CN104215684A - Cell culture fluid sample reagent kit for qualitative and quantitative analysis detection and high-throughput screening of small molecules and preparation of cell culture fluid sample reagent kit - Google Patents
Cell culture fluid sample reagent kit for qualitative and quantitative analysis detection and high-throughput screening of small molecules and preparation of cell culture fluid sample reagent kit Download PDFInfo
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- CN104215684A CN104215684A CN201410455751.2A CN201410455751A CN104215684A CN 104215684 A CN104215684 A CN 104215684A CN 201410455751 A CN201410455751 A CN 201410455751A CN 104215684 A CN104215684 A CN 104215684A
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Abstract
The invention relates to preparation of cell culture fluid sample reagent kit for the qualitative and quantitative analysis detection and the high-throughput screening of small molecules. The preparation method comprises the following steps of enabling the temperature of a cell culture fluid sample to the room temperature, and adequately thawing the cell culture fluid sample; adding mixed solution-basic lysate with the identical volumes at the temperature of minus 20 DEG C to obtain a mixed solution, and enabling the mixed solution to react for 5min in a water bath; centrifuging; collecting liquid supernatant, wherein the basic lysate is QL1, and the QL1 comprises the following components: 10 to 30 percent of methanol, 70 to 90 percent of acetonitrile and 1 to 7 percent of formic acid. The preparation method of the reagent kit is simple, and the cost is relatively low.
Description
Technical field
This technology belongs to analytical chemistry applications field, is specifically related to surface laser and resolves field of mass spectrometry.
Background technology
It is a kind of novel soft ionization biological mass spectrometry that development in recent years is got up that surface laser is resolved mass spectrum, wherein take ground substance assistant laser desorption ionization flight time mass spectrum (MALDI-TOF-MS) as most important example, and the present invention obtained Nobel chemistry Prize.Mainly by two parts, formed: substance assistant laser desorpted ionized ion gun (MALDI) and time of flight mass analyzer (TOF).The principle of MALDI is with Ear Mucosa Treated by He Ne Laser Irradiation sample and substrate formed cocrystallization film, matrix absorbs energy and passes to biomolecule from laser, in ionization process, proton translocation is obtained to proton to biomolecule or from biomolecule, and make the process of biomolecule ionization.Therefore it is a kind of soft ionization technology, is applicable to the mensuration of potpourri and biomacromolecule.The principle of TOF is that ion accelerates to fly over dirft tube under electric field action, is detected the mass-to-charge ratio (M/Z) of measuring ion is directly proportional to the flight time of ion according to the flight time difference that arrives detecting device, detects ion.That MALDI-TOF-MS has is highly sensitive, accuracy is high and resolution high, for the fields such as life science provide a kind of strong analytical test means.
Yet, with all mass spectrums of resolving based on surface laser headed by ground substance assistant laser desorption ionization flight time mass spectrum, all need sample preparation on special target plate, its operation be by sample dispersion in substrate molecule and form crystal.When with Ear Mucosa Treated by He Ne Laser Irradiation crystal, matrix absorbs energy from laser, sample desorption, between matrix-sample, there is electric charge transfer and make ionized sample molecule, the sample of ionization flies over the tof tube of vacuum under electric field action, according to different being detected of the flight time that arrives detecting device, the ratio (M/Z) of the quality electric charge by ion is directly proportional to analyze ion to the flight time of ion, and records the molecular weight of sample molecule.Focal issue is just that the molecular weight of general substrate molecule is at 1000-3000da, has caused excessive background mass spectra peak.This has just determined that all mass spectrophotometry that this type of is resolved based on surface laser can only be applicable to large Molecular Detection, such as protein, long-chain polypeptide, nucleic acid, macromolecular material etc., and can not be applied to little Molecular Detection (molecular weight is less than 1000da), the advantage of uniqueness such as high sensitivity/high flux of the mass spectrophotometry of resolving based on surface laser in other words cannot be put to good use on small molecule analysis detection. and molecular weight to be less than the little molecule of 1000da be the class chemical products to the most important tool using value of the mankind, more than 95% Medicine small molecule for example, all amino acid, vitamin etc. and the healthy relevant chemical substance of human lives.
Currently available technology also rests on spectroscopic methodology detection for little Molecular Detection and screening, for example fluorescence radiation detects or derives substrate that can fluoroscopic examination, but the little molecule molecular structure of this type of testing requirement target has photolytic activity group, another drawback is exactly that often photometry sensitivity is lower, can't detect the little molecular conecentration of trace.Liquid phase mass spectrum (LC-MS) and gaseous mass spectrum (GC-MS) start to be applied in micromolecular detection and high flux screening.But need to consume a large amount of manpowers and solvent consumptive material, and a sample needs quite to grow a process to going out result again from being prepared into mass spectrophotometry, different as requested, analyzing a sample needs 30-60 minute from sample preparation to taking result, can not meet the high flux screening requirement of modern biological medicine development far away.
Summary of the invention
Goal of the invention: find simple, the lower-cost sample preparation methods of a kind of preparation method, reduce expenses and the time.
Technical scheme: a kind of little molecule qualitative and quantitative analysis detection and high flux screening cell culture fluid sample reagent box of being applied to, sample by equal volume forms with basic lysate, basic lysate is QL1, and QL1 material forms and volume ratio is: 10-30% methyl alcohol, 70-90% second cyanogen, 1-7% formic acid.
Be applied to a preparation for little molecule qualitative and quantitative analysis detection and high flux screening cell culture fluid sample reagent box, cell culture fluid sample is placed to room temperature, fully thaw; Mixed solution-basic the lysate that adds-20 degree equal volume reacts 5 minutes in 50 degree water-baths; Centrifugal; Collect supernatant; Basic lysate is QL1, and QL1 material forms and volume ratio is: 10-30% methyl alcohol, 70-90% second cyanogen, 1-7% formic acid.
Preferred version is that QL1 material forms and volume ratio is: 20% methyl alcohol, 80% second cyanogen, 5% formic acid.
Beneficial effect: 1, preparation method is simple; 2, cost is lower.
Accompanying drawing explanation
Fig. 1 utilizes the mass spectrogram of the cell pyrolysis liquid that the present invention detects.
Embodiment
The basic lysate QL1 of cell culture fluid sample preparation reagents box and sample preparation methods
Cell culture fluid sample (single centrifuge tube, 96 hole porous plates, 384 hole porous plates) is placed to room temperature 10 minutes, fully thaw.Mixed solution-basic lysate the QL1 (material volume ratio is: 10-30%, 70-90% second cyanogen, 1-7% formic acid) that adds-20 degree equal volume, acts on 5 minutes, and act on 5 minutes in 50 degree water-bath.After centrifugal 10 minutes of 3000rmp, collect supernatant.
Be illustrated in figure 1 Medicine small molecule was injected in cell nutrient solution after a few days, detect this micromolecular metabolism and dynamics data after clasmatosis, the product analysis of cell lysis.We can carry out rapid screening small-molecule drug from the method.We detect sample simultaneously by the method for MALDI and ESI, the molecular ion peak of verapamil (Verapamil sheet) Verapamil can not be detected.Our sensitivity reaches nanomole rank, and can complete the flux of sample every days 9000.
Claims (3)
1. one kind is applied to little molecule qualitative and quantitative analysis detection and high flux screening cell culture fluid sample reagent box, it is characterized in that, sample by equal volume forms with basic lysate, basic lysate is QL1, and QL1 material forms and volume ratio is: 10-30% methyl alcohol, 70-90% second cyanogen, 1-7% formic acid.
2. be applied to a preparation for little molecule qualitative and quantitative analysis detection and high flux screening cell culture fluid sample reagent box, it is characterized in that, cell culture fluid sample is placed to room temperature, fully thaw; Mixed solution-basic the lysate that adds-20 degree equal volume reacts 5 minutes in 50 degree water-baths; Centrifugal; Collect supernatant; Basic lysate is QL1, and QL1 material forms and volume ratio is: 10-30% methyl alcohol, 70-90% second cyanogen, 1-7% formic acid.
3. the preparation that is applied to little molecule qualitative and quantitative analysis detection and high flux screening cell culture fluid sample reagent box according to claim 1, is characterized in that, QL1 material forms and volume ratio is: 20% methyl alcohol, 80% second cyanogen, 5% formic acid.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410455751.2A CN104215684A (en) | 2014-09-09 | 2014-09-09 | Cell culture fluid sample reagent kit for qualitative and quantitative analysis detection and high-throughput screening of small molecules and preparation of cell culture fluid sample reagent kit |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410455751.2A CN104215684A (en) | 2014-09-09 | 2014-09-09 | Cell culture fluid sample reagent kit for qualitative and quantitative analysis detection and high-throughput screening of small molecules and preparation of cell culture fluid sample reagent kit |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102914462A (en) * | 2012-11-22 | 2013-02-06 | 上海化工研究院 | High-throughput method for extracting carotene in biological sample |
| US20140234873A1 (en) * | 2006-11-24 | 2014-08-21 | Agency For Science, Technology And Research | Apparatus for processing a sample in a liquid droplet and method of using the same |
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2014
- 2014-09-09 CN CN201410455751.2A patent/CN104215684A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140234873A1 (en) * | 2006-11-24 | 2014-08-21 | Agency For Science, Technology And Research | Apparatus for processing a sample in a liquid droplet and method of using the same |
| CN102914462A (en) * | 2012-11-22 | 2013-02-06 | 上海化工研究院 | High-throughput method for extracting carotene in biological sample |
Non-Patent Citations (4)
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| JOSHUA D. RABINOWITZ 等: "Acidic Acetonitrile for Cellular Metabolome Extraction from Escherichia coli", 《ANAL. CHEM.》, vol. 79, no. 16, 15 August 2007 (2007-08-15), pages 6167 - 6173 * |
| 刘永霞 等: "雄激素非依赖前列腺癌细胞系代谢组学的初步研究", 《分析化学》, vol. 39, no. 3, 31 March 2011 (2011-03-31), pages 305 - 311 * |
| 施洁瑕 等: "应用代谢组学探讨芫花酯甲对人肝细胞L02的毒性作用", 《中国药理学与毒理学杂志》, vol. 27, no. 4, 31 August 2013 (2013-08-31), pages 704 - 709 * |
| 沈国林 等: "超高效液相串联质谱法同时定量检测6个细胞色素P450酶探针代谢产物", 《分析化学》, vol. 41, no. 4, 30 April 2013 (2013-04-30), pages 488 - 493 * |
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Application publication date: 20141217 |
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