CN114235979B - Mass spectrometry analysis method and application of lipid ion mobility of marine microorganisms - Google Patents
Mass spectrometry analysis method and application of lipid ion mobility of marine microorganisms Download PDFInfo
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Abstract
The invention provides a method for analyzing lipid ion mobility mass spectrum of marine microorganisms and application thereof, and molecular structural characterization is carried out on lipid molecules of the marine microorganisms by utilizing ion mobility mass spectrum technology. The present invention improves upon existing lipid extraction procedures and replaces chloroform-based processes with methyl tertiary butyl ether. And by adopting ultra-high performance liquid chromatography, the analysis time is shortened. The data is acquired by adopting an ion mobility mass spectrometry method, and the flow of the ion mobility is optimized. Meanwhile, the ion mobility technology can also distinguish isotacticity and isomerism. According to the invention, an automated data processing flow is used for carrying out deep analysis on ion mobility mass spectrum data, a lipid molecular structure database is further constructed, and the characteristic of the chromatographic mass spectrum is annotated by combining the lipid molecular structure database, so that a semi-automated data processing flow is finally achieved. The invention effectively improves the limitation of the prior art for characterizing organic matters generated by marine microorganisms, and allows the possibility of high-efficiency and large-scale research.
Description
Technical Field
The invention relates to the fields of marine microbiology and marine metabonomics, in particular to a mass spectrometry method for lipid ion mobility of marine microorganisms and application thereof.
Background
Numerous single-cell planktonic microorganisms exist in seawater, and although their individuals are small, the biochemical reactions are single, but the variety and number are numerous. The different species of microorganisms interact to form a rather complex microbiota. They play a great role in the important processes of marine ecosystem, geochemical element circulation, etc. Recently, marine planktonic microorganisms such as microalgae and cyanobacteria have attracted much attention. This is because these organisms can perform photosynthesis, can directly absorb carbon in the atmosphere, and settle into their biological substances. Thus, the process can help slow global warming and can produce many natural products of application value. Planktonic marine archaea have been considered rare organisms that survive only extreme temperatures, pressures, or salinity, however they are now found in almost all marine environments and are ubiquitous members of the abundance of marine plankton. Marine archaea differs from other microorganisms in the lipids of their cell membranes, a biochemical trait that also becomes an important tool to study marine archaea and to distinguish them from other microorganisms. At present, how to interact with other marine plankton (such as algae and blue algae) by using marine archaea is discussed, and then the key problem of how to further lock carbon in the atmosphere in the ocean is presented. Thus, the development of an effective method to analyze the lipids of marine plankton has a critical effect on marine microbiology and marine metabolomics.
In the prior art, different types of mass spectrometry techniques have been used to analyze marine microorganism lipidosome. Currently, the most effective method for studying lipids of marine microorganisms is liquid chromatography-mass spectrometry (LC-MS). However, existing mass spectrometry techniques have difficulty coping with chemical diversity of lipid molecules and lack specific methods for analyzing isomerised lipid molecules. Furthermore, the data are usually collected and analyzed manually by the students studying oceanography, which also causes problems such as low efficiency, uncertainty or false positive. And different students may have different interpretations of the same data, resulting in poor repeatability. One reason for this is the previous lack of a molecular structure database built for the lipid groups of marine plankton for use in the processing analysis of mass spectrometry data.
Accordingly, there is a need in the art for improvement.
Disclosure of Invention
In view of the shortcomings of the prior art, the invention aims to provide a mass spectrometry method for analyzing lipid ion mobility of marine microorganisms and application thereof, and aims to solve the problems that the prior art marine microorganism lipid analysis method is insufficient in coping with chemical diversity of lipid molecules and can not realize automatic and standardized analysis data.
The technical scheme of the invention is as follows:
in a first aspect, the present invention provides a method for mass spectrometry of lipid ion mobility of marine microorganisms, comprising the steps of:
lipid extraction: extracting a lipid sample from marine microorganisms to be detected by using methyl tertiary butyl ether;
and a data acquisition step: analyzing the lipid sample by utilizing an ion mobility mass spectrometry method and adopting a positive ion mode and a negative ion mode by taking methanol and ethanol as mobile phases, and collecting data;
And a data analysis step: constructing a lipid molecular structure database, and annotating mass spectrum characteristics of lipid molecules in the marine microorganisms to be detected by utilizing the acquired data based on the assistance of the lipid molecular structure database.
The lipid ion mobility mass spectrometry method for the marine microorganisms comprises the following steps:
capturing marine microorganisms to be detected on a polyvinylidene fluoride membrane;
secondly, placing the polyvinylidene fluoride film obtained in the first step in a clean test tube, adding methyl tertiary 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 centrifugally separating to obtain a water phase and an organic phase;
fourth, 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 steps, and combining all the collected organic phases;
Fifth, all the organic phases obtained in the fourth step are dried with a centrifugal concentrator, after which the dried lipid sample is stored or analyzed directly.
The method for analyzing the lipid ion mobility mass spectrum of the marine microorganism comprises the following steps of: solvent removal and sample reorganization.
According to the marine microorganism lipid ion mobility mass spectrometry method, the data acquisition step adopts reverse phase ultra-high performance liquid chromatography for analysis.
The marine microorganism lipid ion mobility mass spectrometry method specifically comprises the following steps:
Chromatographic separation: using AQUITY UPLC system equipped with ACE Excel 2SuperC column, solvent A is 100% methanol, solvent B is 100% ethanol, and strong washing injection is 2-propanol; the linear gradient starts with 100% solvent a, after 4 minutes of hold, then increases to 50% solvent B at 10 minutes, further increases to 99% solvent B at 30 minutes, holds for another 4 minutes, returns to 100% solvent B at 34.1 minutes, and re-equilibrates 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: using WATERS SYNAPT G-Si system equipped with electrospray source, controlled by MassLynx software, data acquisition using HDMS or HDMS E in extended dynamic range mode, operating in resolution mode; wherein the mass analyzer was mass calibrated with sodium iodide solution using leucine-enkephalin as a chain solution.
The lipid ion mobility mass spectrometry method for the marine microorganisms comprises the following steps of:
The capillary voltage is 2.8kV and 2.2kV, and positive and negative ion modes are respectively adopted; 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 6.5Bar; the DC bias voltage of the catcher is 60V, and the outlet of the catcher DE is 3V; IMS wave speed is 500m/s, wave height is 40V, transfer wave speed is 179m/s, and wave height is 4V; the lipid sample was reconstituted in 150 μl methanol; the lipid sample volume of the injection system was 10 μl; the scanning time is 0.4s; data were obtained in a continuous process from 50Da to 2000Da,3.5min to 34 min.
According to the mass spectrometry method for the lipid ion mobility of the marine microorganisms, the data acquisition adopts a data independent acquisition mode.
The method for analyzing the lipid ion mobility mass spectrum of the marine microorganism comprises the following steps of:
firstly, constructing a series of lipid molecular structures and storing the lipid molecular structures as MOL files; inputting Progenesis SDF Studio the MOL file to generate an SFD file, and obtaining the lipid molecular structure database; the lipid molecular structure database is then used to assist in annotating the mass spectral features of the compound molecules.
According to the method for analyzing the lipid ion mobility mass spectrum of the marine microorganism, based on the mass spectrum characteristics of the obtained compound molecules, a fragmentation mass spectrum database and an ion mobility collision cross-sectional area database can be obtained by utilizing the lipid molecular structure database.
In a second aspect, the invention also provides the use of a method for lipid ion mobility mass spectrometry of marine microorganisms, wherein the method as described in any one of the above is applied to lipid analysis of marine microorganisms.
The beneficial effects are that: the invention provides a method for analyzing lipid ion mobility mass spectrum of marine microorganisms, which utilizes ion mobility mass spectrum (IM-MS) technology to carry out molecular structural characterization on lipid molecules of the marine microorganisms. The invention improves the existing lipid extraction procedure, uses Methyl Tertiary Butyl Ether (MTBE) to replace the common chloroform-based method, reduces toxicity, realizes pollution-free collection of two main liquid phases, and has the same extraction efficiency as the chloroform method. The invention adopts ultra-high performance liquid chromatography (UHPLC) to reduce analysis time. The data is acquired by adopting an ion mobility mass spectrometry method, the flow of the ion mobility is optimized, a positive ion mode and a negative ion mode are adopted, methanol and ethanol are used as mobile phases, interference signals are removed, and background interference is reduced, so that chemical classification is assisted. Meanwhile, the ion mobility technology can also distinguish isotacticity and isomerism. 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 for characterizing organic substances generated by marine microorganisms, and allows the possibility of high-efficiency large-scale research.
Drawings
FIG. 1 is a preferred flow chart of a method for mass spectrometry of lipid ion mobility of marine microorganisms according to an embodiment of the present invention.
Fig. 2 is a comparison of the technical solution in the embodiment of the present invention with the prior art.
Fig. 3 illustrates the adjustment of IMS and TRANSFER T-Wave parameters in TRIWAVE TAP in accordance with an embodiment of the present invention.
FIG. 4 shows a method for constructing a lipid molecular structure library according to an embodiment of the present invention: (A) method one, use Progenesis SDF Studio; method II (B) using DataWarrior.
FIG. 5 is a method of mapping lipid molecular structures according to an embodiment of the present invention.
FIG. 6 is a database of fracture mass spectra obtained in the example of the present invention.
FIG. 7 is a database of ion mobility collisional cross-sectional areas (CCS) obtained according to an embodiment of the invention.
FIG. 8 illustrates the advantage of using ion mobility mass spectrometry in an embodiment of the present invention.
FIG. 9 is a two-dimensional graph of ion mobility spectra of cell lipidosome obtained in the positive ion mode (A) and the negative ion mode (B) in the embodiment of the invention.
FIG. 10 is a chromatographic mass spectrometry characterization of structural isomers of disaccharides GDGTs in the examples of the invention: (a) a reconstitution chromatograph of a structural isomer; (B) chromatographic mass spectrometry features; (C) MS 2 spectrum of structural isomer; (D) ion mobility spectrum of structural isomer.
FIG. 11 is a lipid density map of marine archaea N.maritimus obtained in (A) positive ion mode and (B) negative ion mode in the examples of the present invention.
FIG. 12 is a principle of pedigree annotation in the examples of the present invention, lipid identity was determined by accurate mass, isotope model, fragmentation pattern, retention time.
Detailed Description
The invention provides a mass spectrometry analysis method and application of lipid ion mobility of marine microorganisms, which are used for making the purposes, technical schemes and effects of the invention clearer and more definite, and are further described in detail below. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
In a first aspect, an embodiment of the present invention provides a method for analyzing lipid ion mobility mass spectrum of marine microorganisms, as shown in fig. 1, including the steps of:
S10, lipid extraction: extracting a lipid sample from marine microorganisms to be detected by using methyl tertiary butyl ether;
S20, data acquisition: analyzing the lipid sample by utilizing an ion mobility mass spectrometry method and adopting a positive ion mode and a negative ion mode by taking methanol and ethanol as mobile phases, and collecting data;
S30, data analysis: constructing a lipid molecular structure database, and annotating mass spectrum characteristics of lipid molecules in the marine microorganisms to be detected by utilizing the acquired data based on the assistance of the lipid molecular 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 improvements of the present invention are described in detail below.
In some embodiments, the lipid extraction step specifically comprises:
s101, capturing marine microorganisms to be detected on a polyvinylidene fluoride film;
S102, placing the polyvinylidene fluoride film obtained in the first step in a clean test tube, adding methyl tertiary butyl ether and methanol for extraction, and then rotating the test tube filled with the extracting solution for a preset time;
s103, adding water into the extracting solution obtained in the step S102 to induce phase separation, and then performing vortex centrifugation to obtain a water phase and an organic phase by separation;
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 steps, and combining all the collected organic phases;
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 examples of the present invention used the lipid extraction procedure of methyl tert-butyl ether (MTBE) instead of the conventional chloroform-based method, but the extraction efficiency was comparable to that of the chloroform-based method. In the conventional technique using chloroform extraction, the organic phase containing lipid is mixed in the lower layer, and therefore a certain amount of cell debris is mixed in the bottom. The MTBE is less dense than water, so the organic phase that retains the lipid is in the upper layer, and the hydrophilic compounds and salts are concentrated in the lower aqueous phase. In this way, the lipid-containing upper organic phase can be collected without contacting the aqueous phase or non-extraction residues at the bottom of the extraction tube, thus achieving a pollution-free collection of the two main liquid phases. In addition, MTBE is less toxic than chloroform, which also improves the safety of the lipid extraction process.
In some embodiments, the volume ratio of methyl tertiary 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 step S102 may be rotated for a predetermined time of 1 hour, but is not limited thereto. Can be adaptively adjusted according to the sample amount.
In some embodiments, the clean test tube used in step S104 may be a polytetrafluoroethylene tube, but is not limited thereto.
In some embodiments, the sample storage condition of step S105 may be-30 ℃, but is not limited thereto.
In some embodiments, after the lipid extraction step, further comprising: solvent removal and sample reorganization.
In the conventional method, a nitrogen evaporator is used for removing the solvent, and the method not only contacts toxic organic solvent to introduce pollution, but also needs additional nitrogen supply. The embodiment of the invention adopts the centrifugal vacuum concentrator with the cold trap, so that not only can the toxic organic solvent be effectively removed, but also nitrogen supply is not needed, and high flux can be realized.
In some embodiments, the lipid sample is recombined in methanol.
In the prior art, filtration is usually required after sample reconstitution to remove cell debris and suspicious particles, and this process may lead to further contamination. In the embodiment of the invention, methanol is adopted to recombine the sample, insoluble substances are not contained, and the sample can be precipitated and any suspended particles can be removed after centrifugation.
In some embodiments, the data collection step is analyzed using reverse phase ultra high performance liquid chromatography.
Unlike the prior art, the present embodiments utilize Ultra-high performance liquid chromatography (UHPLC, ultra-High Performance Liquid Chromatography) to reduce analysis time. The column was analyzed by reverse phase liquid chromatography using ACE Excel 2SuperC 18. Moreover, embodiments of the present invention use methanol and ethanol as eluents to reduce background interference. This improvement is important because the prior art uses methanol and isopropanol mobile phases and ammonia modifiers to create additional background interference in the ion mobility mode.
In some embodiments, data is collected using ion mobility mass spectrometry and positive and negative ion modes with methanol and ethanol as mobile phases to reduce background interference.
In some embodiments, the data acquisition step includes chromatographic separation and mass spectrometry.
In some embodiments, the chromatographic separation is specifically performed as follows:
Using AQUITY UPLC system equipped with ACE Excel 2SuperC column (2 μm, 2.1x150mm), solvent a was 100% methanol, solvent B was 100% ethanol, and the power wash injection was 2-propanol; the linear gradient starts with 100% solvent a, after 4 minutes of hold, then increases to 50% solvent B at 10 minutes, further increases to 99% solvent B at 30 minutes, holds for another 4 minutes, returns to 100% solvent B at 34.1 minutes, and re-equilibrates 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 positive ion mode is allowed; and after the modification, positive and negative ion modes can be used for sample analysis.
In some embodiments, the mass spectrometry specific steps are:
data acquisition was controlled by MassLynx software using WATERS SYNAPT G-Si system equipped with Electrospray (ESI) source, HDMS or HDMS E in extended dynamic range mode (ENHANCED DYNAMIC RANGE), operating in resolution mode; wherein the mass analyzer was mass calibrated with sodium iodide solution using leucine-enkephalin as a 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 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 of the lipid sample for analysis are:
The capillary voltage is 2.8kV and 2.2kV, and positive and negative ion modes are respectively adopted; 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 6.5Bar; the DC bias voltage of the catcher is 60V, and the outlet of the catcher DE is 3V; IMS wave speed is 500m/s, wave height is 40V, transfer wave speed is 179m/s, and wave height is 4V; the lipid sample was reconstituted in 150 μl methanol; the lipid sample volume of the injection system was 10 μl; the scanning time is 0.4s; data were obtained in a continuous process from 50Da to 2000Da,3.5min to 34 min.
In some embodiments, after the lipid sample has been reconstituted in 150 μl of methanol, 100 μl is transferred to a sample bottle and the remainder is pooled as QC samples.
In some embodiments, the lipid sample is analyzed simultaneously with the solvent blank, the extraction blank, and the QC sample as corresponding control groups.
In some embodiments, the flow of ion mobility is optimized: beta-L-glyceropyranosyl glyceride (beta-L-gulopyranosyl-CALDARCHAETIDYL-glycidol) is dissolved in methanol, diluted to 10ng/ml, then the solution is directly injected into an ion mobility mass spectrometer, the parameters of the instrument are adjusted to obtain the highest ion mobility resolution and ion transmission rate, the influence of T Wave/push rod aliasing is reduced to the greatest extent, and the parameters of IMS and TRANSFER T-Wave are adjusted in TRIWAVE TAP in the embodiment of the invention shown in figure 3.
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 acquisition has several advantages over DDA, including the ability to collect the intensity of MS 2 spectral lines without distinction, so that MS 2 spectra for all ions can be obtained; more importantly, in the case of using ion mobility techniques (also known as HDMS E),MS2 mass spectrum is comparable to MS 2 mass spectrum obtained with DDA.
In some embodiments, the data analysis step specifically includes:
firstly, constructing a series of lipid molecular structures and storing the lipid molecular structures as MOL files; inputting Progenesis SDF Studio the MOL file to generate an SFD file, and obtaining the lipid molecular structure database; the lipid molecular structure database is then used to assist in annotating the mass spectral features of the compound molecules.
Embodiments of the present invention construct a lipid molecular structure database using an open Structure Data File (SDF) file format to facilitate annotation of mass spectrometry features. The embodiment of the present invention uses Progenesis SDF Studio to build a database (FIG. 4A), and other software such as DataWarrior may also be used to modify existing SFD database files (FIG. 4B).
First, a series of lipid molecular structures were constructed on the software MARVINSKETCH or chembiosdraw et al and saved as MOL files (fig. 5). The MOL file is then entered Progenesis SDF Studio to generate an SFD file, i.e., a molecular structure database in accordance with an embodiment of the present invention. This molecular structure database can then be used Progenesis QI to identify mass spectral features of the compound, including mass errors, isotope similarity, and similarity between experimentally and computer-calculated MS 2 mass spectra (fragmentation mass spectra), all to obtain confidence in the assignments.
In some embodiments, based on the mass spectral characteristics of the resulting compound molecules, a fragmentation mass spectrum database and an ion mobility collision cross-sectional area database can also be obtained using the lipid molecular structure database.
The present example produced another set of fragmentation mass spectrum databases (fig. 6) and ion mobility collision cross-sectional area (Collision cross sections, CCSs) databases (fig. 7) based on experimental results from the lipid molecular structure database. Embodiments of the present invention use an open MSP file format that can be used in addition to Progenesis QI for other mass spectrometry data processing software, such as MS-DIAL. It should be noted that the resulting MS 2 fragmentation mass spectrum database still requires repeated measurements, data accumulation and ongoing corrections to improve coverage and accuracy of lipid molecules and measurement conditions.
The method for analyzing lipid ion mobility mass spectrum of marine microorganism 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 method, and the ion mobility mass spectrum system of main instrument manufacturers (such as Agilent, bruce and SCIEX) can be used. The software and file systems used in embodiments of the present invention are also vendor independent.
The method for analyzing the lipid ion mobility mass spectrum of the marine microorganisms provided by the embodiment of the invention has the key technical point that the ion mobility technology is utilized to remove interference signals and help chemical classification.
Ion mobility is a gas phase electrophoresis technique in which ions are moved in a buffer gas under the influence of an electric field. Since the mobility of ions in a buffer gas depends not only on the mass and charge of the ions, but also on the shape and size of the ions, as well as the nature of the buffer gas, this technique can distinguish heterogeneous compounds of the same mass to charge ratio but with different gas phase geometries or configurations. Background interference is often encountered during lipid analysis, masking the analysis results. These signals are usually synthetic polymers and cannot be completely avoided. Thus, one of the major advantages of using ion mobility spectrometry in general lipid analysis is that interfering ions are separated from lipid ions by additional separation of ion mobility, as shown in fig. 8, 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 associated trend lines corresponding to chemical classes. This enables us to estimate the chemical class of unknown compounds using CCS values and ion mass. Since lipids are relatively large molecules, this results in a relatively large size to mass ratio (size/mass) that allows lipids to be easily distinguished from other molecules in an ion mobility mass spectrum two-dimensional spectrum. The subclasses of lipids may also be distinguished by their ion mobility characteristics. FIG. 9 is a two-dimensional plot of ion mobility versus mass spectrum (drift time-m/z plot) of the cell lipidosome obtained in the (A) positive ion and (B) negative ion modes, showing that lipids are easily distinguished from other molecules.
The method for analyzing the lipid ion mobility mass spectrum of the marine microorganism provided by the embodiment of the invention has the key technical point that the ion mobility technology is utilized to distinguish isotactics and isomers.
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 archaebacteria lipid disaccharide group GDGTs. Disaccharide group GDGTs has two structural isomers, one of which is glycosylated with two hexose molecules at both ends (also known as type I), and the other of which is glycosylated with only one disaccharide molecule on one glycerol molecule (also known as type II). FIG. 10 is a chromatographic mass spectrometry characterization of disaccharide group GDGTs structural isomers ([ M+NH4] +), and the disaccharide group GDGTs isomers of form I and II can be distinguished by ion mobility techniques (FIG. 10D) in addition to chromatography (FIGS. 10A, 10B) and MS 2 mass spectrometry (FIG. 10C), because of their heterogeneous conformations.
The mass spectrometry method for lipid ion mobility of the marine microorganisms provided by the embodiment of the invention has a key innovation point in constructing a lipid molecular structure database.
One feature of an embodiment of the present invention is the use of an automated data preprocessing procedure for mass spectrometry annotation. Taking fig. 11 as an example, fig. 11A and 11B are lipid density maps of marine archaea Nitrosopumilus maritimus obtained in positive and negative modes, respectively. There are a large number of spots in the figure, each representing an ion. It is difficult to accurately annotate each spot as a lipid. This can be improved by automated data preprocessing. Comparison with lipid molecules in the archaea lipid database was made by accurate mass, isotopic similarity and HDMS E MS2 fragmentation mass spectrum, finally annotating mass spectral features (fig. 12). In order to achieve this result, however, a lipid molecular structure database must first be constructed. According to the embodiment of the invention, the lipid molecular structure database is constructed in advance, and then the mass spectrum annotation can be performed by using an automatic data preprocessing program.
The embodiment of the invention also provides an application of the marine microorganism lipid ion mobility mass spectrometry method, wherein 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 single-cell planktonic microorganisms, one of the important characteristics being their unique lipids. Research into the characteristics of liposomes for biological systems remains a number of challenges in the art. Lipids are structurally and chemically diverse. Many lipids are isobaric or isomeric and often cannot be distinguished by measuring mass or even their fragmentation patterns. The technical scheme of the embodiment of the invention utilizes the ion mobility mass spectrometry (IM-MS) technology to carry out molecular structural characterization on the marine planktonic microorganisms, and improves the existing sample processing materials and procedures to extract the lipids of planktonic microorganism cell membranes to be used as the characterization. According to the technical scheme provided by the embodiment of the invention, the automated data processing flow is further used for carrying out deep analysis on the ion mobility data, and the semi-automated data processing flow is carried out by combining a molecular database to annotate the chromatographic mass spectrum characteristics. The technical scheme of the embodiment of the invention effectively improves the limitation of the existing characterization of organic substances generated by marine plankton, and allows the possibility of high-efficiency large-scale research.
In some embodiments, the marine microorganism lipid ion mobility mass spectrometry method can also be applied to extreme archaebacteria, pathogenic bacteria and microorganisms.
The following is a further explanation of the method and application of the present invention for analyzing lipid ion mobility mass spectrum of marine microorganism by using specific examples:
Example 1
Extraction of lipid molecules from marine planktonic microorganisms
Extracting lipid of marine archaea Nitrosopumilus maritimus:
(1) Capturing a certain amount of marine archaea to be detected on a polyvinylidene fluoride (PVDF) film;
(2) Taking a clean Teflon tube, placing a PVDF film captured with marine archaea to be detected in 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 for rotating for one hour;
(4) To the extract, 1ml of water was added 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) - (5) for the lower aqueous phase and biomass, and re-extracting twice;
(7) All organic phases collected were combined and dried with a centrifugal concentrator, after which the dried lipid samples were stored at-30 ℃ until analysis.
Example 2
Analysis of lipid samples extracted from example 1
(1) Recombining the dried lipid sample with 150 μl of methanol, transferring 100 μl to a sample bottle, and collecting the rest to prepare QC sample;
(2) Installing ACE Excel 2SuperC on a UHPLC system;
(3) 100% methanol and 100% ethanol were used 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, a linear gradient starting from 100% A for 4 minutes, then the gradient was increased to 50% B at 10 minutes and further to 99% B at 30 minutes for 4 minutes. The gradient returned to 100% b at 34.1 minutes and was allowed to rebalance for 2 minutes. The flow rate of UHPLC was set to 0.40 mL/min. The column temperature was set at 45 ℃.
(5) The mass analyzer was calibrated with a2 microgram/μl sodium iodide solution mass, and the ion mobility system was calibrated with a waters primary mix IMS/Tof calibration kit.
(6) Samples were analyzed using either HDMS mode or HDMS E mode with extended dynamic range and performed in resolution mode.
(7) Lipid sample analysis can be performed using the following instrument parameters: capillary voltages of 2.8 or 2.2 kv are in positive and negative ion mode. Sample cone 40V, source temperature 120C, cone gas 50L/h, dissolution gas 600L/h, atomization gas 6.5 bar. Trap DC bias 60V,Trap DE exit 3V.IMS wave speed 500m/s, wave height 40V, transfer wave speed 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 is from 40 to 120V, the scan time is 0.4s. Blank and QC were analyzed simultaneously.
Example 3
Pretreatment of the data obtained in example 2
The raw data is processed using Progenesis QI software and auto-deconstructing and alignment are used. Peak pick-up was set to default sensitivity with a minimum peak width of 1.5s. Data were normalized by the default method of software, normalized to all compounds.
The mass spectral features were annotated using Progenesis MetaScope algorithm, and the constructed LIPID pool and other published LIPID structure pools (e.g., the LIPID MAP and BioCyc databases), with a precursor and theoretical fragment tolerance relative mass error range of 5ppm.
Mass spectral characteristics, including mass error, isotope similarity, and similarity between experimentally and computer-calculated MS 2 mass spectra (fragmentation mass spectra), were used to obtain assigned confidence levels.
The software can calculate the similarity of each mass spectral feature and summarize it into an overall confidence score (highest score 60). The most likely lipid molecules were selected when annotating mass spectral features with confidence scores greater than or equal to 47. Lipid molecules with no confidence score of greater than or equal to 47 total scores did not receive comments.
Example 4
Analysis of the results obtained in example 3
(1) The lipid groups of the marine planktonic microorganisms are qualitatively analyzed to find out which lipids neutralize their mass spectrum characteristics, such as CCS value, fragmentation mass spectrum characteristics, and the like.
(2) The biomarker is found by contrast with the lipid group of other marine planktonic microorganisms and is used as a basis for reconnaissance or distinguishing between different marine planktonic microorganisms living in a specific natural marine environment, i.e. also for analysis of a sample of the environment.
(3) The change and cause of the lipid group are found out according to the unused culture conditions. It is inferred from this that the influence on marine microorganisms is estimated due to changes in factors such as the ambient climate, the composition of the microbial community, and interactions between microorganisms of different species.
In summary, the embodiment of the invention provides a method for analyzing lipid ion mobility mass spectrum of marine microorganisms, which utilizes ion mobility mass spectrum (IM-MS) technology to carry out molecular structural characterization on lipid molecules of marine microorganisms. The invention improves the existing lipid extraction procedure, uses Methyl Tertiary Butyl Ether (MTBE) to replace the common chloroform-based method, reduces toxicity, realizes pollution-free collection of two main liquid phases, and has the same extraction efficiency as the chloroform method. The invention adopts ultra-high performance liquid chromatography (UHPLC) to reduce analysis time. The data is acquired by adopting an ion mobility mass spectrometry method, the flow of the ion mobility is optimized, a positive ion mode and a negative ion mode are adopted, methanol and ethanol are used as mobile phases, interference signals are removed, and background interference is reduced, so that chemical classification is assisted. Meanwhile, the ion mobility technology can also distinguish isotacticity and isomerism. 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 ion mobility data, and combines the lipid molecular structure database to carry out a semi-automatic data processing flow to annotate chromatographic mass spectrum characteristics. The technical scheme of the invention effectively improves the limitation of the prior art for characterizing 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 in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.
Claims (4)
1. A method for mass spectrometry of lipid ion mobility of marine microorganisms, comprising the steps of:
lipid extraction: extracting a lipid sample from marine microorganisms to be detected by using methyl tertiary butyl ether;
And a data acquisition step: analyzing the lipid sample by utilizing an ion mobility mass spectrometry method and adopting a positive ion mode and a negative ion mode, methanol and ethanol are used as mobile phases, and acquiring data by adopting an inverse ultra-high performance liquid chromatography, wherein the data acquisition adopts a data independent acquisition mode;
And a data analysis step: firstly, constructing a series of lipid molecular structures, storing the lipid molecular structures as MOL files, inputting Progenesis SDF Studio the MOL files to generate SFD files to obtain a lipid molecular structure database, and annotating mass spectrum characteristics of lipid molecules in the marine microorganisms to be detected by utilizing the acquired data based on the assistance of the lipid molecular structure database; based on the mass spectrum characteristics of the obtained compound molecules, a fragmentation mass spectrum database and an ion mobility collision cross-sectional area database can be obtained by utilizing the lipid molecular structure database;
Both the methanol and the ethanol are modified by 0.1% ammonia water and 0.04% formic acid;
the annotation is performed using an automated data preprocessing program;
the data acquisition step specifically comprises the following steps:
Chromatographic separation: using AQUITY UPLC system equipped with ACE Excel 2 SuperC18 column, solvent A is 100% methanol, solvent B is 100% ethanol, and strong washing injection is 2-propanol; the linear gradient starts with 100% solvent a, after 4 minutes of hold, then increases to 50% solvent B at 10 minutes, further increases to 99% solvent B at 30 minutes, holds for another 4 minutes, returns to 100% solvent B at 34.1 minutes, and re-equilibrates for 2 minutes; the flow rate is 0.40 mL/min, the column temperature is kept at 45 ℃, and the temperature of the sample manager is kept at 7 ℃;
mass spectrometry: using WATERS SYNAPT G-Si system equipped with electrospray source, controlled by MassLynx software, data acquisition using HDMS or HDMS E in extended dynamic range mode, operating in resolution mode; wherein, the mass analyzer uses sodium iodide solution to calibrate the mass, and leucine-enkephalin is used as a chain solution;
The parameters of the lipid sample for analysis were:
Capillary voltages of 2.8 kV and 2.2 kV, positive and negative ion modes, respectively; the sample cone is 40V, and the source temperature is 120 ℃; cone gas is 50L/h, desolventizing gas is 600L/h, atomizer gas is 6.5 Bar; the DC bias of the catcher is 60V, and the DE outlet of the catcher is 3V; IMS wave speed is 500 m/s, wave height is 40V, transfer wave speed is 179 m/s, and wave height is 4V; the lipid sample is recombined in 150 μl of methanol; the lipid sample volume of the injection system was 10 μl; the scan time was 0.4 s; data were obtained in a continuous process from 50 Da to 2000 Da,3.5 min to 34 min.
2. The method for mass spectrometry of lipid ion mobility of marine microorganisms according to claim 1, wherein the lipid extraction step comprises:
capturing marine microorganisms to be detected on a polyvinylidene fluoride membrane;
secondly, placing the polyvinylidene fluoride film obtained in the first step in a clean test tube, adding methyl tertiary 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 centrifugally separating to obtain a water phase and an organic phase;
fourth, 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 steps, and combining all the collected organic phases;
Fifth, all the organic phases obtained in the fourth step are dried with a centrifugal concentrator, after which the dried lipid sample is stored or analyzed directly.
3. The method of claim 1, wherein after the lipid extraction step, further comprising: solvent removal and sample reorganization.
4. Use of a method for mass spectrometry of lipid ion mobility of marine microorganisms, characterized in that the method according to any one of claims 1 to 3 is applied to the lipid analysis of marine microorganisms.
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