CN114235979A

CN114235979A - Marine microorganism lipid ion mobility mass spectrometry method and application

Info

Publication number: CN114235979A
Application number: CN202111346198.5A
Authority: CN
Inventors: 罗启邦; 何炜; 陶建昌; 张传伦
Original assignee: Southwest University of Science and Technology
Current assignee: Southwest University of Science and Technology
Priority date: 2021-11-15
Filing date: 2021-11-15
Publication date: 2022-03-25

Abstract

The invention provides a marine microorganism lipid ion mobility mass spectrometry analysis method and application, and the ion mobility mass spectrometry technology is used for carrying out molecular structural characterization on marine microorganism lipid molecules. The present invention improves upon existing lipid extraction procedures, using methyl tert-butyl ether instead of chloroform based methods. And the ultra-high performance liquid chromatography is adopted, so that the analysis time is reduced. The data acquisition adopts an ion mobility mass spectrometry method, and the flow of the ion mobility is optimized. Meanwhile, the ion mobility technology can also distinguish between isobaric and isomeric forms. The invention uses an automatic data processing flow to carry out deep analysis on the ion mobility mass spectrum data, further constructs a lipid molecular structure database, and annotates the chromatographic mass spectrum characteristics by combining the lipid molecular structure database, and finally achieves a semi-automatic data processing flow. The invention effectively improves the limitation of the prior art on the characterization of organic substances generated by marine microorganisms, and allows the possibility of high-efficiency and large-scale research.

Description

Marine microorganism lipid ion mobility mass spectrometry method and application

Technical Field

The invention relates to the field of marine microbiology and marine metabonomics, in particular to a marine microorganism lipid ion mobility mass spectrometry analysis method and application.

Background

Countless single-cell planktonic microorganisms exist in seawater, and although the individuals are small and the biochemical reaction is single, the species and the number are various. Different species of microorganisms interact to form a rather complex group of microorganisms. They play a great role in the important processes of marine ecosystems, geochemical element circulation and the like. Recently, marine planktonic microorganisms such as microalgae and cyanobacteria have attracted much attention. This is because these organisms can perform photosynthesis, absorb carbon directly from the atmosphere, and settle into their biomass. Thus, the process can help to slow global warming and can produce many natural products of practical value. Planktonic marine archaea were once thought to be rare organisms that survive only extreme temperatures, pressures or salinity, however they are now found in almost all marine environments, being ubiquitous members of the marine planktonic population. Marine archaea differs from other microorganisms in the lipids of their cell membranes, and this biochemical trait also becomes an important tool for studying marine archaea and distinguishing them from other microorganisms. At present, the key problem of how to further lock the carbon in the atmosphere in the sea is to investigate how the marine archaea interacts with other marine plankton (such as algae and cyanobacteria). Therefore, the development of efficient methods to analyze lipids of marine plankton has a critical role in marine microbiology as well as marine metabolomics.

In the prior art, different types of mass spectrometry have been used to analyze the marine microbial lipidome. Currently, the most effective method for studying marine microbial lipids is liquid chromatography-mass spectrometry (LC-MS). However, the existing mass spectrometry techniques are difficult to cope with the chemical diversity of lipid molecules, and there is no specific method for analyzing isomeric lipid molecules. Furthermore, the researchers in oceanography generally use manual methods to analyze and arrange data, which also causes problems of low efficiency, uncertainty or false positive. And different scholars may interpret the same data differently, resulting in poor reproducibility. One reason for this is the lack of previously established molecular structure databases for the lipid panel of marine plankton for processing and analysis of mass spectral data.

Therefore, the prior art has yet to be improved.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide a marine microorganism lipid ion mobility mass spectrometry method and application thereof, and aims to solve the problems that the marine microorganism lipid analysis method in the prior art is insufficient in chemical diversity of lipid molecules and cannot realize automatic and standardized analysis of data.

The technical scheme of the invention is as follows:

in a first aspect, the invention provides a method for mass spectrometric analysis of lipid ion mobility of marine microorganisms, comprising the steps of:

a lipid extraction step: extracting a lipid sample from the marine microorganism to be detected by utilizing methyl tert-butyl ether;

a data acquisition step: analyzing the lipid sample by using an ion mobility mass spectrometry, adopting a positive and negative ion mode, using methanol and ethanol as mobile phases, and collecting data;

and (3) data analysis step: and constructing a lipid molecule structure database, and annotating the mass spectrum characteristics of the lipid molecules in the marine microorganisms to be detected by using the acquired data based on the assistance of the lipid molecule structure database.

The marine microorganism lipid ion mobility mass spectrometry method comprises the following steps of:

firstly, capturing marine microorganisms to be detected on a polyvinylidene fluoride membrane;

secondly, placing the polyvinylidene fluoride membrane obtained in the first step in a clean test tube, adding methyl tert-butyl ether and methanol for extraction, and then rotating the test tube filled with the extracting solution for a preset time;

thirdly, adding water into the extracting solution obtained in the second step to induce phase separation, and then carrying out centrifugal separation to obtain a water phase and an organic phase;

fourthly, collecting the upper organic phase obtained in the third step into a clean test tube, re-extracting the lower aqueous phase twice according to the same step, and combining all the collected organic phases;

in a fifth step, all organic phases obtained in the fourth step are dried using a centrifugal concentrator, after which the dried lipid samples are stored or directly analyzed.

The marine microorganism lipid ion mobility mass spectrometry method comprises the following steps of: solvent removal and sample reconstitution.

The marine microorganism lipid ion mobility mass spectrometry method comprises the step of collecting data, wherein the data are analyzed by adopting reversed phase ultra high performance liquid chromatography.

The marine microorganism lipid ion mobility mass spectrometry method comprises the following data acquisition steps:

and (3) chromatographic separation: using an AQUITY UPLC system equipped with ACE Excel 2SuperC18 column, solvent A is 100% methanol, solvent B is 100% ethanol, and strong washing injection is 2-propanol; the linear gradient started at 100% solvent a, held for 4 minutes, then increased to 50% solvent B at 10 minutes, further increased to 99% solvent B at 30 minutes, held for another 4 minutes, the gradient returned to 100% solvent B at 34.1 minutes, and re-equilibrated for 2 minutes; the flow rate is 0.40mL/min, the column temperature is kept at 45 ℃, and the temperature of the sample manager is kept at 7 ℃;

mass spectrometry analysis: the method uses a Waters Synapt G2-Si system equipped with an electrospray source, and adopts HDMS or HDMS of an extended dynamic range mode for data acquisition under the control of MassLynx software^EOperating in a resolution mode; wherein the mass analyzer is mass calibrated with sodium iodide solution, using leucine-enkephalin as the chain solution.

the capillary voltage is 2.8kV and 2.2kV, which are respectively in a positive and negative ion mode; the sample cone is 40V, and the source temperature is 120 ℃; the cone gas is 50L/h, the desolventizing gas is 600L/h, and the atomizer gas is 6.5 Bar; the DC bias of the trap is 60V, and the DE outlet of the trap is 3V; the IMS wave velocity is 500m/s, the wave height is 40V, the transfer wave velocity is 179m/s, and the wave height is 4V; the lipid sample was reconstituted in 150 μ l methanol; the volume of the lipid sample injected into the system was 10 μ l; the scanning time is 0.4 s; data were acquired in a continuous process from 50Da to 2000Da, 3.5min to 34 min.

The marine microorganism lipid ion mobility mass spectrometry method is characterized in that the data acquisition adopts a data independent acquisition mode.

The marine microorganism lipid ion mobility mass spectrometry method comprises the following data analysis steps:

firstly, constructing a series of lipid molecular structures and storing the lipid molecular structures as MOL files; then inputting the MOL file into Progenisis SDF Studio to generate an SFD file, and obtaining the lipid molecular structure database; the lipid molecular structure database is then used to assist in annotating mass spectral features of compound molecules.

The marine microorganism lipid ion mobility mass spectrometry method comprises the step of obtaining a fracture mass spectrum database and an ion mobility collision cross-sectional area database by utilizing the lipid molecular structure database based on the mass spectrum characteristics of the obtained compound molecules.

In a second aspect, the invention also provides the use of a method for the mass spectrometric analysis of lipid ion mobility of marine microorganisms, wherein the method as defined in any one of the above is used for the analysis of marine microorganism lipids.

Has the advantages that: the invention provides a marine microorganism lipid ion mobility mass spectrometry method, which utilizes an ion mobility mass spectrometry (IM-MS) technology to perform molecular structural characterization on marine microorganism lipid molecules. The invention improves the existing lipid extraction procedure, replaces the common chloroform-based method with methyl tert-butyl ether (MTBE), not only reduces toxicity, but also realizes pollution-free collection of two main liquid phases, and simultaneously has the same extraction efficiency as the chloroform method. The invention adopts ultra-high performance liquid chromatography (UHPLC), thus reducing the analysis time. The data acquisition adopts an ion mobility mass spectrometry method, optimizes the flow of ion mobility, adopts a positive and negative ion mode, takes methanol and ethanol as mobile phases, removes interference signals, reduces background interference and helps chemical classification. Meanwhile, the ion mobility technology can also distinguish between isobaric and isomeric forms. The invention further constructs a lipid molecular structure database, uses an automatic data processing flow to carry out deep analysis on the ion mobility data, and combines the lipid molecular structure database to carry out a semi-automatic data processing flow to annotate the chromatographic mass spectrum characteristics. The technical scheme of the invention effectively improves the limitation of the prior art on the characterization of organic substances generated by marine microorganisms, and allows the possibility of high-efficiency large-scale research.

Drawings

FIG. 1 is a flow chart of a preferred method for mass spectrometry of lipid ion mobility of marine microorganisms in accordance with an embodiment of the present invention.

FIG. 2 is a comparison between the technical solution of the embodiment of the present invention and the prior art.

FIG. 3 shows an embodiment of the present invention adjusting IMS and Transfer T-Wave parameters in Triwave tap.

FIG. 4 shows a method for constructing a library of lipid molecular structures according to an embodiment of the present invention: (A) the first method uses Progenetics SDF Studio; (B) method two, DataWarrior was used.

FIG. 5 is a method for mapping the structure of a lipid molecule according to an embodiment of the present invention.

FIG. 6 is a database of fragmentation mass spectra obtained in accordance with an embodiment of the present invention.

Figure 7 is a database of ion mobility collisional cross-sectional areas (CCS) obtained in accordance with an embodiment of the present invention.

Figure 8 is an advantage of using ion mobility mass spectrometry according to an embodiment of the present invention.

Fig. 9 is a two-dimensional ion mobility spectrum of the celllipid group obtained in the positive ion mode (a) and the negative ion mode (B) according to the example of the present invention.

FIG. 10 is a chromatogram mass spectrum characteristic of the structural isomers of the disaccharide based GDGTs in the example of the invention: (A) reconstruction chromatography of structural isomers; (B) chromatographic mass spectrometry characteristics; (C) MS of structural isomers²A spectrogram; (D) ion mobility spectra of structural isomers.

Fig. 11 is a lipid density map of marine archaea n. maritimas obtained in (a) positive ion mode and (B) negative ion mode in the example of the present invention.

Fig. 12 shows the principle of pedigree annotation in the examples of the present invention, and the identity of lipids was determined by accurate mass, isotope model, fragmentation profile, retention time.

Detailed Description

The invention provides a mass spectrometry method for lipid ion mobility of marine microorganisms and application thereof, and the invention is further described in detail below in order to make the purpose, technical scheme and effect of the invention clearer and more clear. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In a first aspect, embodiments of the present invention provide a method for mass spectrometry of lipid ion mobility of marine microorganisms, as shown in fig. 1, comprising the steps of:

s10, lipid extraction step: extracting a lipid sample from the marine microorganism to be detected by utilizing methyl tert-butyl ether;

s20, data acquisition: analyzing the lipid sample by using an ion mobility mass spectrometry, adopting a positive and negative ion mode, using methanol and ethanol as mobile phases, and collecting data;

s30, data analysis: and constructing a lipid molecule structure database, and annotating the mass spectrum characteristics of the lipid molecules in the marine microorganisms to be detected by using the acquired data based on the assistance of the lipid molecule structure database.

The analysis method provided by the embodiment of the invention mainly comprises the steps of lipid extraction, data acquisition and data processing. As shown in fig. 2, the embodiment of the present invention is technically improved from the above three aspects, respectively. The following describes in detail the improvements of the present invention.

In some embodiments, the lipid extraction step specifically comprises:

s101, capturing marine microorganisms to be detected on a polyvinylidene fluoride membrane;

s102, placing the polyvinylidene fluoride membrane obtained in the first step in a clean test tube, adding methyl tert-butyl ether and methanol for extraction, and then rotating the test tube filled with the extracting solution for preset time;

s103, adding water into the extracting solution obtained in the step S102 to induce phase separation, and then performing vortex centrifugation to separate a water phase and an organic phase;

s104, collecting the upper organic phase obtained in the step S103 into a clean test tube, re-extracting the lower aqueous phase twice according to the same step, and combining all the collected organic phases;

and S105, drying all organic phases obtained in the step S104 by using a centrifugal concentrator, and then storing or directly analyzing the dried lipid sample.

The present examples used a lipid extraction procedure of methyl tert-butyl ether (MTBE) instead of the commonly used chloroform-based method, but the extraction efficiency was comparable to the chloroform-based method. In the prior art, the organic phase containing lipid is in the lower layer, and thus a certain amount of cell debris at the bottom is mixed. MTBE is less dense than water, so the lipid-retaining organic phase is in the upper phase and the hydrophilic compounds and salts are concentrated in the lower aqueous phase. In this way, the upper organic phase containing lipids can be collected without contacting the aqueous phase or the non-extracted residues at the bottom of the extraction tube, thus achieving a contamination-free collection of the two main liquid phases. In addition, MTBE is less toxic than chloroform, which also increases the safety of the lipid extraction process.

In some embodiments, the volume ratio of methyl tert-butyl ether to methanol is 10: 3.

In some embodiments, the test tube used in step S102 may be a teflon tube, but is not limited thereto.

In some embodiments, the predetermined time of the rotation of step S102 may be 1 hour, but is not limited thereto. Can be adjusted adaptively according to the sample amount.

In some embodiments, the clean test tube used in step S104 may be a teflon tube, but is not limited thereto.

In some embodiments, the sample storage conditions of step S105 may be-30 ℃, but are not limited thereto.

In some embodiments, after the lipid extraction step, further comprising: solvent removal and sample reconstitution.

In the conventional method, a nitrogen evaporator is used for solvent removal, and the method not only contacts toxic organic solvents and introduces pollution, but also requires an additional nitrogen supply. The embodiment of the invention adopts the centrifugal vacuum concentrator with the cold trap, so that the toxic organic solvent can be effectively removed, nitrogen supply is not needed, and high flux can be realized.

In some embodiments, the lipid sample is reconstituted in methanol.

In the prior art, after sample recombination, filtration is usually required to remove cellular debris and suspicious particles, and the process may cause further pollution. In contrast, the present example uses methanol to reconstitute the sample, which is generally free of insoluble material, and centrifugation can precipitate and remove any suspended particles.

In some embodiments, the data collection step is analyzed using reverse phase ultra performance liquid chromatography.

Different from the prior art, the embodiment of the present invention uses Ultra-High Performance Liquid Chromatography (UHPLC) to reduce the analysis time. The column was analyzed by reverse phase liquid chromatography using ACE Excel 2super c 18. Furthermore, the present embodiments use methanol and ethanol as eluents to reduce background interference. This improvement is very important because the prior art uses methanol and isopropanol mobile phases and ammonia modifiers that create additional background interference in the ionic mobility mode.

In some embodiments, data is collected using ion mobility mass spectrometry, and using positive and negative ion mode with methanol and ethanol as mobile phases to reduce background interference.

In some embodiments, the data acquisition step comprises chromatographic separation and mass spectrometry.

In some embodiments, the chromatographic separation is specifically performed by:

using an AQUITY UPLC system equipped with an ACE Excel 2SuperC18 column (2 μm, 2.1X 150mm), solvent A being 100% methanol, solvent B being 100% ethanol, and power wash injection being 2-propanol; the linear gradient started at 100% solvent a, held for 4 minutes, then increased to 50% solvent B at 10 minutes, further increased to 99% solvent B at 30 minutes, held for another 4 minutes, the gradient returned to 100% solvent B at 34.1 minutes, and re-equilibrated for 2 minutes; the flow rate was 0.40mL/min, the column temperature was maintained at 45 ℃ and the sample manager temperature was maintained at 7 ℃.

In some preferred embodiments, both solvent a and solvent B are modified with 0.1% ammonia and 0.04% formic acid. In the prior art, the concentration used is higher and only the positive ion mode is allowed; after the modification, the sample can be analyzed in a positive and negative ion mode.

In some embodiments, the mass spectrometry comprises the specific steps of:

HDMS or HDMS with extended Dynamic Range mode (Enhanced Dynamic Range) data acquisition using a Waters Synapt G2-Si system equipped with an Electrospray (ESI) source, controlled by MassLynx software^EOperating in a resolution mode; wherein the mass analyzer is mass calibrated with sodium iodide solution, using leucine-enkephalin as the chain solution.

In some preferred embodiments, the resolution of the resolution mode is > 30000.

In some preferred embodiments, the calibration is performed using a Waters Major Mix IMS/Tof calibration kit.

In some preferred embodiments, the concentration of the sodium iodide solution is 2. mu.g/. mu.l.

In some preferred embodiments, the concentration of leucine-enkephalin is 1 ng/. mu.l.

In some preferred embodiments, the system is controlled by MassLynx software, version 4.2SCN 983.

In some embodiments, the parameters under which the lipid sample is analyzed are:

In some embodiments, after reconstitution of the lipid sample in 150 μ l methanol, 100 μ l is transferred to a sample vial, and the remainder is pooled as a QC sample.

In some embodiments, the lipid sample is analyzed while simultaneously analyzing the solvent blank, extraction blank, and QC sample as a corresponding control group.

In some embodiments, the ion mobility profile is optimized: dissolving beta-L-glyceropyranosyl-caldachiacetyl-glycerol in methanol, diluting to 10ng/ml, directly injecting the solution into an ion mobility mass spectrometer, adjusting parameters of the instrument to obtain the highest ion mobility resolution and ion transmission rate, and reducing the influence of transmission T Wave/push rod aliasing to the maximum extent, wherein FIG. 3 shows that the parameters of IMS and Transfer T-Wave are adjusted in TriWave tap according to the embodiment of the invention.

In some embodiments, the data acquisition employs a data independent acquisition mode.

The embodiment of the invention selects a Data Independent Acquisition (DIA) mode instead of a Data Dependent Acquisition (DDA) mode which is commonly used in the prior art. This is because DIA data collection has several advantages over DDA, including the possibility of collecting MS indiscriminately²Intensity of the spectral line, thus MS of all ions²The spectrogram can be obtained; more importantly, the ion mobility is usedIn the context of technology (also known as HDMS)^E)，MS²Mass of Mass Spectrometry with DDA derived MS²The mass spectrum is comparable to that of the traditional Chinese medicine.

In some embodiments, the data analysis step specifically comprises:

The embodiment of the invention uses an open Structure Data File (SDF) File format to construct a lipid molecular Structure database so as to assist in annotation of mass spectrum characteristics. The embodiment of the invention uses Progenetics SDF Studio to construct a database (as shown in FIG. 4A), and other software such as DataWarrior can also be used for modifying the existing SFD database file (as shown in FIG. 4B).

First, a series of lipid molecular structures were constructed on software such as MarvinSketch or ChemBioDraw and stored as MOL files (fig. 5). Then inputting the MOL file into Progenetics SDF Studio to generate an SFD file, namely the molecular structure database of the embodiment of the invention. This molecular structure database can then be used by Progenetics QI to identify mass spectral characteristics of compounds, including mass error, isotopic similarity, and experimentally and computationally derived MS²The similarity between the mass spectra (fragmentation spectra) is used to obtain confidence in the assignment.

In some embodiments, a fragmentation mass spectrum database and an ion mobility collision cross-sectional area database may also be obtained using the lipid molecule structure database based on mass spectral characteristics of the resulting compound molecules.

The present example generated another set of fracture mass spectrum database (fig. 6) and ion mobility cross-sections (ccs) database (fig. 7) based on experimental results from the lipid molecular structure database. Embodiments of the invention use an open MSP file format that can be used for other mass spectrometry data processing software, such as MS-DIAL, in addition to Progenesis QI. One point to note is that the MS is generated²The fragmentation mass spectrum database still needs to be repeatedly measured, data accumulated and continuously corrected so as to improve the coverage and accuracy of lipid molecules and measurement conditions.

The method for analyzing the lipid ion mobility mass spectrum of marine microorganisms provided by the embodiment of the invention is developed and verified on a Waters ion mobility mass spectrum system, but is not limited to the Waters ion mobility mass spectrum system, and the ion mobility mass spectrum system of main instrument manufacturers (such as Agilent, Bruk and SCIEX) can also be used. The software and file system used in embodiments of the present invention are also vendor independent.

One key technology of the method for analyzing the lipid ion mobility mass spectrum of the marine microorganisms provided by the embodiment of the invention is to remove interference signals by using an ion mobility technology to help chemical classification.

Ion mobility is a gas phase electrophoresis technique in which ions are driven in a buffer gas by an electric field. Since the mobility of ions in the buffer gas depends not only on the mass and charge of the ions, but also on the shape and size of the ions, and the nature of the buffer gas, the technique can distinguish isomeric compounds that have the same mass to charge ratio, but different gas phase geometries or configurations. Background interferences are often encountered during lipid analysis, masking the analysis results. These signals are usually synthetic polymers and cannot be completely avoided. Thus, one of the main advantages of using ion mobility mass spectrometry in general lipid analysis, as shown in fig. 8, is to separate interfering ions from lipid ions by additional separation of ion mobility, thereby purifying the mass spectrum from the interfering ions for more accurate identification of compounds.

One unique feature of ion mobility mass spectrometry data is the presence of a relevant trend line corresponding to a chemical class. This enables us to use the CCS value and the mass of the ion to estimate the chemical class of the unknown compound. Since lipids are relatively large molecules, this results in a relatively large size to mass ratio (size/mass), which makes lipids easily distinguishable from other molecules in an ion mobility mass spectrum two-dimensional spectrum. Subclasses of lipids may also be distinguished by their ion-moving properties. Fig. 9 is a two-dimensional plot of ion mobility spectra (drift time-m/z plot) of the lipid groups obtained in the positive ion (a) and negative ion (B) modes, and it can be seen that lipids are easily distinguished from other molecules.

The marine microorganism lipid ion mobility mass spectrometry provided by the embodiment of the invention has another key technology that the ion mobility technology is used for distinguishing isobaric and isomeric forms.

Many lipids have the same mass (isomers) and are not distinguishable even by their fragmentation spectra. The prior art can only distinguish isomers by chromatography. Ion mobility techniques provide methods other than chromatography. We can verify this with archaea lipid disaccharide based GDGTs. There are two structural isomers of the disaccharide-based GDGTs, one of which is glycosylation with two hexose molecules at both ends (also known as type I), and the other of which is glycosylation with only one dihexose molecule on one of the glycerol molecules (also known as type II). FIG. 10 shows the structural isomer ([ M + NH 4) of the disaccharide based GDGTs]⁺) The type I and type II disaccharide based GDGTs isomers can be obtained by chromatography (FIGS. 10A, 10B), and MS²In addition to the mass spectrum (FIG. 10C) discrimination, ion mobility techniques (FIG. 10D) can be used to distinguish between them because their conformations are not the same.

The marine microorganism lipid ion mobility mass spectrometry provided by the embodiment of the invention has a key innovation point in constructing a lipid molecular structure database.

It is a feature of an embodiment of the present invention to perform mass spectrometry annotations using an automated data preprocessing procedure. Taking fig. 11 as an example, fig. 11A and 11B are respectively a lipid density map of marine archaea nitrosopumulirus maritimus obtained in the positive and negative modes. There are a large number of spots in the figure, each dot representing an ion. However, it is difficult to accurately annotate each spot as a lipid. This can be improved by automated data preprocessing. By precise mass, isotopic similarity and HDMS^E MS²Fragmentation mass spectra were compared to lipid molecules in the archaea lipid database and the mass spectral features were finally annotated (fig. 12). To achieve this result, the lipid molecular structure must first be constructedA database. The embodiment of the invention constructs a lipid molecular structure database in advance, and then mass spectrum annotation can be carried out by using an automatic data preprocessing program.

The embodiment of the invention also provides application of the marine microorganism lipid ion mobility mass spectrometry method, and the analysis method is applied to marine microorganism lipid analysis.

In some embodiments, the marine microorganism is a marine archaea.

Marine archaea have significantly different characteristics from other marine unicellular planktonic microorganisms, one of the important characteristics being their unique lipids. The characterization of liposomes for biological systems remains technically challenging. Lipids are diverse in structure and chemistry. Many lipids are isobaric or isomeric and often cannot be distinguished by measuring mass or even their fragmentation pattern. The technical scheme of the embodiment of the invention utilizes an ion mobility mass spectrometry (IM-MS) technology to perform molecular structural characterization on the marine planktonic microorganisms, and improves the existing sample treatment materials and procedures to extract lipid of planktonic microorganism cell membranes for characterization. According to the technical scheme of the embodiment of the invention, an automatic data processing flow is further used for carrying out deep analysis on the ion mobility data, and a semi-automatic data processing flow is carried out in combination with a molecular database to annotate the chromatographic mass spectrum characteristics. The technical scheme of the embodiment of the invention effectively improves the existing limitation of representing the organic substances generated by the marine plankton, and allows the possibility of high-efficiency large-scale research.

In some embodiments, the marine microorganism lipid ion mobility mass spectrometry method is also applicable to extreme hobbies archaea, pathogenic bacteria, and microorganisms.

The method for analyzing the lipid ion mobility mass spectrum of marine microorganisms and the application thereof are further explained by the following specific examples:

example 1

Extraction of lipid molecules from marine planktonic microorganisms

The lipid of marine archaea Nitrosopumilus maritimus is extracted:

(1) capturing a certain amount of marine archaea to be detected on a polyvinylidene fluoride (PVDF) membrane;

(2) taking a clean Teflon tube, putting a PVDF membrane for capturing marine archaea to be detected into the tube, and adding 4ml of methyl tert-butyl ether (MTBE) and 1.2ml of methanol (10: 3; v/v) for extraction;

(3) placing the Teflon tube filled with the extracting solution on a test tube rotator to rotate for one hour;

(4) to the extract was added 1ml of water to induce phase separation. Swirling the extract and centrifuging to separate polar metabolites from lipids to obtain an aqueous phase and an organic phase;

(5) collecting the upper organic phase in a clean polytetrafluoroethylene tube;

(6) repeating the steps (2) to (5) on the lower-layer water phase and the biomass, and re-extracting twice;

(7) all the collected organic phases were combined and dried with a centrifugal concentrator, after which the dried lipid sample was stored at-30 ℃ until analysis.

Example 2

Lipid samples extracted in example 1 were analyzed

(1) The dried lipid sample was reconstituted with 150. mu.l of methanol, 100. mu.l was transferred to a sample vial, and the remainder was collected to prepare a QC sample;

(2) installing ACE Excel 2SuperC18 on a UHPLC system;

(3) 100% methanol and 100% ethanol as mobile a and B mobile phases and modified with 0.1% ammonia and 0.04% formic acid;

(4) the UHPLC system was set to a gradient with a linear gradient starting at 100% a, held for 4 minutes, then increasing the gradient to 50% B at 10 minutes, and further increasing the gradient to 99% B at 30 minutes, held for 4 minutes. The gradient returned to 100% B at 34.1 minutes and re-equilibrated for 2 minutes. The flow rate of the UHPLC was set at 0.40 mL/min. The column temperature was set at 45 ℃.

(5) The mass analyzer was mass calibrated with 2 μ g/μ L sodium iodide solution and the ion mobility system was calibrated with a waters major mix IMS/Tof calibration kit.

(6) Using HDMS mode or HDMS with extended dynamic range^EThe sample was mode analyzed and performed in resolution mode.

(7) Lipid sample analysis the following instrument parameters can be used: capillary voltage 2.8 or 2.2kv in positive and negative ion mode. Sample cone 40V, source temperature 120C, cone gas 50L/h, dissolved gas 600L/h, atomizing gas 6.5 bar. Trap DC bias 60V, Trap DE exit 3V.IMS wave velocity 500m/s, wave height 40V, transfer wave velocity 179m/s, wave height 4V.

(8) Data were obtained continuously from 50 to 2000Da, from 3.5 minutes to 34 minutes. The transmitted collision energy was from 40 to 120v. the scan time was 0.4 s. Blank and QC were analyzed simultaneously.

Example 3

Data obtained in example 2 were preprocessed

Raw data was processed using prognesis QI software and automated deconstruction and alignment was used. Peak pick-up was set as the default sensitivity with a minimum peak width of 1.5 s. Data were normalized by the default method of the software, normalizing to all compounds.

Mass spectral characteristics were annotated using the Progenesis MetaScope algorithm, with constructed LIPID libraries and other published LIPID structure libraries (e.g., LIPID MAP and BioCyc databases) with relative mass error for precursor and theoretical fragment tolerance of 5 ppm.

Mass spectral features including mass error, isotope similarity, and MS experimentally and computationally derived²The similarity between the mass spectra (fragmentation spectra) is used to obtain confidence in the assignment.

The software can calculate the similarity of each mass spectral feature and summarize it into an overall confidence score (top score 60). When the mass spectrum characteristics are annotated, the most probable lipid molecules are selected, and the confidence coefficient is more than or equal to 47 points. Lipid molecules with no confidence score ≧ 47 did not accept annotation.

Example 4

The results obtained in example 3 were analyzed

(1) And carrying out qualitative analysis on lipid groups of the marine planktonic microorganisms to find out which lipids neutralize mass spectrum characteristics of the lipids, such as CCS values, fracture mass spectrum characteristics and the like.

(2) The biomarkers are found by contrasting the lipid groups of other marine planktonic microorganisms, and are used as a basis for detecting or distinguishing different marine planktonic microorganisms living in a specific natural marine environment, namely, the analysis of a sample of the marine planktonic microorganisms is provided.

(3) The change and cause of the lipid group can be found out according to different culture conditions. It is inferred that the effect on marine microorganisms is estimated due to changes in environmental climate, composition of microbial community, and interaction between microorganisms of different species.

In summary, the embodiment of the present invention provides a method for analyzing marine microorganism lipid ion mobility mass spectrometry, which utilizes an ion mobility mass spectrometry (IM-MS) technique to perform molecular structural characterization on marine microorganism lipid molecules. The invention improves the existing lipid extraction procedure, replaces the common chloroform-based method with methyl tert-butyl ether (MTBE), not only reduces toxicity, but also realizes pollution-free collection of two main liquid phases, and simultaneously has the same extraction efficiency as the chloroform method. The invention adopts ultra-high performance liquid chromatography (UHPLC), thus reducing the analysis time. The data acquisition adopts an ion mobility mass spectrometry method, optimizes the flow of ion mobility, adopts a positive and negative ion mode, takes methanol and ethanol as mobile phases, removes interference signals, reduces background interference and helps chemical classification. Meanwhile, the ion mobility technology can also distinguish between isobaric and isomeric forms. The technical scheme of the invention further constructs a lipid molecular structure database, uses an automatic data processing flow to carry out deep analysis on the ion mobility data, and combines the lipid molecular structure database to carry out a semi-automatic data processing flow to annotate the chromatographic mass spectrum characteristics. The technical scheme of the invention effectively improves the limitation of the prior art on the characterization of organic substances generated by marine microorganisms, and allows the possibility of high-efficiency large-scale research.

It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the foregoing description, and that all such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.

Claims

1. A method for the mass spectrometric analysis of lipid ion mobility of marine microorganisms, comprising the steps of:

2. The method of claim 1, wherein the lipid extraction step comprises:

3. The method of claim 1, further comprising, after the lipid extraction step: solvent removal and sample reconstitution.

4. The method of claim 1, wherein the step of collecting data is performed by reversed phase ultra high performance liquid chromatography.

5. The method of claim 4, wherein the step of collecting data comprises:

6. The method of claim 5, wherein the lipid sample is analyzed using the parameters of:

7. The method of claim 1, wherein the data acquisition is in a data independent acquisition mode.

8. The method of claim 1, wherein the step of analyzing the data specifically comprises:

9. The method of claim 8, wherein a fragmentation mass spectrum database and an ion mobility cross-sectional area database are further generated using the lipid molecule structure database based on mass spectral characteristics of the resulting compound molecules.

10. Use of a method for mass spectrometric analysis of lipid ion mobility of marine microorganisms, characterized in that the method according to any one of claims 1-9 is used for the analysis of lipids of marine microorganisms.