CN105021758A - Chemical derivatization-based phosphatide classification detection and quantification method - Google Patents
Chemical derivatization-based phosphatide classification detection and quantification method Download PDFInfo
- Publication number
- CN105021758A CN105021758A CN201510446589.2A CN201510446589A CN105021758A CN 105021758 A CN105021758 A CN 105021758A CN 201510446589 A CN201510446589 A CN 201510446589A CN 105021758 A CN105021758 A CN 105021758A
- Authority
- CN
- China
- Prior art keywords
- detection
- phosphatide
- ion
- sample
- hemolytic
- 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.)
- Granted
Links
Landscapes
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
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, particularly relate to a kind of based on chemically derived phosphatide classification and Detection and quantivative approach.
Background technology
Phosphatide is not only the important composition composition of cell membrane, and also wide participation regulates multiple vital movement process, comprises energy conversion, matter transportation, information identification and transmission, cell development and differentiation, and Apoptosis etc.The abnormal metabolism of phosphatide and arteriosclerosis, diabetes, obesity, Alzheimer disease and tumour occur also closely related.Therefore, from the angle of " group is learned ", research phospholipid molecule in systematicness, scale ground forms and the Dynamic Variation Analysis of relative quantity under different physiology or pathological state, to the relation disclosed between the physiology of phospholipid metabolism and cell, organ and even body, pathologic process and molecule mechanism thereof, and then excavation, to disease, relevant important biomolecule label occurs, promote that the early diagnosis and therapy of disease has great importance.
At present, along with Ionization Techniques In Mass Spectrometry, particularly electro-spray ionization (electrosprayionization, and substance assistant laser desorpted ionization (matrix assisted laser desorption/ionization ESI), etc. MALDI) development of the ionization techniques of biomacromolecule is suitable for, based on biological mass spectrometry phosphatide qualification and quantitative analysis tech because of features such as its high sensitivity, high resolving power, high accuracy and high flux property, become phosphatide group study main tool.Phospholipid lipid molecular all can utilize ESI-MS to analyze, but different types of phospholipid molecule has different ionization tendencies because of the difference of self structure (mainly polar head) and solution composition.Phosphatid ylcholine (PtdCho) lipoids molecule in electric neutrality, tends to after ionization form adduction H under neutrality or alkaline pH
+positive ion.Although when the solution containing acetate and high voltage, PtdCho also can be formed [M-CH3COO]
-the negative ion of form, but signal is lower.Cytoskeletal protein lipid molecular (PtdEtn) due to containing monoethanolamine polar head, under ph basic conditions, the easy deprotonation of quaternary amine base and carry a net negative charge.Acidic phospholipid lipoids, comprise phosphatidic acid (PtdH), phosphatidyl glycerol (PtdGro), phosphatidylserine (PtdSer), phosphatidylinositols (PtdIns), carry one or more net negative charge under physiological ph conditions, mainly exist with the form of negative ion when Mass Spectrometer Method.Therefore, under normal circumstances, PtdCho positive ion mode detects, PtdEtn, PtdH, PtdGro, PtdSer and PtdIns then detect with negative ion, thus cause in conjunction with positive ion and negative ion mode, thus the growth of analysis time to be caused when utilizing ESI-MS to carry out qualitative and quantitative analysis to the phosphatide group in biological specimen.Meanwhile, in the negative ion mode, the detection sensitivity of part phosphatide is relatively low.
On the other hand, for excavating the important phospholipid molecule and the mechanism of action thereof of being correlated with from different physiology, pathologic process further, need to analyze the change of the relative quantity of phosphatide group under different physiology or pathological state.Much traditional research and utilization radioactive label (as
3h-,
14c-or
32p-mark carbon source or phospholipid head groups) or fluorescence labeling (as 7-nitrobenz-2-oxa-1,3-diazol-4-yl, NBD) precursor, selectivity (a certain lipid specific precursor, as inositol) or non-selective (precursor as common in lipid) metabolic tagged tissue or cell in phosphatide, thus reach quantitative object, but these Measures compare are loaded down with trivial details, and waste time and energy, based on the quantitative analysis tech of ESI/MS because of its feature such as high resolving power, high accuracy, be applied to the quantitative test of cytolipin group more and more.Under optimal conditions (under the concentration conditions of such as pmol/mL or lower), Phospholipids spectrum signal intensity and phospholipid concentration is linear and Ionization Efficiency that is identical type phospholipid molecule is basically identical, has nothing to do with the physical property of fatty acid chain.Therefore, in positive ion and negative ion mode, marking by adding in specific phospholipid species, can carry out quantitatively the phosphatide in biological specimen.
Clark etc. are (see Clark, J., et al.Nat Methods.2011,8,267-72.) report the detection sensitivity utilizing the phosphate group of trimethyl silicane diazomethane (TMS-diazomethane) esterification diphosphoinositide (PIPs) greatly can improve diphosphoinositide, prompting utilizes TMS-diazomethane can make phosphate group esterification, and has and be applied to phosphatide and detect and quantitative Potential feasibility.However, the detection methyl orthophosphoric acid based on trimethyl silicane diazomethane (TMS-diazomethane) being applied to phosphatide still has problems to wait answer.First, due to the complicacy of phospholipid species and structure, the detection whether methyl orthophosphoric acidization based on trimethyl silicane diazomethane (TMS-diazomethane) can be applied to phosphatide (comprising hemolytic phosphatide) it be unclear that, and also has no relevant report.Secondly, whether the derivatization efficiency based on the methyl orthophosphoric acid of trimethyl silicane diazomethane (TMS-diazomethane) is subject to the physics of phospholipid molecule or the impact of chemical property, the fatty acid chain length of such as phospholipid molecule, the saturation degree etc. of fatty acid chain, also it be unclear that.3rd, how the phospholipid molecule that esterification derives utilizes mass spectrum carry out detection and quantitatively also do not understand.
Summary of the invention
In view of this, the invention provides a kind of using trimethyl silicon diazomethane to the phosphate group in phospholipid molecule in sample carry out esterification derive, and utilize mass spectrum, the amount of variety classes phospholipid molecule in sample to be tested is carried out quantitative based on chemically derived phosphatide classification and Detection and quantivative approach.
In order to achieve the above object, the present invention mainly provides following technical scheme:
Provided by the inventionly to comprise the following steps based on chemically derived phosphatide classification and Detection and quantivative approach:
Step 1: extract phosphatide to be detected from sample to be detected;
Step 2: using trimethyl silicon diazomethane carries out esterification to the phosphate group in the phospholipid molecule in sample and derives, reduces the electronegativity of phospholipid molecule;
Step 3: the phospholipid molecule derived through esterification in ionization sample, produce the several ion treating Mass Spectrometer Method, described ionic species to be detected comprises phosphatid ylcholine, phosphatidyl-ethanolamine, phosphatidylserine, phosphatidic acid, phosphatidyl glycerol, phosphatidylinositols, Lysophosphatidylcholone, hemolytic phosphatidyl-ethanolamine, hemolytic phosphatidylserine, lysophosphatidic, hemolytic phosphatidyl glycerol, hemolytic phosphatidylinositols;
Step 4: on described phosphatid ylcholine, phosphatidyl-ethanolamine, phosphatidylserine, Lysophosphatidylcholone, hemolytic phosphatidyl-ethanolamine, hemolytic phosphatidylserine, lysophosphatidic, hemolytic phosphatidyl glycerol, hemolytic phosphatidylinositols, choose adduction H respectively
+ion;
On phosphatidic acid, phosphatidyl glycerol, phosphatidylinositols, choose adduction NH respectively
4 +ion;
According to the cleavage of mass spectrum rule of variety classes ion in described step 3, set different neutrality to lose or precursor ion-scan pattern, utilize mass spectrum to carry out classification and Detection to variety classes ion in described step 3, obtain the amount of the mass ions signal of variety classes ion in described step 3;
Step 5: by comparing with interior mark, in the described step 3 described step 4 obtained, the amount of the mass ions signal of variety classes ion 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 also can be applied to the following technical measures to achieve further.
As preferably, utilize mass spectrum to when variety classes ion carries out classification and Detection in described step 3, select the mode of liquid chromatograph mass spectrography to detect, or, select the mode of direct injected-mass spectrometry to detect.
As preferably, described step 1 is carried out in methyl tert-butyl ether/methanol/water solution system, and in described methyl tert-butyl ether/methanol/water solution system, the volume ratio of methyl tert-butyl ether, methyl alcohol, water is 10: 3: 2.5.
As preferably, described step 2 is carried out in methyl tert-butyl ether/methyl alcohol/1N hydrochloric acid solution system, and in described methyl tert-butyl ether/methyl alcohol/1N hydrochloric acid solution system, the volume ratio of methyl tert-butyl ether, methyl alcohol, 1N hydrochloric acid is 300: 90: 4.
As preferably, described step 3 is carried out in chloroform/methanol/ammonium acetate solution system, and in described chloroform/methanol/ammonium acetate solution system, the concentration of described ammonium acetate is 5 ~ 10mM, and the volume ratio of described chloroform, methyl alcohol is (1 ~ 2): 1.
As preferably, described phosphatidic acid, phosphatidyl-ethanolamine, phosphatidyl glycerol, phosphatidylserine, phosphatidylinositols, lysophosphatidic, hemolytic phosphatidyl-ethanolamine, hemolytic phosphatidyl glycerol, hemolytic phosphatidylserine, hemolytic phosphatidylinositols utilize neutral loss scan to carry out detecting and quantitatively.
As preferably, utilize neutral loss scan to carry out detection to described phosphatidic acid and quantitative time, neutrally to lose for 143Da (126Da+NH
3); Utilize neutral loss scan to carry out detection to described phosphatidyl-ethanolamine and quantitative time, neutral lose for 155Da; Utilize neutral loss scan to carry out detection to described phosphatidyl glycerol and quantitative time, neutral to lose for 203Da (186Da+NH
3); Utilize neutral loss scan to carry out detection to described phosphatidylserine and quantitative time, neutral lose for 213Da; Utilize neutral loss scan to carry out detection to described phosphatidylinositols and quantitative time, neutral to lose for 291Da (274Da+NH
3);
Utilize neutral loss scan to carry out detection to lysophosphatidic and quantitative time, neutral lose for 126Da; Utilize neutral loss scan to carry out detection to hemolytic phosphatidyl-ethanolamine and quantitative time, neutral lose for 155Da; Utilize neutral loss scan to carry out detection to hemolytic phosphatidyl glycerol and quantitative time, neutral lose for 186Da; Utilize neutral loss scan to carry out detection to hemolytic phosphatidylserine and quantitative time, neutral lose for 213Da; Utilize neutral loss scan to carry out detection to hemolytic phosphatidylinositols and quantitative time, neutral to lose for 274Da.
As preferably, described phosphatid ylcholine, Lysophosphatidylcholone utilize precursor ion-scan to carry out detecting and quantitatively.
As preferably, detection is carried out to described phosphatid ylcholine and quantitative time, the matter/lotus ratio of described precursor ion is 198.0 ± 0.5; Detection is carried out to described Lysophosphatidylcholone and quantitative time, the matter/lotus ratio of described precursor ion is 198.0 ± 0.5.
As preferably, described sample to be tested is biological specimen.
As preferably, in described sample to be tested, be added with antioxidant.
As preferably, described antioxidant is dibutyl hydroxy toluene.
As preferably, the addition of described dibutyl hydroxy toluene is 0.01 ~ 0.03% of sample to be tested volume.
As preferably, when adding antioxidant in described sample to be tested, environment temperature is 4 ~ 10 DEG C.
As preferably, the preserving type of described sample to be tested is freezen protective.
As preferably, described interior target selection principle is that each phospholipid species is selected in one and marked.
As preferably, in described, be designated as the isotope-labeled phospholipid molecule that can detect separately,
Or,
Be designated as in described in sample to be tested and do not exist or phosphatide that content is extremely low.
As preferably, described interior target consumption changes according to different samples to be tested.
As preferably, between described step 1 and step 2, also comprise the step of washing.
As preferably, when utilizing mass spectrum to carry out classification and Detection to described step 2 intermediate ion, select direct injected-mass spectrum, described sampling device is syringe pump.
As preferably, when utilizing mass spectrum to carry out classification and Detection to described step 2 intermediate ion, select direct injected-mass spectrum, described sampling device is for automatically receiving spray ion gun direct injected system.
As preferably, in described step 5, compute mode is:
C
phosphatide/ C
interior mark=I
phosphatide/ I
interior mark,
Wherein, C
phosphatideand C
interior markrepresent the interior target concentration of phospholipid molecule and correspondence thereof in sample to be tested respectively and I
phosphatideand I
interior markthen distinguish phospholipid molecule and corresponding interior target mass spectra peak intensity thereof in representative sample.
As preferably, described step 2 is carried out in the fuming cupboard of guard system.
As preferably, described step 2, when the application of trimethyl silicane diazomethane is excessive, selects acetic acid to neutralize excessive trimethyl silicane diazomethane.
Provided by the inventionly based on chemically derived phosphatide classification and Detection and quantivative approach using trimethyl silicon diazomethane, esterification is carried out to the phosphate group in phospholipid molecule in sample to be tested and derive; Through the phospholipid molecule that esterification is derivative in ionization sample, produce the several ion treating Mass Spectrometer Method; Treating that the ion of Mass Spectrometer Method is selected accordingly, according to different phosphate lipid species, select adduction H
+ion or NH
4 +ion; Set different neutrality according to the cleavage of mass spectrum rule of variety classes ion to lose or precursor ion-scan pattern, obtain the amount of the mass ions model of variety classes ion; Finally, by comparing and computing with interior mark, the amount of variety classes phospholipid molecule in sample is obtained.The method detection speed is fast, highly sensitive.
Accompanying drawing explanation
By reading hereafter detailed description of the preferred embodiment, various other advantage and benefit will become cheer and bright for those of ordinary skill in the art.Accompanying drawing only for illustrating the object of preferred implementation, and does not think limitation of the present invention.And in whole accompanying drawing, represent identical parts by identical reference symbol.In the accompanying drawings:
The flow chart of steps based on chemically derived phosphatide classification and Detection and quantivative approach that Fig. 1 provides for the embodiment of the present invention;
Fig. 2 for the embodiment of the present invention provide based in chemically derived phosphatide classification and Detection and quantivative approach, with PtdH, PtdCho, PtdGro and PtdSer are the cleavage of mass spectrum rule of representative explaination esterification phospholipid molecule and the establishment of neutral loss and precursor ion-scan rule.The C17:0/C14:1-PtdH (A) of esterification, C17:0/C14:1-PtdCho (B), C17:0/C14:1-PtdGro (C) and C17:0/C14:1-PtdSer (D) all can produce the fragmention that mass-to-charge ratio is 535, corresponding to charged C17:0/C14:1-DAG (C17:0/C14:1-DAG
+).DAG
+ion also can be considered it is adduction H
+or NH
4 +the neutrality of the esterification phospholipid molecule of ion loses peak, and namely PtdH, PtdCho, PtdGro and PtdSer are neutral respectively loses 143Da (126Da+NH
3), 193Da, 203Da (186+NH
3), the fragment ion peak that 213Da produces.Accordingly, neutral loss scan pattern is utilized to detect above-mentioned phosphatide.Due to the DAG that PtdCho produces
+fragmention signal is more weak, and fragment ion peak (mass-to-charge ratio the is 198) signal corresponding to esterification head group is stronger, therefore, then utilizes precursor ion-scan pattern to detect for PtdCho class phosphatide;
Fig. 3 embodiment of the present invention provide based in chemically derived phosphatide classification and Detection and quantivative approach, with lysoPtdH, lysoPtdGro, lysoPtdSer and lysoPtdIns are the cleavage of mass spectrum rule of the hemolytic phospholipid molecule of representative explaination esterification and the establishment of neutral loss and precursor ion-scan rule.The C17:1-lysoPtdH (A) of esterification, C17:1-lysoPtdGro (B), C17:1-lysoPtdH (C) and C17:1-lysoPtdIns (D) all can produce the fragmention that mass-to-charge ratio is 325, corresponding to charged C17:1-MAG (C17:1-MAG
+).MAG
+ion also can be considered it is adduction H
+or NH
4 +the neutrality of the esterification phospholipid molecule of ion loses peak, i.e. lysoPtdH, lysoPtdGro, lysoPtdSer and the lysoPtdIns neutral fragment ion peak lost 126Da, 186Da, 213Da and 274Da and produce respectively.Accordingly, neutral loss scan pattern is utilized to detect above-mentioned phosphatide;
Fig. 4 embodiment of the present invention provide based in chemically derived phosphatide classification and Detection and quantivative approach, phospholipid molecule esterification derives and loses or the schematic diagram of precursor ion-scan based on neutrality;
Fig. 5 embodiment of the present invention provide based in chemically derived phosphatide classification and Detection and quantivative approach, the esterification of phospholipid molecule derives efficiency evaluation.For the Cho of Ptd, before derivatization, after (A) and derivatization, the mass spectrogram display of the PtdCho of (B) derives efficiency not by the length of phospholipid molecule fatty acid chain and the impact of saturation degree based on the phospholipid molecule esterification of trimethyl silicane diazomethane.
Fig. 6 embodiment of the present invention provide based in chemically derived phosphatide classification and Detection and quantivative approach, in the positive-ion mode, utilize matter/lotus than the precursor ion being 198.0 and carry out detecting fast to phosphatid ylcholine (A) and quantitatively; The neutral loss of utilization is, 155Da, 203Da, 213Da, the phosphatidyl-ethanolamine (B) that 143Da and 291Da is right respectively, phosphatidyl glycerol (C), phosphatidylserine (D), phosphatidic acid (E), and phosphatidylinositols (F) carries out detecting fast with quantitative.
Embodiment
The present invention is for solving prior art Problems existing, provide a kind of using trimethyl silicon diazomethane to the phosphate group in phospholipid molecule in sample carry out esterification derive, and utilize mass spectrum, the amount of variety classes phospholipid molecule in sample to be tested is carried out quantitative based on chemically derived phosphatide classification and Detection and quantivative approach.
Describing based on chemically derived phosphatide classification and Detection and quantivative approach that the embodiment of the present invention provides detects and quantivative approach fast based on chemically derived and phosphatide that is direct injected mass spectrophotometry.The method utilizes trimethyl silicane diazomethane by the phosphate group esterification in phospholipid molecule, reduces the electronegativity of phospholipid molecule, while improving detection sensitivity, realizes all phospholipid species Mass Spectrometer Method in the positive-ion mode, greatly saves the time.By direct injected and electro-spray ionization mode, by phospholipid molecule ionization derivative for esterification, produce the one or more of ion that can be detected by mass spectroscopy, and determine the ion of Mass Spectrometer Method.According to the cleavage of mass spectrum rule of Ionized phospholipid molecule, set different neutrality to lose or precursor ion-scan pattern, in the positive-ion mode, mass spectrum is utilized to detect fast phosphatide and corresponding hemolytic phosphatide (lyso-) (totally 12 kinds) thereof.By comparing with interior target, the amount of the mass ions signal of phospholipid molecule is associated with the amount of phospholipid molecule in sample.The method provided has the sensitivity of enhancing, and completes in the short period of time.
For further setting forth the present invention for the technological means reaching predetermined goal of the invention and take and effect, below in conjunction with accompanying drawing and preferred embodiment, to according to the present invention propose based on chemically derived phosphatide classification and Detection and quantivative approach, its embodiment, structure, feature and effect thereof, be described in detail as follows.In the following description, the not necessarily same embodiment that different " embodiment " or " embodiment " refers to.In addition, special characteristic, structure or feature in one or more embodiment can be combined by any suitable form.
Term "and/or" herein, being only a kind of incidence relation describing affiliated partner, can there are three kinds of relations in expression, such as, A and/or B, concrete is interpreted as: can include A and B simultaneously, can individualism A, also can individualism B, above-mentioned three kinds of any one situations can be possessed.
Provided by the inventionly to comprise the following steps based on chemically derived phosphatide classification and Detection and quantivative approach:
Step 1: extract phosphatide to be detected from sample to be detected;
Step 2: using trimethyl silicon diazomethane carries out esterification to the phosphate group in the phospholipid molecule in sample and derives, reduces the electronegativity of phospholipid molecule;
Step 3: the phospholipid molecule derived through esterification in ionization sample, produce the several ion treating Mass Spectrometer Method, ionic species to be detected comprises phosphatid ylcholine, phosphatidyl-ethanolamine, phosphatidylserine, phosphatidic acid, phosphatidyl glycerol, phosphatidylinositols, Lysophosphatidylcholone, hemolytic phosphatidyl-ethanolamine, hemolytic phosphatidylserine, lysophosphatidic, hemolytic phosphatidyl glycerol, hemolytic phosphatidylinositols;
Step 4: on phosphatid ylcholine, phosphatidyl-ethanolamine, phosphatidylserine, Lysophosphatidylcholone, hemolytic phosphatidyl-ethanolamine, hemolytic phosphatidylserine, lysophosphatidic, hemolytic phosphatidyl glycerol, hemolytic phosphatidylinositols, choose adduction H respectively
+ion;
On phosphatidic acid, phosphatidyl glycerol, phosphatidylinositols, choose adduction NH respectively
4 +ion;
According to the cleavage of mass spectrum rule of variety classes ion in step 3, set different neutrality to lose or precursor ion-scan pattern, utilize mass spectrum to carry out classification and Detection to variety classes ion in step 3, obtain the amount of the mass ions signal of variety classes ion in step 3;
Step 5: by comparing with interior mark, in the step 3 obtain step 4, the amount of the mass ions signal of variety classes ion is associated with the amount of phospholipid molecule in sample, obtains the amount of variety classes phospholipid molecule in sample to be tested.
Wherein, utilize mass spectrum to when variety classes ion carries out classification and Detection in step 3, select the mode of liquid chromatograph mass spectrography to detect, or, select direct injected-mass spectrum.
Wherein, step 1 is carried out in methyl tert-butyl ether/methanol/water solution system, and in methyl tert-butyl ether/methanol/water solution system, the volume ratio of methyl tert-butyl ether, methyl alcohol, water is 10: 3: 2.5.
Wherein, step 2 is carried out in methyl tert-butyl ether/methyl alcohol/1N hydrochloric acid solution system, and in methyl tert-butyl ether/methyl alcohol/1N hydrochloric acid solution system, the volume ratio of methyl tert-butyl ether, methyl alcohol, 1N hydrochloric acid is 300: 90: 4.
Wherein, step 3 is carried out in chloroform/methanol/ammonium acetate solution system, and in chloroform/methanol/ammonium acetate solution system, the concentration of ammonium acetate is 5 ~ 10mM, and the volume ratio of chloroform, methyl alcohol is (1 ~ 2): 1.
Wherein, phosphatidic acid, phosphatidyl-ethanolamine, phosphatidyl glycerol, phosphatidylserine, phosphatidylinositols, lysophosphatidic, hemolytic phosphatidyl-ethanolamine, hemolytic phosphatidyl glycerol, hemolytic phosphatidylserine, hemolytic phosphatidylinositols utilize neutral loss scan to carry out detecting and quantitatively.
Wherein, utilize neutral loss scan to carry out detection to phosphatidic acid and quantitative time, neutral to lose for 143Da (126Da+NH
3); Utilize neutral loss scan to carry out detection to phosphatidyl-ethanolamine and quantitative time, neutral lose for 155Da; Utilize neutral loss scan to carry out detection to phosphatidyl glycerol and quantitative time, neutral to lose for 203Da (186Da+NH
3); Utilize neutral loss scan to carry out detection to phosphatidylserine and quantitative time, neutral lose for 213Da; Utilize neutral loss scan to carry out detection to phosphatidylinositols and quantitative time, neutral to lose for 291Da (274Da+NH
3);
Utilize neutral loss scan to carry out detection to lysophosphatidic and quantitative time, neutral lose for 126Da; Utilize neutral loss scan to carry out detection to hemolytic phosphatidyl-ethanolamine and quantitative time, neutral lose for 155Da; Utilize neutral loss scan to carry out detection to hemolytic phosphatidyl glycerol and quantitative time, neutral lose for 186Da; Utilize neutral loss scan to carry out detection to hemolytic phosphatidylserine and quantitative time, neutral lose for 213Da; Utilize neutral loss scan to carry out detection to hemolytic phosphatidylinositols and quantitative time, neutral to lose for 274Da.
Wherein, phosphatid ylcholine, Lysophosphatidylcholone utilize precursor ion-scan to carry out detecting and quantitatively.
Wherein, detection is carried out to phosphatid ylcholine and quantitative time, the matter/lotus ratio of precursor ion is 198.0 ± 0.5; Detection is carried out to Lysophosphatidylcholone and quantitative time, the matter/lotus ratio of precursor ion is 198.0 ± 0.5.
Wherein, sample to be tested is biological specimen.
Wherein, antioxidant is added with in sample to be tested.
Wherein, antioxidant is dibutyl hydroxy toluene.
Wherein, the addition of dibutyl hydroxy toluene is 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 mark in.
Wherein, be inside designated as the isotope-labeled phospholipid molecule that can detect separately,
Or,
Inside be designated as in sample to be tested and do not exist or phosphatide that content is extremely low.
Wherein, interior target consumption changes according to different samples to be tested.
Wherein, between step 1 and step 2, also comprise the step of washing.
Wherein, when utilizing mass spectrum to carry out classification and Detection to step 2 intermediate ion, select direct injected-mass spectrum, sampling device is syringe pump.
Wherein, when utilizing mass spectrum to carry out classification and Detection to step 2 intermediate ion, select direct injected-mass spectrum, sampling device is for automatically receiving spray ion gun direct injected system.
Wherein, in steps of 5, compute mode is:
C
phosphatide/ C
interior mark=I
phosphatide/ I
interior mark,
Wherein, C
phosphatideand C
interior markrepresent the interior target concentration of phospholipid molecule and correspondence thereof in sample to be tested respectively and I
phosphatideand I
interior markthen distinguish phospholipid molecule and corresponding interior target mass spectra peak intensity thereof in representative sample.
Wherein, step 2 is carried out in the fuming cupboard of guard system.
Wherein, step 2, when the application of trimethyl silicane diazomethane is excessive, selects acetic acid to neutralize excessive trimethyl silicane diazomethane.
As described in the present embodiment of the invention, term " purifying " or " purification " not refer to from sample, remove all substances outside target analytes (one or more of).Replace, purifying refers to can analyze the process of amount of the one or more of target analytes of other component enrichment that quality testing is surveyed by jamming target relative in sample.Herein by multiple method purifying sample, to allow to remove one or more of interfering material.Such as, the one or more of materials of the Mass Spectrometer Method of selected phosphatide parent ion and daughter ion can be disturbed.
As used herein, term " liquid phase chromatography " refers to that the chemical mixture by liquid embarkation is separated into the process of component, and this causes at fixed liquid phase or solid phase ambient dynamic or the distribution of difference when flowing through fixed liquid phase or solid phase due to chemical entities.
As used herein, term " direct injected " in this article refers to and does not need through chromatographic resolution, and directly utilizes syringe pump or other modes, such as automatically receives and sprays ion gun direct injected system (Triversa
advion Biosciences), phosphatide sample is introduced a kind of mass spectrum input mode that mass spectrum carries out analyzing.
As used herein, term " mass spectroscopy " or " MS " refer to the analytical technology by its quality Identification compound.MS refers to the method for filtering, detecting and measure ion based on its mass-to-charge ratio or " m/z ".MS technology generally comprises: (1) ionising compounds, forms charging cpd; (2) molecular weight of measuring tape electric compound, and calculate mass-to-charge ratio.By any suitable method ionization and detection compound.
As used herein, term " under positive ion mode " refers to the mass spectroscopy of those generations and detection of positive ions.
As used herein, term " ionization " or " ionization " refer to such process: generate the analyte ions with the net charge equaling one or more electron unit.Negative ion is the ion of the net negative charge with one or more electron unit, and positive ion is the ion of the clean positive charge with one or more electron unit.
As used herein, term " substance assistant laser desorpted ionization " or " MALDI " refer to such method: wherein non-volatile sample is exposed to laser emission, it makes analyte desorption in sample and ionization by different kinds of ions approach, and this ionization approach comprises photoionization, protonation effect, proton abstraction and cluster decay (cluster decay).For MALDI, mixed by sample with energy absorption matrix, this promotes the desorb of analyte molecule.
As used herein, term " electro-spray ionization " or " ESI " refer to such method: sample solution is added with high positive voltage or the kapillary of negative voltage with low flow velocity (0.1 ~ 10 μ L/min) by end, form drop, when the Coulomb repulsion of drip gauge surface charge reaches the critical point suitable with solution surface tension, the drop containing a large amount of electric charge will be produced.Along with solvent evaporation, droplet retracts, in drop, between electric charge, repulsive force increases, when arriving and surmount to a certain degree, can there is COULOMB EXPLOSION in drop, the excessive charge on removing drop surface, generate less charged droplet, so circulate, finally generating can for the ion of Mass Spectrometer Method.
Suitable detection sample comprises any detection sample that can comprise target analytes.In some preferred implementations, sample is biological specimen, i.e. the sample of biogenetic derivation, as animal tissue, cell culture, blood plasma etc.In order to avoid oxidation or the degraded of phosphatide, antioxidant should be added in sample process process, such as dibutyl hydroxy toluene (BTH, final concentration is 0.01%), and sample is processed immediately at 4 DEG C, or refrigerated storage is also thawed before use completely.
Usually, select an interior target principle according to each phospholipid species, the phosphatide that the isotope-labeled phospholipid molecule that can detect separately or choose in sample does not exist or content is extremely low adds sample as interior mark.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 are as interior mark.In some preferred implementations, according to biological specimen and the sample size of separate sources, add the interior mark of different amount.In particularly preferred embodiments, such as blood plasma (10 μ L), cell (~ 5 × 10
5individual) and tissue (10mg), in often kind, target consumption is 0.4nmol.
Liquid-liquid extraction is that in iipidomic research, Phospholipids extracts use method the most general.(method that (Matyash et al.J Lipid Res.2008,49 (5): 1137-1146.) are reported also has done suitable improvement, for the extraction of phosphatide to list of references of the present invention.Because MTBE density is low, when aqueous phase and organic phase separate, organic phase, on upper strata, which simplify the operation of collecting organic phase, decrease the loss of absorption, and inextractable matrix pellet is in the bottom of centrifuge tube, is easy to remove.
Before esterification is derivative, add the step of washing, one or more of interfering material can be removed as much as possible on the one hand, on the other hand, concentration of hydrochloric acid can be adjusted to preferred concentration.
The present invention chooses the chloroform/methanol (1:1 containing 5mM ammonium acetate, v/v) solution system is as the carrier of the phospholipid molecule direct injected of esterification, and the phospholipid molecule of esterification in this solution system can be ionized and produce the ion that can detect in the positive-ion mode for mass spectrum.The mode of direct injected can choose syringe pump, some preferred embodiment in, utilize and automatically receive spray ion gun direct injected system (Triversa
advion Biosciences), this system is without the need to cleaning and manual operations, automatically continuously, mass spectrum sample introduction can be carried out by electron spray chip, once unattendedly can make 400 samples continuously, and receive upgrading because flow velocity reaches, only need little sample, the electron spray that just can complete long-time stable is tested, each sample new sample suction nozzle and electron spray hole, do not have cross pollution completely.
Above-mentioned containing in chloroform/methanol (1:1, the v/v) solution system of 5mM ammonium acetate, the phospholipid molecule that esterification derives has different band point tendencies.PtdCho, PtdEtn, PtdSer mainly form adduction H
+ion, although PtdH, PtdGro, PtdIns class phosphatide also can form adduction H
+ion, but signal is lower, mainly forms adduction NH
4 +ion.Hemolytic phosphatide mainly forms adduction H
+ion.No matter further Tandem Mass Spectrometry Analysis display is adduction H
+or NH
4 +the esterification phospholipid molecule of ion all can produce a diacylglycerol fragmention (DAG
+), as shown in figure accompanying drawing 1, (mass-to-charge ratio is 678 to the C17:0/C14:1-PtdH of esterification, adduction NH
4 +ion), (mass-to-charge ratio is 718 to C17:0/C14:1-PtdCho, adduction H
+ion), (mass-to-charge ratio is 738 to C17:0/C14:1-PtdGro, adduction NH
4 +ion) and C17:0/C14:1PtdSer (mass-to-charge ratio is 748, the ion of adduction H+) all can produce the fragmention that mass-to-charge ratio is 535, corresponding to charged C17:0/C14:1-DAG (C17:0/14:1-DAG
+).DAG
+ion also can be considered it is adduction H
+or NH
4 +the neutrality of esterification phospholipid molecule of ion lose peak, namely PtdH, PtdEtn, PtdCho, PtdGro, PtdSer and PtdIns are respectively neutral loses 143Da (126Da+NH
3), 155Da, 193Da, 203Da (186Da+NH
3), 213Da, 291Da (274Da+NH
3) fragment ion peak that produces.Accordingly, neutral loss scan pattern is utilized to detect above-mentioned phosphatide.Due to the DAG that PtdCho produces
+fragmention signal is more weak, and fragment ion peak (matter/lotus ratio is 198 ± 0.5) signal corresponding to esterification head group is stronger, therefore, then utilizes precursor ion-scan pattern to detect for PtdCho class phosphatide.Similar, hemolytic phospholipid molecule also can produce a monoacylglycerol fragmention (MAG
+), also can be considered lysoPtdH, lysoPtdEtn, lysoPtdCho, lysoPtdGro, lysoPtdSer and lysoPtdIns are neutral respectively loses 126Da, 155Da, 193Da, 186Da, 213Da, the fragment ion peak that 274Da produces, therefore, utilize neutral loss scan pattern to detect above-mentioned phosphatide, Selection utilization precursor ion-scan pattern carries out the detection of lysoPtdCho too simultaneously.
Embodiment
Sample and reagent preparation
1. reagent: various analytically pure chemical reagent (unless there are specified otherwise) is all purchased from Sigma-Aldrich company (St.Louis, USA); The Milli-Q water that water required for experiment all adopts Mili-Q Hyperpure water manufacturing systems (Milipore company of the U.S.) to produce; Trimethyl silicon based diazomethane (IUPAC name=(diazomethyl)-trimethylsilane, TMS-diazomethane) (2M is dissolved in normal hexane) is purchased from Acros Organics company; Chromatographic grade purity methyl tert-butyl ether (methyl tert-butyl ether, MTBE), methyl alcohol (methanol), chloroform (Choloform), acetic acid (Acetic acid) are J.T.Baker company (Phillipsburg, PA, USA) product.
2. mark in: the isotope-labeled phospholipid molecule that can detect separately can be selected or choose in sample not exist or phosphatide that content is extremely low adds sample as interior mark.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 are as interior mark.
3. solution allocation: washing lotion before (a) common derivatization: methyl tert-butyl ether/methyl alcohol/0.01N hydrochloric acid solution, volume ratio is MTBE:MeOH:0.01N hydrochloric acid=100:30:25, takes off phase; B washing lotion after () derivatization: methyl tert-butyl ether/methanol/water solution, volume ratio is MTBE:MeOH:H
2o=100:30:25, takes off phase.
4. phosphatide extracts
By separate sources biological specimen, such as blood plasma (10 μ L), cell (~ 5 × 10
5individual) and tissue (10mg) etc., put into the eppordorft pipe of organic solvent-resistant corrosion, and mix mark in the phosphatide described in (a), in each, target consumption is 0.4nmol; By 1.3mL methyl tert-butyl ether/methyl alcohol (10:3, v/v) solution, room temperature concussion 1h; Add 0.25mL deionized water toward above-mentioned solution, fully after concussion, room temperature places 10min; 1,000g is visible significantly layering after centrifugal 10 minutes, collects upper solution (organic phase) to new eppordorft pipe; Lower floor's solution then merges with the upper solution of 1.55mL methyl tert-butyl ether/methanol/water (10:3:2.5, v/v/v) solution, extracts once according to above-mentioned again, and collection upper solution and aforesaid upper solution merge; This supernatant collected is carried out vacuum, freeze concentration is dry, be the phospholipid extract of extraction, before further processing ,-80 DEG C of refrigerator freezing collections.
5. the esterification of phosphatide derives
Washing before derivatization: phospholipid extract is heavily dissolved in 682.5 μ L methyl tert-butyl ether/methyl alcohol/2N hydrochloric acid (500:150:32.5, v/v/v), add 250 μ L 0.1N hydrochloric acid, in 4 DEG C centrifugal (6 after abundant concussion, 500 × g) 10 minutes, supernatant is transferred to new eppendorft manage and add 500 μ L methyl tert-butyl ether/methyl alcohol/0.01N hydrochloric acid (100:30:25, v/v/v) the lower phase of solution, wash again once according to above step, and collect upper solution to new eppendorft pipe, be the phospholipid extract after washing (~ 0.5mL).
50 μ L trimethyl silicane diazomethane solutions (2M is dissolved in normal hexane) are added the phospholipid extract after above-mentioned washing (visible significantly yellow), room temperature rotates hatches 20 minutes; Add 3 μ l acetic acid neutralized methylization reactions (visible yellow color takes off to the greatest extent); After neutralization, add the lower phase of 500 μ l methyl tert-butyl ether/methanol/water (100:30:25, v/v/v), fully after concussion, 1,500 × g centrifugal 3 minutes, in collections, phase also repeated above-mentioned washing step once; This supernatant collected is carried out vacuum, freeze concentration is dry, be the phospholipid extract that esterification is derivative, before mass spectrophotometry ,-80 DEG C of refrigerator freezing collections.
6. the mass spectrophotometry of phosphatide esterification derivant
Utilization is equipped with automatically to receive sprays ion gun direct injected system (Triversa
advionBiosciences) TSQ Vantages (ThermoFisher Scientific, Bremen).The internal diameter of spray column received is 5.5 μm.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) by continuing the setting parameter of suction 1 μ L air after rinse Tip head in advance and pipette samples for opening, flow out in mechanical arm moving process to prevent the sample in Tip head;
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 is 217;
B) impact energy of neutral loss or precursor ion-scan is 40eV, dwelling 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.
7. phosphatide is quantitative
Phosphatide in biological specimen quantitatively mainly through with add in mark and carry out reference, calculate the amount of esterification phospholipid molecule, and then the amount of association phosphatide corresponding to sample.Phosphatide esterification derivant quantitatively as follows:
C
phosphatide/ C
interior mark=I
phosphatide/ I
interior mark,
C
phosphatideand C
interior markthe interior target concentration of phospholipid molecule and correspondence thereof in difference representative sample, and I
phosphatideand I
in markthen distinguish phospholipid molecule and corresponding interior target mass spectra peak intensity thereof in representative sample.
8. safety notice
Although TMS-diazomethane is the substitute as a kind of hypotoxic diazomethane at the beginning of using.But suck the damage that excessive TMS-diazomethane also can cause lung and central nervous system.Therefore, advise all carrying out in the fuming cupboard with protection at derivatization or labeling process.Excessive TMS-diazomethane must neutralize with acetic acid.The process of acetic acid neutralization is one and produces the process of nitrogen, must careful operation note protecting when process great amount of samples.
Although describe the preferred embodiments of the present invention, those skilled in the art once obtain the basic creative concept of cicada, then can make other change and amendment to these embodiments.So claims are intended to be interpreted as comprising preferred embodiment and falling into all changes and the amendment of the scope of the invention.
Obviously, those skilled in the art can carry out various change and modification to the present invention and not depart from the spirit and scope of the present invention.Like this, if these amendments of the present invention and modification belong within the scope of the claims in the present invention and equivalent technologies thereof, then the present invention is also intended to comprise these change and modification.
Claims (10)
1., based on chemically derived phosphatide classification and Detection and a quantivative approach, it is characterized in that, comprise the following steps:
Step 1: extract phosphatide to be detected from sample to be detected;
Step 2: using trimethyl silicon diazomethane carries out esterification to the phosphate group in the phospholipid molecule in sample and derives, reduces the electronegativity of phospholipid molecule;
Step 3: the phospholipid molecule derived through esterification in ionization sample, produce the several ion treating Mass Spectrometer Method, described ionic species to be detected comprises phosphatid ylcholine, phosphatidyl-ethanolamine, phosphatidylserine, phosphatidic acid, phosphatidyl glycerol, phosphatidylinositols, Lysophosphatidylcholone, hemolytic phosphatidyl-ethanolamine, hemolytic phosphatidylserine, lysophosphatidic, hemolytic phosphatidyl glycerol, hemolytic phosphatidylinositols;
Step 4: on described phosphatid ylcholine, phosphatidyl-ethanolamine, phosphatidylserine, Lysophosphatidylcholone, hemolytic phosphatidyl-ethanolamine, hemolytic phosphatidylserine, lysophosphatidic, hemolytic phosphatidyl glycerol, hemolytic phosphatidylinositols, choose adduction H respectively
+ion;
On phosphatidic acid, phosphatidyl glycerol, phosphatidylinositols, choose adduction NH respectively
4 +ion;
According to the cleavage of mass spectrum rule of variety classes ion in described step 3, set different neutrality to lose or precursor ion-scan pattern, utilize mass spectrum to carry out classification and Detection to variety classes ion in described step 3, obtain the amount of the mass ions signal of variety classes ion in described step 3;
Step 5: by comparing with interior mark, in the described step 3 described step 4 obtained, the amount of the mass ions signal of variety classes ion is associated with the amount of phospholipid molecule in sample, obtains the amount of variety classes phospholipid molecule in sample.
2. according to claim 1 based on chemically derived phosphatide classification and Detection and quantivative approach, it is characterized in that, utilize mass spectrum to when variety classes ion carries out classification and Detection in described step 3, the mode of liquid chromatograph mass spectrography is selected to detect, or, select the mode of direct injected-mass spectrometry to detect.
3. according to claim 1 based on chemically derived phosphatide classification and Detection and quantivative approach, it is characterized in that, described step 1 is carried out in methyl tert-butyl ether/methanol/water solution system, in described methyl tert-butyl ether/methanol/water solution system, the volume ratio of methyl tert-butyl ether, methyl alcohol, water is 10: 3: 2.5.
4. according to claim 1 based on chemically derived phosphatide classification and Detection and quantivative approach, it is characterized in that, described step 2 is carried out in methyl tert-butyl ether/methyl alcohol/1N hydrochloric acid solution system, in described methyl tert-butyl ether/methyl alcohol/1N hydrochloric acid solution system, the volume ratio of methyl tert-butyl ether, methyl alcohol, 1N hydrochloric acid is 300: 90: 4.
5. according to claim 1 based on chemically derived phosphatide classification and Detection and quantivative approach, it is characterized in that, described step 3 is carried out in chloroform/methanol/ammonium acetate solution system, in described chloroform/methanol/ammonium acetate solution system, the concentration of described ammonium acetate is 5 ~ 10mM, and the volume ratio of described chloroform, methyl alcohol is (1 ~ 2): 1.
6. according to claim 1 based on chemically derived phosphatide classification and Detection and quantivative approach, it is characterized in that, described phosphatidic acid, phosphatidyl-ethanolamine, phosphatidyl glycerol, phosphatidylserine, phosphatidylinositols, lysophosphatidic, hemolytic phosphatidyl-ethanolamine, hemolytic phosphatidyl glycerol, hemolytic phosphatidylserine, hemolytic phosphatidylinositols utilize neutral loss scan to carry out detecting and quantitatively.
7. according to claim 6ly to it is characterized in that based on chemically derived phosphatide classification and Detection and quantivative approach, utilize neutral loss scan to carry out detection to described phosphatidic acid and quantitative time, neutrally to lose for 143Da (126Da+NH
3); Utilize neutral loss scan to carry out detection to described phosphatidyl-ethanolamine and quantitative time, neutral lose for 155Da; Utilize neutral loss scan to carry out detection to described phosphatidyl glycerol and quantitative time, neutral to lose for 203Da (186Da+NH
3); Utilize neutral loss scan to carry out detection to described phosphatidylserine and quantitative time, neutral lose for 213Da; Utilize neutral loss scan to carry out detection to described phosphatidylinositols and quantitative time, neutral to lose for 291Da (274Da+NH
3);
Utilize neutral loss scan to carry out detection to lysophosphatidic and quantitative time, neutral lose for 126Da; Utilize neutral loss scan to carry out detection to hemolytic phosphatidyl-ethanolamine and quantitative time, neutral lose for 155Da; Utilize neutral loss scan to carry out detection to hemolytic phosphatidyl glycerol and quantitative time, neutral lose for 186Da; Utilize neutral loss scan to carry out detection to hemolytic phosphatidylserine and quantitative time, neutral lose for 213Da; Utilize neutral loss scan to carry out detection to hemolytic phosphatidylinositols and quantitative time, neutral to lose for 274Da.
8. according to claim 1ly it is characterized in that based on chemically derived phosphatide classification and Detection and quantivative approach, described phosphatid ylcholine, Lysophosphatidylcholone utilize precursor ion-scan to carry out detecting and quantitatively.
9. according to claim 8ly to it is characterized in that based on chemically derived phosphatide classification and Detection and quantivative approach, detection is carried out to described phosphatid ylcholine and quantitative time, the matter/lotus ratio of described precursor ion is 198.0 ± 0.5; Detection is carried out to described Lysophosphatidylcholone and quantitative time, the matter/lotus ratio of described precursor ion is 198.0 ± 0.5.
10. according to claim 1ly it is characterized in that based on chemically derived phosphatide classification and Detection and quantivative approach, described sample to be tested is biological specimen;
As preferably, in described sample to be tested, be added with antioxidant;
As preferably, described antioxidant is dibutyl hydroxy toluene;
As preferably, the addition of described dibutyl hydroxy toluene is 0.01 ~ 0.03% of sample to be tested volume;
As preferably, when adding antioxidant in described sample to be tested, environment temperature is 4 ~ 10 DEG C;
As preferably, the preserving type of described sample to be tested is freezen protective;
As preferably, described interior target selection principle is that each phospholipid species is selected in one and marked;
As preferably, in described, be designated as the isotope-labeled phospholipid molecule that can detect separately,
Or,
Be designated as in described in sample to be tested and do not exist or phosphatide that content is extremely low;
As preferably, described interior target consumption changes according to different samples to be tested;
As preferably, between described step 1 and step 2, also comprise the step of washing;
As preferably, when utilizing mass spectrum to carry out classification and Detection to described step 2 intermediate ion, select direct injected-mass spectrum, described sampling device is syringe pump;
As preferably, when utilizing mass spectrum to carry out classification and Detection to described step 2 intermediate ion, select direct injected-mass spectrum, described sampling device is for automatically receiving spray ion gun direct injected system;
As preferably, in described step 5, compute mode is:
C
phosphatide/ C
interior mark=I
phosphatide/ I
interior mark,
Wherein, C
phosphatideand C
interior markrepresent the interior target concentration of phospholipid molecule and correspondence thereof in sample to be tested respectively and I
phosphatideand I
interior markthen distinguish phospholipid molecule and corresponding interior target mass spectra peak intensity thereof in representative sample;
As preferably, described step 2 is carried out in the fuming cupboard of guard system;
As preferably, described step 2, when the application of trimethyl silicane diazomethane is excessive, selects acetic acid to neutralize excessive trimethyl silicane diazomethane.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510446589.2A CN105021758B (en) | 2015-07-27 | 2015-07-27 | Chemical derivatization-based phosphatide classification detection and quantification method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510446589.2A CN105021758B (en) | 2015-07-27 | 2015-07-27 | Chemical derivatization-based phosphatide classification detection and quantification method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105021758A true CN105021758A (en) | 2015-11-04 |
| CN105021758B CN105021758B (en) | 2017-05-10 |
Family
ID=54411883
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510446589.2A Active CN105021758B (en) | 2015-07-27 | 2015-07-27 | Chemical derivatization-based phosphatide classification detection and quantification method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105021758B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106645373A (en) * | 2016-09-29 | 2017-05-10 | 中国农业科学院油料作物研究所 | An accurate quantitative analysis method for phosphatides |
| CN107228915A (en) * | 2017-05-12 | 2017-10-03 | 广州大学 | A kind of method for efficiently separating purification glycosyl phosphatidylinositol monoethanolamine |
| CN109358134A (en) * | 2018-12-19 | 2019-02-19 | 武汉绿剑可瑞信科技有限公司 | The pre-treating method and quantitative detecting method of endogenous sugar phosphate compound in a kind of plant sample |
| CN109406687A (en) * | 2018-12-29 | 2019-03-01 | 清华大学 | A kind of high-throughput method for detecting double phosphatide |
| 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 |
| CN114113426A (en) * | 2021-12-27 | 2022-03-01 | 西安血氧生物技术有限公司 | Method for detecting phospholipid in hemoglobin oxygen carrier |
| CN114668860A (en) * | 2022-04-11 | 2022-06-28 | 中国科学技术大学 | Digital lipid nanoparticle and preparation method and application thereof |
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 |
|---|
| ANTHONY D. POSTLE ET AL: "Probing phospholipid dynamics by electrospray ionisation mass spectrometry", 《PROGRESS IN LIPID RESEARCH》 * |
| JONATHAN CLARK ET AL: "Quantification of PtdInsP3 molecular species in cells and tissues by mass spectrometry", 《NATURE METHOD》 * |
| TANXI CAI ET AL: "Profiling and Relative Quantitation of Phosphoinositides by Multiple Precursor Ion Scanning Based on Phosphate Methylation and Isotopic Labeling", 《ANALYTICAL CHEMISTRY》 * |
| 何海波 等: "天然磷脂的色谱法分离、纯化研究进展", 《分析科学学报》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106645373A (en) * | 2016-09-29 | 2017-05-10 | 中国农业科学院油料作物研究所 | An accurate quantitative analysis method for phosphatides |
| CN106645373B (en) * | 2016-09-29 | 2019-03-08 | 中国农业科学院油料作物研究所 | A kind of accurate quantification analysis method of phosphatide |
| 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 |
| CN109358134A (en) * | 2018-12-19 | 2019-02-19 | 武汉绿剑可瑞信科技有限公司 | The pre-treating method and quantitative detecting method of endogenous sugar phosphate compound in a kind of plant sample |
| 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 |
| CN109406687A (en) * | 2018-12-29 | 2019-03-01 | 清华大学 | A kind of high-throughput method for detecting double phosphatide |
| CN114113426A (en) * | 2021-12-27 | 2022-03-01 | 西安血氧生物技术有限公司 | Method for detecting phospholipid in hemoglobin oxygen carrier |
| 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 |
| CN114668860A (en) * | 2022-04-11 | 2022-06-28 | 中国科学技术大学 | Digital lipid nanoparticle and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105021758B (en) | 2017-05-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105021758A (en) | Chemical derivatization-based phosphatide classification detection and quantification method | |
| CN105067697B (en) | A kind of phosphatide classification and Detection and quantitative approach based on stable isotope labeling | |
| Fang et al. | Coupling solid-phase microextraction with ambient mass spectrometry: Strategies and applications | |
| Badu-Tawiah et al. | Chemical aspects of the extractive methods of ambient ionization mass spectrometry | |
| Xue et al. | Recent advances in ambient mass spectrometry imaging | |
| Thiel et al. | Using time-of-flight secondary ion mass spectrometry to study biomarkers | |
| Chen et al. | Application of probe electrospray to direct ambient analysis of biological samples | |
| CN102636553B (en) | Paper-based electrospray ion mobility spectrometry analysis method | |
| CN108828051B (en) | Method for detecting lipid of antarctic krill oil in real time by rapid evaporation ionization mass spectrometry | |
| AU2020239700B2 (en) | Quantitation of tamoxifen and metabolites thereof by mass spectrometry | |
| CN101601805B (en) | Method for detecting peimine and peiminine in Chinese medicament fritillaria extract | |
| CN102354649A (en) | Surface extraction chemical ionization source and surface extraction chemical ionization mass spectrometry method | |
| Yang et al. | A microscale solid-phase microextraction probe for the in situ analysis of perfluoroalkyl substances and lipids in biological tissues using mass spectrometry | |
| CN110887891A (en) | Micro-extraction-nano-liter electrospray ion source system in capillary and application | |
| CN103308641A (en) | High performance liquid chromatography-tandem mass spectrometry measuring method of three amide herbicides in tobacco and tobacco products | |
| CN104245599A (en) | Methods and kits for detection of coenzyme Q10 | |
| US20100264304A1 (en) | Method for detecting volatile species of high molecular weight | |
| Liu et al. | Rapidly detecting tetrabromobisphenol A in soils and sediments by paper spray ionization mass spectrometry combined with isotopic internal standard | |
| D'Ascenzo et al. | Determination of arylphenoxypropionic herbicides in water by liquid chromatography–electrospray mass spectrometry | |
| Miyashita et al. | Single cell analysis by using ICP-MS | |
| CN105548402A (en) | Kit for detecting 8-hydroxyl deoxyguanosine and 8-hydroxyl guanosine in urine through high performance liquid chromatography tandem mass spectrometry technology | |
| CN105527368A (en) | Method for detecting 8-hydroxydeoxyguanosine and 8-hydroxyguanosine in urine by high-performance liquid chromatography tandem mass spectrometry technology | |
| Parshintsev et al. | Analysis of organic compounds in ambient aerosols collected with the particle-into-liquid sampler | |
| CN110208358A (en) | A kind of high-frequency vibration atomization ionization probe device and method | |
| Deng et al. | Nanospray Laser-Induced Plasma Ionization Mass Spectrometry for Rapid and Sensitive Analysis of Polycyclic Aromatic Hydrocarbons and Halogenated Derivatives |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |