chromatography.

[0011] (2) Pretreatment of SARS-CoV-2 S1 protein sample: Dissolve the SARS-CoV-2 S1 protein in water and add urea solution to mix; add dithiothreitol or tri(2-carboxyethyl)phosphine and treat at 37-56°C for 0.5-2h to denature and reduce the S1 protein; then add iodoacetamide and alkylate at room temperature in the dark for 25-40min; add ammonium bicarbonate solution to dilute urea, and finally add trypsin for enzymatic hydrolysis for 12-16h; desalt the obtained peptide using a desalting column and lyophilize to obtain a SARS-CoV-2 S1 peptide sample.

[0012] (3) Constructing a protein database: Download the entire sequence of SARS-CoV-2 protein (P0DTC2) from the website Uniprot (https: / / www.uniprot.org / ), select the amino acid sequence from positions 13–685, which is the SARS-CoV-2 S1 protein sequence; then use the software pFind (http: / / pfind.ict.ac.cn / software / pFind / index.html) to automatically add it to the contaminant library, which is the constructed protein database.

[0013] (4) Construction of a peptide library in PRM mode: The SARS-CoV-2 S1 peptide sample was loaded onto the LC-MS, and a 25 μm inner diameter, 10-15 cm long C18 reverse phase column (filler particle size was 1.9 μm) was used to separate the sample using a 65 min separation gradient. The orbitrap high-resolution mass spectrometer was used in DDA mode for mass spectrometry data acquisition, and Full MS scanning was performed on the Orbitrap. During the acquisition cycle, the 2-7 valence parent ions were fragmented by secondary spectra according to the intensity from high to low, and the original data files of the mass spectra (including primary and secondary spectra) were obtained. The constructed protein database was used as the data retrieval library, and the original files of the mass spectrometry acquisition were retrieved using the software Proteome Discoverer (Thermo Fisher) and Maxquant (https: / / www.maxquant.org / ). Based on the identified peptides, 6-10 high-intensity and high-PSM-number SARS-CoV-2 peptides with the top 12 intensities and PSM numbers were selected. The specific peptides of S1 protein were used to construct a peptide library in PRM mode.

[0014] (5) Identification of peptide samples to be analyzed: The constructed PRM peptide library includes: DIADTTDAVR, SWmESEFR, GWIFGTTLDSK, FASVYAWNR, HTPINLVR, VGGNYNYLYR, NIDGYFK, QIAPGQTGK. The SARS-CoV-2 S1 peptide samples to be analyzed were loaded onto the LC-MS, and the samples were separated by a 65-min separation gradient using a 25 μm inner diameter, 10-15 cm long C18 reverse phase column (filler particle size of 1.9 μm). The target peptides were collected using the constructed PRM peptide library, and the constructed protein database was used as the data retrieval library. Data was retrieved using Proteome Discoverer. If there are two or more peptides in the PRM peptide library, it means that the SARS CoV-2 S1 protein is present in the peptide sample to be analyzed. Otherwise, it means that the SARS CoV-2 S1 protein is not present in the analyzed peptide sample.

[0015] The present invention relates to a method for detecting SARS-CoV-2 S1 protein. In this method, the SARS-CoV-2 S1 protein is pretreated by solution enzymatic hydrolysis to obtain S1 peptides, a C18 chromatographic column is used to separate the samples, and an orbitrap high-resolution mass spectrometer is used for data acquisition. First, data acquisition of the S1 peptide sample is performed in the DDA mode of the mass spectrometer, 6-10 high-intensity, high-PSM-number specific peptides are selected, and a PRM mode peptide library is constructed; finally, the SARS-CoV-2 S1 protein is identified according to the constructed PRM peptide library. This technology can realize high-sensitivity and high-precision detection of SARS-CoV-2 S1 protein, and is expected to become an important supplementary method for nucleic acid detection in the detection of trace viruses in the human body and trace viruses on the surface of goods.

[0016] The present invention also provides a general method for constructing a targeted mass spectrometry parallel reaction monitoring mode to achieve highly sensitive detection and differentiation of other viruses (such as H1N1, MERS, etc.). The present invention has the following advantages:

[0017] (1) The present invention detects the novel coronavirus by directly detecting viral antigens, which can achieve early diagnosis, rapidity, and accuracy compared to methods of detecting RNA and antibodies.

[0018] (2) The present invention combines chromatographic separation technology with high-resolution mass spectrometry detection technology to construct a peptide library in the PRM mode, which not only reduces the impact of interfering substances, but also achieves high-specificity and high-sensitivity detection of the target protein.

[0019] (3) The entire process of the present invention from LC-MS to construction of a peptide library in PRM mode also provides a method template for the specific and highly sensitive detection of other viruses (such as H1N1, MERS, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 :Construction process of the detection method of SARS-CoV-2 S1 protein;

[0021] Figure 2 : 65 min separation gradient on C18 reverse phase separation column on liquid phase;

[0022] Figure 3 : Sequence coverage of 20ng SARS-CoV-2 S1 protein in DDA mode;

[0023] Figure 4 : 8 high-intensity, PSM-rich SARS-CoV-2 S1-specific peptides constructed in the PRM pattern. DETAILED DESCRIPTION

[0024] Example 1

[0025] The whole experimental process is as follows Figure 1 As shown:

[0026] (1) 10 7 The SARS-CoV-2 virus particles were centrifuged at 65000g for 1h to break the virus particles; then the precipitate was suspended by oscillation with 100μl 70% (v / v) formic acid, followed by vigorous oscillation with an equal volume of acetonitrile; finally, the mixture was centrifuged at 16000g for 5min and the supernatant, i.e., the extracted protein solution, was taken out.

[0027] (2) The extracted viral protein solution was purified by size exclusion chromatography using a TOSOH TSK-GELG2000SW gel chromatography column (7.5 mm × 300 mm), a mobile phase of 50 mmol / L phosphate buffer solution (pH 6.8) containing 0.1 mmol / L NaCl, a flow rate of 1 ml / min, and a detection wavelength of 280 nm to obtain purified SARS-CoV-2 S1 protein.

[0028] (3) Dissolve the SARS-CoV-2 S1 protein in water to a concentration of 1 mg / ml. Take 30 μl of the S1 protein aqueous solution and add 24 μl of 8M urea to mix well. Add 1.44 μl of 0.33M dithiothreitol solution and treat at 56°C for 1 hour to denature and reduce the S1 protein. Then add 1.2 μl of 1M iodoacetamide solution and alkylate at room temperature in the dark for 30 minutes. Add 400 μl of 50mM ammonium bicarbonate solution to dilute urea, and then add 1 μg of trypsin for enzymatic hydrolysis for 14 hours. The obtained peptide sample was filtered through a desalting column filled with C18 (filler particle size 5 μm, pore size ) for desalting, and after loading, wash three times with mobile phase A [ultrapure water solution containing 2% (v / v) acetonitrile and 0.1% (v / v) formic acid], and then elute with mobile phase B [ultrapure water solution containing 80% (v / v) acetonitrile and 0.1% (v / v) formic acid]. The desalted peptide sample was lyophilized and reconstituted with 0.1% (v / v) formic acid to obtain the SARS-CoV-2 S1 peptide sample.

[0029] (4) Download the entire sequence of SARS-CoV-2 protein (P0DTC2) from Uniprot (https: / / www.uniprot.org / ), select the amino acid sequence from positions 13–685, which is the S1 protein sequence; automatically add it to the contaminant library through the software pFind (http: / / pfind.ict.ac.cn / software / pFind / index.html), which is the constructed protein database.

[0030] (5) 20 ng of SARS-CoV-2 S1 peptide sample was loaded onto the LC-MS column using a 25 μm inner diameter, 12 cm long C18 reverse phase column (filler particle size was 1.9 μm), mobile phase A: ultrapure water solution containing 0.1% (v / v) formic acid, mobile phase B: ultrapure water solution containing 80% (v / v) acetonitrile and 0.1% (v / v) formic acid, and separation was performed using a 65 min separation gradient (e.g. Figure 2 Sample separation was performed using the DDA mode of orbitrap high-resolution mass spectrometry, and full MS scanning was performed on the Orbitrap. Within a 2s acquisition cycle, the 2-7 valence parent ions were fragmented by secondary spectra according to the intensity from high to low to obtain the original data files of the mass spectrometry (including primary and secondary spectra). The constructed protein database was used as the data retrieval library, and the original files of mass spectrometry acquisition were retrieved using the software Proteome Discoverer (Thermo Fisher) and Maxquant (https: / / www.maxquant.org / ). According to the identified peptides (such as Figure 3 As shown in the sequence coverage diagram, eight high-intensity and high-PSM-number specific peptides of SARS-CoV-2 S1 protein were selected, and a peptide library with PRM mode was constructed (as shown in the sequence coverage diagram). Figure 4 The constructed peptide library of PRM mode includes: DIADTTDAVR, SWmESEFR, GWIFGTTLDSK, FASVYAWNR, HTPINLVR, VGGNYNYLYR, NIDGYFK, QIAPGQTGK.

[0031] (6) 0.1 pg of the SARS CoV-2 S1 peptide sample to be analyzed (approximately 100 SARS CoV-2 virus particles) was separated by a 65 min separation gradient (e.g., 1:100 μg / ml) using a 25 μm inner diameter, 12 cm long C18 reversed phase column (filler particle size: 1.9 μm) with mobile phase A: ultrapure aqueous solution containing 0.1% (v / v) formic acid and mobile phase B: ultrapure aqueous solution containing 80% (v / v) acetonitrile and 0.1% (v / v) formic acid. Figure 2 The sample was separated by using the peptides in the constructed PRM peptide library as the acquisition target for mass spectrometry data acquisition. The constructed protein database was used as the data retrieval library, and Proteome Discoverer was used for retrieval. The SARS CoV-2 S1 protein was successfully identified (sequence coverage was 9%, 6 peptides were identified, and the number of PSMs was 26).

[0032] Example 2

[0033] The operation is as shown in steps (1)-(5) in Example 1.

[0034] (6) 0.02 pg of the SARS CoV-2 S1 peptide sample to be analyzed (approximately 20 SARS CoV-2 virus particles) was separated by a 65 min separation gradient (e.g., 1:100 μg / mL) using a 25 μm inner diameter, 12 cm long C18 reversed phase column (filler particle size: 1.9 μm), with mobile phase A: ultrapure aqueous solution containing 0.1% (v / v) formic acid and mobile phase B: ultrapure aqueous solution containing 80% (v / v) acetonitrile and 0.1% (v / v) formic acid. Figure 2 The sample was separated by using the peptides in the constructed PRM peptide library as the acquisition target for mass spectrometry data acquisition. The constructed protein database was used as the data retrieval library, and Proteome Discoverer was used for retrieval. The SARS CoV-2 S1 protein was successfully identified (sequence coverage was 5%, 2 peptides were identified, and the PSM number was 10).

[0035] Example 3

[0036] The operation is as shown in steps (1)-(5) in Example 1.

[0037] (6) 0.01 pg of the SARS CoV-2 S1 peptide sample to be analyzed (approximately 10 SARS CoV-2 virus particles) was separated by a 65 min separation gradient (e.g., 1:100 μg / ml) using a 25 μm inner diameter, 12 cm long C18 reversed phase column (filler particle size: 1.9 μm), with mobile phase A: ultrapure aqueous solution containing 0.1% (v / v) formic acid and mobile phase B: ultrapure aqueous solution containing 80% (v / v) acetonitrile and 0.1% (v / v) formic acid. Figure 2 The sample was separated by using the peptides in the constructed PRM peptide library as the acquisition target for mass spectrometry data acquisition. The constructed protein database was used as the data retrieval library, and Proteome Discoverer was used for retrieval. The SARS CoV-2 S1 protein was successfully identified (sequence coverage was 4%, 3 peptides were identified, and the PSM number was 15).

Claims

1. A method for detecting SARS-CoV-2 S1 protein, characterized in that: The following steps are involved: (1) SARS-CoV-2 virus particles were disrupted by centrifugation, followed by protein extraction with formic acid and acetonitrile, and finally purification of S1 protein by size exclusion chromatography; (2) Pretreatment of protein samples using solution enzymatic hydrolysis; (3) Use C18 chromatographic column to separate samples, and then use orbitrap high-resolution mass spectrometry to collect data; (4) Use the DDA model to select specific peptides and construct a peptide library for the PRM model; (5) Using the constructed PRM-mode peptide library to identify the SARS-CoV-2 S1 protein in the peptide samples to be analyzed; Step (4) is as follows: download the entire sequence of SARS-CoV-2 protein from Uniprot, select the amino acid sequence from position 13 to 685, which is the S1 protein sequence; automatically add it to the contaminant library through the software pFind, which is the constructed protein database; The SARS-CoV-2 S1 peptide sample was loaded onto the LC-MS. After gradient separation, the DDA mode was used for data acquisition, and the Full MS scan was implemented on the Orbitrap. During the acquisition cycle, the 2-7 valence parent ions were fragmented by secondary spectra from high to low intensity to obtain the raw data file of the mass spectrum. The constructed protein database was used as the data retrieval library, and the original files of mass spectrometry acquisition were retrieved using the software Proteome Discoverer and Maxquant; based on the identified peptides, 6-10 high-intensity and high-PSM-number specific peptides of SARS-CoV-2 S1 protein were selected, and the peptide library of PRM mode was constructed; The peptide library of the PRM mode described in step (5) includes: DIADTTDAVR, SWmESEFR, GWIFGTTLDSK, FASVYAWNR, HTPINLVR, VGGNYNYLYR, NIDGYFK, QIAPGQTGK.

2. The detection method according to claim 1, characterized in that: The process of extracting the protein solution in step (1) is as follows: the SARS-CoV-2 virus particles are centrifuged at a high speed of 60000-80000g for 1-3h to break the virus particles; then the precipitate is suspended by oscillation with 50-80% (v / v) formic acid, and then an equal volume of acetonitrile is added for oscillation; finally, the supernatant is taken out by centrifugation at 14000-16000g for 5-20min, i.e., the extracted protein solution.

3. The detection method according to claim 1, characterized in that: The solution enzymatic hydrolysis method described in step (2) includes: dissolving the SARS-CoV-2 S1 protein in water, adding urea solution and mixing; adding dithiothreitol or tri(2-carboxyethyl)phosphine and treating at 37-56°C for 0.5-2h to denature and reduce the S1 protein; then adding iodoacetamide and alkylating at room temperature in the dark for 25-40min; adding ammonium bicarbonate solution to dilute urea, and finally adding trypsin for enzymatic hydrolysis for 12-16h; desalting the obtained peptide using a desalting column, and lyophilizing to obtain a SARS-CoV-2 S1 peptide sample.

4. The detection method according to claim 1, characterized in that: The C18 chromatographic column described in step (3) comprises: a C18 reverse phase column with an inner diameter of 25 μm and a length of 10-15 cm, and a filler particle size of 1.9 μm.

5. The detection method according to claim 1, characterized in that: In step (5), after the peptide sample to be analyzed is separated using a C18 reverse phase chromatographic column, the peptides in the constructed PRM peptide library are used as the acquisition targets for mass spectrometry data acquisition, and the constructed protein database is used as the data retrieval library, and the software Proteome Discoverer is used for data retrieval; if there are 2 or more peptides in the PRM peptide library, it means that SARS CoV-2 S1 protein exists in the peptide sample to be analyzed, otherwise it means that SARS CoV-2 S1 protein does not exist in the analyzed peptide sample.

Images