CN102262139A - Method for finding and identifying lipid biomarkers of unicellular algae - Google Patents
Method for finding and identifying lipid biomarkers of unicellular algae Download PDFInfo
- Publication number
- CN102262139A CN102262139A CN2011101085944A CN201110108594A CN102262139A CN 102262139 A CN102262139 A CN 102262139A CN 2011101085944 A CN2011101085944 A CN 2011101085944A CN 201110108594 A CN201110108594 A CN 201110108594A CN 102262139 A CN102262139 A CN 102262139A
- Authority
- CN
- China
- Prior art keywords
- lipid
- analysis
- data
- opls
- biomarker
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a method for finding and identifying lipid biomarkers of unicellular algae, which comprises the following steps of: (1) extracting intra-cellular lipid compounds, and carrying out oxidation resistant treatment; (2) extracting lipid compounds by ultra performance liquid chromatography/quadrupole-time-of-flight mass spectrometer (UPLC/Q-TOF-MS) analysis; (3) extracting the analytical data by using Markerlynx software, and leading the analytical data into Simca-p software to carry out orthogonal partial least square discriminatory analysis (OPLS-DA); (4) verifying an OPLS-DA model; (5) screening out potential lipid biomarkers according to the verification result of the OPLS-DA model; and (6) carrying out structure judgment and identification on the potential lipid biomarkers. The universal technical system for finding and identifying lipid biomarkers of unicellular algae, which is established by the invention, can be radially applied to the fields of vegetal and microbial metabolomics analysis, food safety metabolomics analysis and the like.
Description
Technical field
The invention belongs to the bioanalysis chemical field, relate to a kind of method of finding and identifying single-cell algae lipoids biomarker.
Background technology
Lipid compounds all is a micromolecular compound, and they all have close physics and chemical property, and their existence and abundance are most important for important physiological activities such as cellular metabolism regulation and control, energy control, signal conduction.By studying the total fat extract that from unicellular algae, obtains, can obtain the information (lipid metabolism spectrum) of lipid metabolism thing group (lipidome), the composition that has comprised all lipid materials in the cell, interact and function information, it has reflected the overall variation of unicellular alga cells lactones metabolin under specific physiological status, can understand the change that cell is subjected to external interference caused by factors lipid-metabolism path and network in more detail, find and identify the lipid biomarker, can determine to have the bioanalysis of key effect, and help to infer the regulation and control model of crucial metabolic pathway.
The analysis of lipid metabolism thing spectrum requires high sensitivity, high flux and does not have the analytical approach of skewed popularity.Because the complicacy of lipid metabolism thing and living things system up to now, does not still have a kind of metabolism group analytical technology that can satisfy above-mentioned all requirements.Existing nuclear magnetic resonance technique (NMR) possesses fast and the characteristics of no skewed popularity, but its sensitivity is too low.Chromatogram/mass spectrometric hyphenated technique combines the separating power and the mass spectral qualitative function of chromatogram, realizes complex mixture qualitative and quantitative analysis has more accurately also been simplified the pre-treatment process of sample.Gas chromatography (GC-MS) sensitivity is higher, but its research range only limits to analyze volatile material, can't analyze the bigger metabolic product of thermal instability and molecular weight.This defective just in time can be remedied by liquid chromatography/mass spectrometry coupling (LC-MS) technology.Therefore, LC-MS makes the new efficient with uncommon aliphatic compounds and fatty acid (comprising biological membrane key component, lipid signaling molecule) of quick discovery, Sensitive Detection and confirmation obviously improve.Ultra Performance Liquid Chromatography (UPLC) coupling level Four bar time-of-flight mass spectrometry (Q-TOF), both couplings are suitable for the compartment analysis of complex system and the structure of unknown material is identified.
The recognition methods of sample somatotype common pattern is promptly set up the multivariate statistics model based on detected metabolin information, characterizes the relation between the sample on the one hand, screens the potential mark that sample classification is had significant contribution on the other hand.Existing common pattern recognition methods is principal component analysis (PCA) (PCA) and partial least squares discriminant analysis (PLS-DA).Principal component analysis (PCA) (PCA) is a kind of existing no teacher's supervised recognition method commonly used, can describe the difference of sample room intuitively on hyperspace.In pca model, the difference between two groups is not too obvious, and model only can be explained the part raw data.Partial least squares discriminant analysis (PLS-DA) discriminating power is better than PCA, and quadrature offset minimum binary differentiation (OPLS-DA) is a kind of partial least squares discriminant analysis that this advances, and its analytical effect more has superiority than conventional PLS-DA.
Summary of the invention
The objective of the invention is to overcome the prior art above shortcomings, a kind of method of finding and identifying single-cell algae lipoids biomarker is provided.
Technical scheme of the present invention is as follows:
Find and identify the method for single-cell algae lipoids biomarker, its feature comprises the steps:
(1) extraction of unicellular algae lipid within endothelial cells compound and anti-oxidation protection; (2) Ultra Performance Liquid Chromatography/level Four bar-flight time mass spectrum (UPLC/Q-TOF-MS) is analyzed lipid compounds; (3) with Markerlynx extraction and analysis data and import to and carry out quadrature partial least squares discriminant analysis (OPLS-DA) in the Simca-p software; (4) the OPLS-DA model is verified that (5) filter out potential lipid biomarker by the result of OPLS-DA model; (6) potential lipid biomarker is carried out structure deduction and evaluation.
In the above-mentioned method, extract single-cell algae lipid within endothelial cells compound, add antioxidant 2,6 di tert butyl 4 methyl phenol (BHT) in the extract with methyl alcohol-methenyl choloride-aqueous extract.Wherein the volume ratio of extract methyl alcohol, methenyl choloride, water is 1: 1: 0.5, and antioxidant 2,6 di tert butyl 4 methyl phenol (BHT) is 0.01%-0.05%g/mi with the mass volume ratio of extract.Sample is with after extract mixes, and behind oscillator concussion 5-10min, takes off a layer solution at 4-10 ℃ after centrifugal down, dries up with nitrogen, uses 0.5-1ml chromatogram dissolve with methanol again; Described sample is the single-cell algae powder.
In the above-mentioned method, the lipid compounds that extracts in the single-cell algae in the step (1) adopts Ultra Performance Liquid Chromatography and level Four bar time-of-flight mass spectrometry instrument to measure.
In the above-mentioned method, adopt Ultra Performance Liquid Chromatography and level Four bar time-of-flight mass spectrometry instrument to analyze lipid compounds in the step (2), in the Ultra Performance Liquid Chromatography method, mobile phase A is the mixed liquor of water, isopropyl alcohol and formic acid, wherein the volume ratio of water, isopropyl alcohol is 95/5～70/30, and the content of formic acid is always 0.1% (v/v) of mobile phase A; Mobile phase B is the mixed liquor of acetonitrile, isopropyl alcohol and formic acid, and wherein the volume ratio of acetonitrile, isopropyl alcohol is 95/5～70/30, and the content of formic acid is always 0.1% (v/v) of Mobile phase B; Chromatographic column temperature is 35 ℃-40 ℃, and the sample room temperature is 4 ℃-10 ℃, and flow velocity is 0.3-0.4ml/min, and sampling volume is 1-5 μ l.
In the above-mentioned method, the mass spectrophotometry process adopts electron spray (ESI) ion gun positive ion and two kinds of patterns of negative ion to detect respectively.Mass spectrum parameter under the positive and negative ion pattern is as follows: capillary voltage is 2800V-3000V, taper hole voltage is 40-60V, ion source temperature is 95-105 ℃, EFI fog (desolventizing gas) temperature is 340-360 ℃, electron spray airshed (desolventizing airshed) is 580-620l/h, the taper hole airshed is 58-62l/h, and the data acquisition scope is at 100-1000m/z.
In the above-mentioned method, with Markerlynx extraction step (2) by data that Ultra Performance Liquid Chromatography/level Four bar-time of-flight mass spectrometer is collected, and the data of extracting are carried out normalized, import to and carry out the processing of Pareto scale in the Simca-p software, the data after the Pareto scaleization are carried out the quadrature partial least squares discriminant analysis then.
In the above-mentioned method, the OPLS-DA model that step (3) is set up is verified.Verification method is to randomly draw 80% to set up the 0PLS-DA model by the data that Markerlynx extracted, and predicts remaining 20% classification of Data situation by this model.
In the above-mentioned method, by the result of step (3) quadrature partial least squares discriminant analysis, that is: the variable important factor (Variable Importance for the Projection, VIP) value and the s load diagram, filter out potential lipid biomarker; Wherein, variable important factor value (VIP) is greater than 1, the absolute value of ordinate (| p (corr) |) in the s load diagram simultaneously, i.e. and variable projection confidence level, the compound greater than 0.6 is as potential lipid biomarker.
In the above-mentioned method, potential lipid biomarker in the step (5) is carried out the structure deduction and identifies that its foundation is that accurate molecular weight, the possibility element that Masslynx software provides formed, the retention time and the analysis of second order ms (MS/MS) feature fragmention of one-level mass spectrometry precursor ion.
Compared to existing technology, the advantage that has of the present invention is as follows:
1, the present invention uses Ultra Performance Liquid Chromatography (UPLC) coupling level Four bar time-of-flight mass spectrometry instrument (Q-TOF) to be the analyzing and testing means, and both couplings are suitable for the compartment analysis of complex system and the structure of unknown material is identified.UPLC uses granule chromatograph packing material (＜2 μ m) and UHV (ultra-high voltage) transfusion unit (can reach 15,000psi), compare peak capacity with conventional H PLC and increase by 2 times, velocity of separation improves 10 times, and sensitivity improves 3～5 times, and back-pressure improves 8 times, improved the analysis throughput of sample, sensitivity, degree of separation and analysis speed help separating of target compound and competitive ionized impurity, thereby weaken the ion inhibiting effect during again with mass spectrometry.Q-TOF detector sensitivity height, resolution height can reach 20 * 10
-6Mass accuracy, can distinguish the mass peak of the very approaching different lipid molecules of molecular mass.Can obtain the fragmention information of higher quality accuracy by TOF-MS, the MS/MS secondary fragment analytical capabilities that has simultaneously is for identifying that the unknown or still unidentified lipid metabolism molecule are also very helpful.
2, the present invention adopts the mode identification method of quadrature partial least squares discriminant analysis (OPLS-DA), it is a kind of improved partial least squares discriminant analysis (PLS-DA), horizontal ordinate has embodied sample sets differences (the external interference factor causes), ordinate has embodied difference in the sample sets, its discriminating power is better than conventional PCA and PLS-DA, its analysis result will be relevant with classified variable variable and separate with the variable of classified variable quadrature (haveing nothing to do), only filter out the mark relevant, thereby improve reliability and accuracy with classified variable.
3, the discovery that the present invention set up and identify the current techique system of single-cell algae lipoidis biomarker radiation-curablely is applied to plant and fields such as the credit of microbial metabolism group is analysed, the analysis of food security metabolism group.
Description of drawings
Fig. 1 is the OPLS-DA model that 0.5%NaCl coerces the snowfield chlamydomonas of cultivation and the snowfield chlamydomonas contrast that no NaCl cultivates, and wherein Fig. 1 a is the OPLS-DA model of ESI positive ion mode, and Fig. 1 b is the OPLS-DA model of ESI negative ion mode.Triangle is the snow algae that 0.5%NaCl coerces cultivation, the contrast snow algae that square is cultivated for no NaCl, sample of each some expression.
Fig. 2 is the OPLS-DA modelling verification figure that 0.5%NaCl coerces the snowfield chlamydomonas of cultivation and the snowfield chlamydomonas contrast that no NaCl cultivates, and Fig. 2 a is the OPLS-DA modelling verification figure of ESI positive ion mode, and Fig. 2 b is the OPLS-DA modelling verification figure of ESI negative ion mode.The black round dot represents to be used for moulding 80% known sample of OPLS-DA; 20% unknown sample that+symbolic representation is predicted with OPLS-DA, sample of each some expression.
Fig. 3 is the combination figure of s load diagram and VIP value, and wherein Fig. 3 a is ESI positive ion mode figure below, and Fig. 3 b is ESI negative ion mode figure below.Ion that Mass Spectrometer Method arrives of each some expression, blank triangle is represented the confidence level of variable load projection value, black triangle is represented important factor (VIP) value of variable projection.
Embodiment
The invention will be further described below in conjunction with specific embodiment:
Embodiment 1
Discovery and evaluation snowfield chlamydomonas (Chlamydomonas nivalis) adapt to lipid biomarker 1, snowfield chlamydomonas (Chlamydomonas nivalis) cultivation and the results that NaCl coerces environment
The snowfield chlamydomonas is added sterilized NaCl solution after cultivating 6 days, make final concentration reach 0.5% (w/v).0h, 1h are cultivated in continuation, 7h, 15h promptly take a sample, and each is provided with four parallel samples constantly.Algae liquid is centrifugal, and (5000r/min 5min), removes supernatant, and algae mud is placed on for three times in the ultra low temperature freezer after the pre-freeze with pure water washing, and freeze drying gets the algae powder.
2, the extraction of cytolipin compound
The algae powder extracts with methyl alcohol-methenyl choloride-aqueous extract.Elder generation is at methyl alcohol-methenyl choloride-water (1/1/0.5 of 2.5ml, v/v/v) add 0.025% (w/v in the extraction solution, g/ml) antioxidant BHT, accurately take by weighing the algae powder of 10.0mg then, add extract, behind oscillator shake 5min, under 10 ℃ and 5500rpm rotating speed behind the centrifugal 10min, take off a layer solution, dry up, analyze with being used for UPLC-Q-TOF/MS behind the 0.8ml chromatogram dissolve with methanol again with nitrogen.
3, the UPLC-Q-TOF/MS of lipid metabolism spectrum analyzes
Liquid phase analysis adopts the U.S. Acquity of Waters company Ultra Performance Liquid Chromatography system (UPLC); Chromatographic column adopting BEH C18 post (2.1mm * 50mm, 1.7 μ m); Mobile phase A is water/isopropyl alcohol 70/30 (containing 0.1% formic acid); Mobile phase B is acetonitrile/isopropyl alcohol 70/30 (containing 0.1% formic acid); Flow velocity is 0.4ml/min; Sample size is 1 μ l; 40 ℃ of column temperatures, 4 ℃ of sample chamber temperature.Under the positive ion mode, the moving phase elution program is: initial A: the B ratio is (50: 50), 1minA: the B ratio is (15: 85), 1-5min A: the B ratio is (15: 85), 9min A: the B ratio is (0: 100), 9-14min A: the B ratio is (0: 100), 15minA: the B ratio is (50: 50), 15-17minA: the B ratio is (50: 50); Under the negative ion mode, the moving phase elution program is: initial A: the B ratio is (60: 40), 2min A: the B ratio is (20: 80), 2-7min A: the B ratio is (20: 80), 9min A: the B ratio is (0: 100), 9-14min A: the B ratio is (0: 100), 15min A: the B ratio is (50: 50), 15-17min A: the B ratio is (50: 50).
The Micromass Q-TOF of U.S. Waters company series connection quadrupole rod-time of-flight mass spectrometer is adopted in mass spectrophotometry, is furnished with electric spray ion source (ESI).The mass spectrophotometry process adopts ESI source positive ion and two kinds of patterns of negative ion to detect respectively.Mass spectrum parameter under the positive and negative ion pattern is as follows: capillary voltage is 3000V, taper hole voltage is 50V, ion source temperature is 100 ℃, EFI fog (desolventizing gas) temperature is 350 ℃, electron spray airshed (desolventizing airshed) is 600l/h, the taper hole airshed is 60l/h, and the data acquisition scope is at 100-1000m/z.
4, OPLS-DA analyzes
With Markerlynx the total ion current figure that obtains is carried out peak extraction, peak match.Data after the extraction are carried out normalized, again data importing are carried out the processing of Pareto scale in Simca-p, and then carry out quadrature partial least squares discriminant analysis (OPLS-DA).With the OPLS-DA method data that 0.5%NaCl coerces the contrast snowfield chlamydomonas that the snowfield chlamydomonas of cultivation and no NaCl coerce are carried out modeling, the result as shown in Figure 1.(Fig. 1 a) can explain 98.2% classified information to the OPLS-DA model of the ESI positive ion mode of Jian Liing, and the predictive ability of model reaches 91.2% in view of the above; The OPLS-DA model of ESI negative ion mode (Fig. 1 b) can be explained 99.4% classified information, and the predictive ability of model reaches 86.2%.Among Fig. 1, X-direction (linear first principal component) has embodied group difference, and promptly salinity is to the otherness influence of snow algae lipid metabolism spectrum; Y direction (quadrature first principal component) has embodied difference in the group, and promptly same salinity, difference are coerced the time (1h, 7h, 15h) the otherness influence that snow algae lipid metabolism is composed.As can be seen from Figure 1, the lipid metabolism of the contrast snowfield chlamydomonas that snowfield chlamydomonas that 0.5%NaCl coerces and no NaCl coerce spectrum has difference more significantly, on the horizontal ordinate direction tangible classification is arranged in the drawings; The sample of same salinity, difference being coerced the time does not have tangible classification.
5, OPLS-DA modelling verification
Randomly draw 80% data of being extracted by Markerlynx and set up the OPLS-DA model as training set, remaining 20% data import to the accuracy of coming evaluation model in the model of being set up as forecast set.By Fig. 2 a and Fig. 2 b, as can be seen, the model that two kinds of patterns are set up can both correctly be predicted the classification of unknown data, so show the model of being built over-fitting does not take place, and is sane.
6, the screening of potential lipid biomarker
Fig. 3 a and Fig. 3 b have represented the s load diagram of variable under the positive and negative ion pattern and the combination figure of VIP value respectively.In two figure, filter out variable important factor (VIP) value＞1 respectively, simultaneously the compound of load projection value confidence level (| p (corr) |)＞0.6 adapts to the potential lipid biomarker of 0.5%NaCl as the snowfield chlamydomonas in the s load diagram, and potential mark is carried out structure identify.
7, the structure of mark is identified
The main foundation that structure is identified is the accurate molecular weight that provides of Masslynx software, may element form (＜10ppm), the analysis of the retention time of one-level mass spectrometry precursor ion and second order ms (MS/MS) feature fragmention segment and obtaining.Table 1 has provided 14 kinds of lipid biomarkers that positive ion mode is identified down, and table 2 has provided 7 kinds of lipid biomarkers that negative ion mode is identified down.They are:
Betaine-1,2-DG-O-4 '-(N, N, N-trimethyl) homoserine (DGTS, 1,2-diacylglyceryl-3-O-4 '-(N, N, N-trimethyl)-homoserine);
Single galactose diacylglycerol (DGDG) (MGDG, Monogalactosyl-diacylglycerol);
Two galactose diacylglycerol (DGDG)s (DGDG, Digalactosyl-diacylglycerol);
Phosphatidyl-ethanolamine (PE, Phosphatidylethanolamine);
Sulfonic group isorhamnose base two fatty acyl group glycerine (SQDG, sulfoquinovosyldiacylglycerol);
Phosphatidyl glycerol (PG, Phosphatidylglycerol);
Phosphatidylinositols (PI, Phosphatidylinositiol).
14 kinds of lipid biomarkers that table 1 positive ion mode is identified down
Continuous table 1
7 kinds of lipid biological markers that table 2 negative ion mode is identified down
Continuous table 2
Discovery and evaluation snowfield chlamydomonas (Chlamydomonas nivalis) adapt to lipid biomarker 1, snowfield chlamydomonas (Chlamydomonas nivalis) cultivation and the results that NaCl coerces environment
The snowfield chlamydomonas is added sterilized NaCl solution after cultivating 6 days, make final concentration reach 0.5% (w/v).0h, 1h are cultivated in continuation, 7h, 15h promptly take a sample, and each is provided with four parallel samples constantly.Algae liquid is centrifugal, and (5000r/min 5min), removes supernatant, and algae mud is placed on for three times in the ultra low temperature freezer after the pre-freeze with pure water washing, and freeze drying gets the algae powder.
2, the extraction of cytolipin compound
The algae powder extracts with methyl alcohol-methenyl choloride-aqueous extract.Elder generation is at methyl alcohol-methenyl choloride-water (1/1/0.5 of 2.5ml, v/v/v) add 0.05% (w/v in the extraction solution, g/ml) antioxidant BHT, accurately take by weighing the algae powder of 10.0mg then, add extract, behind oscillator shake 8min, under 8 ℃ and 5500rpm rotating speed behind the centrifugal 10min, take off a layer solution, dry up, analyze with being used for UPLC-Q-TOF/MS behind the 1ml chromatogram dissolve with methanol again with nitrogen.
3, the UPLC-Q-TOF/MS of lipid metabolism spectrum analyzes
Liquid phase analysis adopts the U.S. Acquity of Waters company Ultra Performance Liquid Chromatography system (UPLC); Chromatographic column adopting BEH C18 post (2.1mm * 50mm, 1.7 μ m); Mobile phase A is water/isopropyl alcohol 80/20 (containing 0.1% formic acid); Mobile phase B is acetonitrile/isopropyl alcohol 80/20 (containing 0.1% formic acid); Flow velocity is 0.35ml/min; Sample size is 1 μ l; 38 ℃ of column temperatures, 8 ℃ of sample chamber temperature.Under the positive ion mode, the moving phase elution program is: initial A: the B ratio is (50: 50), 1min A: the B ratio is (15: 85), 1-5min A: the B ratio is (15: 85), 9min A: the B ratio is (0: 100), 9-14minA: the B ratio is (0: 100), 15minA: the B ratio is (50: 50), 15-17minA: the B ratio is (50: 50); Under the negative ion mode, the moving phase elution program is: initial A: the B ratio is (60: 40), 2min A: the B ratio is (20: 80), 2-7minA: the B ratio is (20: 80), 9minA: the B ratio is (0: 100), 9-14minA: the B ratio is (0: 100), 15minA: the B ratio is (50: 50), 15-17min A: the B ratio is (50: 50).
The Micromass Q-TOF of U.S. Waters company series connection quadrupole rod-time of-flight mass spectrometer is adopted in mass spectrophotometry, is furnished with electric spray ion source (ESI).The mass spectrophotometry process adopts ESI source positive ion and two kinds of patterns of negative ion to detect respectively.Mass spectrum parameter under the positive and negative ion pattern is as follows: capillary voltage is 2900V, taper hole voltage is 60V, ion source temperature is 95 ℃, EFI fog (desolventizing gas) temperature is 340 ℃, electron spray airshed (desolventizing airshed) is 620l/h, the taper hole airshed is 62l/h, and the data acquisition scope is at 100～1000m/z.
4, OPLS-DA analyzes
With Markerlynx the total ion current figure that obtains is carried out peak extraction, peak match.Data after the extraction are carried out normalized, again data importing are carried out the processing of Pareto scale in simca-p, and then carry out quadrature partial least squares discriminant analysis (OPLS-DA).With the OPLS-DA method data that 0.5%NaCl coerces the contrast snowfield chlamydomonas that the snowfield chlamydomonas of cultivation and no NaCl coerce are carried out modeling, shown in Figure 1 consistent in result and the example 1.
5, OPLS-DA modelling verification
Randomly draw 80% data of being extracted by Markerlynx and set up the 0PLS-DA model as training set, remaining 20% data import to the accuracy of coming evaluation model in the model of being set up as forecast set.Shown in Figure 2 consistent in result and the example 1.
6, the screening of potential lipid biomarker
The combination figure of the s load diagram of variable and VIP value is as shown in Figure 3 in the example 1 under the positive and negative ion pattern, filter out variable important factor (VIP) value＞1, simultaneously the compound of load projection value confidence level (| p (corr) |)＞0.6 adapts to the potential lipid biomarker of 0.5%NaCl as the snowfield chlamydomonas in the s load diagram, and potential mark is carried out structure identify.
7, the structure of mark is identified
The main foundation that structure is identified is the accurate molecular weight that provides of Masslynx software, may element form (＜10ppm), the analysis of the retention time of one-level mass spectrometry precursor ion and second order ms (MS/MS) feature fragmention segment and obtaining.Identify 14 kinds of lipid biomarkers under the positive ion mode, identify 7 kinds of lipid biomarkers under the negative ion mode.Consistent shown in table 1, the table 2 in result and the example 1.
Embodiment 3
Discovery and evaluation snowfield chlamydomonas (Chlamydomonas nivalis) adapt to lipid biomarker 1, snowfield chlamydomonas (Chlamydomonas nivalis) cultivation and the results that NaCl coerces environment
The snowfield chlamydomonas is added sterilized NaCl solution after cultivating 6 days, make final concentration reach 0.5% (w/v).0h, 1h are cultivated in continuation, 7h, 15h promptly take a sample, and each is provided with four parallel samples constantly.Algae liquid is centrifugal, and (5000r/min 5min), removes supernatant, and algae mud is placed on for three times in the ultra low temperature freezer after the pre-freeze with pure water washing, and freeze drying gets the algae powder.
2, the extraction of cytolipin compound
The algae powder extracts with methyl alcohol-methenyl choloride-aqueous extract.Elder generation is at methyl alcohol-methenyl choloride-water (1/1/0.5 of 2.5ml, v/v/v) add 0.05% (w/v in the extraction solution, g/ml) antioxidant BHT, accurately take by weighing the algae powder of 10.0mg then, add extract, behind oscillator shake 8min, under 8 ℃ and 5500rpm rotating speed behind the centrifugal 10min, take off a layer solution, dry up, analyze with being used for UPLC-Q-TOF/MS behind the 1ml chromatogram dissolve with methanol again with nitrogen.
3, the UPLC-Q-TOF/MS of lipid metabolism spectrum analyzes
Liquid phase analysis adopts the U.S. Acquity of Waters company Ultra Performance Liquid Chromatography system (UPLC); Chromatographic column adopting BEH C18 post (2.1mm * 50mm, 1.7 μ m); Mobile phase A is water/isopropyl alcohol 80/20 (containing 0.1% formic acid); Mobile phase B is acetonitrile/isopropyl alcohol 80/20 (containing 0.1% formic acid); Flow velocity is 0.35mL/min; Sample size is 1 μ l; 38 ℃ of column temperatures, 8 ℃ of sample chamber temperature.Under the positive ion mode, the moving phase elution program is: initial A: the B ratio is (50: 50), 1min A: the B ratio is (15: 85), 1-5min A: the B ratio is (15: 85), 9min A: the B ratio is (0: 100), 9-14minA: the B ratio is (0: 100), 15min A: the B ratio is (50: 50), 15-17min A: the B ratio is (50: 50); Under the negative ion mode, the moving phase elution program is: initial A: the B ratio is (60: 40), 2min A: the B ratio is (20: 80), 2-7minA: the B ratio is (20: 80), 9min A: the B ratio is (0: 100), 9-14min A: the B ratio is (0: 100), 15min A: the B ratio is (50: 50), 15-17min A: the B ratio is (50: 50).
The Micromass Q-TOF of U.S. Waters company series connection quadrupole rod-time of-flight mass spectrometer is adopted in mass spectrophotometry, is furnished with electric spray ion source (ESI).The mass spectrophotometry process adopts ESI source positive ion and two kinds of patterns of negative ion to detect respectively.Mass spectrum parameter under the positive and negative ion pattern is as follows: capillary voltage is 2900V, taper hole voltage is 60V, ion source temperature is 95 ℃, EFI fog (desolventizing gas) temperature is 340 ℃, electron spray airshed (desolventizing airshed) is 620l/h, the taper hole airshed is 62l/h, and the data acquisition scope is at 100～1000m/z.
4, OPLS-DA analyzes
With Markerlynx the total ion current figure that obtains is carried out peak extraction, peak match.Data after the extraction are carried out normalized, again data importing are carried out the processing of Pareto scale in simca-p, and then carry out quadrature partial least squares discriminant analysis (OPLS-DA).With the OPLS-DA method data that 0.5%NaCl coerces the contrast snowfield chlamydomonas that the snowfield chlamydomonas of cultivation and no NaCl coerce are carried out modeling, shown in Figure 1 consistent in result and the example 1.
5, OPLS-DA modelling verification
Randomly draw 80% data of being extracted by Markerlynx and set up the 0PLS-DA model as training set, remaining 20% data import to the accuracy of coming evaluation model in the model of being set up as forecast set.Shown in Figure 2 consistent in result and the example 1.
6, the screening of potential lipid biomarker
The combination figure of the s load diagram of variable and VIP value is as shown in Figure 3 in the example 1 under the positive and negative ion pattern, filter out variable important factor (VIP) value＞1, simultaneously the compound of load projection value confidence level (| p (corr) |)＞0.6 adapts to the potential lipid biomarker of 0.5%NaCl as the snowfield chlamydomonas in the s load diagram, and potential mark is carried out structure identify.
7, the structure of mark is identified
The main foundation that structure is identified is the accurate molecular weight that provides of Masslynx software, may element form (＜10ppm), the analysis of the retention time of one-level mass spectrometry precursor ion and second order ms (MS/MS) feature fragmention segment and obtaining.Identify 14 kinds of lipid biomarkers under the positive ion mode, identify 7 kinds of lipid biomarkers under the negative ion mode.Consistent shown in table 1, the table 2 in result and the example 1.
Claims (8)
1. find and identify the method for single-cell algae lipoids biomarker, its feature comprises the steps:
(1) extraction of unicellular algae lipid within endothelial cells compound and anti-oxidation protection; (2) analyze lipid compounds with Ultra Performance Liquid Chromatography-level Four bar time of-flight mass spectrometer; (3) with Markerlynx software extraction and analysis data and import to and carry out the quadrature partial least squares discriminant analysis in the Simca-p software; (4) quadrature offset minimum binary discrimination model is verified; (5) result by quadrature offset minimum binary discrimination model filters out potential lipid biomarker; (6) potential lipid biomarker is carried out structure deduction and evaluation.
2. method according to claim 1, it is characterized in that step (1) methyl alcohol-methenyl choloride-aqueous extract extracts unicellular algae lipid within endothelial cells compound, add antioxidant 2 in the extract, the 6-di-tert-butyl-4-methy phenol, the volume ratio of methyl alcohol, methenyl choloride, water is 1:1:0.5 in the extract, the mass volume ratio of antioxidant 2,6 di tert butyl 4 methyl phenol and extract is 0.01%-0.05% g/ml; Sample is with after extract mixes, and behind oscillator concussion 5-10min, takes off a layer solution at 4-10 ℃ after centrifugal down, dries up with nitrogen, uses 0.5-1ml chromatogram dissolve with methanol again; Described sample is the single-cell algae powder.
3. method according to claim 1, it is characterized in that adopting in the step (2) Ultra Performance Liquid Chromatography and level Four bar time-of-flight mass spectrometry instrument to analyze lipid compounds, in the Ultra Performance Liquid Chromatography method, mobile phase A is the mixed liquor of water, isopropyl alcohol and formic acid, wherein the volume ratio of water, isopropyl alcohol is 95/5 ~ 70/30, and the content of formic acid is always the 0.1%(v/v of mobile phase A); Mobile phase B is the mixed liquor of acetonitrile, isopropyl alcohol and formic acid, and wherein the volume ratio of acetonitrile, isopropyl alcohol is 95/5 ~ 70/30, and the content of formic acid is always the 0.1%(v/v of Mobile phase B); Chromatographic column temperature is 35 ℃-40 ℃, and the sample room temperature is 4 ℃-10 ℃, and flow velocity is 0.3-0.4 ml/min, and sampling volume is 1-5 μ l.
4. as method as described in the claim 3, it is characterized in that the mass spectrophotometry process adopts electric spray ion source positive ion and two kinds of patterns of negative ion to detect respectively; Mass spectrum parameter under the positive and negative ion pattern is as follows: capillary voltage is 2800V-3000V, taper hole voltage is 40-60V, ion source temperature is 95-105 ℃, electron spray temperature degree is 340-360 ℃, the electron spray airshed is 580-620l/h, the taper hole airshed is 58-62l/h, and the data acquisition scope is at 100-1000m/z.
5. method according to claim 1, it is characterized in that step (3) with Markerlynx extraction step (2) by data that Ultra Performance Liquid Chromatography-level Four bar time of-flight mass spectrometer is collected, and the data of extracting are carried out normalized, import to and carry out the processing of Pareto scale in the Simca-p software, the data after the Pareto scaleization are carried out the quadrature partial least squares discriminant analysis then.
6. method according to claim 1, it is characterized in that step (4) verifies quadrature offset minimum binary discrimination model, verification method is to randomly draw 80% to set up quadrature offset minimum binary discrimination model by the data that Markerlynx extracted, and predicts remaining 20% classification of Data situation by this model.
7. method according to claim 1 is characterized in that the result by step (5) quadrature partial least squares discriminant analysis is variable important factor value and s load diagram, filters out potential lipid biomarker; Wherein, variable important factor value is greater than 1, and the absolute value of ordinate is a variable projection confidence level greater than 0.6 compound as potential lipid biomarker in the s load diagram simultaneously.
8. method according to claim 1, it is characterized in that step (6) carries out to potential lipid biomarker that structure is inferred and identify, it is according to being that accurate molecular weight, the element that Masslynx software provides formed, the retention time and the analysis of second order ms feature fragmention of one-level mass spectrometry precursor ion.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101085944A CN102262139A (en) | 2011-04-28 | 2011-04-28 | Method for finding and identifying lipid biomarkers of unicellular algae |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101085944A CN102262139A (en) | 2011-04-28 | 2011-04-28 | Method for finding and identifying lipid biomarkers of unicellular algae |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102262139A true CN102262139A (en) | 2011-11-30 |
Family
ID=45008847
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011101085944A Pending CN102262139A (en) | 2011-04-28 | 2011-04-28 | Method for finding and identifying lipid biomarkers of unicellular algae |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102262139A (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103278576A (en) * | 2013-05-03 | 2013-09-04 | 中国农业科学院北京畜牧兽医研究所 | Serum metabonomic method for screening biomarkers of transgenic animal |
| CN105760713A (en) * | 2014-12-19 | 2016-07-13 | 中教亚航（天津）教育科技发展有限公司 | Tumor cell classifying method based on cell membrane phospholipid composition differences |
| CN106133149A (en) * | 2013-10-09 | 2016-11-16 | 克雷托斯分析有限公司 | Microbiological analysis |
| CN108414635A (en) * | 2018-02-13 | 2018-08-17 | 广西医科大学 | The difference metabolin metabolic pathway and research method of a kind of anti-grease toxicity of rubusoside based on cell metabolism group |
| CN109444316A (en) * | 2018-12-27 | 2019-03-08 | 中国农业大学 | The iipidomics analysis method and application of toxoplasma based on high performance liquid chromatography series connection high resolution mass spectrum |
| CN109580861A (en) * | 2018-10-10 | 2019-04-05 | 中国疾病预防控制中心营养与健康所 | A kind of marker combination and method for measuring honey sample antioxidant activity |
| CN112710765A (en) * | 2019-10-24 | 2021-04-27 | 泰州医药城国科化物生物医药科技有限公司 | Fingerprint detection method of gardenia medicinal material and application thereof |
| CN113125588A (en) * | 2021-03-17 | 2021-07-16 | 广东省农业科学院农业质量标准与监测技术研究所 | Application of metabonomics analysis technology to discrimination of space-time classification of duck dung fragrance single tea |
| CN114942286A (en) * | 2022-05-17 | 2022-08-26 | 复旦大学 | Detection method of hydrophilic polypeptide |
| CN115308340A (en) * | 2022-09-13 | 2022-11-08 | 甘肃农业大学 | Method for detecting cold-resistant marker in winter rape wintering period and application |
| CN116453583A (en) * | 2023-03-16 | 2023-07-18 | 宁夏蓝怡生物工程有限公司 | Method for simulating change of protein nuclear algae corrected by cellular automaton of LUCC (luCC) |
-
2011
- 2011-04-28 CN CN2011101085944A patent/CN102262139A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| 李海英等: "亚心形扁藻中磷脂的UPLC-Q-TOF-MS分析", 《质谱学报》 * |
| 梁晓萍等: "蟾酥急性毒性的代谢组学研究", 《大中药产业健康发展战略研讨会》 * |
| 陈德莹等: "光合膜膜脂在八种海洋硅藻中的分析研究", 《海洋学报》 * |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103278576A (en) * | 2013-05-03 | 2013-09-04 | 中国农业科学院北京畜牧兽医研究所 | Serum metabonomic method for screening biomarkers of transgenic animal |
| CN103278576B (en) * | 2013-05-03 | 2014-12-24 | 中国农业科学院北京畜牧兽医研究所 | Serum metabonomic method for screening biomarkers of transgenic animal |
| CN106133149A (en) * | 2013-10-09 | 2016-11-16 | 克雷托斯分析有限公司 | Microbiological analysis |
| CN105760713A (en) * | 2014-12-19 | 2016-07-13 | 中教亚航（天津）教育科技发展有限公司 | Tumor cell classifying method based on cell membrane phospholipid composition differences |
| CN108414635A (en) * | 2018-02-13 | 2018-08-17 | 广西医科大学 | The difference metabolin metabolic pathway and research method of a kind of anti-grease toxicity of rubusoside based on cell metabolism group |
| CN109580861A (en) * | 2018-10-10 | 2019-04-05 | 中国疾病预防控制中心营养与健康所 | A kind of marker combination and method for measuring honey sample antioxidant activity |
| CN109580861B (en) * | 2018-10-10 | 2021-04-23 | 中国疾病预防控制中心营养与健康所 | Marker composition and method for determining antioxidant activity of honey sample |
| CN109444316A (en) * | 2018-12-27 | 2019-03-08 | 中国农业大学 | The iipidomics analysis method and application of toxoplasma based on high performance liquid chromatography series connection high resolution mass spectrum |
| CN112710765A (en) * | 2019-10-24 | 2021-04-27 | 泰州医药城国科化物生物医药科技有限公司 | Fingerprint detection method of gardenia medicinal material and application thereof |
| CN113125588A (en) * | 2021-03-17 | 2021-07-16 | 广东省农业科学院农业质量标准与监测技术研究所 | Application of metabonomics analysis technology to discrimination of space-time classification of duck dung fragrance single tea |
| CN114942286A (en) * | 2022-05-17 | 2022-08-26 | 复旦大学 | Detection method of hydrophilic polypeptide |
| CN115308340A (en) * | 2022-09-13 | 2022-11-08 | 甘肃农业大学 | Method for detecting cold-resistant marker in winter rape wintering period and application |
| CN115308340B (en) * | 2022-09-13 | 2023-11-14 | 甘肃农业大学 | Method for detecting cold-resistant marker in winter rape overwintering period and application |
| CN116453583A (en) * | 2023-03-16 | 2023-07-18 | 宁夏蓝怡生物工程有限公司 | Method for simulating change of protein nuclear algae corrected by cellular automaton of LUCC (luCC) |
| CN116453583B (en) * | 2023-03-16 | 2024-05-31 | 宁夏蓝怡生物工程有限公司 | Method for simulating change of nuclear protein algae corrected by LUCC cellular automaton |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102262139A (en) | Method for finding and identifying lipid biomarkers of unicellular algae | |
| Zhou et al. | Strategies for large-scale targeted metabolomics quantification by liquid chromatography-mass spectrometry | |
| Wong et al. | An overview of label-free quantitation methods in proteomics by mass spectrometry | |
| Draper et al. | Flow infusion electrospray ionisation mass spectrometry for high throughput, non-targeted metabolite fingerprinting: a review | |
| Allwood et al. | Metabolomic technologies and their application to the study of plants and plant–host interactions | |
| Jones et al. | Using metabolic profiling to assess plant-pathogen interactions: an example using rice (Oryza sativa) and the blast pathogen Magnaporthe grisea | |
| Duportet et al. | The biological interpretation of metabolomic data can be misled by the extraction method used | |
| Aszyk et al. | Main strategies, analytical trends and challenges in LC-MS and ambient mass spectrometry–based metabolomics | |
| Pan et al. | Untargeted metabolomic analysis of Chinese red wines for geographical origin traceability by UPLC-QTOF-MS coupled with chemometrics | |
| Danzi et al. | To metabolomics and beyond: a technological portfolio to investigate cancer metabolism | |
| Rubert et al. | Metabolic fingerprinting based on high-resolution tandem mass spectrometry: a reliable tool for wine authentication? | |
| Mousavi et al. | Development of high throughput 96-blade solid phase microextraction-liquid chromatrography-mass spectrometry protocol for metabolomics | |
| CN104297355A (en) | Simulative-target metabonomics analytic method based on combination of liquid chromatography and mass spectrum | |
| Kim et al. | Mass spectrometry-based metabolomics | |
| Tsednee et al. | A sensitive LC‐ESI‐Q‐TOF‐MS method reveals novel phytosiderophores and phytosiderophore–iron complexes in barley | |
| CN105067697B (en) | A kind of phosphatide classification and Detection and quantitative approach based on stable isotope labeling | |
| Zhang et al. | In situ characterisation of phytohormones from wounded Arabidopsis leaves using desorption electrospray ionisation mass spectrometry imaging | |
| CN110320303A (en) | A kind of Efficiency for Soil Aquifer Treatment metabonomic analysis methods based on UPLC-MS | |
| CN113552247A (en) | Liquid chromatography-mass spectrometry non-target analysis method for unknown components of sample | |
| CN103245716A (en) | Quick high-sensitivity microbiological identification method based on micromolecular metabolic substance spectral analysis | |
| Moco et al. | Metabolomics: Going deeper, going broader, going further | |
| Zhang et al. | Rapidly improved determination of metabolites from biological data sets using the high-efficient TransOmics tool | |
| Qi et al. | Global metabolic profiling using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry | |
| Deng et al. | Quality assessment and origin tracing of Guangdong Liangcha granules using direct mass spectrometry fingerprinting | |
| Højer-Pedersen et al. | The yeast metabolome addressed by electrospray ionization mass spectrometry: Initiation of a mass spectral library and its applications for metabolic footprinting by direct infusion mass spectrometry |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20111130 |