阅读说明：本技术 一种基于13c代谢全位标记脂质结合同位素稀释质谱法的全脂质定量方法 (Based on13Full-lipid quantitative method of C metabolism full-position labeled lipid combined isotope dilution mass spectrometry ) 是由 魏芳 吴邦富 吕昕 陈洪 黄凤洪 于 2021-07-19 设计创作，主要内容包括：本发明公开了一种基于～(13)C代谢全位标记脂质作为内标结合同位素稀释质谱法的全脂质定量方法,该方法是利用生物代谢合成～(13)C代谢全位标记脂质,并结合同位素稀释质谱法建立的一种全脂质一对一绝对定量方法。其特征在于利用～(13)C全标记底物(如CO-(2)、葡萄糖或碳酸(>99%)等)作为唯一碳源加入到诸如微生物、细胞、植物和动物培养基或膳食等中,通过生物代谢过程制备大量高标记效率的～(13)C标记(全位标记)脂质；然后将这些标记脂质作为内标,结合同位素稀释质谱法对实际生物样本中脂质进行一对一的定量分析。本发明具有制备过程简单、可操作性强、定量准确性高、线性动态范围广等优点,同时解决了目前商业化同位素标记脂质标准品缺乏且价格昂贵的难题。(The invention discloses a method based on 13 A full-lipid quantitative method using C metabolism full-position labeled lipid as internal standard and combining isotope dilution mass spectrometry is disclosed, which uses biological metabolism synthesis 13 C metabolizes the whole labeled lipid, and combines the isotope dilution mass spectrometry to establish a whole lipid one-to-one absolute quantitative method. Which is characterized by utilizing 13 C fully-labelled substrate (e.g. CO) 2 Glucose or carbonic acid: (>99%), etc.) as a sole carbon source into media or meals such as microorganisms, cells, plants and animals, producing large quantities of a high labeling efficiency by a process of biological metabolism 13 C-labeled (all-position labeled) lipids; then, the labeled lipids are used as internal standards, and the lipids in the actual biological samples are subjected to one-to-one quantitative analysis by combining isotope dilution mass spectrometry. The invention has the advantages of simple preparation process, strong operability and accurate quantificationHigh performance, wide linear dynamic range and the like, and solves the problems of lack of commercial isotope labeled lipid standard products and high price at present.)

1. Based on¹³Full-lipid quantitative method using C metabolism full-position labeled lipid as internal standard and combining isotope dilution mass spectrometryIs characterized by utilizing¹³C full-mark substrate is used as a unique carbon source to be added into a biological system and is prepared through a biological metabolism process¹³C-labeled lipid; then the said¹³C-labeled lipid is used as an internal standard, and isotope dilution mass spectrometry is combined to perform one-to-one quantitative analysis on the lipid in the actual biological sample.

2. Based on¹³A full-lipid quantitative method of C metabolism full-position labeling lipid combined isotope dilution mass spectrometry is characterized by comprising the following steps:

1) will be provided with¹³C full-mark substrate as sole carbon source added into biological culture medium or diet for biological metabolism preparation¹³Labeling C-position with lipid, and extracting¹³C-holo-labeled lipids; at the same time contain¹²General media or dietary culture biological preparation of C carbon source¹²C lipids, and extracting¹²C lipids; the above-mentioned¹²C carbon source and¹³c all-labeled substrates are the same substance except that no labeling is carried out¹³C, marking;

2) subjecting the extract obtained in the step 1) to ultra performance liquid chromatography tandem mass spectrometry¹²C lipid,¹³C, carrying out lipidomics data acquisition on the full-position labeled lipid;

3) using database spectrogram matching and isotope theoretical bias pairs¹²C lipids and¹³c all-labeled lipid qualitative analysis and¹³c, calculating marking efficiency;

4) extracting lipid in an actual sample to be detected, and performing data acquisition and qualitative lipid analysis according to the steps 2) and 3) to obtain the type information of the lipid in the actual sample;

5) to be analyzed with the actual sample and¹³a standard substance of lipids present in all of the C-labeled lipids (the C element in the lipid standard substance is¹²C, subsequently called¹²C lipid standards) were configured into gradient standard solutions of different concentrations to¹³C all-bit labeled lipid is used as an internal standard and added into the gradient standard solution in equal quantity to establish¹²The concentration of C lipid standard is plotted on the abscissa¹²C lipid standards and corresponding¹³C, the isotope dilution mass spectrum calibration curve of each lipid class with the ratio of the peak areas of the extracted ion chromatogram of the all-site labeled lipid as the ordinate;

6) when the lipid in the actual sample is quantitatively analyzed, the same amount as that in step 4) is added¹³C all-site labeled lipid as internal standard, and corresponding target lipid¹³Substituting the peak area ratio of the C all-site labeled lipid internal standard into the isotope dilution mass spectrum calibration curve of the C all-site labeled lipid internal standard so as to obtain the absolute content of the target lipid in the actual sample.

3. A method according to claim 1 or 2, based on¹³A full-lipid quantitative method of C metabolism full-position labeling lipid combined isotope dilution mass spectrometry is characterized by utilizing¹³C full-label substrate as the sole carbon source to culture biological systems including microorganisms, cells, plants and animals, by the organism's own uptake of said substances¹³C full-labeled substrate metabolism is performed to replace the original C element, so that all lipids in a biological system are realized¹³And C, marking.

4. A method according to claim 1 or 2, based on¹³A full-lipid quantitative method of C-metabolism full-position labeling lipid combined isotope dilution mass spectrometry, characterized in that¹³C fully labeled substrate comprises CO₂Glucose or carbonate; lipids include glycerides, phospholipids, sphingolipids, glycolipids, fatty acids, sterol lipids, and various lipid metabolism intermediates.

5. A method according to claim 2¹³The full-lipid quantitative method of C metabolism full-position labeling lipid combined isotope dilution mass spectrometry is characterized in that in the step 3),¹²c the database used in the qualitative analysis of lipids is MS-DIAL built-in lipid database, the ion mass-to-charge ratio of the primary and secondary mass spectra: (m/z) The precision of the method is set to be +/-0.01 and +/-0.05 Da, and meanwhile, the secondary spectrogram matching scoring threshold is set to be 70-90%;

and, the results of the qualitative analysis are from the primary mass spectrum of the molecular ionsm/zAccuracy, retention time and isotope mode threeFurther screening is carried out on each dimension; wherein the content of the first and second substances,m/zscreening criteria for accuracy are error<5ppm, retention time screening criterion error<5% and the screening criteria for isotopic pattern is difference<10%。

6. A method according to claim 2¹³The full-lipid quantitative method of C metabolism full-position labeled lipid combined isotope dilution mass spectrometry is characterized in that the method in the step 3) is¹³Qualitative analysis of C-holo-labeled lipids mainly by mapping¹²C lipid retention time and¹³c introduction caused primary mass spectrum molecular ions and secondary mass spectrum fragment ionsm/zThe difference is subjected to orientation judgment whilem/zWithin 5 ppm.

7. A method according to claim 2¹³The full-lipid quantitative method of C metabolism full-position labeled lipid combined isotope dilution mass spectrometry is characterized in that the method in the step 3) is¹³The labeling efficiency of the C all-position labeled lipid is obtained by simulation calculation through isotope mode distribution simulation software, and the labeling efficiency is above 95%.

8. A method according to claim 2¹³The full-lipid quantitative method of C metabolism full-position labeling lipid combined isotope dilution mass spectrometry is characterized in that in the step 4),¹²c lipid standards and corresponding¹³When C all-position labeled lipid is extracted from ion chromatographic peak, analyte first-order molecular ions are selectedm/zExtracted ion chromatographic peaks corresponding to the monoisotopic peak and the fully-labeled isotopic peak.

9. A method according to claim 2¹³The full-lipid quantitative method of C metabolism full-position labeled lipid combined isotope dilution mass spectrometry is characterized in that in the step 4), the standard solution¹²The gradient concentration range of the C lipid standard covers the concentration of the target lipid in the actual sample in step 5).

Technical Field

The invention relates to the use of biological metabolic synthesis¹³C metabolism is completeA full-lipid one-to-one absolute quantitative method is established by site-labeling lipid and combining isotope dilution mass spectrometry, and belongs to the field of analysis and detection.

Technical Field

Lipids are a generic term for a complex class of compounds with hydrophobic or amphiphilic properties, comprising a number of different classes and subclasses (class/subclasses) classified as basic building blocks based on backbone structures, head groups or fatty acid acyl chains. As one of the most important biomolecules, lipids have various biological functions, such as major components constituting cell membranes, energy storage, and signal transduction involved in various physiological and biochemical processes of the body. Meanwhile, lipid metabolism disorder in organisms is closely related to various diseases and is a potential biomarker for various diseases such as inflammation, cancer, endocrine dyscrasia and the like. Therefore, analyzing the composition and content of lipids in biological samples and monitoring the lipid changes of living bodies in different physiological states or pathological processes are of great significance for understanding the biological functions of lipids and the occurrence and development of diseases.

Lipidomics analysis technology based on an advanced mass spectrometry platform is one of the mainstream technologies of lipid analysis at present, and can realize composition and structure analysis of hundreds of lipids in a biological sample. Depending on the purpose, lipidomics can be divided into targeted lipidomics to analyze a few classes of defined lipids and non-targeted lipidomics to analyze all potential lipid classes without bias. Currently, most non-targeted lipidomic analysis still stays at a relative quantitative level, i.e. only the relative peak intensity change of the target compound in the experimental group relative to the control group can be obtained, and the specific concentration of each lipid can not be determined. This severely hampers the definition of disease marker thresholds and the comparison and normalization of different laboratory analytical data in more practical clinical studies in lipidomics. The reason why the absolute quantification of non-targeted lipidomics is difficult is mainly that the number of non-targeted identification lipid types is large, the concentration range is wide, the mass spectrum response of different lipids is different, but the absolute quantification of each lipid by adding commercially purchased isotope-labeled lipid is expensive and unrealistic. Therefore, how to obtain as much isotopically labeled lipid as possible is critical. At present, the isotope-labeled lipid is obtained mainly by a chemical synthesis method, and a series of organic chemical synthesis reactions and separation and purification methods are designed to obtain the stable isotope-labeled lipid with high purity. However, this method is time consuming, labor intensive and expensive. Some problems can be solved to a certain extent by a method of introducing isotopic groups by chemical derivatization, but the chemical derivatization can only be performed on lipids containing special reactive groups, such as phospholipids, fatty acids, sterol esters and the like. And these methods are generally only suitable for targeted analysis.

Disclosure of Invention

The invention aims to solve the technical problem of providing a full-lipid absolute quantitative method which is established by taking metabolic all-bit labeled lipid as an internal standard and combining an isotope dilution mass spectrometry, and can realize one-to-one absolute quantitative analysis of lipid in a biological sample.

The technical scheme adopted by the invention for solving the problems is as follows:

based on¹³A full-lipid quantitative method of C-metabolism full-position labeled lipid combined Isotope Dilution Mass Spectrometry (IDMS) is characterized by utilizing¹³C full-mark substrate is used as a unique carbon source to be added into a biological system and is prepared through a biological metabolism process¹³C-labeled lipid; then the said¹³C-labeled lipid is used as an internal standard, and isotope dilution mass spectrometry is combined to perform one-to-one quantitative analysis on the lipid in the actual biological sample.

The invention is based on¹³A full-lipid quantitative method of C metabolism full-position labeling lipid combined isotope dilution mass spectrometry comprises the following steps:

1)¹³c-holo-labeled lipids and¹²c preparation of common lipids: will be provided with¹³C full-mark substrate is used as a unique carbon source to be added into a biological culture medium or diet, and the high-mark efficiency is prepared through a biological metabolism process¹³C-labeled (all-position labeled) lipid, and extracting¹³C-holo-labeled lipids; at the same time contain¹²General media or dietary culture biological preparation of C carbon source¹²C lipidAnd extracting¹²C lipids; the above-mentioned¹²C carbon source and¹³c all-labeled substrates are the same substance except that no labeling is carried out¹³C, marking;

2) and (3) collecting lipidomics data of the labeled biological samples: performing ultra performance liquid chromatography (UPLC-Q-TOF-MS) on the extracted substance in the step 1) by using an ultra performance liquid chromatography tandem time-of-flight mass spectrometry (UPLC-Q-TOF-MS)¹³C-holo-labeled lipid,¹²C, carrying out lipidomics data acquisition on common lipids;

3) lipid profiling and labeling efficiency calculation: using database spectrogram matching and isotope theoretical bias pairs¹²C common lipid and¹³c all-labeled lipid qualitative analysis and¹³c, calculating marking efficiency;

4) qualitative analysis of actual sample lipid to be detected: extracting lipid in the actual sample to be detected, and performing data acquisition and qualitative lipid analysis according to the steps 2) and 3) to obtain the type information of the lipid in the actual sample.

5) Establishing an IDMS calibration curve: to be analyzed with the actual sample and¹³a standard substance of lipids present in all of the C-labeled lipids (the C element in the lipid standard substance is¹²C, subsequently called¹²C lipid standards) were configured into gradient standard solutions of different concentrations to¹³C, adding the full-position labeled lipid serving as an internal standard into the gradient standard solution in equal quantity, and performing mass spectrum lipidomics data acquisition; then, in¹²The concentration of C lipid standard is plotted on the abscissa¹²C lipid standards and corresponding¹³C, establishing an IDMS calibration curve of each lipid class by taking the ratio of the peak areas of the extracted ion chromatograms of the all-bit labeled lipids as a vertical coordinate;

6) quantitative analysis of lipids in actual samples: when the lipid in the actual sample is quantitatively analyzed, the same amount as that in step 4) is added¹³C all-site labeled lipid as internal standard, and corresponding target lipid¹³Substituting the peak area ratio of the C all-bit labeled lipid internal standard into the IDMS calibration curve so as to obtain the absolute content of the target lipid in the actual sample.

Is pressed onThe above scheme is utilized¹³Culture biological systems using C-holo-labeled substrates as the sole carbon source include microorganisms, cells, plants, animals, and the like, particularly through the uptake of these by the organism itself¹³C fully-labelled substrate (e.g. CO)₂Glucose or carbonate, etc.) to replace the original C element, thereby realizing the metabolism of all lipids in the biological system¹³C label including glyceride, phospholipid, sphingolipid, glycolipid, fatty acid, sterol lipid and various lipid metabolism intermediate products.

According to the above scheme, the mass spectrum to be used needs to be a high resolution mass spectrum (such as Q-TOF-MS), and the Data acquisition mode is a Data Dependent Acquisition (DDA) or a SWATH (sequential dependent acquisition of all the electronic fragments). The mass spectrum used for data acquisition can also be other types of high resolution mass spectra, such as Orbitrap.

According to the scheme, in the step 1), the method for extracting the lipid is a liquid-liquid extraction method.

According to the scheme, in the step 2),¹²c common lipid and¹³the C all-position labeled lipid can be used for respectively and independently carrying out mass spectrum data acquisition and data analysis, and can also be mixed for carrying out. When the analysis is carried out in a mixed manner,¹²c common lipid and corresponding¹³The isotope peak distribution mode of the C-labeled lipid can form a characteristic symmetric peak in the same spectrogram, and can be used as a basis for qualitative judgment of the lipid, so that a qualitative false positive result caused by introducing impurities or instrument signal residues in the pretreatment process is avoided.

According to the scheme, in the step 3), the¹²C, during the qualitative analysis of common lipid, the used database is an MS-DIAL built-in LipidPlast lipid database, the m/z precision of primary Mass Spectrum (MS) molecular ions and secondary mass spectrum (MS/MS) fragment ions is set to be +/-0.01 and +/-0.05 Da, and meanwhile, the spectrogram matching scoring threshold is set to be 70-90%. According to the precision and the spectrogram matching scoring threshold value, the accuracy (error) of the primary molecular ion m/z is further determined<5ppm), retention time (error)<5%) and isotopic pattern (differences)<10%) manual screening in three dimensions to improve characterizationAccuracy, avoiding false positive results.

According to the scheme, in the step 3),¹³qualitative analysis of C-holo-labeled lipids mainly by mapping¹²C common lipid retention time and¹³c, performing directional judgment on the m/z difference between the primary mass spectrum molecular ions and the secondary mass spectrum fragment ions caused by introduction, wherein the m/z deviation is within 5 ppm.¹²C common lipid and¹³c all-bit labeled lipid exists in one-to-one correspondence relationship, and¹³the presence of C-holo-labeled lipids may assist¹²And C, judging a qualitative result in the common lipid sample, and eliminating a false positive result. At the same time, the user can select the desired position,¹²c should not be present in the common lipid sample¹³C-holo-labeled lipids.

According to the above scheme, in step 3), for¹³When the C-holo-labeled lipid is qualitatively analyzed, the reason is that¹³After C labeling, both the MS and MS/MS fragment ions m/z of lipids changed, while the MS-DIAL built-in lipidosome database or other commercial databases only contain common lipid information, and therefore cannot be used alone¹³C-labeled samples that were subjected to lipid database matching. In this case, the present invention utilizes the lipids of the biological sample obtained and characterized under normal culture conditions (i.e., the lipids of the biological sample¹²C common lipid), by subjecting it to¹³C-labeled lipids are subjected to m/z calculations for the corresponding primary and secondary ions, i.e., one for each additional¹³C isotope atom, the m/z of which is increased 1.003355 correspondingly, thereby obtaining¹³Theoretical information of the primary and secondary fragment ions m/z of the lipid after C-holo-labeling. Combining information such as retention time and the like, and then utilizing analysis software PeakView to carry out analysis on the information¹³C-labeled lipids for search, and the method¹³Qualitative judgment of C-labeled lipid, according to the distribution mode of lipid MS isotope, using IsoPro 3.0 software to perform simulation calculation¹³C labeling efficiency.

According to the scheme, in the step 3),¹³the labeling efficiency of the C all-position labeled lipid is obtained by simulation calculation through isotope pattern distribution simulation software IsoPro, and the labeling efficiency is above 95%.

According to the scheme, in the step 4),¹²the C lipid standard must be a standardAnalyzing the actual sample and¹³c-holo-site labeling of lipids coexisting in the lipids, and therefore, metabolic labeling targets can be reasonably selected according to the lipid coverage in the actual analysis sample. When there is no corresponding target lipid to be quantified in the actual analysis sample¹³And C, taking the full-labeled lipid as an internal standard, and selecting the full-labeled lipid with the most similar structure and capable of basically realizing co-elution as the internal standard.

According to the scheme, in the step 4),¹²c lipid standards and corresponding¹³When C all-position labeled lipid is used for extracting ion chromatographic peaks, analyte primary mass spectrum molecular ions m/z are selected to correspond to monoisotopic peaks m₀The extracted ion chromatographic peak of (1).

According to the above scheme, in step 4), the standard solution¹²The gradient concentration range of the C lipid standard covers the concentration of the target lipid in the actual sample in step 5). In general, the concentration range of each lipid standard is preferably set to 0.001 to 500. mu.g/mL. The lipid standard may be prepared as a gradient solution alone or in combination, and preferably prepared in a chloroform/methanol-2: 1(v/v) solvent.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention obtains a large amount of the products by a biological metabolism marking method¹³C all-position labeled lipid, high labeling efficiency, simple preparation process, strong operability, lower cost than chemical synthesis and wider lipid coverage, and solves the problems of lack of commercial isotope labeled lipid standard products and high price at present;

2. the hair is prepared¹³The C all-bit labeled lipid is used as an internal standard, and the isotope dilution mass spectrometry which is a well-known quantitative method is combined, so that one-to-one quantitative analysis of the lipid in the sample can be realized, the quantitative accuracy is high, and the limitation that the lipid internal standard is lacked or an additional chemical derivation process is needed in the traditional lipid quantitative analysis is effectively overcome.

Drawings

FIG. 1: general (A) and¹³c, a schematic flow chart of qualitative analysis of lipid in the labeled sample (B);

FIG. 2: general and¹³c labelSecondary mass spectrum of marking lipid (DGDG 34: 3);

FIG. 3: calculation Using IsoPro¹³The labeling efficiency of C metabolic marker lipids (DGDG 34:3 and MGDG 34: 4);

FIG. 4: schematic experimental and analytical flow diagrams based on an ID-MS lipid absolute quantification method;

FIG. 5: lipid linear equation and linear correlation comparison thereof: based on¹²Area of C peak and based on¹²C and¹³c peak area ratio (A and B) SQDG 34: 3; (C and D) MGDG 34: 6; (E and F) DGDGDG 36: 0;

FIG. 6: phaeodactylum tricornutum cultured under different conditions (normal, nitrogen-deficient, phosphorus-deficient) contains 11 different lipid contents.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.

In the present invention are concerned with¹³The C metabolic marker cultured organism can adopt:

1) yarrowia lipolytica (Yarrowia lipolytica). As an unconventional yeast, yarrowia lipolytica can utilize various cheap substances as substrates required by growth, synthesize various nutrient substances required by self life activities in a large amount and generate various metabolites, and has the characteristics of high propagation speed, large biomass and rich grease.

After the strain seed liquid is cultured to a logarithmic phase by an LB culture medium, repeatedly cleaning and centrifuging for several times under an aseptic condition, then re-suspending, inoculating into a synthetic culture medium according to one thousandth of the volume of the culture medium, after the strain seed liquid is cultured to a stable phase, centrifuging to remove the culture liquid, and performing vacuum freeze drying to obtain thalli.

Composition of the synthetic medium: adding 5g glucose (common or common) into 1L deionized water¹³C fully labeled glucose), 5g (NH)₄)₂SO₄、3g KH₂PO₄、0.5g MgSO₄·7H₂O, 15mg EDTA, 1mL Trace Salt solution and 100. mu.L Vitamin solution. Wherein the volume of the Trace salt solution: every 100mL of the solution contains 45mg of ZnSO₄·7H₂O、3mg CoCl₂·6H₂O、 10mg MnCl₂·4H₂O、3mg CuSO₄·5H₂O、45mg CaCl₂·2H₂O、30mg FeSO₄·7H₂O、4mg NaMoO₄·2H₂O、10mg H₃BO₄1mg KI. Vitamin solution: each 50mL of the solution contains 2.5mg of biotin, 50mg of calcium pantothenate, 50mg of nicotinic acid, 1250mg of inositol, 50mg of thiamine nicotinate, 50mg of pyridine hydrochloride, and 50mg of p-aminobenzoic acid. The dissolved medium was adjusted to pH 6 with 6M HCl solution and KOH solution. Sterilizing at 121 deg.C for 20min, sterilizing glucose alone at 115 deg.C, sterilizing Trace salt solution and Vitamin solution with 0.22 μm filter, and mixing with the main solution.

2) Spirulina platensis (Spirulina platensis). As a marine plant of blue algae which performs photosynthesis, Spirulina platensis can synthesize various lipids including glycolipids in large amounts using simple nutrients.

Culturing the spirulina platensis seed liquid with an f/2 culture medium to a logarithmic phase, repeatedly cleaning and centrifuging for several times under an aseptic condition, then resuspending, inoculating into a fresh f/2 culture medium according to one thousandth of the volume of the culture medium, standing and culturing to a stable phase, centrifuging to remove the culture liquid, and performing vacuum freeze drying to obtain thalli.

f/2 culture medium: adding NaNO 0.075g into artificial seawater per 1L₃、0.057g NaH₂PO₄·2H₂O、0.03g Na₂SiO₃·9H₂O, 1mL of trace element storage solution and 1mL of vitamin storage solution. Wherein the artificial seawater comprises: 20.758g (2.07%) NaCl, 0.587g (0.06%) KCl, 0.17g NaHCO were added to 1L deionized water₃(general or¹³C full mark NaHCO₃)、0.0746g NaBr、0.0225g H₃BO₃、0.0027g NaF、5.5g(0.55％)Na₂SO₄、 9.395g(0.94％)MgCl₂·6H₂O、1.316g CaCl₂·2H₂O、0.0214g SrCl₂·6H₂And O, dissolving, and sterilizing at 121 ℃ for 20min for later use. Storage solution of trace elements: 9.8mg of CuSO₄·5H₂O、22mg ZnSO₄·7H₂O、 10mg CoCl₂·6H₂O、0.18g MnCl₂·4H₂O、6.3mg Na₂MoO₄·2H₂O、4.36g Na₂EDTA、3.15g FeCl₃·6H₂O、1L H₂And O, filtering and sterilizing. Vitamin storage solution prepared by dissolving 1mg biotin and 1mg vitamin B in 1L deionized water₁₂200mg of vitamin B₁And then, the mixture was filtered and sterilized by a 0.22 μm filter.

The invention is as described¹³The C metabolic marker is not limited to the above two, and others are administered by ingestion¹³C fully marking substrate or nutrient component and then replacing original by biological metabolism¹²C, organisms such as cells, plants or animals may be suitable for use in the present invention.

Neutralization of common biological samples according to the invention¹³The method for extracting the lipid in the C metabolism marker biological sample is a liquid-liquid extraction method, and comprises the following extraction methods and processes: referring to the study by Bligh and Dyer et al (Dyer,1959), the lipid extraction of biological samples was performed as follows: grinding the freeze-dried sample into fine powder by using a mortar, weighing about 10 mg in a 12mL glass threaded tube, adding 1mL deionized water, adding 2.5mL methanol, vortex, uniformly mixing for several minutes, and placing in a refrigerator at-20 ℃ overnight to precipitate the protein. After taking out every other day, 1.25mL of chloroform was added, and after vortexing for 10min, 1.25mL of chloroform was added, and vortexing for 10 min. Then 1.25mL of deionized water was added, vortexed for 10min, and centrifuged at 5000 rpm/min at 4 ℃ for 20 min. The body of the threaded tube was then tilted and the tube was carefully pipetted down into a clean glass tube, taking care not to puncture the middle protein layer. Then adding 2mL of chloroform, repeating the extraction process, combining the supernatants, drying with nitrogen, redissolving with 1mL of chloroform/methanol (1:1) solution, filtering with 0.22 μm organic phase filter membrane, and placing in a sample injection vial for loading.

The present invention relates to the following instrument analysis conditions, but not limited thereto, as long as the liquid chromatography tandem high resolution mass spectrometry method capable of realizing non-targeted lipid analysis in a biological sample can be used.

The instrument used was the UPLC 30A system (Shimadzu Corporation, Japan) tandem TripleTOF 6600 system (AB SCIEX, USA). Liquid phase separation conditions: a chromatographic column: kinetex C18 column (100 mm. times.2.1 mm,2.6 μm) (Phenomenex, USA), and precolumn Security Guard precolumn (Phenomenex, USA) with the same core material; mobile phase: phase A: methanol/acetonitrile/water (1:1:1, V/V/V); phase B: isopropanol/acetonitrile (5:1, V/V), both containing 5mmol/L ammonium acetate; gradient elution procedure is 0-0.5min, 80% phase A; 0.5-1.5min, 60% phase A; 1.5-3 min, 40% phase A; 3-13min, 2% of phase A; 13-13.1min, 80% phase A; 13.1-17min, 80% phase A. Mass spectrometry conditions: the data acquisition mode performed in positive electrospray ionization (ESI +) and negative electrospray ionization (ESI-) modes is a data-dependent acquisition mode (DDA). The external calibration input system (CDS) automatically calibrates the mass spectral mass accuracy every 8 samples. In positive ion mode, the MS parameters are: the cluster removing voltage is set to be 80V, the collision energy is set to be 30V, the ion jet voltage is set to be +5500V, and the mass range is 50-1200 m/z. Gas 1 and gas 2 of the ion source were set at 50 psi. Curtain gas was set at 35psi, interface heater temperature was set at 600 ℃; in the negative ion mode, the declustering voltage is set to-80V, the collision energy is set to-30V, the ion jet voltage is set to-4500V, and the mass range is 50-1200 m/z. The data acquisition Software was AB SCIEX analysis TF 1.7Software (AB SCIEX, USA).

After mass spectrum data acquisition, data analysis is carried out on secondary spectrogram matching through an MS-DIAL built-in database LipidMass, and meanwhile, the m/z precision of primary mass spectrum molecular ions and secondary mass spectrum fragment ions is +/-0.01 and +/-0.05 Da. The qualitative accuracy score was set to 80%. To improve the confidence of identification and reduce false positive results, the m/z accuracy (error) of primary molecular ions is improved by using PeakView<5ppm), retention time (error)<5%) and isotopic pattern (differences)<10%) was performed on the identified lipids in MS-DIAL for data human evaluation and further validation and screening of the identified lipids in MS-DIAL.¹³C-labeled lipid passage¹²C common lipid retention time (C)¹³C-labeled lipids and conjugates¹²C retention time of common lipids) and¹³MS and MS/MS ion m/z difference due to C introduction (¹³The difference M/z between MS and MS/M ions resulting from C introduction is 1.003355n, n represents a lipidMedium carbon number) and the deviation of m/z of the molecular ions of the primary mass spectrum is within 5 ppm.¹³The labeling efficiency of the C-holo-labeled lipid was calculated by simulation using the isotopic pattern distribution simulation software IsoPro.

The lipid quantification method related to the present invention is isotope dilution mass spectrometry, gold standard and measurement framework established according to the bioanalytical method verification guidelines of the U.S. Food & Drug Administration (FDA) or European Medicines Administration (EMA), and isotope dilution mass spectrometry based on multi-external standard calibration of matrix matching and an internal standard method is the quantification method with the highest measurement grade in mass spectrometry-based analysis. Isotope dilution mass spectrometry is a method in which an isotope element of a known mass is added as a diluent to an analysis element, and the difference in isotope abundance before and after mixing is analyzed by mass spectrometry, thereby calculating the concentration of the element in an analysis sample. For lipidomics absolute quantitative analysis, isotope-labeled lipid can be used as an internal standard and added into an analysis sample to obtain the response ratio of target analysis lipid and isotope-labeled lipid, meanwhile, a series of gradient solutions are prepared in a linear dynamic range by using a common lipid standard, isotope-labeled lipid with equal mass is added, an external calibration curve is established according to the response ratio and the concentration ratio of the two, and the absolute concentration of the lipid in the sample can be calculated by substituting the response ratio obtained from the analysis sample into the calibration curve. When the amounts of the isotope-labeled lipids added to the sample and the external standard solution are the same, even the isotope-labeled lipids with uncertain concentrations can be used as the internal standard, and in this case, the concentration ratio only needs to be changed into the external standard concentration.

The quantitative analysis process of isotope dilution mass spectrometry of the present invention is as follows:

1) preparing a standard mixed mother solution and a mixed gradient solution: taking each kind of standard mother liquor with a proper volume, preparing a standard series gradient standard mixed mother liquor solution, wherein the specific concentration of each kind of lipid is as follows: 0.0001. mu.g/mL, 0.001. mu.g/mL, 0.005. mu.g/mL, 0.01. mu.g/mL, 0.05. mu.g/mL, 0.1. mu.g/mL, 0.5. mu.g/mL, 1. mu.g/mL, 10. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL, and 500. mu.g/mL. The lipid standard is prepared by using a chloroform/methanol-2: 1(v/v) solvent.

2) Taking several clean sample vials, adding into each vial¹³C metabolizing 20 mu L of the whole-position labeled lipid extract, respectively adding 100 mu L of standard substance mixed gradient solution with different concentrations after nitrogen drying, and repeating each gradient for three times. And then, the mixture is swirled, mixed uniformly and transferred to a lining pipe to be arranged on a machine.

3) Drawing an IDMS calibration curve: the abscissa of the IDMS calibration curve is¹²Concentration of C Standard (μ g/mL), ordinate is¹²C lipid standards and corresponding¹³C ratio of extracted ion chromatographic peak areas of the whole labeled lipid, where the analyte molecular ion is selected to correspond to m₀(natural monoisotopic peak and all-position labeled isotopic peak) m/z extracted ion chromatographic peak. The effectiveness of the analysis method is evaluated by measuring the linearity, limit of detection (LOD), limit of quantitation (LOQ), etc. of each standard according to the united states Food and Drug Administration (FDA) bioanalysis guidelines. The signal-to-noise ratios of 3:1 and 10:1 are used to estimate LODs and LOQs, respectively, while the correlation coefficient (R) is used²) As a linear judgment basis. For quantitative analysis of lipid in actual sample, only equal amount of lipid is added¹³C, marking the lipid in a whole position, obtaining the ratio of corresponding peak areas, and inputting the ratio into an IDMS standard curve to obtain the concentration of the lipid in an actual sample.

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, steps or conditions of the present invention may be made without departing from the spirit and substance of the invention.

Example 1

Based on¹³The glycolipid quantitative method of C metabolism holosite labeling lipid combined isotope dilution mass spectrometry comprises the following steps:

1)¹³preparation of C-metabolic all-position labeled glycolipids: to be provided with¹³C full mark NaHCO₃Culturing Spirulina with the above culture conditions and flow as the only carbon source, centrifuging after a stabilization period, freeze drying, and extracting by liquid-liquid extraction method¹³All position of CLabeling glycolipids. And is as follows¹²C substrate NaHCO₃The lipid was extracted by culturing spirulina cells as a control (the culture conditions were the same).

2) Qualitative analysis of spirulina lipid and¹³c, marking efficiency calculation: will be a general sum¹³Respectively and independently performing mass spectrum lipidomics data acquisition on the C all-position labeled spirulina lipid samples, and performing qualitative analysis and labeling calculation according to the analysis process and the labeling calculation method¹³C, calculating labeling efficiency.

TABLE 1¹³C-labeled Spirulina medium glycolipid information and labeling rate thereof

As can be seen from the data in Table 1, 15 SQDGs, 12 DGDGs and 22 MGDGs in Spirulina cells were labeled¹³The labeling efficiency of C is between 97.5 and 98.6 percent. These results indicate that metabolically labeled lipids can serve as ideal internal standards for lipid quantification, and can solve the problem of the current lack of isotopically labeled glycolipid commercial standards.

3) And (3) preparing a glycolipid standard substance mixed mother liquor: taking glycolipid standard substance to prepare 500 mu g/mL of mixed standard substance mother solution, and diluting to obtain a gradient concentration solution: 0.0001. mu.g/mL, 0.001. mu.g/mL, 0.005. mu.g/mL, 0.01. mu.g/mL, 0.05. mu.g/mL, 0.1. mu.g/mL, 0.5. mu.g/mL, 1. mu.g/mL, 10. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL. The lipid standard is prepared by using a chloroform/methanol-2: 1(v/v) solvent.

Taking clean sample injection vials, and adding each vial¹³C metabolizing 20 mu L of the whole-position labeled lipid extract, adding 100 mu L of standard substance mixed gradient solution after nitrogen drying, and repeating each gradient for three times. And then, the mixture is swirled, mixed uniformly and transferred to a lining pipe to be arranged on a machine.

4) Drawing an IDMS calibration curve: the abscissa of the IDMS calibration curve is¹²Concentration of C Standard (. mu.s)g/mL) of the ordinate¹²C lipid standards and corresponding¹³C ratio of extracted ion chromatographic peak areas of the whole labeled lipid, where the analyte molecular ion is selected to correspond to m₀(natural monoisotopic peak and all-position labeled isotopic peak). Using a correlation coefficient (R)²) As a linear judgment basis. The results are shown in Table 2.

TABLE 2 utilization of¹²Area of C peak and¹²C/¹³linear equation and linear correlation comparison of glycolipid quantification method established by C peak area ratio

As can be seen from Table 2, the glycolipids are based on¹²Linear coefficient of C common lipid peak area calibration curve¹³The C-labeled lipid is low before calibration, and the linear equation curve is shown in FIG. 5, so that when the lipid concentration gradually rises to a certain value, the mass spectrum response peak area gradually tends to be saturated and does not linearly increase any more, which is probably because the interaction and aggregation among lipid ions are enhanced at a high concentration, and an inhibition effect exists, so that the mass spectrum response is influenced. Through¹³After the peak area of the C-labeled lipid is calibrated, the linear coefficient of a calibration curve is obviously improved, about 0.99 can be still achieved in a high-concentration investigation range, and the quantitative accuracy and the precision are obviously improved.

Example 2

Based on¹³The quantitative analysis of the lipid contained in phaeodactylum tricornutum by the C metabolism holohedral labeling lipid combined isotope dilution mass spectrometry method comprises the following steps:

1)¹³preparation and analysis of C-metabolism holo-labeled lipids: are respectively provided with¹³C Total glucose and NaHCO₃Culturing yarrowia lipolytica and Spirulina as the sole carbon source under the above culture conditions and process, centrifuging after a stabilization period, freeze drying, and extracting by liquid-liquid extraction¹³C-holo-labeled lipids. And is as follows¹²C carbon source culture medium for culturing yarrowia lipolyticaYeast and Spirulina thallus extracted lipid as control (the control group only differs by adopting common glucose and NaHCO respectively₃As the sole carbon source). Will be a general sum¹³Carrying out mass spectrum lipidomics data acquisition on the whole-bit labeled lipid samples independently or after mixing, and carrying out qualitative analysis and labeling calculation according to the analysis process and the labeling calculation method¹³The efficiency of labeling C was calculated and the results are shown in Table 3 and Table 1.

TABLE 3¹³Information on major lipid classes in C-labeled yarrowia lipolytica and their labeling rates

Thus, in yarrowia lipolytica, a coexisting common sum was identified¹³C marks lipid totally 18 types, 288 types, and most of the lipid has the marking efficiency of more than 99 percent. Can be used as a good internal standard for the quantification of common lipids.

2) Preparing a lipid standard substance mixed mother liquor: taking TAG (16:0-18:1-16:0), TAG (18:2-18:2-18:2), TAG (20:1-20:1-20:1), DAG (16:0-18:1), DAG (18:1-18:1), LPC 18:1, LPE 16:0, PC (16:0-18:1), PC (18:1-18:1), PE (16:0-18:1), PE (18:1-18:1), PG (16:0-18:1), PG (18:1-18:1), PS (16:0-18:1), PS (18:1-18:1), PI (16:0-18:1), PI (18:1-18:1), PA (16:0-18:1), PA (18:0-18:1), (16:0-18:1), PE (18:1-18:1), Cer (d18:1/16:0), Cer (d18:1/17:0), FA 18:2 and FA 22:1 were prepared as mother liquors of mixed standards at a concentration of 100. mu.g/mL each other except PI (10. mu.g/mL) and diluted to: gradient solutions of 0.0001. mu.g/mL, 0.001. mu.g/mL, 0.005. mu.g/mL, 0.01. mu.g/mL, 0.05. mu.g/mL, 0.1. mu.g/mL, 0.5. mu.g/mL, 1. mu.g/mL, 10. mu.g/mL, 50. mu.g/mL. Each gradient was repeated three times. And then, the mixture is swirled, mixed uniformly and transferred to a lining pipe to be arranged on a machine. Wherein the lipid standard substance is prepared by using chloroform/methanol-2: 1(v/v) solvent.

3) Drawing an IDMS calibration curve: the abscissa of the IDMS calibration curve is¹²Concentration of C Standard (μ g/mL), ordinate is¹²C standard and corresponding¹³C all-position labeled lipid extracted ion chromatographic peakArea ratio, where analyte molecular ion is selected to correspond to m₀(natural monoisotopic peak and all-position labeled isotopic peak) m/z extracted ion chromatographic peak. Using a correlation coefficient (R)²) As a linear judgment basis. The results are shown in Table 4.

TABLE 4 utilization of¹²Area of C peak and¹²C/¹³linear equation and linear correlation comparison of various lipid quantification methods established by C peak area ratio

It can be seen that in the process of passing¹³After the peak area of the C-labeled lipid is calibrated, the correlation coefficient of a linear equation of almost all lipids is obviously improved from about 0.90 or even below to more than 0.99, the linear range of quantitative analysis of the lipids is improved, more accurate quantification is provided for the quantitative analysis of high-content lipids in a sample, and meanwhile, the analysis of low-concentration lipids is not influenced.

4) Obtaining the Phaeodactylum tricornutum thallus in normal and phosphorus/nitrogen-deficient culture: inoculating 8mL (inoculum size 1%) of Phaeodactylum tricornutum strain liquid cultured to logarithmic phase into a conical flask containing 800mL of f/2 culture medium, ventilating at 22 deg.C with air pump, and culturing at 50 μmol phosns m^-2·s^-1Culturing for about 7 days under the conditions of light intensity and light and dark cycle of 12h/12h (the algae liquid is dark brown), and starting to culture in the absence of phosphorus and nitrogen. Centrifuging at 4 deg.C and 9000rpm/min for 10min to obtain thallus, removing original bacteria liquid, collecting thallus part as normal culture Phaeodactylum tricornutum thallus, and adding phosphorus/nitrogen f/2 culture medium (without adding Na) into the rest thallus₂PO₃Salt or NaNO₃Salt) and then the nitrogen/phosphorus elements caused by the original culture medium are removed as much as possible before the heavy suspension. Finally, the cells were further cultured for approximately 5 to 6 days according to the previous conditions, centrifuged and collected, and stored at-20 ℃.

5) Mixing the obtained in 1)¹³Adding C-labeled glycolipid and common lipid as internal standard into normal and phosphorus/nitrogen-deficient cultured Phaeodactylum tricornutum thallus, extracting lipid by liquid-liquid extraction method, and using the obtained extractAll-lipidome qualitative and quantitative analysis was performed on IDMS standard curves for each lipid class. To verify the accuracy of the quantification method, an internal deuterated lipid standard of known concentration for routine quantification was added to the assay sample. The composition is as follows: 15:0-18:1-d7-PE, 15:0-18:1-d7-PS, 15:0-18:1-d 7-PG, 15:0-18:1-d7-PI, 15:0-18:1-d7-PA, 18:1(d7) Lyso PE, 15:0-18:1-d7-PC, 18:1(d7) Lyso PC, d18:1-18:1(d9) SM, 15:0-18:1-d7 DG, 18:1(d7) Chol Ester, 15:0-18:1(d7) -15:0, 18:1(d7) MAG, C15 Ceramide-d7(d18:1-d7/15:0), C16:0-d 4-FA. The actual concentrations were compared with the concentrations calculated using this method, and the results are shown in table 5.

TABLE 5 quantitative method accuracy verification results

From the results in Table 5, it is clear that¹³The C metabolism holohedral labeled lipid is used as an internal standard and combined with isotope dilution mass spectrometry to ensure that the measured concentration and the real concentration in the quantitative analysis of the lipid in the actual sample are very close, which indicates that the lipid concentration in the actual sample obtained by the quantitative method is real and reliable. The invention carries out quantitative analysis on 436 lipids including 18 (sub) classes, mainly glyceride (DAG and TAG), phospholipid (LPC, PC, PA, PE, PG, PI and CL), glycolipid (MGDG, SQDG, DGGA, DGDGDG and LDGTS), Cer, FFA and ASG of the phaeodactylum tricornutum thallus cultured normally and in a phosphorus/nitrogen deficiency way according to the quantitative method, and provides a good method and a good tool for accurate kinetic modeling analysis of the metabolic process of an organism in quantitative biology.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, many modifications and changes can be made without departing from the inventive concept of the present invention, and these modifications and changes are within the protection scope of the present invention.