CN117554531A - Specific peptide fragments and methods for detecting novel coronavirus delta strains - Google Patents
Specific peptide fragments and methods for detecting novel coronavirus delta strains Download PDFInfo
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Abstract
The present invention relates to specific peptide fragments and methods for detecting novel coronavirus delta strains. The present invention provides a method for detecting novel coronavirus delta strains comprising detecting the presence of a specific peptide fragment of amino acid sequence LQNVVNQNAQALNTLVK (SEQ ID No. 1) in a sample by tandem mass spectrometry using liquid chromatography. The invention also provides a specific peptide segment for detecting novel coronavirus delta strain, which has an amino acid sequence of LQNVVNQNAQALNTLVK (SEQ ID No. 1), a mass-to-charge ratio of the ion pair parent ion of 934.02271 +/-0.02 Da, and a mass-to-charge ratio of the child ion of 455.26108 +/-0.02 Da and/or 356.19305 +/-0.02 Da.
Description
Technical Field
The present invention relates to the field of novel coronavirus detection, in particular to specific peptide fragments and methods for detecting novel coronavirus delta strains, and more particularly to specific peptide fragments and methods for detecting novel coronavirus delta strains using liquid chromatography tandem mass spectrometry.
Background
The transmission enhancement of Delta variant viruses is associated with key mutations in the S protein (e.g., D614G, L452R, T478K, D950N, etc.), as compared to the original strain virus pedigree, with key mutations in the S protein portion of the Delta variant having 8 major mutation sites. These mutations result in structural changes in the S protein, facilitating viral fusion and viral replication, while increased affinity of the S protein and ACE is believed to be a key cause of reduced vaccine efficacy (Di Giacomo, S. Et al, preliminary report on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike mutation T478K, J Med Virol (2021) 93 (9): 5638-5643; planas, D. Et al, reduced sensitivity of SARS-CoV-2 variant Delta to antibody neutralization. "Nature (2021) 596 (7871): 276-280; shi A, C. And X. Xie, making sense of Spike D614G in SARS-CoV-2 transmission,Sci China Life Sci (2021) 64 (7): 1062-1067).
Taken together, the significant enhancement in transmissibility and pathogenicity of Delta variants raised concerns about re-prevalence of Delta variants in susceptible populations (including vaccinated individuals), the emergence and global prevalence of Delta variants with high pathogenicity and high transmissibility further increased the necessity and urgency of rapid detection and identification method development. Meanwhile, as the most critical preventive means, the novel vaccine developed for the variant strain also needs a rapid means to distinguish the variant strain from other strain type vaccines, so the development of a detection method for Delta strain specificity identification is particularly critical.
The existing real-time fluorescent quantitative PCR (qPCR) for identifying SARS-CoV-2 variant strain utilizes TaqMan probe RT-qPCR technology to quickly identify SARS-CoV-2 main variant strain, firstly according to the gene sequences of original strain and Delta variant strain in NCBI GenBank, respectively designing specific primer sequences, then using virus genome RNA extraction kit to respectively extract new coronavirus original and variant strain RNA or artificially synthesizing mutant strain RNA fragment and making sequencing verification. And then, the reaction system and conditions are properly optimized, and whether the two specific primers can distinguish the original strain gene sequences from the variant strain gene sequences is judged according to the PCR result.
qPCR methods can be used to characterize variant viruses, but suffer from the following drawbacks: (1) long time consumption, and a plurality of uncontrollable influencing factors: the time from sample processing to result reporting is 6-8 hours, most virus detection units do not have gene sequencing conditions, and the samples need to be submitted to third-party sequencing companies for substitution measurement, so that the sample path transfer and possible on-machine queuing waiting time are slow, and the uncontrollable influence factors are more; (2) the detection sensitivity is low: before the on-machine test of gene sequencing, gene amplification is needed, and the sequencing can be influenced by slightly poor amplification effect on low-abundance nucleic acid samples; (3) high detection cost: the new coronavirus gene sequencing comprises complicated steps of primer design, nucleic acid extraction and the like, so that the cost of detection materials and manpower is high, and the detection results are easily deviated due to careless operation due to the operation of a plurality of complicated steps; (4) low specificity: since SARS-CoV-2 is always under evolutionary variation, detection failure may result if the new mutation site is not included in the sequence length of the PCR method study design. The defects can influence the detection timeliness, flux and cost of the virus variant strain when the molecular sequencing method is applied to the prevention and control of the new coronavirus epidemic situation, and can lead to result deviation, mislead or delay the prevention and control work process of the new coronavirus epidemic situation due to the fact that the detection specificity of the variant strain is not sensitive enough.
The liquid chromatography tandem mass spectrometry technology has the advantages of high selectivity, high sensitivity and the like in the aspect of protein detection and analysis, and is very suitable for qualitative research of targeted proteins in complex biological matrixes. The invention provides a mass spectrum method for rapidly and accurately identifying a Delta subline by identifying an analysis result of a specific mutation site through mass spectrum of a variant strain.
Disclosure of Invention
The present application aims to solve at least one of the above technical problems existing in the prior art. To this end, one of the main objects of the present invention is to provide a method for detecting novel coronavirus delta strains by detecting and identifying novel coronavirus delta strains by detection of specific peptide fragments of the delta strains using liquid chromatography tandem mass spectrometry.
It is also an object of the present invention to provide a specific peptide fragment for detecting novel coronavirus delta strains.
In one aspect, the invention provides a method of detecting a novel coronavirus delta strain comprising detecting the presence of a specific peptide fragment of amino acid sequence LQNVVNQNAQALNTLVK (SEQ ID No. 1) in a sample by tandem mass spectrometry using liquid chromatography. The invention provides a specific peptide segment for detecting novel coronavirus delta strain, wherein the amino acid sequence of the specific peptide segment is LQNVVNQNAQALNTLVK (SEQ ID No. 1).
Other aspects of the invention also relate to the use of the above specific peptide fragments in the detection, prevention and/or treatment of novel coronavirus delta strains and kits comprising the same.
The method for detecting the novel coronavirus delta strain is particularly used for identifying the specific peptide segment of the novel coronavirus delta strain by utilizing a liquid chromatography tandem mass spectrometry, and has the advantages of high sensitivity, good specificity and high accuracy. The specific peptide segment also has the characteristics of strong specificity, high stability and the like in the detection of novel coronavirus delta strain. Compared with the detection method of the novel coronavirus delta strain in the prior art, the technical scheme of the invention does not need to design primers or prepare antibodies, can carry out specific identification on the novel coronavirus delta strain at the protein level, provides a new thought for the specific identification of the novel coronavirus delta strain, and has important significance for the identification of the novel coronavirus variant strain, the research and development of vaccines and the quality control.
Drawings
FIG. 1 shows SDS-PAGE protein bands of sample 1 of the virus to be tested in example 1 of the present invention.
FIG. 2 shows SDS-PAGE protein bands of sample 2 of the virus to be tested in example 1 of the present invention.
FIG. 3 is a total ion flow diagram of protein sample 1 in example 2 of the present invention.
FIG. 4 is a total ion flow diagram of protein sample 2 in example 2 of the present invention.
FIG. 5 shows the results of protein coverage of Delta strain S of protein sample 1 in example 3 of the present invention.
FIG. 6 is a parent ion mass spectrum of peptide LQNVVNQNAQALNTLVK in protein sample 1 of example 3 of the present invention.
FIG. 7 is a mass spectrum of the second-order fragments of peptide LQNVVNQNAQALNTLVK in protein sample 1 of example 3 of the present invention.
FIG. 8 shows the results of protein coverage of Delta strain S of protein sample 2 in example 3 of the present invention.
FIG. 9 shows the results of protein coverage of HB02 strain S of protein sample 2 in example 3 of the invention.
FIG. 10 is a parent ion mass spectrum of peptide LQDVVNQNAQALNTLVK in protein sample 2 of example 3 of the present invention.
FIG. 11 is a mass spectrum of the second-order fragments of peptide LQDVVNQNAQALNTLVK in protein sample 2 of example 3 of the present invention.
Detailed Description
Hereinafter, embodiments of the present invention will be described in more detail to aid understanding of the present invention. It should be understood that the description of these embodiments is for illustrative purposes only and is not intended to limit the scope of the invention as claimed in any way.
During the pandemic of the new coronavirus (SARS-CoV-2, COVID-19), some mutations in the viral sequence have appeared that have raised public health concerns, including Alpha strain, beta strain, delta strain, and more recently Omicron-BA.1, BA.5, XBB strain, etc. Wherein, compared with the original strain virus pedigree, the Delta variant Spike protein (Spike protein, S protein) has key mutation of 10 main mutation sites, and the transmission enhancement of the Delta variant virus is related to the key mutation in the S protein (such as D614G, L452R, T478K, D950N and the like). The D950N mutation was found to be unique to the Delta strain by analysis of the sequences of the various major line variants, see Table 1 below. The inventors of the present invention have further studied intensively, and found that a specific peptide fragment (also referred to as a specific peptide fragment) including a D950N mutation can be specifically detected by using a liquid chromatography tandem mass spectrometry, and identification of a novel coronavirus Delta strain can be effectively achieved, thereby completing the present invention.
TABLE 1 Spike protein mutation sites of SARS-CoV-2 Alpha, beta, delta, omicron-BA.1, omicron-BA.5, omicron-XBB variant
Accordingly, in one aspect, the invention provides a method of detecting a novel coronavirus delta strain comprising detecting the presence of a specific peptide fragment of amino acid sequence LQNVVNQNAQALNTLVK (SEQ ID No. 1) in a sample by tandem mass spectrometry using liquid chromatography. In a preferred embodiment, the specific peptide fragment of SEQ ID No.1 has a parent ion mass to charge ratio of 934.02271.+ -. 0.02 Da and a child ion mass to charge ratio of 455.26108.+ -. 0.02 Da and/or 356.19305.+ -. 0.02 Da as determined by liquid chromatography tandem mass spectrometry.
In some embodiments, the methods of the invention for detecting a novel coronavirus delta strain comprise the steps of:
preparing a sample and performing enzymolysis treatment to separate and collect novel coronavirus S protein;
performing liquid chromatography tandem mass spectrometry on the sample subjected to enzymolysis to determine whether the specific peptide fragment exists.
In some embodiments, in the methods of the invention for detecting novel coronavirus delta strains, the step of preparing a sample and performing an enzymatic hydrolysis treatment comprises:
performing preseparation on a sample to be detected by SDS-PAGE;
the pre-separated samples were decolorized, then subjected to disulfide reduction and alkylation, and treated with trypsin at 37 ℃.
In a preferred embodiment, the pre-separated sample is decolorized and then subjected to disulfide bond reduction with 5-25 mM dithiothreitol solution, 11-50 mM iodoacetamide alkylation.
In some embodiments, in the methods of the invention for detecting novel coronavirus delta strains, liquid chromatography is performed under conditions comprising:
analytical column: c-18 resin packed column;
mobile phase: mobile phase a was 0.1% formic acid in water and mobile phase B was 80% acetonitrile and 0.1% formic acid in water;
flow rate: 0.20-0.30 mu L/min.
In some embodiments, in the methods of the invention for detecting novel coronavirus delta strains, mass spectrometry is performed under conditions comprising:
the mass-charge ratio of the four-stage mass spectrum connected in series with the liquid chromatograph is 200-2000, and the resolution is 70000.
In some embodiments, in the methods of the invention for detecting novel coronavirus delta strains, after performing a liquid chromatography tandem mass spectrometry assay, further retrieval and identification is performed by a novel coronavirus database.
In another aspect, the invention provides a specific peptide for detecting a novel coronavirus delta strain, wherein the specific peptide has the amino acid sequence LQNVVNQNAQALNTLVK (SEQ ID No. 1). In a preferred embodiment, the specific peptide fragment of SEQ ID No.1 has a parent ion mass to charge ratio of 934.02271.+ -. 0.02 Da, and a child ion mass to charge ratio of 455.26108.+ -. 0.02 Da and/or 356.19305.+ -. 0.02 Da, as determined by liquid chromatography tandem mass spectrometry.
Other aspects of the invention also relate to the use of the above specific peptide fragments in the detection, prevention and/or treatment of novel coronavirus delta strains and kits comprising the same.
Abbreviations
SARS-CoV-2: severe acute respiratory syndrome coronavirus 2, severe acute respiratory syndrome coronavirus 2
COVID-19: coronavirus disease 2019, novel coronavirus infection
VOC variant of concern, variants of interest
qPCR (quantitative polymerase chain reaction) Real-time fluorescence quantitative Polymerase Chain Reaction (PCR) of Real-time Quantitative PCR
MS Mass Spectrometry, mass Spectrometry
ELISA: enzyme linked immunosorbent assay ELISA
Examples
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 Pre-separation of novel coronavirus sample proteins to be tested
Two novel coronavirus samples to be tested (stock solution obtained after virus inactivation and purification) were obtained from Beijing biological product research all of the Limited companies, and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after lysis and denaturation treatment. After gel staining and decoloration, protein band distribution is observed, and the result shows that the main protein components of the two virus samples are three proteins with molecular weights similar to that of S protein, N protein and M protein (figure 1 and figure 2).
Example 2 liquid chromatography tandem mass spectrometry detection of novel coronavirus samples to be tested
The specific method for detecting the liquid chromatography tandem mass spectrometry comprises the following steps:
1. sample preparation
Using the SDS-PAGE pretreated samples obtained in example 1, the protein bands of interest were excised from the gel and cut to 1 mm x 1 mm size, and after decolorization, the disulfide bonds were reduced sequentially with 5 mM Dithiothreitol (DL-DTT) solution, 11 mM Iodoacetamide (IAM) solution for alkylation, and sequencing grade modified trypsin (Promega) was added for overnight cleavage at 37 ℃. The cleavage reaction was quenched by addition of acid, extracted 2 times in 1% trifluoroacetic acid (trifluoroacetic acid, TFA), 50% acetonitrile in water for 30 minutes each time, the extracts combined and concentrated in a vacuum centrifuge concentrator. After the extract was evaporated, it was reconstituted with 0.1% TFA solution and checked on an engine.
2. Liquid chromatography tandem mass spectrometry detection
The high performance liquid chromatography detection conditions of the sample to be detected are as follows:
analytical column: a self-made fused silica capillary column (75 μm inside diameter, 150 mm length; upchurch, oak Harbor, WA, U.S.A.) packed with a C-18 resin (300A, 5 μm, varian, lexington, mass.).
Mobile phase: mobile phase a was 0.1% formic acid in water and mobile phase B was 80% acetonitrile and 0.1% formic acid.
The peptide fragments were separated using a Thermo-Dionex Ultimate 3000 HPLC system, flow rate 0.30. Mu.L/min, 120 min gradient elution.
The mass spectrum detection conditions were as follows:
the liquid phase system described above was directly connected in series with a Q exact combination quadrupole orbitrapTM mass spectrometer (Thermo Fisher Scientific, bremen, germany). Q exact mass spectrometer was run in data dependent acquisition mode using Xcalibur 2.1.2 software, with a single full scan in Orbitrap at 300-1800 mass to charge ratio (m/z), 70000 resolution, followed by separation and fragmentation of the 20 most abundant ions by high energy collision dissociation (HCD) (nce=30%).
3. Data processing
The MS/MS spectra obtained from each LC-MS/MS run were retrieved for the novel coronavirus database using Proteome Discoverer (PD 1.4 edition, thermo-Fisher Scientific, U.S.).
The search criteria were set as follows: the complete trypsin specificity is required; allowing two missed cuts; the ureido (C) is set as a fixed modification; oxidation (M) is set to a variable modification; the parent ion mass error for all MSs collected in the Orbitrap mass analyzer was 20 ppm; for all MS2 mass spectra acquired, the fragment ion mass error was 0.02 Da; the false discovery rate (False discovery rate, FDR) of protein identification was 0.01.
After the S protein bands of two novel coronavirus samples to be detected are respectively processed, detection analysis is carried out by using LC-MS/MS to obtain total ion flow diagrams, which are respectively shown in fig. 3 and 4. The liquid chromatography tandem mass spectrometry detection result is searched by a novel coronavirus database, and the result shows that the protein samples 1 and 2 are compared with novel coronavirus S protein scoring results are 5034.77 and 7184.76 respectively, which show that the protein samples are highly likely to be novel coronavirus S proteins.
Example 3S protein coverage and Delta Strain-specific peptide analysis of novel coronavirus samples to be tested
The sample is processed by adopting trypsin for enzyme digestion, and partial peptide fragments can not be covered due to the problems of the length of the peptide fragments and the ionization capacity of the peptide fragments after enzyme digestion. Thus, the S protein coverage of the sample protein was analyzed and it was determined whether the sample was a novel coronavirus Delta strain by comparing whether the retrieved sample protein matched the specific peptide segment containing the characteristic site 950N of the Delta strain.
The detection result of the liquid chromatography tandem mass spectrometry of the sample 1 in the example 2 is searched by taking the novel coronavirus Delta strain S protein sequence as a template, and the result shows that the detection coverage rate of the sample is 67.35% (see FIG. 5). According to the search comparison result of the S protein sequence of the novel coronavirus Delta strain, the peptide fragment spectrogram of the sample 1 covers and matches with the 950N characteristic site of the Delta strain, and the PSM is 153, so that the sample 1 can be confirmed to be derived from the novel coronavirus Delta strain. The specific peptide LQNVVNQNAQALNTLVK (SEQ ID No. 1) with a mass to charge ratio of 934.032271 Da for parent ions was detected by liquid chromatography tandem mass spectrometry, with the parent ion mass spectrum referring to fig. 6, the secondary fragment mass spectrum and the daughter ion mass to charge ratio referring to fig. 7, and is summarized in table 2 below.
The detection result of the liquid chromatography tandem mass spectrometry of the sample 2 in the example 2 is searched by taking the novel coronavirus Delta strain S protein sequence as a template, and the result shows that the detection coverage rate of the sample is 51.30% (see FIG. 8). According to the search comparison result of the S protein sequence of the novel coronavirus Delta strain, the peptide fragment spectrogram of the sample 2 is not matched with the specific peptide fragment LQNVVNQNAQALNTLVK (SEQ ID No. 1) of the Delta strain. Thus, it was confirmed that sample 2 was not derived from the novel coronavirus Delta strain.
The detection result of the liquid chromatography tandem mass spectrometry of the sample 2 in the example 2 was searched by using the novel coronavirus HB02 strain S protein sequence as a template, and the result shows that the detection coverage rate of the sample is 58.21% (see FIG. 9). According to the result of the search and comparison of the S protein sequence of the novel coronavirus HB02 strain, the peptide fragment spectrum of sample 2 at the above Delta strain characteristic site is shown as 950D, and the PSM thereof is 62. The peptide LQDVVNQNAQALNTLVK (SEQ ID No. 2) with a mass to charge ratio of 934.51691 Da of parent ion was detected by liquid chromatography tandem mass spectrometry, the parent ion mass spectrum of which is shown in fig. 10, the secondary fragment mass spectrum and the daughter ion mass to charge ratio are shown in fig. 11, and are summarized in table 2 below. Therefore, it was further confirmed that sample 2 was not derived from the novel coronavirus Delta strain, but from the novel coronavirus HB02 strain.
TABLE 2 summary of parent and daughter ion mass to charge ratios of peptide fragments in protein samples 1 and 2
Claims (6)
1. A method for detecting a novel strain of coronavirus delta comprising detecting the presence of a specific peptide fragment having an amino acid sequence of LQNVVNQNAQALNTLVK (SEQ ID No. 1) in a sample by tandem mass spectrometry using liquid chromatography,
preferably, the mass-to-charge ratio of the parent ion of the specific peptide fragment of SEQ ID No.1 is 934.02271 + -0.02 Da, and the mass-to-charge ratio of the child ion is 455.26108 + -0.02 Da and/or 356.19305 + -0.02 Da, as determined by liquid chromatography tandem mass spectrometry.
2. The method of claim 1, wherein the method comprises the steps of:
preparing a sample and performing enzymolysis treatment to separate and collect novel coronavirus S protein;
performing liquid chromatography tandem mass spectrometry on the sample subjected to enzymolysis to determine whether the specific peptide fragment exists.
3. The method of claim 2, wherein the preparing and enzymatically treating the sample comprises:
performing preseparation on a sample to be detected by SDS-PAGE;
decolorizing the pre-separated sample, performing disulfide bond reduction and alkylation treatment, treating with trypsin at 37deg.C,
preferably, the pre-separated sample is decolorized and then subjected to disulfide bond reduction with 5 mM to 25 mM dithiothreitol solution, 11 mM to 50 mM iodoacetamide alkylation.
4. The method of claim 2, wherein liquid chromatography is performed under conditions comprising:
analytical column: c-18 resin packed column;
mobile phase: mobile phase a was 0.1% formic acid in water and mobile phase B was 80% acetonitrile and 0.1% formic acid in water;
flow rate: 0.20 Mu L/min to 0.30 mu L/min.
5. The method of claim 2, wherein mass spectrometry is performed under conditions comprising:
the mass-charge ratio of the four-stage mass spectrum connected in series with the liquid chromatograph is 200-2000, and the resolution is 70000.
6. A specific peptide fragment for detecting a novel coronavirus delta strain, wherein the amino acid sequence of the specific peptide fragment is LQNVVNQNAQALNTLVK (SEQ ID No. 1),
preferably, the mass-to-charge ratio of the parent ion of the specific peptide fragment of SEQ ID No.1 is 934.02271 + -0.02 Da, and the mass-to-charge ratio of the child ion is 455.26108 + -0.02 Da and/or 356.19305 + -0.02 Da, as determined by liquid chromatography tandem mass spectrometry.
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Citations (2)
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| CN112225782A (en) * | 2020-09-29 | 2021-01-15 | 中国食品药品检定研究院 | Specific peptide segment and method for determining content of structural protein in COVID-19 vaccine |
| CN116804047A (en) * | 2022-03-17 | 2023-09-26 | 北京科兴中维生物技术有限公司 | Method for detecting S protein content in novel coronavirus vaccine |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112225782A (en) * | 2020-09-29 | 2021-01-15 | 中国食品药品检定研究院 | Specific peptide segment and method for determining content of structural protein in COVID-19 vaccine |
| CN116804047A (en) * | 2022-03-17 | 2023-09-26 | 北京科兴中维生物技术有限公司 | Method for detecting S protein content in novel coronavirus vaccine |
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| KANTAPHON SUDDHAPAS ET AL.: "Evaluation of Variant-Specific Peptides for Detection of SARS-CoV-2 Variants of Concern", JOURNAL OF PROTEOME RESEARCH, vol. 21, 15 September 2022 (2022-09-15), pages 1 * |
| SAKSHI RAJORIA ET AL.: "Detection of Mutant Peptides of SARS-CoV-2 Variants by LC/MS in the DDA Approach Using an In-House Database", JOURNAL OF PROTEOME, vol. 22, 31 December 2023 (2023-12-31), pages 1 - 3 * |
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