CN114720618A - Analysis method of Phaffia yeast metabolite - Google Patents
Analysis method of Phaffia yeast metabolite Download PDFInfo
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- 238000000034 method Methods 0.000 claims abstract description 21
- 238000001514 detection method Methods 0.000 claims abstract description 16
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
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Abstract
The invention discloses an analysis method of a Phaffia yeast metabolite, which comprises the following steps: extracting metabolites from the Phaffia yeast fermentation liquor as a sample; performing liquid chromatography-electrospray ionization-triple quadrupole flight time mass spectrometry combined detection on a sample to obtain mass spectrometry data; selecting a liquid metabonomics database from the MS-Dial for downloading; analyzing the mass spectrum data of the steps through MS-Dial to obtain a total ion peak diagram; and (4) identifying the metabolic products of the phaffia rhodozyma by using the total ion peak diagram of the step and the database of the step. The method improves the accuracy of detecting the targeting property, improves the detection sensitivity, does not need isotope tracing, and has simple operation and wide applicability.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an analysis method of a Phaffia yeast metabolite.
Background
Phaffia is a microorganism belonging to the genus Phaffia, is an extremely important industrial strain, and can synthesize various natural products having biological activities. Astaxanthin, fatty acid, fucoxanthin, lutein and the like produced by the phaffia rhodozyma have higher utilization values in the aspects of food health care, medical treatment, beauty treatment and the like.
The metabolite characterization is always a big difficulty of metabonomics, and because the Phaffia rhodozyma metabolites are complex and the traditional microbial metabolism database has fewer samples, the Phaffia rhodozyma metabolites are inaccurate in characterization and high in false positive.
Disclosure of Invention
In order to solve the problems, the invention provides a method for analyzing the metabolic products of Phaffia rhodozyma, which improves the detection targeting property, overcomes the defect of inaccurate analysis qualitative property of a common method, improves the detection sensitivity, does not need isotope tracing, and has simple operation and wide applicability.
In order to achieve the above object, an embodiment of the present invention in one aspect provides a method for analyzing a metabolite of Phaffia yeast, comprising the steps of:
(1) extracting metabolites from the Phaffia yeast fermentation liquor as a sample;
(2) performing liquid chromatography-electrospray ionization-triple quadrupole flight time mass spectrometry combined detection on a sample to obtain mass spectrometry data;
(3) selecting a liquid metabonomics database from the MS-Dial for downloading;
(4) analyzing the mass spectrum data in the step (2) through MS-Dial to obtain a total ion peak diagram;
(5) and (4) identifying the Phaffia rhodozyma metabolites by using the total ion peak image in the step (4) and the database in the step (3).
According to the method for analyzing the Phaffia yeast metabolites, the LC-Qtof-MS liquid chromatography-mass spectrometry technology is used for mass spectrometry, mass spectrometry data are transferred into MS-Dial software, an All public MS/MS database is selected for data, and the data are analyzed by matching the mass spectrometry data with the database, so that the detection targeting property is improved, and the defect that the analysis qualitative performance is not accurate in a general method is overcome; meanwhile, the metabolites are quantitatively detected according to the abundance of the specific ion, so that the detection sensitivity is greatly improved, and a new method for researching the natural active metabolites of the phaffia rhodozyma is provided.
In addition, the method for analyzing the metabolite of the Phaffia yeast according to the above embodiment of the present invention may further have the following additional technical features:
optionally, in step (5), the Phaffia rhodozyma metabolites are identified by matching mass spectrum peaks and retention times in the total ion peak pattern to the database.
Alternatively, in step (2), the operating conditions of the liquid chromatography are: the sample feeding amount is 5-10 mu L, the column temperature is 25-35 ℃, and the flow rate is 0.2-0.4 mL/min; chromatographic mobile phase A: 0.1% formic acid water, B: acetonitrile; the chromatographic gradient elution procedure was as follows: 0-1 min, 90% A; for 1-3 min, changing A from 90% to 50%; changing A linearly from 50% to 10% for 3-6 min; maintaining A at 10% for 6-9 min; for 9-11 min, changing A from 10% to 50%; and (3) changing A linearly from 50% to 90% in 11-12 min.
Optionally, in step (2), electrospray ionization is detected in a positive ion mode and a negative ion mode.
Optionally, in step (2), ESI source conditions are: capillary voltage: 3-3.30 kV; sampling cone: 40; seismic source migration: 80; source temperature: 100 ℃; desolventizing temperature: 250-300 ℃; air flow of the air curtain: 50L/H; desolventizing agent gas flow: 59850L/H; positive ion scan m/range: 50-1200 Da; negative ion scanning m/z range: 50-1200 Da.
Optionally, the method further comprises the step (6) of calibrating the instrument and the software by using L-glutamic acid, imidazole, glucose and sucrose standard products.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a total ion flow diagram of Phaffia rhodozyma metabolites under the detection condition of positive ion and sensitivity mode by taking formic acid water (0.01% formic acid) as a mobile phase according to an embodiment of the invention;
FIG. 2 is a total ion flow diagram of Phaffia rhodozyma metabolites under the detection condition of negative ions and sensitivity mode by taking formic acid water (0.01% formic acid) as a mobile phase according to the embodiment of the invention;
FIG. 3 is a graph of total ion peaks of Phaffia yeast metabolites processed by MS-Dial software in negative ion mode according to an embodiment of the present invention;
FIG. 4 is a graph of total ion peaks of Phaffia yeast metabolites processed by MS-Dial software in positive ion mode according to an embodiment of the present invention;
FIG. 5 is a comparison of anion mode Phaffia rhodozyma metabolite spectra data with MS-Dial anion database for qualitative substances according to an embodiment of the present invention;
FIG. 6 is a comparison of positive ion mode Phaffia rhodozyma metabolite spectra data with an MS-Dial positive ion database for qualitative substances according to an embodiment of the present invention;
FIG. 7 is a mass spectrum of a standard L-glutamic acid according to an embodiment of the present invention;
FIG. 8 is a mass spectrum of an imidazole standard according to an embodiment of the present invention;
FIG. 9 is a mass spectrum of a glucose standard according to an embodiment of the present invention;
FIG. 10 is a mass spectrum of sucrose standards according to an embodiment of the present invention.
Detailed Description
The technical solution of the present invention is illustrated by specific examples below. It is to be understood that one or more method steps mentioned in the present invention do not exclude the presence of other method steps before or after the combination step or that other method steps may be inserted between the explicitly mentioned steps; it should also be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.
In order to better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the invention have been shown, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
Fermentation medium: 20g/L of glucose, 1.0-1.5 g/L of monopotassium phosphate, 0.1-0.3 g/L of sodium chloride, 0.5-0.8 g/L of magnesium sulfate heptahydrate, 0.1-0.3 g/L of calcium chloride dihydrate, 0.2-0.4 g/L of yeast powder and pH of 6.
The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.
Examples
And (3) metabolite extraction:
JMU-MVP14 Phaffia yeast strain is added into a fermentation culture medium with the inoculation amount of 9% for fermentation for 5 days, after centrifugal liquid nitrogen grinding, 80 mg-100 mg is weighed, 200 mu L of precooled water and 800 mu L of precooled methanol/acetonitrile (1:1, v/v) are added, the mixture is mixed evenly, ultrasonic treatment is carried out in ice bath for 60min at minus 20 ℃ for 1h to precipitate protein, 16000g is carried out, centrifugation is carried out for 20min at 4 ℃, and supernatant is obtained. And (5) volatilizing the supernatant in a high-speed vacuum concentration centrifugal machine to obtain a sample.
Sample detection:
in the mass spectrometric detection, 500. mu.L of a reagent such as acetonitrile (merck, 1.00029.2500) or methanol (merck, 1.06035.2500) was added to the sample obtained by the evaporation.
Mass spectrum: i-class Plus/Xevo G0-XS TOF (Waters); and (3) chromatography: acquity uplc class 1; ultra high performance liquid chromatographs (Waters); column BE527 (Waters); reagent: acetonitrile (merck, 1.00029.2500), formic acid (alatin, F301957). Mass spectrometry was performed using a combination of positive ion mode and sensitivity mode, and a combination of negative ion mode and sensitivity mode, respectively.
Each 1 group of 2 samples to be tested, each group of 1 replicate. This example was performed for quality control, and 1 QC sample, which was a sample of an equal mix of all samples, was prepared simultaneously. QC samples were used to balance the chromatography-mass spectrometry system and determine instrument status and to evaluate system stability throughout the experiment.
Using a liquid chromatography-electrospray ionization-triple quadrupole time-of-flight mass spectrometry coupled technique, the instrument is configured to: ultra-high performance liquid chromatography as a separation system; an electrospray ionization system is used as an ion source; a triple quadrupole mass spectrometer was used as the detector.
Specific parameters using the liquid chromatography separation system were as follows: the sample injection amount in the ultra-high performance liquid chromatography is 5-10 mu L, the column temperature is 25-35 ℃, and the flow rate is 0.2-0.4 mL/min; chromatographic mobile phase A: 0.1% formic acid water, B: acetonitrile; the chromatographic gradient elution procedure was as follows: 0-1 min, 90% A; for 1-3 min, changing A from 90% to 50%; changing A linearly from 50% to 10% for 3-6 min; maintaining A at 10% for 6-9 min; for 9-11 min, changing A from 10% to 50%; and (3) changing A linearly from 50% to 90% in 11-12 min.
Each sample was tested in positive and negative ion mode using electrospray ionization (ESI), respectively. Samples were separated by UPLC and analyzed by mass spectrometry using a Qtof-MS mass spectrometer. The ESI source conditions are as follows: capillary voltage: 3-3.30 kV; sampling cone: 40; seismic source migration: 80; source temperature: 100 ℃; desolventizing temperature: 250-300 ℃; air flow of the air curtain: 50L/H; desolventizing agent gas flow: 59850L/H. TOF MS positive ion scan m/z range: 50-1200 Da, TOF MS negative ion scanning m/z range: 50-1200 Da, TOF MS scanning accumulation time of 0.1-0.5 s/spectra, and product ion scan accumulation time of 0.03 s/spectra.
The results are shown in fig. 1 and fig. 2, and fig. 1 is a total ion flow diagram of the Phaffia yeast metabolite under the detection condition of positive ion and sensitivity mode by taking formic acid water (0.01% formic acid) as a mobile phase. FIG. 2 is a total ion flow diagram of Phaffia yeast metabolites under the detection conditions of negative ions and sensitivity mode by taking formic acid water (0.01% formic acid) as a mobile phase.
And (3) analyzing sample data:
(1) the format conversion was performed on raw-formatted mass spectrometry data obtained by Q-tof-MS LC-MS of waters. Firstly, introducing mass spectrum data into MSconvert software to convert the mass spectrum data into an mzML format, and then introducing the obtained mzML format data into Reinforcs Analysis Base File Converter software to convert the mzML format into abf format data.
(2) The MS-Dial selects the hydrometabolomics database for downloading, and the present example downloads the All public MS/MS (13,303unique components) Positive database and the All public MS/MS (12,879unique components) Negative database for subsequent analysis.
(3) The abf-formatted data obtained in step (1) were analyzed by MS-Dial 4.7. Clicking new project to import the data after format conversion into MS-Dial, selecting corresponding options according to the mass spectrum type and mass spectrum conditions, and selecting centroid date and selecting positive and negative ion modes from mass spectrum data date type (MS1)/date type (MS/MS) options obtained by waters. And clicking next, entering an Analysis file pages interface to import abf format data, respectively selecting blank, standard, QC and sample according to the sample type, and clicking next to enter an Analysis parameter setting interface. Selecting mass spectrum conditions on a Date collection interface, setting the error range of a primary mass spectrum as 0.01Da, setting the error range of a secondary mass spectrum as 0.025Da, setting the scanning time as 0.6-12 min after eliminating solvent peak-out time and pollution peaks, and setting the scanning ranges of the primary mass spectrum and the secondary mass spectrum as 50-1200 Da according to the sample condition. The type of Adduct possibly existing in sample data is selected on an Adduct interface according to factors such as a sample processing method, a mobile phase, a chromatographic column and the like, and the M + H is selected in a positive ion mode according to the sample processing condition in the embodiment+、M+NH3、M+CH3OH+H+、M+Na+The adduct form is selected from M-H, M-H2O-H, M + H-H2O adduct form in a negative ion mode. The Alignment option selects reference Alignment quality control QC sample data, the retention time error is set to 0.05min, and the primary mass spectrum error is 0.015 Da. Inputting solvent ion peaks and contaminant ion peaks to be eliminated, selecting 1000amplitude as minimum Peak height in Peak detection option, selecting 0.1Da as mass slice width, selecting linear weighted moving average as smoothing method, selecting 3scan as smoothing level, and selecting minimum Peak height in Peak detection optionThe peak width was selected to be5 scan. And (3) selecting the database downloaded in the step (2) to be imported in an Identification option, setting the retention time error to be 100min, setting the accuracy of the primary mass spectrum to be 0.01Da, setting the accuracy of the secondary mass spectrum to be 0.05Da, and setting the Identification fraction cut-off value to be 70%.
(4) And (4) obtaining a comparison result with the database through the step (3), and finding ion peaks, Peak-off time, charge-to-mass ratio and the like of all scanned substances through a Peak spot viewer window. Selecting the Alignment navigator option to click on the show table displays all identified materials, including the time to peak, charge-to-mass ratio, adduct type, error, etc. for each material.
The results are shown in FIG. 3 and FIG. 4, in which FIG. 3 is a graph of total ion peaks of Phaffia yeast metabolites processed by MS-Dial software in negative ion mode, and FIG. 4 is a graph of total ion peaks of Phaffia yeast metabolites processed by MS-Dial software in positive ion mode.
(5) And finally confirming the qualitative accuracy according to the scoring result of software by analyzing and comparing the peak-out time, the mass-to-charge ratio and the characteristic peak of the database.
The results are shown in FIG. 5 and FIG. 6, in which FIG. 5 shows 1036 kinds of substances qualitatively obtained by comparing the mass spectrum data of the Phaffia rhodozyma metabolic substances with the MS-Dial negative ion database; FIG. 6 shows 1622 substances characterized by a positive ion mode Phaffia rhodozyma metabolite spectra data vs MS-Dial negative ion database.
Results are shown in Table 1, key Phaffia metabolites identified by the MS-Dial software database.
(6) Finally, calibration is carried out in a positive and negative ion mode by using L-glutamic acid, imidazole, glucose and sucrose standard products in a mobile phase of formic acid (0.01 percent formic acid) and acetonitrile to prevent problems of spectral peak drift, accuracy reduction and the like, thereby improving the reliability of data.
As shown in fig. 7-10, fig. 7 is a mass spectrum of L-glutamic acid standard, fig. 8 is a mass spectrum of imidazole standard, fig. 9 is a mass spectrum of glucose standard, and fig. 10 is a mass spectrum of sucrose standard.
TABLE 1 Key Phaffia metabolites identified by MS-Dial software database
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the terminology used in the description presented above should not be understood as necessarily referring to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples described in this specification can be combined and combined by those skilled in the art.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (6)
1. A method for analyzing a Phaffia yeast metabolite, which is characterized by comprising the following steps:
(1) extracting metabolites from the fermentation liquor of the Phaffia yeast as a sample;
(2) performing liquid chromatography-electrospray ionization-triple quadrupole flight time mass spectrometry combined detection on a sample to obtain mass spectrometry data;
(3) selecting a liquid metabonomics database from the MS-Dial for downloading;
(4) analyzing the mass spectrum data in the step (2) through MS-Dial to obtain a total ion peak diagram;
(5) and (4) identifying the Phaffia rhodozyma metabolites by using the total ion peak image in the step (4) and the database in the step (3).
2. The assay of claim 1, wherein in step (5), the Phaffia metabolites are identified by matching mass spectral peaks and retention times in the total ion peak profile to a database.
3. The analytical method of claim 1, wherein in step (2), the operating conditions of the liquid chromatography are: the sample feeding amount is 5-10 mu L, the column temperature is 25-35 ℃, and the flow rate is 0.2-0.4 mL/min; chromatographic mobile phase A: 0.1% formic acid water, B: acetonitrile; the chromatographic gradient elution procedure was as follows: 0-1 min, 90% A; for 1-3 min, changing A from 90% to 50%; changing A linearly from 50% to 10% for 3-6 min; maintaining A at 10% for 6-9 min; for 9-11 min, changing A from 10% to 50%; and (3) changing A linearly from 50% to 90% in 11-12 min.
4. The method of claim 1, wherein in step (2), electrospray ionization is detected in a positive ion mode and a negative ion mode.
5. The analytical method according to claim 1 or 4, wherein in the step (2), the ESI source conditions are: capillary voltage: 3-3.30 kV; sampling cone: 40; seismic source migration: 80; source temperature: 100 ℃; desolventizing temperature: 250-300 ℃; air flow of the air curtain: 50L/H; desolventizing agent gas flow: 59850L/H; positive ion scan m/range: 50-1200 Da; negative ion scanning m/z range: 50-1200 Da.
6. The assay of claim 1, further comprising step (6) calibrating the instrument and software using L-glutamic acid, imidazole, glucose, sucrose standards.
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