CN106324159A - Pretreatment method for detection and analysis of micromolecular metabolites in adherent cells - Google Patents
Pretreatment method for detection and analysis of micromolecular metabolites in adherent cells Download PDFInfo
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- CN106324159A CN106324159A CN201510386828.XA CN201510386828A CN106324159A CN 106324159 A CN106324159 A CN 106324159A CN 201510386828 A CN201510386828 A CN 201510386828A CN 106324159 A CN106324159 A CN 106324159A
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Abstract
The invention provides a pretreatment method which is suitable for detection and analysis of micromolecular metabolites of all components in adherent cells. The method is characterized in that: (a) cells are cleaned by using rapid washing; (b) cells are instantly quenched by using liquid nitrogen; (c) cell walls are thoroughly removed by water-bath ultrasonic treatment; (d) after freeze drying, an appropriate extract is employed for extracting intracellular metabolites, after filtering, intracellular micromolecular metabolites are analyzed. The method is used for determining metabolites in cells, and the pretreatment method has the advantages of simple and fast method, less loss of samples, and good reappearance; the method is especially suitable for pretreatment of samples in batch and samples of trace amounts, prior tedious pretreatment processes of adherent cells are changed, and rapid, simple and high efficient batch treatment for micromolecular metabolites of intracellular all components can be carried out.
Description
Technical field
The present invention is the new method of intracellular products pre-treatment, specifically establishes a kind of use
The pre-treatment means of simple and quick original position, complete cell cleaning, cell cancellation, cell take off wall and crush
And the pre-treating method that little molecule Metabolite extracts, it is simple and efficient that the method has pre-treating method,
Sample loss is few, favorable reproducibility, is particularly suited for batch and the advantage of micro-example.
Background technology
Metabolism group is that the low-molecular-weight metabolism all to a certain biology or cell grown up in recent years are produced
Thing carries out a new disciplines of qualitative and quantitative analysis, and its utilization in environmental toxicology becomes the most day by day
For the focus of research, its research can help people to be best understood from biosystem to environmental change
Reaction, the accumulation of certain specific metabolite may indicate that the defect of certain path, certain signal respond path
Activate or the dynamic change strengthening certain biomolecule internal or metabolite of certain biosynthesis pathway
Can be as the mark of toxic damages.Especially recently as the development of cellular metabolism group, profit
The method cultivated with cell, evaluates environment by the situation of change of detection cell small molecule metabolites dirty
The toxic action of dye thing becomes new direction (Liu Peng, Piao Fengyuan, Wang Yanyan, the flood of environmental toxicology development
Rock, metabonomic technology and the application in toxicology thereof, Dalian Medical Univ's journal, 30 (2008)
565-569；A.Kortenkamp,Ten Years of Mixing Cocktails:A Review of
Combination Effects of Endocrine-Disrupting Chemicals,Environ Health Persp,
115(2007)98-105).Complete cellular metabolism group includes the outer metabolite of born of the same parents and the detection of intracellular metabolism
Two parts, relative to the detection of the outer metabolite of born of the same parents, the detection of intracellular metabolite and constituent to be answered
Miscellaneous many, the change of intracellular metabolite more can be caused reacting cells environmental stimulus to external world intuitively
The composition distribution situation of panorama metabolite, is more beneficial for finding biomarker, therefore by detection point
The metabonomic analysis application of analysis intracellular metabolite relatively extensively (Panopoulos, A.D., Yanes, O.,
Ruiz,S.,Kida,Y.S.,et al.,Cell Res 2012,22,168-177；Chrysanthopoulos,P.
K.,Goudar,C.T.,Klapa,M.I.,Metab Eng 2009,12,212-222；Ruiz-Aracama,
A.,Peijnenburg,A.,Kleinjans,J.,Jennen,D.,et al.,BMC Genomics 2011,
12,1-19).The most conventional cell model includes the animal cell models such as rat, mice, human body cell
Such as human cancer cell, human liver cell and induction stem cell etc., the common feature of these cells is adherent growth,
It is referred to as attached cell, the flow process that traditional process to this type of cell secondary is relatively complicated, concrete
Including cell cancellation, the steps such as cell takes off wall and processes, metabolite extraction, the pre-treatment side of application now
Formula is varied, and different pre-treating methods all exists certain shortcoming, and forefathers are also before cell
Process aspect has done a series of related work, such as the improvement of cell quenching method, cell cleaning solution
Selection, (Leon, the Zacarias such as selection of cell extraction solvent；Garcia-Canaveras,Juan C.；
Teresa Donato,Maria；Mammalian cell metabolomics:Experimental design and
Sample preparation ELECTROPHORESIS 2013,19 (34), 2762-2775), but above-mentioned
All of extracting method have selected cell scraper or adding the clearing up of pancreatin, pancreatin the most without exception
Addition make the state of cell change, and clean pancreatin the substantial amounts of cell of process possible loss,
Cell scraper needs to expend substantial amounts of manpower, and repeatability is very poor, and these all can make follow-up sample
Collimation receives impact, the cell cultivated especially for 96 orifice plates, and scraper is difficult to operation, pancreatin
Addition is difficult to control to, and rarely has report that the 96 such few cells of orifice plate are carried out metabolite the most so far and divides
Analysis, for the series of problems of above-mentioned appearance, finds a kind of simple and quick, and favorable reproducibility does not introduces
The attached cell pre-treating method of allogenic material and beneficially Mass Spectrometer Method is particularly important.Especially
Or else cell is made thoroughly to take off wall for having weight while utilizing extraneous instrument and not introducing external source chaff interference
The researching value wanted.
Summary of the invention
It is an object of the invention to development a kind of simple and efficient, sample loss is few, favorable reproducibility, and fits
The pre-treating method analyzed for batch and the detection of micro-example attached cell intracellular metabolite product, for solving
Above-mentioned technical problem, the technical solution adopted in the present invention is:
Utilize the most quickly pre-treatment means, do not introduce the external source of any interference metabolite detection
Component, reaches the high efficiency extraction to attached cell intracellular metabolite product, comprises the following steps: attached cell
Clean with ultra-pure water, the cell after cleaning pour liquid nitrogen into, after liquid nitrogen volatilizees, add ultra-pure water,
Supersound process, extracts intracellular metabolite, is analyzed extract after lyophilization.
Concretely comprise the following steps:
A) attached cell discarding cell culture fluid, add ultra-pure water, piping and druming is cleaned cell, is discarded liquid
Body；When ultra-pure water cleans attached cell, in order to effectively remove dead cell and residual cell culture fluid,
It is unlikely to again to damage normal cell.The piping and druming of rifle head is wanted quickly, uniformly.
B) cell after cleaning is quickly poured into liquid nitrogen, pours during liquid nitrogen thing in cell plates to be guaranteed into residual
The water stayed is drawn clean, makes cell moment cancellation；
C) after liquid nitrogen volatilizees, add the ultra-pure water of certain volume, supersound process under water bath condition, a side
Face cell can be met water and be risen brokenly, and on the other hand cell can take off wall thoroughly；
D) cell after de-wall is broken is dried by freezer dryer quick freeze, after being dried now
Dried object be cell fragmentation thing, then use suitable solvent extraction cell metabolite, filtering and impurity removing
Enter to analyze to intracellular small molecule metabolites afterwards.
The attached cell that the method relates to include regular growth cultivate in can be adherent zooblast
And human body cell, such as mice, rat relevant cell, human cancer cell, human liver cell, human body is induced
Versatile stem cell etc.；
The volume of the ultra-pure water added in step a) is unsuitable too many, but guarantees again to clean fully, its
Volume is the 1/2-2/3 that Tissue Culture Plate adds cell culture fluid volume, (24 orifice plates add cultivation liquid measure 1.0mL,
12 orifice plates add cultivation liquid measure 2.0mL, and 6 orifice plates add cultivation liquid measure 2.5mL, and 4 orifice plates add cultivation liquid measure 5.0
mL)；
The usage amount of step b) liquid nitrogen is to be immersed in liquid nitrogen by all attached cells；
Step c) water-bath cell makes cell rise brokenly and the water consumption of ultra-pure water of de-wall too much can cause freezing
Taking a substantial amount of time time dry, cross and cell can be made at least to take off wall not exclusively, its volume is step a)
Tissue Culture Plate adds the 1/3-1/2 of cell culture fluid volume, (24 orifice plates add cultivation liquid measure 1.0mL, 12
Orifice plate adds cultivation liquid measure 2.0mL, and 6 orifice plates add cultivation liquid measure 2.5mL, and 4 orifice plates add cultivation liquid measure 5.0
mL)；
Step d) holds the container masking foil of cell and covers when putting into freezer dryer, prevent freezing
The loss of cell component when being dried, and by pricking upper pore above masking foil, it is beneficial to moisture evaporation；
In step a), ultra-pure water clean cell time be the 6-10 second, overlong time can affect normally
The state of cell, quickly discards liquid sucking-off after washing, to prevent cell water suction from rising brokenly；.
After adding liquid nitrogen cancellation cell in step b), in liquid nitrogen volatilization process, cell is put in refrigerator, treats
Liquid nitrogen is evaporated completely, and takes out and carries out next step operation, easily condenses the globule under room temperature environment in culture plate,
And normal temperature environment can affect the change of cell component.
In step c), during water bath sonicator, water-bath is ice-water bath, mainly for preventing cell component from changing because of temperature
Becoming and change, the general ice bag that adds can supersonic frequency be 40-60kHz, and ultrasonic time is
3-5min。
During described in step d), solvent is the aqueous solution that volume fraction is 80%-85% methanol, this extract
Can farthest dissolve the component in cell.
Present invention have the advantage that
(1) establish attached cell dead cell and cell surface residual cell broth out is quick
Method for washing；
(2) cell component is made preferably to rest on specific moment by the quick cancellation of liquid nitrogen；
(3) quickly ice-water bath makes cell take off wall and water suction is risen brokenly metabolite precipitation；
(4) methanol/water makes cell component rapid extraction.
The present invention is the new method of intracellular products pre-treatment, and specifically a kind of employing is simple
The pre-treatment means of quick in situ complete cell cleaning, cell cancellation, cell rupture and cellular metabolism
The pre-treating method that composition obtains, it is simple and efficient that the method has pre-treating method, and sample loss is few,
Favorable reproducibility, is particularly suited for batch and the advantage of micro-example.
Accompanying drawing explanation
The total ion current figure of Fig. 1 HepG2 cell full constituent small molecule metabolites Q-TRAP detection；
Under wherein a is Q-TRAP detection HepG2 cell full constituent small molecule metabolites positive ion mode
Total ion current figure, b be Q-TRAP detect HepG2 cell full constituent small molecule metabolites anion
Total ion current figure under pattern.
Detailed description of the invention
The present invention is further described by example in detail below, but is not intended to the present invention
Embodiment 1:
Small molecule metabolite assay in human liver cancer cell HepG2 attached cell:
Taking the human liver cancer cell HepG2 of the logarithmic (log) phase that six orifice plates are cultivated, the volume of cell culture fluid is every
Hole 3mL. adds the ultra-pure water of 1.5mL in every hole after cell culture fluid sucking-off being discarded, and utilizes rifle head to blow and beat
Rapid cleaning cell 7 seconds, discards rapid for liquid sucking-off, is quickly poured into liquid nitrogen in the cell after cleaning,
The liquid level of liquid nitrogen not have the bottom of culture plate to make cell moment cancellation, then culture plate is put into refrigerator
In, after liquid nitrogen volatilizees, every hole adds the ultra-pure water of 1mL, ice bath, and supersonic frequency is 40kHz, super
Sonication 3min, makes cell take off wall rapidly and thoroughly；Tissue Culture Plate masking foil after de-wall covers
(prick upper aperture with pin on masking foil and be conducive to moisture evaporation) puts into freezer dryer, after being dried
Every hole adds the aqueous solution extraction intracellular metabolite of the methanol of 1mL volume fraction 85%, filters with syringe needle
Intracellular full constituent small molecule metabolites is entered Ultra Performance Liquid Chromatography Tandem Mass Spectrometry Analysis after filtering by device.
Due to the difference of metabolite molecular property, it is divided into two kinds of scan patterns of negative ions, chromatographic condition
As described below.Positive ion mode: chromatographic column with ACQUITY UPLC BEH C8 (1.7 μm,
2.1 × 100mm), mobile phase A is the super water containing formic acid 0.1% (v/v), and B phase is containing formic acid 0.1%
(v/v) acetonitrile.Eluent gradient elution program is: be initiated with 90%A, drops to 60% during 3min,
Drop to 0% during 15min and maintain 5min, during 20.1min, returning to 90%A, balancing 3min；Flow velocity
For 0.35mL/min, column temperature is 50 DEG C, and sample size is 10 μ L.
Negative ion mode: chromatographic column ACQUITY UPLC HSS T3 (1.8 μm, 2.1 × 100mm),
Mobile phase A is the ultra-pure water containing NH4HCO35mmol/L, and B phase is containing NH4HCO35mmol/L
Methanol.Eluent gradient elution program is: is initiated with 98%A and maintains 1min, declines during 3min
To 58%, drop to 0% during 12min and maintain 4min, during 16.1min, returning to 98%A, balancing 4min；
Flow velocity is 0.35mL/min, and column temperature is 50 DEG C, and sample size is 10 μ L.
Under above chromatographic condition, use AB Sciex UHPLC/Q-trap to filtering out nearly thousand to from
Son is to carrying out quantitative analysis.First with non-Scheduled MRM (without retention time) pattern, dwell
Time is 5msec, and capillary voltage positive ion mode is 4000V, and negative ion mode is-3500V, hair
Capillary temperature is 350 DEG C, atomization gas (N2) pressure position 40psi, it is dried gas (N2) flow velocity is 9L/min。
Respectively obtain the EIC spectrogram under negative ions pattern, extract each Ion-pair chromalography peak and obtain accurately
Retention time, then by Scheduled MRM (withing a hook at the end the time) pattern, cell sample is carried out
Analyze.The MultiQuant software of obtained initial data Analyst is carried out batch processing, leads
Go out the data crossed through Internal standard correction methods, next data are carried out statistical analysis.
Using the method to detect nearly thousand kinds of components of six orifice plate human liver cancer cell HepG2, metabolism is composed
Figure is as shown in Figure 1.
Claims (8)
1. the pre-treating method that in an attached cell, small molecule metabolite detection is analyzed, it is characterised in that:
Comprise the following steps:
Attached cell ultra-pure water cleans, and pours liquid nitrogen in the cell after cleaning, after liquid nitrogen volatilizees
Add ultra-pure water, supersound process, extract intracellular metabolite after lyophilization, extract is analyzed.
2. according to the pre-treating method described in claim 1, it is characterised in that: comprise the following steps:
A) attached cell discarding cell culture fluid, add ultra-pure water, piping and druming is cleaned cell, is discarded liquid
Body；
B) cell after cleaning pours liquid nitrogen into, make cell moment cancellation；
C) after liquid nitrogen volatilizees, add ultra-pure water, supersound process under water bath condition, make cell take off wall；
D) cell freezing after de-wall is dried, with solvent extraction intracellular metabolite, institute after extract filtration
Obtain liquid to be analyzed.
3. according to the pre-treating method described in claim 1 or 2, it is characterised in that:
Described attached cell be cell cultivate in can be adherent zooblast or human body cell.
4. according to the pre-treating method described in claim 2, it is characterised in that:
The 1/2-3/4 that volume is germinal cell culture fluid volume of the ultra-pure water added in step a)；
The usage amount of step b) liquid nitrogen is to be immersed in liquid nitrogen by all attached cells；
The volume of the ultra-pure water added in step c) is that step a) Tissue Culture Plate adds cell culture liquid
Water bath condition described in long-pending 1/3-1/2 is ice-water bath；
Solvent described in step d) is the aqueous solution of methanol.
5. according to the pre-treating method described in claim 2, it is characterised in that: in step a), super
The time of pure water cleaning cell is the 6-10 second, is discarded by liquid immediately after washing.
6. according to the pre-treating method described in claim 2, it is characterised in that: step b) adds
After liquid nitrogen cancellation cell, cell is put in refrigerator by liquid nitrogen volatilization process, treat that liquid nitrogen is evaporated completely, take
Go out to carry out next step operation.
7. according to the pre-treating method described in claim 2, it is characterised in that: step c) supersonic frequency
Rate is 40-60kHz, and ultrasonic time is 3-5min.
8. according to the pre-treating method described in claim 4, it is characterised in that: molten belonging to step d)
Agent i.e. volume fraction is in the aqueous solution of 80%-85% methanol.
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Cited By (1)
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| CN112540133A (en) * | 2020-11-02 | 2021-03-23 | 暨南大学 | Detection and analysis method for marine microalgae intracellular metabolome |
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Application publication date: 20170111 |