CN103529159B - Method for pretreating research sample in blood non-target metabonomics - Google Patents
Method for pretreating research sample in blood non-target metabonomics Download PDFInfo
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Abstract
The invention discloses a method for pretreating a research sample in blood non-target metabonomics. Polar and non-polar substances in serum can be extracted at the same time by adopting the method. Compared with an existing common sample pretreatment method, the method has the advantages that the number of extracted metabolites is remarkably increased, protein precipitate is dissolved by acetonitrile and water mixed solution, residues dried by nitrogen is re-dissolved, so that the dissolubility of non-polar substance lysophosphatide can be reduced on the one hand, and the high mass spectrum matrix effect interference caused by high-concentration lysophosphatide can be reduced; on the other hand, the experiment operation steps are reduced, the experiment errors are reduced, and a sample treated by adopting the method can be detected by a UPLC-Q-TOF MSMS (ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry) as an analysis platform to obtain more metabolites in blood. Therefore, changes of blood metabolism can be favorably observed comprehensively, more reliable differential metabolites or biomarkers can be discovered, metabolic changes and change mechanism of a body can be further observed and researched, and the development of blood non-target metabonomics can be promoted.
Description
Technical field
The present invention relates to a kind of sample-pretreating method of new blood non-target mark metabolism group research, particularly relate to a kind of a kind of method simultaneously extracting the sample pre-treatments of blood Semi-polarity and nonpolar metabolic product being newly analysis platform with UPLC-Q-TOF MSMS.
Background technology
Non-target plasma metabolism group is after comprehensively investigating living things system irriate or disturbance, utilize a series of technological means and detection method to characterize the dynamic change of metabolite concentration and kind in blood, observe the physiological and pathological change that the change of the metabolic pathway of body is corresponding with body.In order to clearly understand the change of human body integral metabolism, should detect metabolic products all in body fluid as far as possible, this just needs a kind of method to extract all metabolic products in blood as far as possible.Now conventional blood sample pre-treating method mainly contains: organic solvent extraction, solid phase extraction and both mixing methods.The cardinal principle of these methods is the protein removed in blood, and the metabolic product extracted in blood carries out LC-MS analysis detection.But the weak point of this several method is the part metabolic product extracted in blood, and organic solvent extraction is apolar substance (lipid) mainly, and solid phase extraction removes the lysophosphatide of blood middle high concentration, and to the polar material in blood extract little.Therefore current sample-pretreating method can not extract polarity in blood and nonpolar metabolic product simultaneously, the demand of all metabolic products in metabonomic analysis blood can not be met, also just can not the concrete change that changes of comprehensive and systematic observation plasma metabolism and difference.
Summary of the invention
Technical matters to be solved by this invention to overcome in prior art the shortcoming only paid close attention to and extract apolar substance in blood or polar material, provides a kind of new simple and quick sample-pretreating method that simultaneously can extract polarity in blood and apolar substance.
In order to achieve the above object, the technology used in the present invention means are:
The pre-treating method of a kind of blood non-target mark of the present invention metabolism group study sample, is characterized in that comprising the following steps:
(1)-80 DEG C of blood preserved are taken out, 4 DEG C thaw after vortex 1-2 minute, get mixing blood plasma 250 microlitre in 2 milliliters of centrifuge tubes, add 750 microliter methanol, centrifugal 10-15 minute at vortex 1-3 minute, 12000rpm4 DEG C;
(2) get the supernatant solution of the centrifugal rear acquisition of step (1) in another one 2ml centrifuge tube, nitrogen dries up, and remaining solid residue is in order to again redissolving;
(3) lower floor's albumen precipitation of centrifugal for step (1) rear acquisition is smashed to pieces, add mixed solution 350 microlitre of acetonitrile and water, after vortex 1-3 minute, centrifugal 10-15 minute at 12000rpm4 DEG C, to remove the protein in blood plasma, and the polar material extracted in serum and do not extract apolar substance completely;
(4) in the centrifuge tube after the supernatant nitrogen poured in step (2) after centrifugal for step (3) being dried up, the remaining solid residue redissolved in centrifuge tube, centrifugal 10-15 minute at leaving standstill 5-10 minute, 12000rpm4 DEG C after vortex 1-3 minute;
(5) collect step (4) centrifugal after the supernatant that obtains, be the blood non-target mark metabolism group study sample after process.
In the present invention, preferably, in acetonitrile and water mixed solution, the volume ratio of acetonitrile and water is 1:1.
According to the sample after method process of the present invention, can be used as the study sample of non-target plasma metabolism group, enter ULPLC/Q-TOF MSMS and detect, and then obtain data.
Compared to prior art, the pre-treating method of a kind of blood non-target mark metabolism group study sample of the present invention has the following advantages:
1, adopt method of the present invention can extract polarity in serum and apolar substance simultaneously, compared with now conventional sample-pretreating method, the metabolic product quantity showed increased of extraction, as shown in Figure 1;
2, pass through again to extract the polarity in precipitating proteins and residue apolar substance, ensure the metabolic product obtaining maximum quantity;
3, with acetonitrile and water mixed solution (volume ratio of preferred acetonitrile and water is 1:1) first soluble protein sediment, residue after the nitrogen that redissolves again dries up, reduce the solubleness of apolar substance lysophosphatide on the one hand, and then the high mass spectrum matrix effect interference that reduction high concentration lysophosphatide causes, decrease laboratory operating procedures on the other hand, reduce experimental error;
4, solution is repeatedly through high speed centrifugation, removes the impact of the large particulate matters such as protein on chromatographic column as much as possible, without membrane filtration, directly can carry out chromatographic resolution and detection;
5, the sample after adopting method process of the present invention can detect the metabolic product in more blood, more be conducive to the change comprehensively observing plasma metabolism, find more reliable difference metabolic product or biomarker, can further observe and study metabolic alterations and the change mechanism of body, promote the progress of blood non-target mark metabolism group.
Accompanying drawing explanation
Fig. 1 is the base peak ion current chromatogram of serum sample.
A: extracting method of the present invention; B: now conventional sample-pretreating method.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
The pre-treatment of embodiment 1 blood non-target mark metabolism group study sample
Carry out according to following steps:
(1)-80 DEG C of blood preserved are taken out, 4 DEG C thaw after vortex 2 minutes, get mixing blood plasma 250 microlitre in 2 milliliters of centrifuge tubes, add 750 microliter methanol, vortex 2 minutes, at 12000rpm4 DEG C centrifugal 10 minutes;
(2) get the supernatant solution of the centrifugal rear acquisition of step (1) in another one 2ml centrifuge tube, nitrogen dries up, and remaining solid residue is in order to again redissolving;
(3) lower floor's albumen precipitation of centrifugal for step (1) rear acquisition is smashed to pieces, add mixed solution 350 microlitre of acetonitrile and water, in acetonitrile and water mixed solution, the volume ratio of acetonitrile and water is 1:1, vortex is after 2 minutes, at 12000rpm4 DEG C centrifugal 10 minutes, to remove the protein in blood plasma, and the polar material extracted in serum and do not extract apolar substance completely;
(4) in the centrifuge tube after the supernatant nitrogen poured in step (2) after centrifugal for step (3) being dried up, the remaining solid residue redissolved in centrifuge tube, vortex left standstill 10 minutes after 2 minutes, at 12000rpm4 DEG C centrifugal 10 minutes;
(5) collect step (4) centrifugal after the supernatant that obtains, be the blood non-target mark metabolism group study sample after process.
The Mass Spectrometer Method of sample after embodiment 2 pre-treatment
1, reagent
Acetonitrile, methyl alcohol (chromatographically pure), formic acid (chromatographically pure), ultrapure water is produced by pure water instrument.
2, chromatographic separation condition:
Liquid chromatography is Waters, US Acquity Ultra Performance Liquid Chromatography system.Chromatographic column is (BEH) C18 post (1.8 μm, 2.1 × 100mm).Liquid phase chromatogram condition: mobile phase A is distilled water (formic acid solution of 0.1%), Mobile phase B is acetonitrile, flow velocity is that 0.35mL/min. is with linear gradient elution, start gradient is 2% acetonitrile and maintains 0.5min, 0.5-1.5min acetonitrile 2%-20%, 1.5-6.0min acetonitrile 20%-70%, 6.0-10.0min acetonitrile 70%-98%, 10.0-12.0min acetonitrile 98% maintains 2min, and then in 2min, ethane nitrile content falls back 2%, balance chromatographic column 2min.Each sample size is 2 μ L, and column temperature 35 DEG C, auto injection actuator temperature maintains 4 DEG C.
3, Mass Spectrometer Method condition:
Mass spectrometer is Waters company Micromass Q-TOF series connection quadrupole time-of-flight mass spec-trometry instrument, is furnished with electric spray ion source (ESI).Mass spectrum detects with positive ion mode, and positive ion mode condition is: capillary voltage 3000V (negative ion mode, capillary voltage is 2800V); Taper hole voltage 35V; Potential energy 6eV; Desolventizing gas velocity 650L/h; Taper hole gas velocity 50L/h; Desolventizing temperature 320 DEG C; Source temperature 110 DEG C.Use full scan pattern, mass range is 50 – 1000amu.Collect data with the Centroid pattern of Lock Spray and guarantee accuracy and reappearance; Single point correction is made, flow velocity 8 μ L/min with the LEK of 200pg/ml concentration (fixing mass-to-charge ratio: positive ion 556.2771/ negative ion 554.2615).Lock spray frequency is 0.48s, LEK mean scan more than 10 times to correct.
4, sample: the blood sample after the process of embodiment 1 method
5, result
The base peak ion current chromatogram of serum sample, as shown in Figure 1, A: extracting method of the present invention; B: now conventional sample-pretreating method, concrete steps can see document:
1.Comparative Analysis of Sample Preparation Methods To Handle the Complexityof the Blood Fluid Metabolome:When Less Is More.Sara Tulipani,Rafael Llorach,Mireia Urpi-Sarda,and Cristina Andres-Lacueva.Anal.Chem.2013,85,341-348；
2.UPLC-MS-Based Analysis of Human Plasma for Metabonomics Using SolventPrecipitation or Solid Phase Extraction Filippos Michopoulos,Lindsay Lai,Helen Gika,Georgios Theodoridis,and Ian Wilson.Journal of Proteome Research2009,8,2114–2121。
As can be seen from Fig. 1 result, adopt method process of the present invention to extract polarity in blood and nonpolar metabolic product simultaneously, the metabolic product quantity showed increased extracted, chromatographic peak in figure A is than the chromatographic peak showed increased in figure B, particularly add the quantity of polar material, the chromatographic peak before retention time 0.6-5 minute significantly increases and increases.Therefore, use blood sample pre-treating method of the present invention, the metabolic product in more blood can be detected, more be conducive to the change comprehensively observing plasma metabolism, find more reliable difference metabolic product or biomarker, can further observe and study metabolic alterations and the change mechanism of body, thus promote the progress of blood non-target mark metabolism group.
Claims (1)
1. a pre-treating method for blood non-target mark metabolism group study sample, is characterized in that comprising the following steps:
(1)-80 DEG C of blood preserved are taken out, 4 DEG C thaw after vortex 1-2 minute, get mixing blood plasma 250 microlitre in 2 milliliters of centrifuge tubes, add 750 microliter methanol, centrifugal 10-15 minute at vortex 1-3 minute, 12000rpm 4 DEG C;
(2) get the supernatant solution of the centrifugal rear acquisition of step (1) in another one 2ml centrifuge tube, nitrogen dries up, and remaining solid residue is in order to again redissolving;
(3) lower floor's albumen precipitation of centrifugal for step (1) rear acquisition is smashed to pieces, add mixed solution 350 microlitre of acetonitrile and water, after vortex 1-3 minute, centrifugal 10-15 minute at 12000rpm 4 DEG C, to remove the protein in blood plasma, and the polar material extracted in serum and do not extract apolar substance completely; In the mixed solution of wherein acetonitrile and water, the volume ratio of acetonitrile and water is 1:1;
(4) in the centrifuge tube after the supernatant nitrogen poured in step (2) after centrifugal for step (3) being dried up, the remaining solid residue redissolved in centrifuge tube, centrifugal 10-15 minute at standing 5-10 minute, 12000rpm 4 DEG C after vortex 1-3 minute;
(5) collect step (4) centrifugal after the supernatant that obtains, be the blood non-target mark metabolism group study sample after process.
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| CN105424429B (en) * | 2015-11-04 | 2018-03-06 | 哈尔滨医科大学 | A kind of sample-pretreating method of the non-target metabolism group research of formalin-fixed tissue |
| CN109425669B (en) * | 2017-09-01 | 2022-09-16 | 中国民用航空局民用航空医学中心 | Method for screening biomarkers related to fatigue degree in human body fluid by liquid chromatography-mass spectrometry |
| CN112525631A (en) * | 2020-10-16 | 2021-03-19 | 华南农业大学 | Sample preparation method for non-targeted metabonomics and non-targeted lipidomics research of shrimp meat samples |
| CN113156028A (en) * | 2021-04-30 | 2021-07-23 | 厦门市迈理奥科技有限公司 | Derivatization method of carbonyl metabolites and efficient non-targeted metabonomics analysis method |
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| High-Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS)-Based Drug Metabolite Profiling;Ian D. Wilson;《Methods in Molecular Biology》;20111231;第708卷;第180页3.3节 * |
| 代谢组学血浆样品前处理及其快速高分辨液相色谱-质谱分析方法研究;徐婧 等;《分析化学》;20111231;第39卷(第12期);1793-1797 * |
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