RU2748540C1 - Method for detecting sars-cov-2 virus by mass spectrometry - Google Patents

Abstract

FIELD: biochemistry.SUBSTANCE: invention relates to the field of biochemistry. A method for identifying SARS-CoV-2 virus in a sample, which includes the following steps is described: (a) sample preparation of a biological sample using the enzyme trypsin to hydrolyze proteins and obtain a mixture of polypeptides; (b) mass spectrometric analysis of the resulting mixture of polypeptides in order to identify at least two peptides selected from SEQ ID NO: 1-19; (c) if at least two peptides having the sequence SEQ ID NO: 1-19 are detected in the sample, at least one of which is a peptide selected from: SEQ ID NO: 1, SEQ ID NO: 3 - SEQ ID NO: 9, SEQ ID NO: 12 - SEQ ID NO: 19, SARS-CoV-2 virus in the sample is considered to be identified. The invention solves the problem of accelerating and increasing the reliability of the procedure for identifying the SARS-CoV-2 virus in patients. The problem is solved by the proposed method, which consists in identifying the SARS-CoV-2 virus using mass spectrometric analysis based on the developed panel of N-protein peptides (nucleocapsid).EFFECT: accelerating and increasing reliability of the procedure for identifying the SARS-CoV-2 virus in patients.6 cl, 5 dwg, 1 tbl, 5 ex

Description

The invention relates to medicine, namely to clinical diagnostics, to a method for identifying the SARS-CoV-2 virus in samples of various biological origin. In the field of medicine, the method solves the problem of accelerating and increasing the reliability of the procedure for identifying the SARS-CoV-2 virus in COVID 19 patients. It can be used for express analysis of various samples in order to determine the content of the virus in them as an addition / confirmation of an urgent PCR test or as an independent study.

Prior art

The emergence of diseases caused by the new coronavirus (2019-nCoV) has posed challenges for healthcare professionals and doctors related to the rapid diagnosis and clinical management of patients with this infection. To date (December 23, 2020), according to the WHO [1], more than 76 million cases of COVID-19 and more than 1,702,1280 deaths have been registered [1, 2]. Fast and timely diagnosis, including during the incubation period, before the onset of symptoms, allows you to prevent further spread of the virus and adjust treatment measures.

Currently, a test based on the polymerase chain reaction (PCR) method is mainly used to detect the SARS-CoV-2 virus in patients. Despite the high sensitivity of this method, there are a number of limitations and disadvantages of this technique. In total, from the moment of sampling to the issuance of results, it takes about 3 hours of real time, of which 1 hour 40 minutes is amplification and this time does not depend on the number of samples. In addition, if the internal control sample is in error, the entire sample preparation procedure (including sample collection) must be repeated again. An important aspect of the technique is also the professionalism of the staff involved in sample preparation and method setting. Also, during the analysis, false positive and false negative results are not excluded, associated, for example, with the mutation of viral proteins or the detection of the virus at relatively late cycles (40 + cycle). In this regard, the time for the issuance of results in many medical institutions can take up to 48 hours at a rather high cost of diagnostics. Thus, the existing method of laboratory diagnostics - a test based on the polymerase chain reaction (PCR) method - has a number of drawbacks in terms of the reliability and speed of the SARS-CoV-2 virus identification procedure, which can lead to untimely or incorrect diagnosis and delay in the appointment of treatment. This increases the likelihood of adverse disease outcomes.

In this regard, additional tests are needed based on modern high-precision methods, in particular, mass spectrometry, in order to provide an objective and reproducible basis for the selection of patients with clinically significant disease. Due to the insufficient diagnostic value of one biomarker, it is necessary to develop a panel of biomarkers and, on its basis, a monitoring method that will reliably identify the SARS-CoV-2 virus and distinguish the conditions of patients with good clinical sensitivity and specificity, as well as give an accurate prediction of the likelihood of COVID-19 disease.

The SARS-CoV-2 virus contains four structural proteins included in the virion particle known as the S (spine), E (envelope), M (membrane), and N (nucleocapsid) proteins; protein N is linked to an RNA molecule, and proteins S, E and M together create the viral envelope. From the data obtained earlier for SARS-CoV [3], which has a structure similar to that of SARS-CoV-2, it can be concluded that the number of copies of proteins E, M and N is much higher (about 10 times) than the number of copies protein S. Proteins E and M are relatively short, nonspecific, proteins closely bound to the membrane, which complicates their isolation, detection and identification. Thus, it is more promising to develop a detection methodology based on the N-protein [4].

Quantitative proteomic analysis by mass spectrometry methods can serve as a basis for new, high-precision and specific methods of differential diagnostics [5]. In the case of a known target, “targeted” mass spectrometric approaches based on monitoring of multiple / parallel reactions (MRM / PRM) can be used to quantify changes in the protein / peptide panel. A targeted quantitative approach for monitoring a target protein is characterized by the speed of obtaining results, fast sample preparation, and a decrease in the influence of external parameters of the experiment, such as the size and shape of the sample. The latter factor is overcome, among other things, by adding an isotopically labeled internal standard [5].

In the article Ihling S. et al. [6] described a method according to which the method of chromatography-mass spectrometry analyzed precipitated with acetone protein precipitate in a solution for rinsing the throat. In 2 out of three samples obtained from patients diagnosed with COVID-19, one peptide RPQGLPNNTASWFTALTQHGK from the N-protein (nucleocapsid) of the SARS-CoV-2 virus was identified. The disadvantages of this work are several stages of protein hydrolysis (which complicates the sample preparation procedure), a very dilute sample (solution for gargling), a small sample of samples (3 samples without a control group), and as the main result, the authors demonstrate only one repeating peptide, which was identified not in all samples (in 2 out of 3). Moreover, according to the Uniprot database (https://www.uniprot.org/), it is known that this peptide is also typical for other mammalian SARS viruses, and not only for SARS-CoV-2 (including Bat coronavirus HKU3 (BtCoV), Bat coronavirus Rp3 / 2004 (BtCoV / Rp3 / 2004), etc.). Thus, the data obtained do not allow us to propose any reproducible approach based on a reliable peptide panel for the detection of the SARS-CoV-2 virus. Also, a serious disadvantage of the described method is the lack of data on virus inactivation, which is important for the safety of the operator performing sample manipulations.

Thus, despite the popularity of a number of methods for diagnosing the SARS-CoV-2 virus, today they all have drawbacks, and there remains a need on the market for a fast and effective method for detecting the SARS-CoV-2 virus.

Disclosure of invention

The problem solved by the invention is to develop a method for reliable identification of the SARS-CoV-2 virus in samples of various origins, based on the analysis of the N-protein (nucleocapsid) of the SARS-CoV-2 virus.

The problem is solved when implementing the proposed method for identifying the SARS-CoV-2 virus in samples of various origins, including the use of the panel of N-protein peptides (nucleocapsid) of the SARS-CoV-2 virus developed by the authors of the invention.

More specifically, the task is solved when implementing a method for identifying the SARS-CoV-2 virus in a sample, which includes the following steps:

(a) sample preparation of a biological sample using the trypsin enzyme to hydrolyze proteins and obtain a mixture of polypeptides

(b) mass spectrometric analysis of the resulting mixture of polypeptides in order to identify at least two peptides selected from SEQ ID NO: 1-19;

(c) if at least two peptides are identified in the sample having a sequence selected from SEQ ID NO: 1-19, at least one of which is a peptide selected from: SEQ ID NO: 1, SEQ ID NO: 3 - SEQ ID NO: 9, SEQ ID NO: 12 - SEQ ID NO: 19, the SARS-CoV-2 virus in the sample is considered identified.

Also, to ensure biological safety when working with a potentially contagious biological object, before the sample is treated with trypsin, the SARS-CoV-2 virus in the sample is inactivated.

In some private embodiments of the invention, the SARS-CoV-2 virus is inactivated by heating the sample to 65 ° C for at least 30 minutes. followed by the addition of isopropanol.

In some embodiments, SARS-CoV-2 virus in a sample is considered identified when at least two peptides selected from SEQ ID NO: 1, SEQ ID NO: 3 - SEQ ID NO: 9, SEQ ID NO: 12 - SEQ ID NO: 19.

In some embodiments, the sample is a throat swab, nasopharyngeal swab, blood, plasma, serum, bronchial lavage, amniotic fluid, placental tissue, saliva, tear, washout or scraping from food, waste, surface of materials: clothing, furniture, items of individual or general use, waste water, air sample.

In some embodiments, the mass spectrometric analysis is of the type of parallel reaction monitoring (PRM), multiple monitoring (MRM), MRM ³ , selected response monitoring (SRM), DDA (data collection depending on data), DIA (data collection independent of data) or iMALDI.

In some embodiments, the SARS-CoV virus N protein is further mass spectrometrically analyzed in the sample using a panel of isotopically labeled peptides.

As a result of the implementation of the method, the following technical results are achieved:

- a method for reliable identification of the SARS-CoV-2 virus was developed, based on the analysis of a panel of peptides of the N-protein of the virus;

- the proposed method has high sensitivity and specificity, provided through the use of a unique panel of peptides of the N-protein SARS-CoV;

- to carry out the proposed method for identifying the SARS-CoV-2 virus, samples of various origins can be used, including biological samples (tissues, physiological fluids (blood, urine, saliva, etc.), smears from the skin, nasopharynx, bronchial lavage, amniotic liquid, placental tissue, etc.), waste products (food, waste, for example, sewage, etc.), environmental objects (air, surfaces of materials (clothing, common items), etc.);

- the method is widely available for carrying out in laboratory and clinical conditions, quickly introduced and implemented;

- the proposed method provides a reduction in the diagnostic time while reducing the likelihood of technical errors during laboratory manipulations;

- the method can be carried out very quickly, in less than 1 hour,

- the developed method can be used for express analysis of various samples in order to determine the content of the virus in them as an addition / confirmation of a test based on polymerase chain reaction (PCR) or other methods of analysis, as well as as an independent study.

Detailed description of the invention

Definitions and terms

The following terms and definitions apply in this document unless otherwise indicated. References to techniques used in describing the present invention refer to well known techniques, including modifying these techniques and replacing them with equivalent techniques known to those skilled in the art.

In the description of this invention, the terms "includes" and "including" are interpreted as meaning "includes, among other things". These terms are not intended to be construed as “consists only of”.

The term "sample" refers to samples of various origins, including both biological samples and waste products (waste, for example, sewage, etc.), food, environmental objects (air, surfaces of materials (clothes, furniture, objects general use)), etc.

The term "biological sample" ("biosample") refers to any component obtained or isolated from a patient and includes a smear (scraping of the epithelium) from the pharynx, a swab (scraping of the epithelium) from the nasopharynx, blood, plasma, blood serum, urine, bronchial lavage, amniotic fluid, placental tissue, saliva, tears.

As used herein, the term "patient" refers to a person. However, the patient can also be an animal such as a bird or mammal. Specific animals include rat, mouse, dog, cat, cow, sheep, horse, pig, or primates. The patient can also be a transgenic animal.

The term "patient with COVID-19" refers to patients in whom the SARS-CoV-2 coronavirus has been diagnosed by alternative methods (such as a PCR-based test (RT-qPCR); for example, as described in [3]).

The term "isotopically labeled peptides"("stable isotope-labeled standard peptides (SIS)") refers to peptides that have been synthesized using stable isotope-labeled amino acids to produce a peptide that has a greater mass than the corresponding unlabeled target peptide for use. as an internal standard for determining the amount of a target peptide in a sample. Suitable isotopes are non-radioactive chemical isotopes that do not decay spontaneously. For example, stable isotopes that can be used to study biological systems include the isotopes ¹⁸ O, ¹³ C, ¹⁵ N, ² H, and ³² S.

Unless otherwise specified, technical and scientific terms in this application have the standard meanings generally accepted in the scientific and technical literature.

Brief Description of Figures

FIG. 1. The amino acid sequence of the N-protein of the coronavirus SARS-CoV (P0DTC9 | NCAP_SARS2), on which (bold + underlining) peptides included in the peptide panel developed by the inventors are highlighted.

The O (210) label denotes oxidized methionine, which is a variable modification of one of the peptides included in the proposed panel of peptides according to the invention.

FIG. 2a and 2b. Examples of mass spectra of fragmentation of tryptic peptides N-protein (P0DTC9 | NCAP_SARS2) of SARS-CoV-2 coronavirus identified by the proposed method in epithelial swabs from the nasopharynx obtained from COVID-19 patients.

FIG. H. Table 1: Panel of N-protein peptides (P0DTC9 | NCAP_SARS2) of SARS-CoV-2 coronavirus identified by the method of the invention using PEAKS Studio software in epithelial nasopharyngeal swabs obtained from patients with COVID-19. Samples 1-5 were taken from 5 patients with COVID-19 (1-3 were prepared according to protocol 1; 4-5 were prepared according to protocol 2). Samples 6-8 are negative controls (healthy individuals). Two replicate analyzes were performed for each sample. The numbers in the table correspond to the number of spectra ("spectral counts") for each peptide. Lines for peptides with missed cleavages are marked in gray.

FIG. 4. Table 2: Panel of N-protein peptides (P0DTC9 | NCAP_SARS2) of SARS-CoV-2 coronavirus identified by the proposed method using MaxQuant software in epithelial nasopharyngeal swabs obtained from patients with COVID-19. Samples 1-5 were taken from 5 patients with COVID-19 (1-3 were prepared according to protocol 1; 4-5 were prepared according to protocol 2). Samples 6-8 are negative controls (healthy individuals). Two replicate analyzes were performed for each sample. The numbers in the table correspond to the number of spectra ("spectral counts") for each peptide. Lines for peptides with missed cleavages are marked in gray.

FIG. 5. Identification by the proposed method of the SARS-CoV-2 coronavirus by N-protein peptides (P0DTC9 | NCAP_SARS2) in a sample of placental tissue obtained from a patient with COVID-19. The blue lines mark the tryptic peptides identified by the proposed method.

The proposed approach to identifying the SARS-CoV-2 virus in samples of various origins is based on mass spectrometric analysis in samples after specific sample preparation. The advantage of this method is the ability to detect viral proteins in complex biological samples. The peptide panel proposed by the authors of this invention and the methods of sample preparation used make it possible to identify the SARS-CoV-2 virus with high accuracy in samples of various origins, including complex samples containing a large amount of proteins of various origins.

The proposed method includes the following steps:

- inactivation of the virus and disinfection of the surface of the test tubes,

- precipitation of total protein and its hydrolysis by trypsin,

- mass spectrometric analysis and identification of tryptic peptides of the SARS-CoV-2 virus.

Before inactivation of the virus and disinfection of the surface of the tubes, all work must be carried out in accordance with the rules of biosafety level III [7].

Virus inactivation can be done as follows:

• Transfer 0.25 ml of the analyzed material (sample) into a polypropylene (PP) Eppendorf tube with a volume of 1.5-2.0 ml.

• Inactivate by heating at 65 ° C for at least 30 minutes (water bath or solid-state thermostat with thermoblock) [8] (it is important that during heat treatment all parts of the tube are heated, and thus inactivation of all, even “inaccessible” surfaces).

• After incubation, add 0.75 ml of 100% isopropanol to obtain a 75% solution and allow the sample to stand for 10 minutes at room temperature [9].

• Treat tube surfaces with 70% isopropanol or 1% sodium hypochlorite: spill from a jet wash bottle and incubate for at least 15 minutes without wiping.

As a result of this procedure, the virus is inactivated, and the surface of the tube becomes safe.

At the next stage, precipitation of the total protein and its hydrolysis with trypsin are carried out. The use of trypsin (as well as other specific proteases such as Arg-C, Arg-C, Glu-C, Lys-C) to hydrolyze proteins in mass spectrometric analysis is well known in the art. The choice of an enzyme for hydrolysis depends on many parameters, including the structure of the analyzed protein, the presence of other proteins in the images. The optimal combination of sample preparation method and trypsinolysis developed by the authors of the invention ensures high efficiency of N-protein detection in samples.

Sample preparation can be carried out according to the "standard" or "express" protocols, which provide the most complete coverage of the N-protein sequence. In some cases, for the most accurate analysis of the sample, repeated assays using different trypsinolysis techniques should be used to maximize the detection of peptides included in the N-protein peptide panel of the invention.

For the "standard" protocol (1), inactivated biosamples are lyophilized and dissolved in 50 mM ammonium bicarbonate buffer containing 0.1% Rapigest (Waters). Reduced with dithiothreitol (20 mM) for 30 min. at 500 ° C, alkylated with iodoacetamide (50 mM) for 45 minutes at room temperature in the dark, and then hydrolyzed with trypsin for 4 hours at 37 ° C. The reaction is stopped by adding formic acid to a final concentration of 0.5%.

In the case of the "express" protocol (2), inactivated biosamples are cooled for two hours at -200 ° C and centrifuged at 20,000 g for 20 minutes. The precipitate is dissolved in 50 mM ammonium bicarbonate buffer containing 0.1% Rapigest (Waters) and hydrolyzed with trypsin for 4 hours at 37 ° C. The reaction is stopped by adding formic acid to a final concentration of 0.5%.

As a result of the studies carried out, it was found that the use of the "express" protocol (2) allows better detection of the N-protein than the more thorough "standard" protocol usually used for preparing a protein sample (1). Probably, such a non-obvious difference is explained by the fact that when using the "express" protocol (2), a significantly smaller amount of peptides from the so-called "host proteins" are detected in the samples, which are initially present in the sample under study and for their effective detection, more careful sample preparation is usually required. including steps: reduction, alkylation, deglycosylation and other preparatory steps for good coverage of the sequence.

Other options for sample preparation are also possible. In particular, accelerated trypsin digestion can be used.

The "fast" protocol (3) can be implemented using immobilized trypsin. Immobilization of trypsin can be carried out on the surface of magnetic microspheres, for example, carboxyl functionalized magnetic microspheres (BioMag Plus carboxyl) can be used. Immobilization is carried out by a reaction between a succinimide group on the surface of microspheres and an amine group on trypsin, in accordance with the protocol [10]. Using this protocol allows trypsinolysis to be performed within 15 minutes. The use of the "fast" protocol of trypsinolysis (3) or other known fast protocols can significantly speed up the overall analysis time.

At the next stage, mass spectrometric analysis and identification of tryptic peptides of the SARS-CoV-2 virus are carried out.

Mass spectrometry is well known to those skilled in the art as a powerful tool for the analysis, identification and quantification of various types of molecules (classes of compounds). The method is based on the ionization of the molecule, leading to the formation of a "molecular ion", on the basis of which analysis and identification is carried out by measuring the ratio of mass to charge and the intensity of the ion current from the corresponding molecular ion. Depending on the nature of the detected molecule (for example, protein or non-protein origin), the complexity of the sample (mixture or pure substance) and the research problem (for example, qualitative or quantitative analysis), an appropriate ionization and mass spectrometry method is selected.

Recent advances in peptide and protein analysis by mass spectrometry (MS) are the result of the development of advanced ionization techniques such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). Electrospray Ionization (ESI) involves the ionization at atmospheric pressure of a liquid sample. During the electrospray process, highly charged droplets are created, which, when evaporated, create ions characteristic of the molecules contained in the solution. With the ESI method, it has become possible to use mass spectrometry in tandem with liquid chromatography.

Liquid chromatography combined with mass spectrometry (LC-MS) is a powerful tool for analyzing and quantifying protein in complex biosamples. Since peptides obtained from cleavage of the protein of interest and other proteins of the biosample may have the same or similar nominal mass, additional fragmentation (MS / MS or MS2) often results in a unique fragment of the peptide of interest. The combination of a specific parent peptide and its unique fragment is used to selectively monitor and quantify the concentration of the peptide and its corresponding protein. This approach is called “Multiple Response Monitoring” (MRM), also referred to as “Selected Response Monitoring” (SRM), and is the most widely used method for quantifying protein. Another approach is parallel response monitoring (PRM).

To develop a quantitative method for a specific panel of proteins, the corresponding tryptic peptides are selected and synthesized. Usually, a pair of standard peptides is synthesized: an unlabeled "light" peptide (NAT), which is completely analogous to a natural peptide, and a paired, labeled with stable isotopes "heavy" peptide (SIS). For quantitative analysis, a calibration curve is constructed for each pair of peptides (NAT / SIS) by diluting a variable amount of light peptide in the required concentration range, with the addition of the same amount of SIS peptide as an internal standard for signal normalization and quantitative assessment of the concentration of unlabeled peptides. To control the accuracy of the calibration curve, additional quality control (QC) samples prepared by diluting the standard peptides are used. The obtained calibration curves for all peptide pairs can be used further for the quantitative analysis of the corresponding natural proteins and peptides. All of these approaches can be used to identify and quantify viral N-protein (P0DTC9 | NCAP_SARS2) in a sample. For example, an appropriate panel of isotopically labeled peptides can be used, in which the C-terminal lysine or arginine is labeled with ¹³ C and ¹⁵ N isotopes: K (+8 Da; 6x 13C, 2x 15N), R (+10 Da; 6x 13C, 4x 15N).

In a particular embodiment of the invention, the peptides obtained as a result of hydrolysis by trypsin in the previous step can be analyzed in LC-MS / MS mode using a Dionex Ultimate3000 nanoflow high-performance liquid chromatographic (nano-HPLC) system (Thermo Fisher Scientific, USA) connected to Time-of-flight mass spectrometer TIMS TOF Pro (Bruker Daltonics, USA) operating in the mode of trapped ion mobility spectrometry (TIMS). The loaded sample volume is 2 μl per injection. HPLC separation was carried out using a packed emitter column (C18, 25 cm x 75 μm, 1.6 μm) using a gradient elution method. Mobile phase A - 0.1% formic acid in water; mobile phase B - 0.1% formic acid in acetonitrile. Separation of nano-HPLC is achieved at a flow rate of 400 nl / min. using a 40-minute gradient from 4% to 90% of phase B.

The skilled person will understand that other methods for identifying peptides using mass spectrometry can be used.

As a result of the studies carried out, described in more detail in the "examples" section below, a panel of N-protein peptides (P0DTC9 | NCAP_SARS2) of the SARS-CoV-2 coronavirus was developed, having the sequences SEQ ID NO: 1-19, which can be used as panels of biomarkers for performing reliable identification of the SARS-CoV-2 virus, with good clinical sensitivity and specificity. FIG. 1, showing the coverage of the N-protein sequence by the peptides of the panel, shows the amino acid sequence of the N-protein of the SARS-CoV coronavirus (P0DTC9 | NCAP_SARS2), in which the peptides included in the peptide panel developed by the inventors are highlighted in bold and underlined. Table 1 shows a list of all peptides included in the panel developed by the authors.

It is important to note that almost all peptides in the panel are unique to SARS-CoV-2. Such unique peptides are indicated in Table 1 in bold and underlined.

At the same time, as the studies have shown, the detection in a sample of only one peptide included in the developed panel of the N-protein (P0DTC9 | NCAP_SARS2) of the SARS-CoV-2 coronavirus is insufficient for reliable detection of the SARS-CoV-2 virus in the sample. For more accurate identification of the virus, it is necessary to detect at least 2 peptides included in the developed panel of N-protein peptides, and at least one peptide detected in the sample should be unique for SARS-CoV-2, i.e. is a peptide selected from: SEQ ID NO: 1, SEQ ID NO: 3 - SEQ ID NO: 9, SEQ ID NO: 12 - SEQ ID NO: 19.

Thus, as a result of the conducted studies, a panel of unique easily detectable peptides obtained as a result of hydrolysis of the SARS-CoV-2 nucleocapsid protein was developed. The developed peptide panel can be used as targets for creating highly specific and sensitive methods for detecting the SARS-CoV-2 virus in samples based on mass spectrometry, including the use of parallel reaction monitoring (PRM) or rapid parallel mass spectrometric approaches based on immunoprecipitation and laser desorption / ionization in the presence of a matrix (iMALDI), allowing very quickly to check for the presence of virus in the sample.

The described approach demonstrates reliable identification of the N-protein in samples even at the lowest viral loads and low viral content using a fairly simple sample preparation procedure.

At the same time, the use of fast trypsinolysis procedures (~ 15 min), fast mass spectrometry procedures (~ 15 min) and the use of automation of sample preparation make it possible to carry out the analysis very quickly, in less than 1 hour (60 min).

The following examples of the implementation of the method are given in order to disclose the characteristics of the present invention, and should not be construed as in any way limiting the scope of the invention.

Example 1. Collection of samples and inactivation of the virus

All procedures for the collection, transportation and preparation of samples were carried out in accordance with the restrictions and protocols when working with microorganisms of the level of biological safety III pathogenicity (hazard). "

To detect the SARS-CoV-2 virus, scrapings of the mucous membrane of the lower part of the nasopharynx and the posterior wall of the oropharynx were used. Samples were collected using a sterile velor swab with a plastic applicator. The tampon was inserted along the outer wall of the nose to a depth of 2-3 cm to the lower shell, and after the rotational movement was performed, it was removed along the outer wall of the nose. After taking the material, the tampon was placed to the point of destruction in a sterile disposable tube with a transport medium, and the end of the probe was broken off. Oropharyngeal swabs were taken with dry sterile viscose tampons with rotational movements along the surface of the tonsils, palatine arches and the posterior wall of the oropharynx. To increase the concentration of the virus, nasopharyngeal and oropharyngeal swabs are placed in one tube, which is then sealed and labeled.

Epithelial nasopharyngeal swab samples were collected from 5 patients with RT-qPCR confirmed COVID-19 infection. Negative control samples were taken from 3 healthy individuals.

Inactivation of the virus in the collected samples was carried out in accordance with the method described above.

Example 2. Preparation of samples for mass spectrometry

After inactivation of the virus, sample preparation of the selected samples was carried out in accordance with the "standard" (1) or "express" (2) protocols described above, as a result of which precipitation of the total protein contained in the samples was achieved and its hydrolysis with trypsin.

Sample preparation of part of biosamples obtained from patients with COVID-19 infection confirmed by RT-qPCR method was carried out in accordance with protocol (1) (patients 1-3), sample preparation of another part of biosamples obtained from patients with COVID-19 infection (patients 4-5), as well as biosamples taken from healthy individuals, was carried out according to protocol (2) (see Fig. 3 and 4).

Example 3. LC-MS / MS method (combination of liquid chromatography and tandem mass spectrometry)

Peptides after trypsin digestion were analyzed in duplicate on a Dionex Ultimate 3000 nano-HPLC system (Thermo Fisher Scientific, USA) connected to a TIMS TOF Pro mass spectrometer (Bruker Daltonics, USA) using the method of data collection using parallel accumulation and Sequential fragmentation (PASEF) in DDA mode (data-driven acquisition). The electrical spray source (ESI) settings were as follows: capillary voltage - 4500 V, end plate displacement potential - 500 V, dry gas flow rate - 3.0 L / min at 180 ° C. The measurements were carried out in the mass / charge (m / z) range from 100 to 1700. The ion mobility range included values from 0.60 to 1.60 V⋅s / cm ² (1 / k0, where k0 is the ion mobility). The total cycle time was set to 1.16 s and the number of PASEF MS / MS scans was 10. For small sample quantities, the total cycle time was set to 1.88 s.

FIG. 2 shows examples of mass spectra of fragmentation of tryptic peptides (Fig. 2a - RPQGLPNNTASWFTALTQHGK peptide, Fig. 2b - DGIIWVATEGALNTPK peptide).

Example 4. Data Analysis

The data obtained were analyzed using the PEAKS Studio 8.5 software and MaxQuant version 1.6.7.0 using the following parameters: the error in measuring the mass of the parent ion - 20 ppm; fragment mass error - 0.03 Da. Methionine oxidation and carbamidomethylation of cysteine residues were identified as possible variable modifications and up to 3 variable modifications per peptide were allowed. The search was carried out using the Swissprot SARS-COV-2 database, and the human protein database was selected as the database for "contamination". The FDR thresholds for all stages were set at 0.01 (1%) or lower.

As a result of the analysis performed, peptides related to approximately 1000-1500 proteins were identified in each sample. At the same time, peptides related to the N-protein (P0DTC9 | NCAP_SARS2) of the SARS-CoV-2 coronavirus were registered in a number of samples (Fig. 1). The reliability of the data was confirmed by carrying out the detection in several repetitions, as well as using 2 different programs for data analysis. At the same time, the analysis showed that all samples in which peptides related to the N-protein were identified were taken from patients with COVID-19 infection, previously confirmed by the RT-qPCR method. In addition, in none of the control samples taken from healthy individuals, peptides related to the N-protein were detected (Fig. 3-4). Thus, the analysis made it possible to identify a panel of N-protein peptides (P0DTC9 | NCAP_SARS2) of the SARS-CoV-2 coronavirus, having the sequences SEQ ID NO: 1-19, which can be used as a panel of biomarkers for reliable identification of the SARS-CoV virus -2, with good clinical sensitivity and specificity.

The results of an additional analysis carried out on a larger number of samples showed that the detection in the sample of only one peptide included in the N-protein panel (P0DTC9 | NCAP_SARS2) of the SARS-CoV-2 coronavirus is insufficient for reliable detection of the SARS-CoV-2 virus in the sample. ... It is necessary to detect at least 2 peptides included in the developed panel of N-protein peptides, with the detection of at least one peptide unique to SARS-CoV-2, shown in Table 1.

Example 5. Application of the developed panel of N-protein peptides for identification of SARS-CoV-2 virus in a sample

For the analysis, a sample of placental tissue obtained from a patient with COVID-19 infection, previously confirmed by the RT-qPCR method, was taken. After inactivation of the virus and carrying out sample preparation, including the precipitation of the total protein contained in the sample and its hydrolysis with trypsin, the analysis of tryptic peptides using mass spectrometry was carried out and the results of mass spectrometry were compared with the developed panel of peptides. As a result of the analysis, the peptides DGIIWVATEGALNTPK (SEQ ID: NO: 5) and NPANNAAIVLQLPQGTTLPK (SEQ ID: NO 6) included in the developed panel were identified in the sample, indicating the presence of the COVID-19 virus in the analyzed sample (Fig. 5).

Although the invention has been described with reference to the disclosed embodiments, it should be apparent to those skilled in the art that the specific experiments described in detail are provided for the purpose of illustrating the present invention only and should not be construed as in any way limiting the scope of the invention. It should be understood that various modifications are possible without departing from the spirit of the present invention.

Cited documents:

[1] Coronavirus disease (COVID-2019) situation reports https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports

[2] WHO Coronavirus Disease (COVID-19) Dashboard https://covid19.who.int/

[3] Virological assessment of hospitalized patients with COVID-2019 https://www.nature.com/articles/s41586-020-2196-x

[4] Mass-Spectrometric Detection of SARS-CoV-2 Virus in Scrapings of the Epithelium of the Nasopharynx of Infected Patients via Nucleocapsid N Protein. Nikolaev EN, Indeykina Ml, Brzhozovskiy AG, Bugrova AE, Kononikhin AS, Starodubtseva NL, Petrotchenko EV, Kovalev GI, Borchers CH, Sukhikh GT // J Proteome Res. 2020 Nov 6; 19 (11): 4393-4397. doi: 10.1021 /acs.jproteome.0c00412.

[5] Gaither C, Popp R, Mohammed Y, Borchers CH. Determination of the concentration range for 267 proteins from 21 lots of commercial human plasma using highly multiplexed multiple reaction monitoring mass spectrometry // Analyst. 2020 May 18; 145 (10): 3634-3644. doi: 10.1039 / c9an01893j.

[6] Ihling C, Tänzler D, Hagemann S, Kehlen A, Hüttelmaier S, Arlt C, Sinz A. Mass Spectrometric Identification of SARS-CoV-2 Proteins from Gargle Solution Samples of COVID-19 Patients // J Proteome Res. 2020 Nov 6; 19 (11): 4389-4392. doi: 10.1021 / acs.jproteome.0c00280. Epub 2020 Jun 23.

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[9] Rabenau, H. F., J. Cinatl, B. Morgenstern, G. Bauer, W. Preiser, and H. W. Doerr. 2005. Stability and inactivation of SARS coronavirus // Med. Microbiol. Immunol. (Berl.). 194: 1-6.

[10] Sun L, Li Y, Yang P, Zhu G, Dovichi NJ. High efficiency and quantitatively reproducible protein digestion by trypsin-immobilized magnetic microspheres. J Chromatogr A. 2012; 1220: 68.

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LIST OF SEQUENCES

<110> AUTONOMOUS NON-PROFIT EDUCATIONAL ORGANIZATION OF HIGHER EDUCATION

"SKOLKOVO INSTITUTE OF SCIENCE AND TECHNOLOGY"

<120> Method for detecting SARS-CoV-2 virus by mass spectrometry

<130> 428398

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Ile Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg

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<212> PRT

<213> SARS coronavirus

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Arg Pro Gln Gly Leu Pro Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys

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<212> PRT

<213> SARS coronavirus

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Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg

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<212> PRT

<213> SARS coronavirus

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Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys

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<212> PRT

<213> SARS coronavirus

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Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys

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Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu Asp Arg

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<221> MOD_RES

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<223> oxidation

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<213> SARS coronavirus

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Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu Asp

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Arg Leu Asn Gln Leu Glu Ser Lys

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Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile Arg

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Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala Ile Lys

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Leu Asp Asp Lys Asp Pro Asn Phe Lys

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Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser Thr Gln Ala

1 5 10

<---

Claims (10)

1. A method for identifying the SARS-CoV-2 virus in a sample, which includes the following steps: (a) sample preparation of a biological sample using the enzyme trypsin to hydrolyze proteins and obtain a mixture of polypeptides; (b) mass spectrometric analysis of the resulting mixture of polypeptides in order to identify at least two peptides selected from SEQ ID NO: 1-19; (c) if at least two peptides having the sequence SEQ ID NO: 1-19 are detected in the sample, at least one of which is a peptide selected from: SEQ ID NO: 1, SEQ ID NO: 3 - SEQ ID NO: 9, SEQ ID NO: 12 - SEQ ID NO: 19, SARS-CoV-2 virus in the sample is considered to be identified. 2. The method according to claim 1, wherein the SARS-CoV-2 virus is considered identified when at least two peptides selected from: SEQ ID NO: 1, SEQ ID NO: 3 - SEQ ID NO: 9, SEQ ID NO: 12 - SEQ ID NO: 19. 3. The method according to claim 1, in which, before the sample is treated with trypsin, the SARS-CoV-2 virus is inactivated in the sample. 4. The method according to claim 3, wherein the SARS-CoV-2 virus is inactivated by heating the sample to 65 ° C for at least 30 minutes, followed by the addition of isopropanol. 5. The method according to claim 1, wherein the sample is a throat swab, nasopharyngeal swab, blood, plasma, blood serum, urine, bronchial lavage, amniotic fluid, placental tissue, saliva, skin swab, washout from the surface of the object, waste water sample or air sample. 6. The method of claim 1, wherein the mass spectrometric analysis is of the type of parallel reaction monitoring (PRM), multiple monitoring (MRM), MRM ³ , selected response monitoring (SRM), DDA (data-dependent data collection), DIA (data collection independent of data) or iMALDI. 7. The method of claim 1, further comprising mass spectrometric quantitative analysis of the SARS-CoV virus N protein in the sample using a panel of isotopically labeled peptides.

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