CN105021758B - Chemical derivatization-based phosphatide classification detection and quantification method - Google Patents
Chemical derivatization-based phosphatide classification detection and quantification method Download PDFInfo
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- 238000011002 quantification Methods 0.000 title abstract 2
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- 230000007935 neutral effect Effects 0.000 claims abstract description 19
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- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 claims abstract description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 113
- 150000003904 phospholipids Chemical class 0.000 claims description 71
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Abstract
The present invention discloses a chemical derivatization-based phosphatide classification detection and quantification method, and belongs to the quantitative detection method technical field. According to the method, trimethylsilyldiazomethane is used for methylation derivation of a phosphate group in a phosphatide molecule in a to-be-tested sample; a plurality of ions to be tested by mass spectrometer are produced by the methylation derivatived phosphatide molecule in the ionized sample; in the selection of corresponding ions to be tested by the mass spectrometer, the ions added with H <+> or NH4 <+> are selected by different phosphatide kinds; different neutral loss or precursor ion scanning models can be set according to mass spectrometer fragmentation rules of different kinds of ions, and amounts of mass spectrometer ion models of different kinds of ions can be obtained; and finally, by comparison with the internal standard and calculation, the amounts of different types of phosphatide molecules in the sample can be obtained. The method is fast in detecting speed and high in sensitivity.
Description
Technical field
The present invention relates to phosphatide quantitative detecting method technical field, more particularly to a kind of based on chemically derived phosphatide point
Class is detected and quantitative approach.
Background technology
Phosphatide is not only the important composition composition of cell membrane, and also wide participation adjusts various vital movement processes, including energy
Amount conversion, matter transportation, information identification and transmission, cell development and differentiation, and Apoptosis etc..The abnormal metabolism of phosphatide with
Arteriosclerosis, diabetes, obesity, Alzheimer diseases and tumour occur also closely related.Therefore, from the angle of " group is learned "
Degree sets out, and systematicness, scale ground research phospholipid molecule are constituted under different physiology or pathological state and the dynamic of relative quantity becomes
Change analysis, to disclosing phospholipid metabolism and relation and its molecule machine between physiology, the pathologic process of cell, organ or even body
Reason, and then excavate the important biomolecule label related to disease generation, the early diagnosis and therapy of promotion disease has important
Meaning.
At present, with Ionization Techniques In Mass Spectrometry, particularly electro-spray ionization (electrosprayionization,
ESI) and substance assistant laser desorpted ionization (matrix assisted laser desorption/ionization,
) etc. MALDI it is suitable for the development of the ionization techniques of large biological molecule, the phosphatide identification and quantitative analysis skill based on biological mass spectrometry
The features such as art is because of its high sensitivity, high-resolution, high accuracy and high flux property, has become the main work of phosphatide group research
Tool.Phospholipid lipid molecular is analyzed using ESI-MS, but different types of phospholipid molecule because of self structure (mainly
Polar head) and the difference of solution composition there are different ionization tendencies.Phosphatid ylcholine (PtdCho) lipoids molecule exists
It is in electroneutral under neutral or alkaline pH, tends to form adduction H after ionization+Cation.Although containing acetate
In the case of solution and high voltage, PtdCho can also be formed [M-CH3COO]-The anion of form, but signal is than relatively low.
Cytoskeletal protein lipid molecular (PtdEtn) is because containing monoethanolamine polar head, under ph basic conditions, quaternary amine base holds
Easily deprotonation and carry a net negative charge.Acidic phospholipid lipoids, including phosphatidic acid (PtdH), phosphatidyl glycerol
(PtdGro), phosphatidylserine (PtdSer), phosphatidylinositols (PtdIns), carries under physiological ph conditions one or more
Net negative charge, in Mass Spectrometer Method mainly in the form of anion.Therefore, it is generally the case that PtdCho cation moulds
Formula is detected, and PtdEtn, PtdH, PtdGro, PtdSer and PtdIns are then detected with anion, so as to cause to utilize
ESI-MS will be with reference to cation and negative ion mode, so as to lead when carrying out qualitative and quantitative analysis to the phosphatide group in biological specimen
Cause the growth of analysis time.Meanwhile, in the negative ion mode, the detection sensitivity of part phosphatide is relatively low.
On the other hand, it is further to excavate the important phospholipid molecule and its effect machine related from different physiology, pathologic process
Reason, the change for needing the relative quantity to phosphatide group under different physiology or pathological state is analyzed.Many traditional research profits
With radioactive label (such as3H-,14C- or32P- mark carbon source or phospholipid head groups) or fluorescence labeling (such as 7-nitrobenz-
2-oxa-1,3-diazol-4-yl, NBD) precursor, it is selective (a certain lipid specific precursor, such as inositol) or non-selective
Phosphatide in (the common precursor such as lipid) metabolic tagged tissue or cell, so as to reach quantitative purpose, but these methods
It is comparatively laborious, and waste time and energy, the features such as the quantitative analysis tech based on ESI/MS is because of its high-resolution, high accuracy, more
To be applied to the quantitative analysis of cytolipin group more.(such as pmol/mL or lower concentration conditions under optimal conditions
Under), phosphatide mass signal intensity and phospholipid concentration be linear and Ionization Efficiency basic of identical type phospholipid molecule
Cause, it is unrelated with the physical property of fatty acid chain.Therefore, in cation and negative ion mode, by adding specific phospholipid species
Internal standard, the phosphatide in biological specimen can be carried out quantitatively.
Clark etc. (referring to Clark, J., et al.Nat Methods.2011,8,267-72.) is reported and is utilized front three
The phosphate group of base silicon diazomethane (TMS-diazomethane) esterification diphosphoinositide (PIPs) is greatly improved
The detection sensitivity of diphosphoinositide, points out that using TMS-diazomethane phosphate group esterification can be made, and has
It is applied to phosphatide detection and quantitative Potential feasibility.Even so, will be based on trimethyl silicane diazomethane (TMS-
Diazomethane methyl orthophosphoric acid) is applied to the detection of phosphatide still has problems to wait answer.Firstly, since phosphatide kind
Whether the complexity of class and structure, the methyl orthophosphoric acidization based on trimethyl silicane diazomethane (TMS-diazomethane) can answer
Detection for phosphatide (including hemolytic phosphatide) is unclear, also has no related report.Secondly, based on trimethyl silicane diazonium
Whether the derivatization efficiency of the methyl orthophosphoric acid of methane (TMS-diazomethane) is subject to the physically or chemically property of phospholipid molecule
The impact of matter, the fatty acid chain length of phospholipid molecule, saturation degree of fatty acid chain etc., it is also unclear.3rd, esterification
How derivative phospholipid molecule is carried out detection and quantitatively also not understood using mass spectrum.
The content of the invention
In view of this, the present invention provides a kind of application trimethyl silicane diazomethane to the phosphate in phospholipid molecule in sample
Group carries out esterification and derives, and utilizes mass spectrum, the amount of variety classes phospholipid molecule in sample to be tested is carried out quantitative based on change
Learn derivative phosphatide classification and Detection and quantitative approach.
In order to achieve the above object, present invention generally provides following technical scheme：
What the present invention was provided is comprised the following steps based on chemically derived phosphatide classification and Detection and quantitative approach：
Step 1：Phosphatide to be detected is extracted from sample to be detected；
Step 2：Esterification is carried out using trimethyl silicane diazomethane to the phosphate group in the phospholipid molecule in sample to spread out
It is raw, reduce the electronegativity of phospholipid molecule；
Step 3：Through phospholipid molecule derived from esterification in ionization sample, the several ion for treating Mass Spectrometer Method is produced,
The ionic species to be detected includes that phosphatid ylcholine, phosphatidyl-ethanolamine, phosphatidylserine, phosphatidic acid, phosphatidyl are sweet
Oil, phosphatidylinositols, Lysophosphatidylcholone, hemolytic phosphatidyl-ethanolamine, hemolytic phosphatidylserine, hemolytic phosphorus
Resin acid, hemolytic phosphatidyl glycerol, hemolytic phosphatidylinositols；
Step 4：In the phosphatid ylcholine, phosphatidyl-ethanolamine, phosphatidylserine, Lysophosphatidylcholone, molten
Courageous and upright phosphatidyl-ethanolamine, hemolytic phosphatidylserine, lysophosphatidic, hemolytic phosphatidyl glycerol, hemolytic phosphatide
On acyl inositol, adduction H is chosen respectively+Ion；
On phosphatidic acid, phosphatidyl glycerol, phosphatidylinositols, adduction NH is chosen respectively4 +Ion；
According to the cleavage of mass spectrum rule of variety classes ion in the step 3, set it is different it is neutral lose or precursor from
Sub- scan pattern, classification and Detection is carried out using mass spectrum to variety classes ion in the step 3, obtains different in the step 3
The amount of the mass ions signal of species ion；
Step 5：By comparing with internal standard, in the step 3 that the step 4 is obtained the mass spectrum of variety classes ion from
The amount of subsignal is associated with the amount of phospholipid molecule in sample, obtains the amount of variety classes phospholipid molecule in sample.
The object of the invention to solve the technical problems can also be applied to the following technical measures to achieve further.
Preferably, when carrying out classification and Detection to variety classes ion in the step 3 using mass spectrum, from liquid phase color
The mode of spectrum-mass spectrometry detects, or, detected from the mode of direct injected-mass spectrometry.
Preferably, the step 1 is carried out in methyl tertiary butyl ether(MTBE)/methanol/water solution system, the methyl- tert
In butyl ether/methanol/water solution system, methyl tertiary butyl ether(MTBE), methyl alcohol, the volume ratio of water are 10: 3: 2.5.
Preferably, the step 2 is carried out in methyl tertiary butyl ether(MTBE)/methyl alcohol/1N hydrochloric acid solution systems, the first
In base tertbutyl ether/methyl alcohol/1N hydrochloric acid solution systems, methyl tertiary butyl ether(MTBE), methyl alcohol, the volume ratio of 1N hydrochloric acid are 300: 90: 4.
Preferably, the step 3 is carried out in chloroform/methanol/ammonium acetate solution system, the chloroform/methanol/
In ammonium acetate solution system, the concentration of the ammonium acetate is 5～10mM, and the chloroform, the volume ratio of methyl alcohol are (1～2): 1.
Preferably, the phosphatidic acid, phosphatidyl-ethanolamine, phosphatidyl glycerol, phosphatidylserine, phosphatidylinositols,
Lysophosphatidic, hemolytic phosphatidyl-ethanolamine, hemolytic phosphatidyl glycerol, hemolytic phosphatidylserine, hemolytic phosphorus
Acyl inositol carries out detecting and quantitative using neutral loss scan.
Preferably, when carrying out detecting and being quantitative to the phosphatidic acid using neutral loss scan, it is 143Da that neutrality is lost
(126Da+NH3)；When carrying out detecting and being quantitative to the phosphatidyl-ethanolamine using neutral loss scan, neutrality is lost and is
155Da；When carrying out detecting and being quantitative to the phosphatidyl glycerol using neutral loss scan, it is 203Da (186Da+ that neutrality is lost
NH3)；When carrying out detecting and being quantitative to the phosphatidylserine using neutral loss scan, it is 213Da that neutrality is lost；Utilize
When neutral loss scan carries out detecting and being quantitative to the phosphatidylinositols, it is 291Da (274Da+NH that neutrality is lost3)；
When carrying out detecting and being quantitative to lysophosphatidic using neutral loss scan, it is 126Da that neutrality is lost；In utilization
Property lose scanning to hemolytic phosphatidyl-ethanolamine carry out detection and it is quantitative when, neutrality lose be 155Da；Swept using neutral loss
Retouch to hemolytic phosphatidyl glycerol carry out detection and it is quantitative when, neutrality lose be 186Da；Using neutral loss scan to hemolytic
When phosphatidylserine carries out detecting and being quantitative, it is 213Da that neutrality is lost；Using neutral loss scan to hemolytic phosphatidyl-4
When alcohol carries out detecting and being quantitative, it is 274Da that neutrality is lost.
Preferably, the phosphatid ylcholine, Lysophosphatidylcholone are detected using precursor ion-scan and determined
Amount.
Preferably, when carrying out detecting and being quantitative to the phosphatid ylcholine, the matter/lotus ratio of the precursor ion is
198.0±0.5；When carrying out detecting and being quantitative to the Lysophosphatidylcholone, the matter/lotus ratio of the precursor ion is
198.0±0.5。
Preferably, the sample to be tested is biological specimen.
Preferably, being added with antioxidant in the sample to be tested.
Preferably, the antioxidant is dibutyl hydroxy toluene.
Preferably, the addition of the dibutyl hydroxy toluene is the 0.01～0.03% of sample to be tested volume.
Preferably, when adding antioxidant in the sample to be tested, environment temperature is 4～10 DEG C.
Preferably, the preserving type of the sample to be tested is freezen protective.
Preferably, the interior target selection principle is each phospholipid species selects an internal standard.
Preferably, the isotope-labeled phospholipid molecule that can individually detect is designated as in described,
Or,
The phosphatide that do not exist or content extremely low is designated as in sample to be tested in described.
Preferably, the interior target consumption changes according to different samples to be tested.
Preferably, between the step 1 and step 2, also including the step of washing.
Preferably, when carrying out classification and Detection to step 2 intermediate ion using mass spectrum, from direct injected-mass spectrum, institute
Sampling device is stated for syringe pump.
Preferably, when carrying out classification and Detection to step 2 intermediate ion using mass spectrum, from direct injected-mass spectrum, institute
State sampling device and spray ion gun direct injected system to receive automatically.
Preferably, in the step 5, computing mode is：
CPhosphatide/CInternal standard=IPhosphatide/IInternal standard,
Wherein, CPhosphatideAnd CInternal standardPhospholipid molecule and its corresponding interior target concentration are represented in sample to be tested respectively and IPhosphatideAnd IInternal standard
Then distinguish phospholipid molecule and its corresponding interior target mass spectra peak intensity in representative sample.
Preferably, the step 2 is carried out in the fume hood with guard system.
Preferably, the step 2, when the application of trimethyl silicane diazomethane is excessive, from acetic acid to excessive front three
Base silicon diazomethane is neutralized.
The present invention provide based on chemically derived phosphatide classification and Detection and quantitative approach application trimethyl silicane diazomethane
Carry out esterification to the phosphate group in phospholipid molecule in sample to be tested to derive；Through phosphatide derived from esterification in ionization sample
Molecule, produces the several ion for treating Mass Spectrometer Method；Select in the ion for treating Mass Spectrometer Method accordingly, according to different phosphatide kinds
Class, selects adduction H+Ion or NH4 +Ion；Different neutrality are set according to the cleavage of mass spectrum rule of variety classes ion
Lose or precursor ion-scan pattern, obtain the amount of the mass ions model of variety classes ion；Finally, by with internal standard ratio
Compared with and computing, obtain the amount of variety classes phospholipid molecule in sample.The method detection speed is fast, and sensitivity is high.
Description of the drawings
By the detailed description for reading hereafter preferred embodiment, various other advantages and benefit is common for this area
Technical staff will be clear from understanding.Accompanying drawing is only used for illustrating the purpose of preferred embodiment, and is not considered as to the present invention
Restriction.And in whole accompanying drawing, it is denoted by the same reference numerals identical part.In the accompanying drawings：
Fig. 1 is provided in an embodiment of the present invention based on flow process the step of chemically derived phosphatide classification and Detection and quantitative approach
Figure；
Fig. 2 be it is provided in an embodiment of the present invention based on chemically derived phosphatide classification and Detection and quantitative approach in, with PtdH,
PtdCho, PtdGro and PtdSer be the cleavage of mass spectrum rule of representative explaination esterification phospholipid molecule and it is neutral lose and precursor from
The establishment of sub- scanning rule.The C17 of esterification:0/C14:1-PtdH (A), C17:0/C14:1-PtdCho (B), C17:0/C14:
1-PtdGro (C) and C17:0/C14:1-PtdSer (D) can produce the fragment ion that mass-to-charge ratio is 535, corresponding to powered
C17:0/C14:1-DAG(C17:0/C14:1-DAG+)。DAG+Ion is also considered as adduction H+Or NH4 +The esterification of ion
The neutral of phospholipid molecule loses peak, i.e. PtdH, and PtdCho, PtdGro and PtdSer are neutral respectively to lose 143Da (126Da+
NH3), 193Da, 203Da (186+NH3), the fragment ion peak that 213Da is produced.Accordingly, using neutral loss scan pattern to upper
State phosphatide to be detected.Due to the DAG that PtdCho is produced+Fragment ion signal is weaker, and corresponds to esterification head group
Fragment ion peak (mass-to-charge ratio is 198) signal it is stronger, therefore, for PtdCho classes phosphatide then utilize precursor ion-scan mould
Formula is detected；
Fig. 3 it is provided in an embodiment of the present invention based on chemically derived phosphatide classification and Detection and quantitative approach in, with
LysoPtdH, lysoPtdGro, lysoPtdSer and lysoPtdIns are the hemolytic phospholipid molecule that esterification is explained in representative
Cleavage of mass spectrum rule and the neutral establishment lost with precursor ion-scan rule.The C17 of esterification:1-lysoPtdH (A), C17:
1-lysoPtdGro (B), C17:1-lysoPtdH (C) and C17:1-lysoPtdIns (D) can produce mass-to-charge ratio be 325 it is broken
Piece ion, corresponding to powered C17:1-MAG(C17:1-MAG+)。MAG+Ion is also considered as adduction H+Or NH4 +Ion
The neutral of esterification phospholipid molecule loses peak, i.e. lysoPtdH, in lysoPtdGro, lysoPtdSer and lysoPtdIns difference
Property lose 126Da, 186Da, 213Da and 274Da produce fragment ion peak.Accordingly, using neutral loss scan pattern to upper
State phosphatide to be detected；
Fig. 4 is provided in an embodiment of the present invention based in chemically derived phosphatide classification and Detection and quantitative approach, phospholipid molecule
Esterification derives and based on neutral loss or the schematic diagram of precursor ion-scan；
Fig. 5 is provided in an embodiment of the present invention based in chemically derived phosphatide classification and Detection and quantitative approach, phospholipid molecule
The derivative efficiency evaluation of esterification.By taking the Cho of Ptd as an example, before derivatization after (A) and derivatization the PtdCho of (B) mass spectrogram
Show the derivative efficiency of phospholipid molecule esterification based on trimethyl silicane diazomethane by phospholipid molecule fatty acid chain length and
The impact of saturation degree.
Fig. 6 it is provided in an embodiment of the present invention based on chemically derived phosphatide classification and Detection and quantitative approach in, in cation
Under pattern, phosphatid ylcholine (A) is used for quickly detecting and quantitatively using the precursor ion that matter/lotus ratio is 198.0；In utilization
Property lose be, 155Da, 203Da, 213Da, 143Da and 291Da respectively to phosphatidyl-ethanolamine (B), phosphatidyl glycerol (C),
Phosphatidylserine (D), phosphatidic acid (E), and phosphatidylinositols (F) are used for quickly detecting and quantitatively.
Specific embodiment
The present invention is to solve the problems, such as prior art, there is provided one kind is using trimethyl silicane diazomethane in sample
Phosphate group in phospholipid molecule carries out esterification and derives, and utilizes mass spectrum, to variety classes phospholipid molecule in sample to be tested
Amount carries out quantitative based on chemically derived phosphatide classification and Detection and quantitative approach.
It is provided in an embodiment of the present invention to be described based on chemistry based on chemically derived phosphatide classification and Detection and quantitative approach
The phosphatide quick detection and quantitative approach of derivative and direct injected mass spectral analysis.The method utilizes trimethyl silicane diazomethane by phosphorus
Phosphate group esterification in fat molecule, reduces the electronegativity of phospholipid molecule, while improving detection sensitivity, realizes all phosphorus
Lipid species Mass Spectrometer Method in the positive-ion mode, greatlys save the time.By direct injected and electro-spray ionization mode,
Phospholipid molecule derived from esterification is ionized, the one or more of ions that can be detected by mass spectrography are produced, and is determined for matter
The ion of spectrum detection.According to the cleavage of mass spectrum rule of the phospholipid molecule of ionization, different neutral loss or precursor ion are set
Scan pattern, in the positive-ion mode, using mass spectrum to phosphatide and its corresponding hemolytic phosphatide (lyso-) (totally 12 species)
It is used for quickly detecting.By comparing with interior target, the amount of the mass ions signal of phospholipid molecule is associated with into phosphatide point in sample
The amount of son.The method for being provided has enhanced sensitivity, and is completed in a relatively short time.
Further to illustrate the present invention to reach technological means and effect that predetermined goal of the invention is taken, below in conjunction with
Accompanying drawing and preferred embodiment, to according to proposed by the present invention, based on chemically derived phosphatide classification and Detection and quantitative approach, it has
Body embodiment, structure, feature and its effect, describe in detail as after.In the following description, different " embodiment " or " real
Apply example " refer to be not necessarily same embodiment.Additionally, the special characteristic, structure or feature in one or more embodiments can by appoint
What suitable form combination.
The terms "and/or", only a kind of incidence relation of description affiliated partner, represents there may be three kinds of passes
System, for example, A and/or B is specifically interpreted as：A and B can simultaneously be included, can be with individualism A, it is also possible to individualism
B, can possess above-mentioned three kinds of any one situations.
What the present invention was provided is comprised the following steps based on chemically derived phosphatide classification and Detection and quantitative approach：
Step 1：Phosphatide to be detected is extracted from sample to be detected；
Step 2：Esterification is carried out using trimethyl silicane diazomethane to the phosphate group in the phospholipid molecule in sample to spread out
It is raw, reduce the electronegativity of phospholipid molecule；
Step 3：Through phospholipid molecule derived from esterification in ionization sample, the several ion for treating Mass Spectrometer Method is produced,
Ionic species to be detected includes phosphatid ylcholine, phosphatidyl-ethanolamine, phosphatidylserine, phosphatidic acid, phosphatidyl glycerol, phosphorus
Acyl inositol, Lysophosphatidylcholone, hemolytic phosphatidyl-ethanolamine, hemolytic phosphatidylserine, lysophosphatidic,
Hemolytic phosphatidyl glycerol, hemolytic phosphatidylinositols；
Step 4：In phosphatid ylcholine, phosphatidyl-ethanolamine, phosphatidylserine, Lysophosphatidylcholone, hemolytic
Phosphatidyl-ethanolamine, hemolytic phosphatidylserine, lysophosphatidic, hemolytic phosphatidyl glycerol, hemolytic phosphatidyl-4
On alcohol, adduction H is chosen respectively+Ion；
On phosphatidic acid, phosphatidyl glycerol, phosphatidylinositols, adduction NH is chosen respectively4 +Ion；
According to the cleavage of mass spectrum rule of variety classes ion in step 3, the different neutral loss of setting or precursor ion are swept
Pattern is retouched, classification and Detection is carried out to variety classes ion in step 3 using mass spectrum, obtain the matter of variety classes ion in step 3
The amount of spectrum ion signal；
Step 5：By comparing with internal standard, the step of step 4 is obtained 3 in variety classes ion mass ions signal
Amount is associated with the amount of phospholipid molecule in sample, obtains the amount of variety classes phospholipid molecule in sample to be tested.
Wherein, using mass spectrum to variety classes ion carries out classification and Detection in step 3 when, from liquid phase chromatogram-mass spectrometry combination
Mode detects, or, from direct injected-mass spectrum.
Wherein, step 1 is carried out in methyl tertiary butyl ether(MTBE)/methanol/water solution system, and methyl tertiary butyl ether(MTBE)/methyl alcohol/
In water solution system, methyl tertiary butyl ether(MTBE), methyl alcohol, the volume ratio of water are 10: 3: 2.5.
Wherein, step 2 is carried out in methyl tertiary butyl ether(MTBE)/methyl alcohol/1N hydrochloric acid solution systems, and methyl tertiary butyl ether(MTBE)/
In methyl alcohol/1N hydrochloric acid solution systems, methyl tertiary butyl ether(MTBE), methyl alcohol, the volume ratio of 1N hydrochloric acid are 300: 90: 4.
Wherein, step 3 is carried out in chloroform/methanol/ammonium acetate solution system, chloroform/methanol/ammonium acetate solution body
In system, the concentration of ammonium acetate is 5～10mM, and chloroform, the volume ratio of methyl alcohol are (1～2): 1.
Wherein, phosphatidic acid, phosphatidyl-ethanolamine, phosphatidyl glycerol, phosphatidylserine, phosphatidylinositols, hemolytic phosphorus
Resin acid, hemolytic phosphatidyl-ethanolamine, hemolytic phosphatidyl glycerol, hemolytic phosphatidylserine, hemolytic phosphatidylinositols
Carry out detecting and quantitative using neutral loss scan.
Wherein, when carrying out detecting and being quantitative to phosphatidic acid using neutral loss scan, it is 143Da (126Da+ that neutrality is lost
NH3)；When carrying out detecting and being quantitative to phosphatidyl-ethanolamine using neutral loss scan, it is 155Da that neutrality is lost；Using neutrality
When loss scanning carries out detecting and being quantitative to phosphatidyl glycerol, neutrality loss is 203Da (186Da+NH3)；Using neutral loss
When scanning carries out detecting and being quantitative to phosphatidylserine, it is 213Da that neutrality is lost；Using neutral loss scan to phosphatidyl-4
When alcohol carries out detecting and being quantitative, it is 291Da (274Da+NH that neutrality is lost3)；
When carrying out detecting and being quantitative to lysophosphatidic using neutral loss scan, it is 126Da that neutrality is lost；In utilization
Property lose scanning to hemolytic phosphatidyl-ethanolamine carry out detection and it is quantitative when, neutrality lose be 155Da；Swept using neutral loss
Retouch to hemolytic phosphatidyl glycerol carry out detection and it is quantitative when, neutrality lose be 186Da；Using neutral loss scan to hemolytic
When phosphatidylserine carries out detecting and being quantitative, it is 213Da that neutrality is lost；Using neutral loss scan to hemolytic phosphatidyl-4
When alcohol carries out detecting and being quantitative, it is 274Da that neutrality is lost.
Wherein, phosphatid ylcholine, Lysophosphatidylcholone are detected and quantitatively using precursor ion-scan.
Wherein, when carrying out detecting and being quantitative to phosphatid ylcholine, the matter/lotus ratio of precursor ion is 198.0 ± 0.5；To molten
When courageous and upright phosphatid ylcholine carries out detecting and being quantitative, the matter/lotus ratio of precursor ion is 198.0 ± 0.5.
Wherein, sample to be tested is biological specimen.
Wherein, it is added with antioxidant in sample to be tested.
Wherein, antioxidant is dibutyl hydroxy toluene.
Wherein, the addition of dibutyl hydroxy toluene is the 0.01～0.03% of sample to be tested volume.
Wherein, when adding antioxidant in sample to be tested, environment temperature is 4～10 DEG C.
Wherein, the preserving type of sample to be tested is freezen protective.
Wherein, interior target selection principle is that each phospholipid species selects an internal standard.
Wherein, the isotope-labeled phospholipid molecule that can individually detect inside is designated as,
Or,
The phosphatide not existed or content is extremely low is inside designated as in sample to be tested.
Wherein, interior target consumption changes according to different samples to be tested.
Wherein, between step 1 and step 2, also including the step of washing.
Wherein, when carrying out classification and Detection to step 2 intermediate ion using mass spectrum, from direct injected-mass spectrum, sampling device is
Syringe pump.
Wherein, when carrying out classification and Detection to step 2 intermediate ion using mass spectrum, from direct injected-mass spectrum, sampling device is
Automatically receive and spray ion gun direct injected system.
Wherein, in steps of 5, computing mode is：
CPhosphatide/CInternal standard=IPhosphatide/IInternal standard,
Wherein, CPhosphatideAnd CInternal standardPhospholipid molecule and its corresponding interior target concentration are represented in sample to be tested respectively and IPhosphatideAnd IInternal standard
Then distinguish phospholipid molecule and its corresponding interior target mass spectra peak intensity in representative sample.
Wherein, step 2 is carried out in the fume hood with guard system.
Wherein, step 2, when the application of trimethyl silicane diazomethane is excessive, from acetic acid to excessive trimethyl silicane diazonium
Methane is neutralized.
As described in the present embodiment of the invention, term " purifying " or " purification " is not necessarily referring to remove target analytes (from sample
Kind or it is more kinds of) outside all substances.Replace, purifying is referred to relative to may interfere with target analysis analyte detection in sample
Other components are enriched with the process of the amount of one or more of target analytes.Sample can be purified by various methods herein, with
Just allow to remove one or more of interfering materials.For example, the Mass Spectrometer Method of selected phosphatide parent ion and daughter ion can be disturbed
One or more of materials.
As used herein, term " liquid chromatography " refers to the mistake that component is separated into by the chemical mixture of liquid embarkation
Journey, this be when fixed liquid phase or solid phase are flowed or flow through around fixed liquid phase or solid phase due to chemical entities difference distribution and
Cause.
As used herein, term " direct injected " in this article refers to need not move through chromatographic isolation, and directly using note
Pump or other modes are penetrated, is such as received automatically and is sprayed ion gun direct injected system (TriversaAdvion
Biosciences), phosphatide sample is introduced into a kind of mass spectrum input mode that mass spectrum is analyzed.
As used herein, term " mass spectrography " or " MS " refer to the analytical technology by its quality Identification compound.MS refers to base
In the method that its mass-to-charge ratio or " m/z " are filtered, detected and measure ion.MS technologies are generally comprised：(1) ionising compounds, form
Charging cpd；(2) molecular weight of charging cpd is measured, and calculates mass-to-charge ratio.Can be by any appropriate method ionization
And detection compound.
As used herein, term " under positive ion mode " refers to that those generate and detect the mass spectrography of cation.
As used herein, term " ionization " or " ionization " refers to such process：Generate and have equal to one or more
The analyte ions of the net charge of electron unit.Anion be the net negative charge with one or more electron units from
Son, and cation is the ion of the net positive charge with one or more electron units.
As used herein, term " substance assistant laser desorpted ionization " or " MALDI " refer to such method：Wherein will be non-
Volatility sample is exposed to laser emission, and it passes through different kinds of ions approach makes the analyte desorption in sample and ionization, should be from
Sub-ization approach includes photoionization, protonation effect, proton abstraction and cluster decay (cluster decay).For
MALDI, sample is mixed with energy absorption matrix, and this promotes the desorption of analyte molecule.
As used herein, term " electro-spray ionization " or " ESI " refer to such method：Sample solution is with low flow velocity (0.1
～10 μ L/min) pass through capillary of the end added with high positive voltage or negative voltage, drop is formed, when the storehouse of droplet surface electric charge
When logical sequence repulsion reaches the critical point suitable with solution surface tension, the drop containing a large amount of electric charges will be produced.As solvent evaporates,
Droplet retracts, repulsive force increase between electric charge in drop, when reaching and surmounting to a certain degree, drop can occur COULOMB EXPLOSION, remove
Remove the excessive charge of droplet surface, generate less powered droplet, so circulation, ultimately produce be available for Mass Spectrometer Method from
Son.
Appropriate detection sample includes that any detection sample of target analytes can be included.In some preferred embodiments
In, sample is biological specimen, i.e. the sample of biogenetic derivation, such as animal tissue, cell culture, blood plasma.In order to avoid phosphatide
Oxidation or degraded, should add antioxidant during sample process, such as dibutyl hydroxy toluene (BTH, it is final concentration of
0.01%), and by sample process immediately at 4 DEG C, or completely stored frozen and using front defrosting.
Usually, an interior target principle is selected according to each phospholipid species, it is isotope-labeled by what is can individually detected
Phospholipid molecule or is chosen do not exist in sample or phosphatide that content is extremely low adds sample as internal standard.The present invention chooses C17:0/
C14:1-PtdCho, C17:0/C14:1-PtdEtn, C17:0/C14:1-PtdGro, C17:0/C14:1-PtdSer, C17:0/
C14:1-PtdH, C17:0/C14:1-PtdIns, C17:1-lysoPtdCho, C17:1-lysoPtdEtn, C17:1-
LysoPtdGro, C17:1-lysoPtdSer, C17:1-lysoPtdH and C17:1-lysoPtdIns is used as internal standard.It is excellent at some
In selecting embodiment, according to the biological specimen and sample size of separate sources, different amounts of internal standard is added.In particularly preferred enforcement
In mode, such as blood plasma (10 μ L), cell (～5 × 105It is individual) and tissue (10mg), every kind of interior target consumption is 0.4nmol.
Liquid-liquid extraction is that Phospholipids extracts most commonly used method in iipidomic research.Bibliography of the present invention
((Matyash et al.J Lipid Res.2008,49 (5)：1137-1146.) report method and be suitably modified,
For the extraction of phosphatide.Because MTBE density is low, when water phase and organic phase separate, organic phase which simplify receipts on upper strata
The operation of collection organic phase, reduces the loss of absorption, and bottom of the inextractable matrix pellet in centrifuge tube, it is easy to go
Remove.
Before esterification is derivative, the step of increased washing, on the one hand can remove as far as possible one or more of
Interfering material, on the other hand, concentration of hydrochloric acid can be adjusted to preferred concentration.
The present invention chooses the chloroform/methanol (1 containing 5mM ammonium acetates:1, v/v) phospholipid molecule of the solution system as esterification
The carrier of direct injected, the phospholipid molecule of esterification can be ionized and produce and be available for mass spectrum in cation in the solution system
The ion detected under pattern.The mode of direct injected can choose syringe pump, some preferred embodiment in, using receiving automatically
Spray ion gun direct injected system (TriversaAdvion Biosciences), the system is without the need for cleaning
And manual operations, continuously mass spectrum sample introduction can be carried out automatically by electron spray chip, once unattended can continuously make 400 samples
Product, and because flow velocity reaches nanoliter level, only need little sample, so that it may complete the electron spray experiment of long-time stable, each sample
Product new sample suction nozzle and electron spray hole, completely without cross pollution.
In the above-mentioned chloroform/methanol (1 containing 5mM ammonium acetates:1, v/v) in solution system, phospholipid molecule derived from esterification
It is inclined to different band points.PtdCho, PtdEtn, PtdSer primarily form adduction H+Ion, although PtdH, PtdGro,
PtdIns classes phosphatide can also form adduction H+Ion, but signal is relatively low, mainly forms adduction NH4 +Ion.Hemolytic
Phosphatide primarily forms adduction H+Ion.Further Tandem Mass Spectrometry Analysis show, either adduction H+Or NH4 +The first of ion
Esterification phospholipid molecule can produce a diacylglycerol fragment ion (DAG+), as shown in figure accompanying drawing 1, the C17 of esterification:0/
C14:(mass-to-charge ratio is 678 to 1-PtdH, adduction NH4 +Ion), C17:0/C14:(mass-to-charge ratio is 718 to 1-PtdCho, adduction H+'s
Ion), C17:0/C14:(mass-to-charge ratio is 738 to 1-PtdGro, adduction NH4 +Ion) and C17:0/C14:1PtdSer (mass-to-charge ratioes
For 748, the ion of adduction H+) fragment ion that mass-to-charge ratio is 535 can be produced, corresponding to powered C17:0/C14:1-DAG
(C17:0/14:1-DAG+)。DAG+Ion is also considered as adduction H+Or NH4 +Ion esterification phospholipid molecule neutrality
Peak, i.e. PtdH are lost, PtdEtn, PtdCho, PtdGro, PtdSer and PtdIns are neutral respectively to lose 143Da (126Da+
NH3), 155Da, 193Da, 203Da (186Da+NH3), 213Da, 291Da (274Da+NH3) produce fragment ion peak.Accordingly,
Above-mentioned phosphatide is detected using neutral loss scan pattern.Due to the DAG that PtdCho is produced+Fragment ion signal compares
It is weak, and fragment ion peak (matter/lotus ratio is 198 ± 0.5) signal for corresponding to esterification head group is stronger, therefore, for
PtdCho classes phosphatide is then detected using precursor ion-scan pattern.Similar, hemolytic phospholipid molecule can also produce one
Monoacylglycerol fragment ion (MAG+), lysoPtdH, lysoPtdEtn, lysoPtdCho, lysoPtdGro are also can be considered,
LysoPtdSer and lysoPtdIns is neutral respectively to lose 126Da, 155Da, 193Da, 186Da, 213Da, and it is broken that 274Da is produced
Piece quasi-molecular ions, therefore, above-mentioned phosphatide is detected using neutral loss scan pattern, at the same similarly Selection utilization precursor from
Sub- scan pattern carries out the detection of lysoPtdCho.
Embodiment
It is prepared by sample and reagent
1. reagent：Various analytically pure chemical reagent (unless there are specified otherwise) are purchased from Sigma-Aldrich companies
(St.Louis, USA)；Water required for experiment is produced using Mili-Q Hyperpure water manufacturing systems (Milipore companies of the U.S.)
Milli-Q water；Trimethyl silicon substrate diazomethane (IUPAC name=(diazomethyl)-trimethylsilane, TMS-
Diazomethane) (2M is dissolved in n-hexane) is purchased from Acros Organics companies；Chromatographic grade purity methyl tertiary butyl ether(MTBE)
(methyl tert-butyl ether, MTBE), methyl alcohol (methanol), chloroform (Choloform), acetic acid (Acetic
Acid) it is J.T.Baker companies (Phillipsburg, PA, USA) product.
2. internal standard：Do not exist or content in the optional isotope-labeled phospholipid molecule that can individually detect or selection sample
Extremely low phosphatide adds sample as internal standard.The present embodiment chooses C17:0/C14:1-PtdCho, C17:0/C14:1-PtdEtn,
C17:0/C14:1-PtdGro, C17:0/C14:1-PtdSer, C17:0/C14:1-PtdH, C17:0/C14:1-PtdIns,
C17:1-lysoPtdCho, C17:1-lysoPtdEtn, C17:1-lysoPtdGro, C17:1-lysoPtdSer, C17:1-
LysoPtdH and C17:1-lysoPtdIns is used as internal standard.
3. solution allocation：Washing lotion before (a) common derivatization：Methyl tertiary butyl ether(MTBE)/methyl alcohol/0.01N hydrochloric acid solutions, volume ratio
Example is MTBE:MeOH:0.01N hydrochloric acid=100:30:25, remove phase；Washing lotion after (b) derivatization：Methyl tertiary butyl ether(MTBE)/methyl alcohol/
The aqueous solution, volume ratio is MTBE:MeOH:H2O=100:30:25, remove phase.
4. phosphatide is extracted
By separate sources biological specimen, such as blood plasma (10 μ L), cell (～5 × 105It is individual) and tissue (10mg) etc., it is put into
The eppordorft pipes of organic solvent-resistant corrosion, and the phosphatide internal standard described in (a) is mixed, each interior target consumption is
0.4nmol；By 1.3mL methyl tertiary butyl ether(MTBE)s/methyl alcohol (10:3, v/v) solution, room temperature concussion 1h；Add toward above-mentioned solution
0.25mL deionized waters, after fully shaking, room temperature places 10min；Visible obvious layering after 1,000g centrifugation 10 minutes, collects
Upper solution (organic phase) to new eppordorft is managed；Lower floor's solution then with 1.55mL methyl tertiary butyl ether(MTBE)s/methanol/water (10:
3:2.5, v/v/v) upper solution of solution merges, and extracts again once according to above-mentioned, collects upper solution and aforesaid upper solution
Merge；The supernatant collected is carried out into vacuum, freeze concentration to be dried, the phospholipid extract as extracted further is being processed
Before, -80 DEG C of refrigerator freezing collections.
5. the esterification of phosphatide derives
Wash before derivatization：Phospholipid extract is dissolved in 682.5 μ L methyl tertiary butyl ether(MTBE)s/methyl alcohol/2N hydrochloric acid (500 again:
150:32.5, v/v/v) 250 μ L 0.1N hydrochloric acid, are added, fully shaking is centrifuged (6,500 × g) 10 minutes after 4 DEG C, by supernatant
It is transferred to new eppendorft and manages and add 500 μ L methyl tertiary butyl ether(MTBE)s/methyl alcohol/0.01N hydrochloric acid (100:30:25, v/v/v)
The lower phase of solution, washed once again according to above step, and collect upper solution to new eppendorft pipes, after as washing
Phospholipid extract (～0.5mL).
50 μ L trimethyl silicane diazomethane solutions (2M is dissolved in n-hexane) are added into the phospholipid extract after above-mentioned washing
(visible obvious yellow), room temperature rotation incubation 20 minutes；Add 3 μ l acetic acid neutralized methylizations reaction (visible yellow color takes off to the greatest extent)；
After neutralization, 500 μ l methyl tertiary butyl ether(MTBE)s/methanol/water (100 is added:30:25, v/v/v) lower phase, after fully shaking, 1,500
× g be centrifuged 3 minutes, in collections phase and repeat above-mentioned washing step once；The supernatant collected is carried out into vacuum, freeze concentration to do
It is dry, as phospholipid extract derived from esterification, before mass spectral analysis, -80 DEG C of refrigerator freezings collections.
6. the mass spectral analysis of phosphatide esterification derivative
Ion gun direct injected system (Triversa is sprayed using equipped with receiving automaticallyAdvion
Biosciences TSQ Vantages (ThermoFisher Scientific, Bremen)).Receive spray column internal diameter be 5.5 μ
m.Software control system is ChipSoftTM Software (Advion Biosciences), and parameter is as follows:
A) positive ion mode, ionizing voltage is 1.25kV；
B) gas backpressure is 0.5psi；
C) parameter setting for inhaling 1 μ L air will be continued after advance rinse Tip heads and pipette samples to open, to prevent Tip
Sample in head flows out in mechanical arm moving process；
The parameter of mass spectrometer system is as follows：
A) temperature of ion transfer tube is 190 DEG C, and the RF Amplitude of S-Lens are 217；
B) neutral to lose or the impact energy of precursor ion-scan is 40eV, dwelling time are 500ms, mass spectrometric resolution
Rate is 1Da；
C) each neutrality is lost or precursor ion-scan collection 3min；
D) mass spectrum mass range is set as m/z 400-1200.
7. phosphatide is quantitative
Phosphatide in biological specimen is quantitative main by carrying out reference with the internal standard for adding, and calculates esterification phospholipid molecule
Amount, and then associate the amount of phosphatide corresponding to sample.Phosphatide esterification derivative it is quantitative as follows：
CPhosphatide/CInternal standard=IPhosphatide/IInternal standard,
CPhosphatideAnd CInternal standardPhospholipid molecule and its corresponding interior target concentration in difference representative sample, and IPhosphatideAnd IInternal standardThen generation respectively
Phospholipid molecule and its corresponding interior target mass spectra peak intensity in table sample sheet.
8. safety notice
Although TMS-diazomethane is as a kind of substitute of hypotoxic diazomethane at the beginning of using.But inhale
Entering excessive TMS-diazomethane can also cause the damage of lung and central nervous system.Therefore, it is suggested that in derivatization or mark
The Cheng Jun that records a demerit is carried out in the fume hood with protection.Excessive TMS-diazomethane must be neutralized with acetic acid.In acetic acid
The process of sum be one generation nitrogen process, process great amount of samples when must careful operation and note protect.
, but those skilled in the art once know basic creation although preferred embodiments of the present invention have been described
Property concept, then can make other change and modification to these embodiments.So, claims are intended to be construed to include excellent
Select embodiment and fall into having altered and changing for the scope of the invention.
Obviously, those skilled in the art can carry out the essence of various changes and modification without deviating from the present invention to the present invention
God and scope.So, if these modifications of the present invention and modification belong to the scope of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to comprising these changes and modification.
Claims (21)
1. it is a kind of based on chemically derived phosphatide classification and Detection and quantitative approach, it is characterised in that to comprise the following steps：
Step 1：Phosphatide to be detected is extracted from sample to be detected；
Step 2：Esterification is carried out to the phosphate group in the phospholipid molecule in sample using trimethyl silicane diazomethane to derive, drop
The electronegativity of low phospholipid molecule；
Step 3：Through phospholipid molecule derived from esterification in ionization sample, the several ion for treating Mass Spectrometer Method is produced, it is described
Ionic species to be detected includes phosphatid ylcholine, phosphatidyl-ethanolamine, phosphatidylserine, phosphatidic acid, phosphatidyl glycerol, phosphorus
Acyl inositol, Lysophosphatidylcholone, hemolytic phosphatidyl-ethanolamine, hemolytic phosphatidylserine, lysophosphatidic,
Hemolytic phosphatidyl glycerol, hemolytic phosphatidylinositols；
Step 4：In the phosphatid ylcholine, phosphatidyl-ethanolamine, phosphatidylserine, Lysophosphatidylcholone, hemolytic
Phosphatidyl-ethanolamine, hemolytic phosphatidylserine, lysophosphatidic, hemolytic phosphatidyl glycerol, hemolytic phosphatidyl-4
On alcohol, adduction H is chosen respectively+Ion；
On phosphatidic acid, phosphatidyl glycerol, phosphatidylinositols, adduction NH is chosen respectively4 +Ion；
According to the cleavage of mass spectrum rule of variety classes ion in the step 3, the different neutral loss of setting or precursor ion are swept
Pattern is retouched, classification and Detection is carried out to variety classes ion in the step 3 using mass spectrum, obtain variety classes in the step 3
The amount of the mass ions signal of ion；
Step 5：By comparing with internal standard, the mass ions letter of variety classes ion in the step 3 that the step 4 is obtained
Number amount be associated with the amount of phospholipid molecule in sample, obtain the amount of variety classes phospholipid molecule in sample；
Wherein,
The step 3,
Ionization using equipped with receive automatically spray ion gun direct injected system, receive spray column internal diameter be 5.5 μm；Parameter is as follows:
A) positive ion mode, ionizing voltage is 1.25kV；
B) gas backpressure is 0.5psi；
C) parameter setting for inhaling 1 μ L air will be continued after advance rinse termination and pipette samples to open, in case in not-go-end head
Sample flows out in mechanical arm moving process；
The parameter of mass spectrometer system is as follows：
A) temperature of ion transfer tube is 190 DEG C, and the high frequency amplitude of S lens is 217；
B) impact energy of neutral loss or precursor ion-scan is 40eV, and residence time is 500ms, and mass resolution is 1Da；
C) each neutrality is lost or precursor ion-scan collection 3min；
D) mass spectrum mass range is set as m/z 400-1200；
The step 4,
Phosphatid ylcholine, phosphatidyl-ethanolamine, phosphatidylserine primarily form adduction H+Ion；Phosphatidic acid, phosphatidyl are sweet
Oil, phosphatidylinositols primarily form adduction NH4 +Ion.
2. it is according to claim 1 based on chemically derived phosphatide classification and Detection and quantitative approach, it is characterised in that described
Step 1 is carried out in methyl tertiary butyl ether(MTBE)/methanol/water solution system, the methyl tertiary butyl ether(MTBE)/methanol/water solution body
In system, methyl tertiary butyl ether(MTBE), methyl alcohol, the volume ratio of water are 10: 3: 2.5.
3. it is according to claim 1 based on chemically derived phosphatide classification and Detection and quantitative approach, it is characterised in that described
Step 2 is carried out in methyl tertiary butyl ether(MTBE)/methyl alcohol/1N hydrochloric acid solution systems, the methyl tertiary butyl ether(MTBE)/methyl alcohol/1N salt
In acid solution system, methyl tertiary butyl ether(MTBE), methyl alcohol, the volume ratio of 1N hydrochloric acid are 300: 90: 4.
4. it is according to claim 1 based on chemically derived phosphatide classification and Detection and quantitative approach, it is characterised in that described
Step 3 is carried out in chloroform/methanol/ammonium acetate solution system, described in the chloroform/methanol/ammonium acetate solution system
The concentration of ammonium acetate is 5～10mM, and the chloroform, the volume ratio of methyl alcohol are (1～2): 1.
5. it is according to claim 1 based on chemically derived phosphatide classification and Detection and quantitative approach, it is characterised in that described
Phosphatidic acid, phosphatidyl-ethanolamine, phosphatidyl glycerol, phosphatidylserine, phosphatidylinositols, lysophosphatidic, hemolytic phosphorus
Acyl monoethanolamine, hemolytic phosphatidyl glycerol, hemolytic phosphatidylserine, hemolytic phosphatidylinositols are swept using neutral loss
Retouching carries out detecting and quantitative.
6. it is according to claim 5 based on chemically derived phosphatide classification and Detection and quantitative approach, it is characterised in that to utilize
When neutral loss scan carries out detecting and being quantitative to the phosphatidic acid, it is 143Da that neutrality is lost；Using neutral loss scan to institute
State phosphatidyl-ethanolamine carry out detection and it is quantitative when, neutrality lose be 155Da；It is sweet to the phosphatidyl using neutral loss scan
When oil carries out detecting and being quantitative, it is 203Da that neutrality is lost；The phosphatidylserine is detected using neutral loss scan
With it is quantitative when, neutrality lose be 213Da；When carrying out detecting and being quantitative to the phosphatidylinositols using neutral loss scan, in
Property lose be 291Da；
When carrying out detecting and being quantitative to lysophosphatidic using neutral loss scan, it is 126Da that neutrality is lost；Lost using neutrality
When mistake scanning carries out detecting and being quantitative to hemolytic phosphatidyl-ethanolamine, neutrality loss is 155Da；Using neutral loss scan pair
When hemolytic phosphatidyl glycerol carries out detecting and being quantitative, it is 186Da that neutrality is lost；Using neutral loss scan to hemolytic phosphatide
When acyl serine carries out detecting and being quantitative, it is 213Da that neutrality is lost；Hemolytic phosphatidylinositols is entered using neutral loss scan
When row detects and be quantitative, it is 274Da that neutrality is lost.
7. it is according to claim 1 based on chemically derived phosphatide classification and Detection and quantitative approach, it is characterised in that described
Phosphatid ylcholine, Lysophosphatidylcholone are detected and quantitatively using precursor ion-scan.
8. it is according to claim 7 based on chemically derived phosphatide classification and Detection and quantitative approach, it is characterised in that to institute
State phosphatid ylcholine carry out detection and it is quantitative when, the matter/lotus ratio of the precursor ion is 198.0 ± 0.5；To the hemolytic phosphorus
When phosphatidylcholine carries out detecting and being quantitative, the matter/lotus ratio of the precursor ion is 198.0 ± 0.5.
9. it is according to claim 1 based on chemically derived phosphatide classification and Detection and quantitative approach, it is characterised in that described
Sample to be detected is biological specimen.
10. it is according to claim 1 based on chemically derived phosphatide classification and Detection and quantitative approach, it is characterised in that institute
State in sample to be detected and be added with antioxidant.
11. is according to claim 10 based on chemically derived phosphatide classification and Detection and quantitative approach, it is characterised in that institute
Antioxidant is stated for dibutyl hydroxy toluene.
12. is according to claim 11 based on chemically derived phosphatide classification and Detection and quantitative approach, it is characterised in that institute
The addition for stating dibutyl hydroxy toluene is the 0.01～0.03% of sample to be tested volume.
13. according to arbitrary described based on chemically derived phosphatide classification and Detection and quantitative approach in claim 10～12, its
It is characterised by, when adding antioxidant in the sample to be detected, environment temperature is 4～10 DEG C.
14. is according to claim 1 based on chemically derived phosphatide classification and Detection and quantitative approach, it is characterised in that institute
The preserving type for stating sample to be detected is freezen protective.
15. is according to claim 1 based on chemically derived phosphatide classification and Detection and quantitative approach, it is characterised in that institute
State interior target selection principle and select an internal standard for each phospholipid species.
16. is according to claim 15 based on chemically derived phosphatide classification and Detection and quantitative approach, it is characterised in that institute
The isotope-labeled phospholipid molecule that can individually detect is designated as in stating,
Or,
The phosphatide that do not exist or content extremely low is designated as in sample to be tested in described.
17. according to claim 15 or 16 based on chemically derived phosphatide classification and Detection and quantitative approach, its feature exists
In the interior target consumption changes according to different samples to be tested.
18. is according to claim 1 based on chemically derived phosphatide classification and Detection and quantitative approach, it is characterised in that
Between the step 1 and step 2, also including the step of washing.
19. is according to claim 1 based on chemically derived phosphatide classification and Detection and quantitative approach, it is characterised in that
In the step 5, computing mode is：
CPhosphatide/CInternal standard=IPhosphatide/IInternal standard,
Wherein, CPhosphatideAnd CInternal standardPhospholipid molecule and its corresponding interior target concentration are represented in sample to be tested respectively and IPhosphatideAnd IInternal standardThen divide
Phospholipid molecule and its corresponding interior target mass spectra peak intensity in other representative sample.
20. is according to claim 1 based on chemically derived phosphatide classification and Detection and quantitative approach, it is characterised in that institute
State step 2 is carried out in the fume hood with guard system.
21. is according to claim 1 based on chemically derived phosphatide classification and Detection and quantitative approach, it is characterised in that institute
Step 2 is stated, when the application of trimethyl silicane diazomethane is excessive, in carrying out to excessive trimethyl silicane diazomethane from acetic acid
With.
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| CN107228915A (en) * | 2017-05-12 | 2017-10-03 | 广州大学 | A kind of method for efficiently separating purification glycosyl phosphatidylinositol monoethanolamine |
| CN110243917A (en) * | 2018-03-09 | 2019-09-17 | 中国科学院天津工业生物技术研究所 | A kind of method of quick detection small molecule compound |
| US20210310999A1 (en) * | 2018-09-10 | 2021-10-07 | Shimadzu Corporation | Biological membrane phosphoinositide separation method |
| CN109358134B (en) * | 2018-12-19 | 2021-06-01 | 武汉绿剑可瑞信科技有限公司 | Pretreatment method and quantitative detection method for endogenous phosphosaccharide compounds in plant sample |
| CN109406687B (en) * | 2018-12-29 | 2021-03-23 | 清华大学 | Method for detecting double phospholipids at high flux |
| CN114113426B (en) * | 2021-12-27 | 2024-04-26 | 西安血氧生物技术有限公司 | Method for detecting phospholipid in hemoglobin oxygen carrier |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008051790A (en) * | 2006-03-15 | 2008-03-06 | Noguchi Inst | Method for trace mass analysis |
| CN101374555A (en) * | 2005-04-06 | 2009-02-25 | 圣路易斯华盛顿州立大学 | Methods for measuring the metabolism of neurally derived biomolecules in vivo |
| CN102473208A (en) * | 2009-07-02 | 2012-05-23 | 百奥科瑞茨生命科学公司 | Method for normalization in metabolomics analysis methods with endogenous reference metabolites |
-
2015
- 2015-07-27 CN CN201510446589.2A patent/CN105021758B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101374555A (en) * | 2005-04-06 | 2009-02-25 | 圣路易斯华盛顿州立大学 | Methods for measuring the metabolism of neurally derived biomolecules in vivo |
| JP2008051790A (en) * | 2006-03-15 | 2008-03-06 | Noguchi Inst | Method for trace mass analysis |
| CN102473208A (en) * | 2009-07-02 | 2012-05-23 | 百奥科瑞茨生命科学公司 | Method for normalization in metabolomics analysis methods with endogenous reference metabolites |
Non-Patent Citations (4)
| Title |
|---|
| Probing phospholipid dynamics by electrospray ionisation mass spectrometry;Anthony D. Postle et al;《Progress in Lipid Research》;20071231;第46卷;200-224 * |
| Profiling and Relative Quantitation of Phosphoinositides by Multiple Precursor Ion Scanning Based on Phosphate Methylation and Isotopic Labeling;Tanxi Cai et al;《Analytical Chemistry》;20141126;第87卷;513-521 * |
| Quantification of PtdInsP3 molecular species in cells and tissues by mass spectrometry;Jonathan Clark et al;《Nature Method》;20110331;第8卷(第3期);267-272 * |
| 天然磷脂的色谱法分离、纯化研究进展;何海波 等;《分析科学学报》;20040229;第20卷(第1期);97-102 * |
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