WO2024040840A1 - Construction method for intestinal microorganism-related metabolite mass spectrometry database and use thereof - Google Patents

Abstract

The present invention provides a construction method for an intestinal microorganism-related metabolite mass spectrometry database and a use thereof. The construction method comprises the following steps: (1) collecting a standard substance of an intestinal microorganism-related metabolite, and inputting basic information into a library building system; (2) feeding the standard substance of the intestinal microorganism-related metabolite into a chromatograph mass spectrometer, carrying out chromatographic elution testing and mass spectrometry testing, and collecting and obtaining the retention time and spectrogram information; and (3) importing the collected retention time and spectrogram information into the library building system, integrating the information, and constructing to obtain the intestinal microorganism-related metabolite mass spectrometry database. The mass spectrometry database constructed by the present invention is currently the most comprehensive intestinal microorganism-related metabolite mass spectrometry database, comprising 405 metabolites. The database may be used for rapid and accurate identification of intestinal microorganism-related metabolites of biological samples from different sources, reducing the cost of detection, greatly improving detection speed and efficiency, and having significant application value.

Description

Construction method and application of intestinal microbiome-related metabolite mass spectrometry database

This application requests the priority of the Chinese patent application submitted to the State Intellectual Property Office on August 23, 2022, with the application number 202211012492.7 and the invention title "A method for constructing an intestinal microorganism-related metabolic mass spectrometry database and its application" , the entire contents of which are incorporated herein by reference.

Technical field

The invention belongs to the field of intestinal microorganism-related metabolites and relates to a method for constructing an intestinal microorganism-related metabolite mass spectrum database and its application.

Background technique

In recent years, the interaction between gut microbial structure and its metabolite profile and host metabolism has been studied in various animal models and human populations. Experimental data suggest that gut microbe-related metabolites have a huge impact not only on gut health, but also on many other systems in the body, including the brain. Intestinal microbial imbalance is closely related to the occurrence of many diseases, such as irritable bowel syndrome, tumors, renal failure, neurological diseases, obesity and diabetes, etc. Intestinal microbial metabolites serve as complementary host receptors and become key signaling molecules. Microbial metabolomics can be used to monitor the changes and effects of these "signaling molecules".

At present, the complex system between intestinal microorganisms and the host is studied. When the human body and intestinal microorganisms participate in the co-metabolism of food or exogenous substances, they produce a large number of small molecule metabolites, some of which are harmful to host cells and intestinal microorganisms. Metabolites that play a key role in the transmission of bacterial information. A comprehensive survey of literature in related fields found nearly 300 common microbial-host metabolism-related products, including bile acids, short-chain fatty acids, amino acids, benzoyl and phenyl derivatives, indole derivatives, lipids, choline, and phenols. Classes, lipids, vitamins, hormones and polyamines, etc., play an important role in the host. However, most literature studies only focus on the development of targeted quantitative methods for a certain type of metabolites. This model will increase costs, such as the need to purchase high-purity standards and isotope re-labeling of the metabolites of concern. At the same time, it will also cause low detection throughput and low depth.

Metabolomics can solve the above two problems (high cost, detection throughput and depth). In metabolomics, the mass spectrometry detection platform is a relatively comprehensive, continuously dynamic, and non-irritating analysis method that can comprehensively and deeply collect metabolome data. Combined with functions such as high-throughput database retrieval, it is possible to quickly and accurately identify and annotate a large number of metabolic substances, solving throughput and depth issues. Establish a mass spectrometry database suitable for the identification and annotation of intestinal microbial-related metabolites for qualitative and quantitative identification and confirmation. This will solve the problem of purchasing standards that consume financial resources.

However, there are currently no reports on intestinal microbial-related metabolic mass spectrometry databases in the existing technology.

Therefore, how to establish a suitable mass spectrometry database for the identification and annotation of intestinal microbial-related metabolites and use it for high-throughput identification of intestinal microbial-related metabolites in non-targeted or broadly targeted metabolomics research. Identification and annotation have become urgent problems in this field.

Contents of the invention

In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a method for constructing an intestinal microorganism-related metabolic mass spectrometry database and its application. The intestinal microorganism-related metabolic mass spectrometry database constructed by the present invention is currently the most comprehensive intestinal microorganism-related metabolic mass spectrometry database. Metabolic mass spectrometry database contains 405 metabolites, covering fatty acids (Fatty acids), organic acids (Organic Acids), benzene (Benzene), polyamines (Catecholamines), vitamins and coenzymes (Vitamins and Cofactors) ), Peptides, Nucleotides, Bile acids, Choline, Carbohydrates, Amino Acids, Indoles (Indole), photidylethanolamines (phoethanolamines), glycerides (Glycerides) and others (Others).

In order to achieve the purpose of this invention, the present invention adopts the following technical solutions:

In a first aspect, the present invention provides a method for constructing an intestinal microorganism-related metabolic mass spectrometry database. The construction method includes the following steps:

(1) Collect standards for intestinal microbial-related metabolites and enter basic information into the database construction system;

(2) Inject the intestinal microbial related metabolite standard into the chromatography mass spectrometer, perform chromatography elution detection and mass spectrometry detection, and collect retention time and spectrum information;

(3) Import the collected retention time and spectrum information into the database construction system, integrate the information, and construct the intestinal microorganism-related metabolic mass spectrometry database.

Preferably, the database building system is the mzvault database building system.

Preferably, step (1), after entering the basic information into the library construction system, also includes calculating the precise molecular weight of the standard and storing it in the library construction system.

Preferably, the basic information includes the name, molecular formula, CAS number, category, mol file (a file commonly used in this field to record the structure of metabolites) of the standard substance, and its location in public databases and/or public websites. serial number.

Preferably, the basic information also includes Simplified molecular input line entry system (SMILES) Description.

Preferably, the public database includes Human Metabolome Database (HMDB), Kyoto Encyclopedia of Genes and Genomes (KEGG), The Small Molecule Pathway Database (SMPDB) ) or any one or a combination of at least two of the databases (METLIN) used to describe the characteristics of known metabolites, and the public website includes a chemical search engine (ChemSpider).

Preferably, in step (2), the sample injection refers to mixing a standard substance with a solvent to obtain a standard substance solution, and then injecting the sample. The solvent includes any one of methanol, isopropyl alcohol or dimethyl sulfoxide. Or at least a combination of two.

Among them, the solvent used for the standards of highly polar metabolites is preferably methanol, and the solvent used for the standards of conventional polar metabolites is preferably isopropyl alcohol.

The strong polarity in the present invention means that the XLogP hydrophobic constant is less than 0, and the conventional polarity means that the XLogP hydrophobic constant is between 0 and 5.

Preferably, dimethyl sulfoxide can be added to methanol or isopropyl alcohol to help dissolve.

Preferably, the concentration of the standard solution is 2-5 mg/mL, such as 2 mg/mL, 2.5 mg/mL, 3 mg/mL, 3.5 mg/mL, 4 mg/mL, 4.5 mg/mL, 5 mg/mL, etc.

Preferably, according to the polarity of the standard, different modes are selected for chromatographic elution detection. Standards of conventional polar metabolites are detected in the conventional polarity mode, and standards of highly polar metabolites are detected in the strong polarity mode. Perform testing.

In the conventional polarity mode, the chromatographic column used is a BEH C18 column, and in the strong polarity mode, the chromatographic column used is a BEH Amide column.

Preferably, in the conventional polarity mode, the mobile phase includes phase A and phase B. Phase A is a 0.05%-0.15% formic acid aqueous solution (formic acid is dissolved in water, and the mass percentage of formic acid is 0.05%-0.15%), and phase B is The phase is 0.05%-0.15% formic acid acetonitrile solution (formic acid is dissolved in pure acetonitrile, and the mass percentage of formic acid is 0.05%-0.15%).

Specific numerical values in the above 0.05%-0.15% include, for example, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, etc.

Preferably, in the conventional polarity mode, the elution method of the chromatography is gradient elution, and the gradient is set as: 0-0.5 min, the volume ratio of mobile phase A is 97%-99%; 0.5-10 min, The volume proportion of mobile phase A is 1%-3%; at 10-16min, the volume proportion of mobile phase A is 1%-3%; at 16-16.1min, the volume proportion of mobile phase A is 97%- 99%; at 16.1-18 minutes, the volume proportion of mobile phase A is 97%-99%.

Specific numerical values in the above 97% to 99% include, for example, 97%, 97.2%, 97.4%, 97.6%, 97.8%, 98%, 98.2%, 98.4%, 98.6%, 98.8%, 99%, etc.

Specific values in the above 1%-3% include, for example, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, etc.

Preferably, in the strong polarity mode, the mobile phase includes phase A and phase B, phase A is 20-30 mmol/L mM ammonium formate aqueous solution, and phase B is 100% acetonitrile.

Specific values in the above 20-30mM include 20mM, 21mM, 22mM, 23mM, 24mM, 25mM, 26mM, 27mM, 28mM, 29mM, 30mM, etc.

Preferably, in the strong polarity mode, the elution method of the chromatography is gradient elution, and the gradient is set as: 0-0.5 min, the volume ratio of mobile phase A is 4%-6%; 0.5-7 min, The volume proportion of mobile phase A is 30%-40%; at 7-8 minutes, the volume proportion of mobile phase A is 55%-65%; at 8-9 minutes, the volume proportion of mobile phase A is 55%-65 %; at 9-9.1min, the volume proportion of mobile phase A is 4%-6%; at 9.1-12min, the volume proportion of mobile phase A is 4%-6%.

Specific values in the above 4%-6% include 4%, 4.2%, 4.4%, 4.6%, 4.8%, 5%, 5.2%, 5.4%, 5.6%, 5.8%, 6%, etc.

Specific values in the above 30%-40% include, for example, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, etc.

Specific numerical values in the above-mentioned 55%-65% include 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, etc.

Preferably, the mass spectrometry detection includes positive ion scanning mode and negative ion scanning mode.

Preferably, the positive ion scanning mode includes the following three addition modes: [M+H]+, [M+NH4]+ and [M+Na]+, and the negative ion scanning mode is [M-H]-.

Preferably, in the mass spectrometry detection, the NCE (normalized collision energy) in the mass spectrometry parameters is set to 10, 20, 30, 40, 50 and 60 (6 windows).

Preferably, the chromatography mass spectrometer is a Q Exactive combined quadrupole Orbitrap mass spectrometer.

In a second aspect, the present invention provides an intestinal microorganism-related metabolite mass spectrometry database, which is established by the method of constructing an intestinal microorganism-related metabolite mass spectrometry database as described in the first aspect.

In a third aspect, the present invention provides a method for detecting intestinal microorganism-related metabolites in biological samples, the method comprising the following steps:

(1) Establish an intestinal microbe-related metabolite mass spectrometry database according to the construction method of the intestinal microbe-related metabolite mass spectrometry database as described in the first aspect;

(2) Mix the biological sample with the extraction reagent to extract the metabolites in the biological sample;

(3) Inject the extracted metabolites into the chromatography mass spectrometer, perform chromatography elution detection and mass spectrometry detection, and collect data;

(4) Import the data into the database search software to search the database, and match it with the information in the intestinal microbial-related metabolism mass spectrometry database established in step (1). According to the degree of matching, the intestinal microbial-related metabolism in the biological sample is obtained. identification results of the object.

Preferably, the biological sample includes any one of plasma, serum, urine, feces, cerebrospinal fluid, saliva or tissue.

Preferably, the chromatographic elution detection includes two detections: detection in conventional polarity mode and strong polarity mode respectively, and the detection conditions are consistent with those used in the construction method of step (1).

Preferably, the database search software includes CD (Compound Discoverer) software.

The numerical range described in the present invention not only includes the point values listed above, but also includes any point value between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention will not exhaustively enumerate the ranges. Specific point values included.

Compared with the existing technology, the present invention has the following beneficial effects:

For the first time, the present invention constructs a high-throughput identification and annotation mass spectrometry database of intestinal microbial-related metabolites in metabolomics research and launches its application (detection of actual biological samples). Through the metabolite name, retention time, molecular formula, Precursor ion and fragment ion accurate mass information, categories, HMDB ID, KEGG ID, isotope abundance ratio, etc. can be used to quickly identify and annotate intestinal microbial-related metabolites in biological samples, providing effective technical support and high-level support for metabolomics research. repeatability and reliability.

The mass spectrometry database constructed by the present invention is currently the most comprehensive mass spectrometry database related to intestinal microorganism-related metabolism, including 405 kinds of metabolites, and can be used to quickly and accurately conduct intestinal microbial metabolism of biological samples from different sources, such as tissues, feces, etc. Object identification reduces detection costs and greatly improves detection speed and efficiency. Based on the information in the database of the present invention, a broadly targeted metabolomics method based on tandem quadrupole mass spectrometry can also be developed, which has important application value.

The standard operating procedures for high-throughput identification of intestinal microbial-related metabolites and construction of annotation mass spectrometry database in the metabolomics technology mentioned in the present invention are based on the Thermo Scientific Q Exactive combined quadrupole Orbitrap mass spectrometer (UPLC-HR- MS); this database also supports exporting in multiple formats such as .txt, .msp, .csv, etc., which is suitable for comparison by various metabolomics analysis software based on this spectral library; this database has also developed an html web page format , for quick search of metabolites.

It is worth mentioning that this invention proposes for the first time that when collecting standard information, the normalized collision energy (NCE) in the mass spectrometry conditions is set to: 10, 20, 30, 40, 50, 60, realizing a single injection. At the same time, the collection of primary and secondary spectra generated under 6 different collision energies (10eV, 20eV, 30eV, 40eV, 50eV, 60eV) is completed. The advantages of this setting are: (1) Using different collision energies, more fragment ions can be obtained, more spectral information can be collected, and the database information established can be more comprehensive and credible, which is conducive to matching with actual complex samples. (2) The collection of 6 different collision energies is completed in one injection, which avoids multiple injections, greatly shortens the detection time, and saves biological samples.

Description of drawings

Figure 1 is the interface for inputting metabolite standard information in the mass spectrometry database in the mzvault library construction system.

Figure 2 is the UPLC HR-MS high-resolution mass spectrometry method setting interface during the library construction process.

Figure 3 is a statistical diagram of the classification of 405 metabolites in the intestinal microorganism-related metabolite mass spectrometry database constructed by the present invention.

Figure 4 is the UPLC HR-MS high-resolution mass spectrometry data acquisition interface during the detection of the sample to be tested.

Figure 5 is the original mass spectrum data diagram of the sample to be tested (the upper picture is the conventional polar chromatography mode; the lower picture is the strong polar chromatography mode).

Figure 6 is the identification parameter setting interface of the CD library search system during the identification process of metabolites in the sample to be tested.

Figure 7 is the identification result interface of the CD library search system during the identification process of metabolites in the sample to be tested.

Figure 8 is a comparison diagram of L-tyrosine matching mirror images between the sample to be tested and the database constructed by the present invention.

Figure 9 is a comparison diagram of L-phenylalanine matching mirror images between the sample to be tested and the database constructed by the present invention.

Detailed ways

The technical solution of the present invention will be further described below through specific implementations. Those skilled in the art should understand that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

In the following examples, unless otherwise specified, all reagents and consumables were purchased from conventional reagent manufacturers in the field; unless otherwise specified, the experimental methods and technical means used were conventional methods and means in the field.

Example 1

This embodiment provides a method for constructing an intestinal microorganism-related metabolic mass spectrometry database. The specific steps are as follows:

1. Enter basic information on intestinal microbiome-related metabolites.

The collected information on intestinal microbial related metabolites includes Chinese name, English name, molecular formula, CAS number, metabolite biological category, metabolite category subcategory, mol file, HMDB ID, KEGG ID, ChemSpider ID, SMILES Description, etc. , enter the newly created mass spectrometry database in the mzvault library construction system. At the same time, the mzvault library construction system will calculate the precise molecular weight of the metabolite based on the molecular formula of the metabolite and the natural abundance ratio of each element in the molecule, and save it in the mass spectrometry database background, waiting for the standard Compare molecular weights when importing spectra. See Figure 1 for the input interface for metabolite standard information in the mass spectrometry database in the mzvault library building system.

2. Dissolve the standard of a single intestinal microbial-related metabolite in a solvent and prepare a standard solution with a concentration of 2-5 mg/mL (adjust appropriately according to the mass spectrum signal size of each standard), among which the strongest solution is The solvent for polar metabolite standards is methanol, and the solvent for conventional polar metabolite standards is isopropyl alcohol. For metabolite standards with particularly low solubility, appropriate amounts of dimethyl sulfoxide (DMSO) can be added to promote dissolution.

3. Set the ultra-high performance liquid chromatography parameters to determine the chromatographic retention time of the metabolite standard.

3.1. Chromatographic column: Acquity BEH C18 column (100mm×2.1mm×1.8μm) is used for conventional polar metabolite standards; Acquity BEH Amide column (100mm×2.1mm×1.7μm) is used for highly polar metabolite standards; The polarity information of each standard is shown in Table 1.

3.2. Mobile phase: For conventional polar metabolite standards, mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: 0.1% formic acid acetonitrile solution; for highly polar metabolite standards, mobile phase A: 25mM ammonium formate aqueous solution ( pH 9.0); mobile phase B: pure acetonitrile.

3.3. Chromatographic gradient:

The conventional polar metabolite standard gradient is as follows:

Time(min)

Mobile phase A

Mobile phase B

0	98	2
0.5	98	2
10	2	98
16	2	98
16.1	98	2
18	98	2

The gradient of highly polar metabolite standards is as follows:

Time(min)	Mobile phase A	Mobile phase B
0	5	95
0.5	5	95
7	35	65
8	60	40
9	60	40
9.1	5	95
12	5	95

3.4. Other conditions: flow rate for conventional polar metabolite standards in positive and negative ion modes: 0.3mL/min; injection volume: 5μL; column temperature: 40°C; highly polar metabolite standards in positive and negative ion modes Product selection flow rate: 0.4mL/min; injection volume: 5μL; column temperature of the chromatographic column: 50°C.

4. Optimize the mass spectrometry conditions of the high-resolution mass spectrometer to determine the parent ion and fragment ion spectra of metabolite standards.

4.1. Ion source parameters: electrospray ion source (ESI source); Spray Voltage voltage: 3.5kV (positive ion mode)/2.8kV (negative ion mode); ion source (Vaporizer Temp) temperature: 350°C; ion transmission Tube temperature (ITT Temp): 275°C; Sheath Gas flow rate: 40arb; Aux Gas flow rate: 10arb.

4.2. Monitoring mode: All metabolite standard solutions select the data-dependent scanning DDA (Full MS-ddMS2 (Top N)) mode in positive ion (ESI ⁺ ) and negative ion (ESI ^- ) modes respectively.

4.3. General settings: acquisition time: 18min or 12min (consistent with the chromatographic gradient time); ionization (Polarity) mode: positive ions or negative ions (consistent with the ion source settings).

4.4. First-level mass spectrometry parameters: first-level resolution: 70,000; maximum injection time: 100ms; scanning mass-to-nucleus ratio range: 70-1050Da.

4.5. Secondary mass spectrometry parameters: secondary resolution: 17,500; maximum injection time: 50ms; scanning mass-to-nucleus ratio range: 70-1050Da; Top N: 10 (select the top 10 ions with response intensity to enter the mass spectrometer); isolation window: 1.5m/z; normalized collision energy (NCE): 10, 20, 30, 40, 50, 60; dynamic exclusion time: 4.0s.

The mass spectrometry method setting interface is shown in Figure 2.

5. Perform metabolite standard mass spectrum detection.

According to the polar characteristics of the metabolite standard (see Table 1 for details), select the most appropriate chromatographic and mass spectrometric conditions for mass spectrometry detection of the metabolite standard solution.

6. Construct a metabolite standard mass spectrum database.

After the metabolite standard mass spectrum detection step is completed, use the Xcalibur system to import the different fragment ion spectra of the precursor ions and product ions of the metabolite standard under CE collision energy into the corresponding metabolite column in the mzvault system. At this time, the mass spectrum and retention time of the metabolite exist in the mzvault system. Repeating the above steps, the present invention finally constructed a mass spectrometry database of 405 intestinal microorganism-related metabolites (named Microbial metabolite library). The statistical diagram of metabolites in the database is shown in Figure 3, and the detailed information is summarized in Table 1.

Table 1

Example 2

This embodiment provides a method for detecting intestinal microbial-related metabolites in plasma samples, specifically as follows:

Plasma samples were collected from healthy volunteers in the endocrinology department of the hospital. The volunteers knew and agreed before collection. The collection and use of plasma samples complied with the ethical norms and principles that must be followed in the medical field.

1. Preparation of samples to be tested.

Accurately remove 100 μL of plasma, add pure methanol for protein precipitation, vortex to mix, and let stand at -80°C for 2 hours for extraction of intestinal microbial-related metabolites. After the extraction is completed, centrifuge at 4°C and 12,000 rpm for 10 minutes. The supernatant is evenly divided into two parts for freeze-drying. Before loading on the machine, add 100 μL of 75% acetonitrile solution to one freeze-dried sample to reconstitute the strong polarity present in the sample. For metabolites, add 100 μL of 30% acetonitrile solution to another freeze-dried sample to reconstitute the conventional polar metabolites present in the sample, and transfer the reconstituted solution into an injection vial for use as a sample to be tested.

2. Mass spectrometry detection of the sample to be tested.

Since the intestinal microbial-related metabolites contained in biological samples have greatly different polarities, the same sample to be tested is separated through two different polarity (strong polarity or conventional polarity) chromatographic conditions, and finally injected into the mass spectrometer for analysis. analyze. The chromatographic condition settings of the two polarities were consistent with those used for the standard product in Example 1. In the mass spectrometry conditions, the NCE (normalized collision energy) in the mass spectrometry parameters is set to 20, 40, 60 (1 window. In actual acquisition, the instrument will select an optimal mixing energy based on different ions, which is equivalent to from 20-60 Select an energy within the interval to give the ion), and the other conditions are consistent with those used for the standard substance in Example 1.

The data collection interface of the sample is shown in Figure 4, and the original spectrum collected is shown in Figure 5 (the upper picture is the spectrum in the conventional polar chromatography mode, and the lower picture is the spectrum in the strong polar chromatography mode).

3. Identification of mass spectrometry results of the sample to be tested.

After the mass spectrometry detection of the sample to be tested is completed, the raw mass spectrometry data is imported into the CD (Compound Discoverer) system to start the mass spectrometry database search (see Figure 6 for the parameter interface), and identification of intestinal microbial-related metabolites is performed. During the database search process, the CD system Preset identification parameters (primary/secondary mass number error less than 5ppm; secondary spectral library matching greater than 70; retention time error less than 0.5min, etc.), search for various intestinal microorganisms in the mass spectrometry raw data of the sample to be tested The parent ion mass-to-charge ratio, product ion mass-to-charge ratio, retention time, isotope information, etc. of the relevant metabolites are compared with the information entered in the intestinal microbial-related metabolic mass spectrometry database that we have constructed in Example 1, and then Judgment criteria based on the identification results set in the system. Specifically, when the CD system searches the library, the system will calculate the mzvault best match score (best match score in the library) of each intestinal microbial-related metabolite in the sample to be tested based on the matching results. When mzvault best match score ≥ 70, It can be confirmed as a positive result, that is, the sample to be tested contains this intestinal microbial-related metabolite, and the information related to the intestinal microbial-related metabolite that meets the identification standards can be screened out, such as metabolite name, retention time, molecular formula, molecular weight, Deviation, mzvault best match score value and peak response intensity of metabolites identified in the sample (see Figure 7 for the identification results interface).

Identification results: The plasma sample to be tested finally identified 241 intestinal microbial-related metabolites (mzvault best match score ≥ 70) through the intestinal microbial-related metabolite mass spectrometry database. In order to save space, the specific information of the metabolites will not be repeated. Only List the HDMB ID, and the information on related metabolites can be found in Table 1 for correspondence. The HDMB IDs of the identified metabolites are as follows: HMDB0000500, HMDB0000292, HMDB0000221, HMDB0060038, HMDB0011637, HMDB0002340, HMDB0000812, HMDB0004370, HMDB0003164, HMDB0028929, HMDB0000893, HMDB0000267, HMDB0001201, HMDB0031321, HMDB0000535, HMDB0000156, HMDB0006409, HMDB0000975, HMDB0004095, HMDB0000613, HMDB0000807, HMDB0000157, HMDB0012328, HMDB0013713, HMDB0002274, HMDB0002285, HMDB0002581, HMDB0060002, HMDB0001341, HMDB0003447, HMDB0012252, HMDB0003405, HMDB0 002117, HMDB0059999, HMDB0000626, HMDB0000619, HMDB0000867, HMDB0002003, HMDB0001397, HMDB0002006, HMDB0001432, HMDB0059773, HMDB0240254, HMDB0000128, HMDB000203 5. HMDB0029738, HMDB0002092, HMDB0000719, HMDB0003072, HMDB0000176, HMDB0000962, HMDB0059805, HMDB0003152, HMDB0000407, HMDB0002189, HMDB0000740, HMDB0001488, HMDB0 000045, HMDB0000056, HMDB0010319, HMDB0000235, HMDB0013751, HMDB0000472, HMDB0002231, HMDB0001624, HMDB0004259, HMDB0000306, HMDB0000752, HMDB0001476, HMDB000051 7. HMDB0000511, HMDB0000094, HMDB0001885, HMDB0000254, HMDB0002199, HMDB0000215, HMDB0000673, HMDB0000122, HMDB0000194, HMDB0000792, HMDB0002259, HMDB0000763, HMDB0 006116, HMDB0000002, HMDB0002579, HMDB0002486, HMDB0000073, HMDB0002100, HMDB0002586, HMDB0000423, HMDB0000637, HMDB0000064, HMDB0000168, HMDB0000557, HMDB002941 2. HMDB0003229, HMDB0000686, HMDB0000466, HMDB0000822, HMDB0000357, HMDB0061741, HMDB0002226, HMDB0000400, HMDB0001873, HMDB0000661, HMDB0000193, HMDB0000043, HMDB0 002925, HMDB0000881, HMDB0001972, HMDB0002243, HMDB0003345, HMDB0002991, HMDB0000734, HMDB0000840, HMDB0000097, HMDB0000806, HMDB0000426, HMDB0000821, HMDB000051 0. HMDB0001401, HMDB0000230, HMDB0000700, HMDB0000696, HMDB0000951, HMDB0001276, HMDB0033724, HMDB0000019, HMDB0000068, HMDB0000929, HMDB0007098, HMDB0011530, HMDB0 000755, HMDB0000502, HMDB0000733, HMDB0002215, HMDB0012162, HMDB0000895, HMDB0000689, HMDB0002183, HMDB0000910, HMDB0000020, HMDB0000098, HMDB0001999, HMDB000156 5. HMDB0000134, HMDB0094657, HMDB0000638, HMDB0004437, HMDB0000883, HMDB0000022, HMDB0002172, HMDB0000555, HMDB0000008, HMDB0000232, HMDB0000467, HMDB0004461, HMDB0 001901, HMDB0000150, HMDB0000303, HMDB0000243, HMDB0002302, HMDB0002641, HMDB0011631, HMDB0000163, HMDB0033193, HMDB0000904, HMDB0000158, HMDB0001256, HMDB001367 8. HMDB0002642, HMDB0000663, HMDB0000132, HMDB0034297, HMDB0002580, HMDB0000532, HMDB0001900, HMDB0034146, HMDB0000708, HMDB0000606, HMDB0003633, HMDB0000058, HMDB0 000892, HMDB0000033, HMDB0000161, HMDB0000707, HMDB0002068, HMDB0000048, HMDB0000682, HMDB0000484, HMDB0029739, HMDB0000452, HMDB0001866, HMDB0000917, HMDB000016 7. HMDB0004989, HMDB0000191, HMDB0000187, HMDB0000152, HMDB0033774, HMDB0000210, HMDB0000617, HMDB0011567, HMDB0000716, HMDB0008923, HMDB0000039, HMDB0000172, HMDB0 000192, HMDB0001190, HMDB0000182, HMDB0001160, HMDB0000195, HMDB0000326, HMDB0000631, HMDB0000729, HMDB0000162, HMDB0000205, HMDB0000902, HMDB0000244, HMDB000014 8. HMDB0003320, HMDB0000669, HMDB0000259, HMDB0000202, HMDB0000159, HMDB0000679, HMDB0000849, HMDB0000946, HMDB0000491, HMDB0061740, HMDB0000620, HMDB0000721, HMDB0 000925, HMDB0033567, HMDB0004073, HMDB0000896, HMDB0000634, HMDB0000667, HMDB0000139, HMDB0000518, HMDB0000138.

At the same time, taking the identified L-tyrosine (HMDB0000158) and L-phenylalanine (HMDB0000159) as examples, the matching results of the sample to be tested and the database constructed by the present invention are interpreted in detail: the metabolites in the sample to be tested are processed before After processing, it is separated by chromatography and finally enters the mass spectrometer to collect the secondary spectrum. After the data collection is completed, the collected secondary spectrum data is matched with the intestinal microorganism-related metabolite database constructed by the present invention through CD software. The results are finally given in the form of a metabolite matching mirror image comparison chart (see Figure 8 and Figure 9). The upper half of the mirror image comparison chart is the secondary mass spectrum of the unknown metabolite actually collected in the sample to be tested, and the lower half is The secondary spectrum of the reference metabolite in the database constructed for the present invention; through the metabolite matching mirror image comparison chart, it can be seen intuitively that there are more fragment ions matching the sample to be tested and the corresponding metabolite in the established database, and the result can be letter.

The applicant declares that the present invention uses the above embodiments to illustrate the construction method and application of an intestinal microorganism-related metabolic mass spectrometry database of the present invention, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must Implementation relies on the above embodiments. Those skilled in the art should understand that any improvements to the present invention, equivalent replacement of raw materials of the product of the present invention, addition of auxiliary ingredients, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention. These simple modifications All belong to the protection scope of the present invention.

In addition, it should be noted that each of the specific technical features described in the above-mentioned specific embodiments can be combined in any suitable manner without conflict. In order to avoid unnecessary repetition, the present invention combines various possible combinations. The combination method will not be further explained.

Claims (10)

1. A method for constructing an intestinal microorganism-related metabolic mass spectrometry database, characterized in that the construction method includes the following steps:

   (1) Collect standards for intestinal microbial-related metabolites and enter basic information into the database construction system;

   (2) Inject the intestinal microbial related metabolite standard into the chromatography mass spectrometer, perform chromatography elution detection and mass spectrometry detection, and collect retention time and spectrum information;

   (3) Import the collected retention time and spectrum information into the database construction system, integrate the information, and construct the intestinal microorganism-related metabolic mass spectrometry database.
2. The method for constructing an intestinal microorganism-related metabolic mass spectrometry database as claimed in claim 1, characterized in that in step (2), the sampling means mixing a standard substance with a solvent to obtain a standard substance solution, and then injecting the sample, The solvent includes any one or a combination of at least two of methanol, isopropyl alcohol or dimethyl sulfoxide.
3. The method for constructing an intestinal microorganism-related metabolic mass spectrometry database according to claim 2, wherein the concentration of the standard solution is 2-5 mg/mL.
4. The construction method of intestinal microorganism-related metabolite mass spectrometry database as claimed in claim 1, characterized in that, according to the polarity of the standard substance, different modes are selected for chromatographic elution detection, and the standard of conventional polar metabolites is The standard products of highly polar metabolites are detected using the conventional polarity mode, and the standards of highly polar metabolites are detected using the highly polar mode;

   In the conventional polarity mode, the chromatographic column used includes a BEH C18 column, and in the strong polarity mode, the chromatographic column used includes a BEH Amide column.
5. The construction method of intestinal microorganism-related metabolic mass spectrometry database according to claim 1, characterized in that, in the mass spectrometry detection, the NCE in the mass spectrometry parameters is set to 6 windows, which are 10, 20, 30, 40, 50 and 60.
6. The construction method of intestinal microorganism-related metabolic mass spectrometry database as claimed in claim 1, characterized in that the chromatography mass spectrometer is a Q Exactive combined quadrupole Orbitrap mass spectrometer.
7. An intestinal microorganism-related metabolism mass spectrometry database, characterized in that the intestinal microorganism-related metabolism mass spectrometry database is composed of the method for constructing an intestinal microorganism-related metabolism mass spectrometry database according to any one of claims 1-6 Build to get.
8. A method for detecting intestinal microorganism-related metabolites in biological samples, characterized in that the method includes the following steps:

   (1) Establish an intestinal microorganism-related metabolism mass spectrometry database according to the construction method of the intestinal microorganism-related metabolism mass spectrometry database as described in any one of claims 1-6;

   (2) Mix the biological sample with the extraction reagent to extract the metabolites in the biological sample;

   (3) Inject the extracted metabolites into the chromatography mass spectrometer, perform chromatography elution detection and mass spectrometry detection, and collect data;

   (4) Import the data into the database search software to search the database, and match it with the information in the intestinal microbial-related metabolism mass spectrometry database established in step (1). According to the degree of matching, the intestinal microbial-related metabolism in the biological sample is obtained. identification results of the object.
9. The method of claim 8, wherein the biological sample includes any one of plasma, serum, urine, feces, cerebrospinal fluid, saliva or tissue.
10. The method for detecting intestinal microorganism-related metabolites in biological samples according to claim 8 or 9, characterized in that the chromatographic elution detection includes two detections, and the detection on both sides is respectively in the conventional polar mode and the strong Detection was performed in polar mode, and the detection conditions were consistent with those used in the construction method of step (1).

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