Exosome mass spectrum analysis method adopting continuous filtration

A spectrum analysis and physical fitness technology, applied in the field of medical experimental analysis, can solve problems such as expensive, easy to block or block, and the accuracy of the results cannot meet the requirements.

Inactive Publication Date: 2020-07-07
苏州先觉生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Ultracentrifugation is currently the "gold standard" of exosome isolation methods. However, ultracentrifugation technology has the disadvantages of time-consuming, expensive, and large sample loss. Membrane filtration technology can separate and purify exosomes. When the sample size Larger use of filtration membranes to separate and purify exosome vesicles is easy to block or block, and whether exosomes pass through the filtration membrane is easy to adhere and cannot be eluted
[0005] In the prior art, the Chinese invention patent No. ZL2016105973232, titled "Extraction Method of Exosomes in Human Body Fluids" discloses "a. Preparation of body fluid samples: first extract human body fluids under sterile conditions and dilute them with PBS buffer. Then preliminarily remove the cell components and cell fragments in the body fluid by centrifugal screening, and make a body fluid sample

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  • Exosome mass spectrum analysis method adopting continuous filtration
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  • Exosome mass spectrum analysis method adopting continuous filtration

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Such as Figure 1-4 The shown method of exosome mass spectrometry using continuous filtration of the present invention comprises the following steps:

[0029] (1) extract the liquid sample to be tested after preparing the liquid to be tested, the preparation of the liquid sample to be tested in the present invention can adopt the routine preparation method of the medical laboratory, and obtain the one that meets the requirements of the medical experiment;

[0030] (2) The liquid sample to be tested is filtered through three layers of a primary filter membrane, a secondary filter membrane, and a tertiary filter membrane. For filtering extracellular vesicles with a size larger than 300-450nm, the three-stage filter membrane mainly maintains extracellular vesicles mainly exosomes, and at the same time filters out impurities such as particles or proteins smaller than 30nm. Exosomes are mainly concentrated on the three-stage filter membrane, and exosome samples are directly...

Embodiment 2

[0042] On the basis of the exosome mass spectrometry method using continuous filtration described in Example 1, the following specific experiments were carried out:

[0043] 2.1 Equipment source:

[0044] Ultracentrifuge: Optima XE-90 Ultracentrifuge (BECKMAN COULTER); MALDI-TOF mass spectrometer: Bruker Microflex LRF MALDI-TOF massspectrometer (Bremen, Germany).

[0045] 2.1 Experimental materials:

[0046] 20 mg sinapinic acid, 1 ml 50% acetonitrile, 0.1% trifluoroacetic acid, 49.9% (v / v) deionized water;

[0047] Standards CM, cytochrome C and myoglobin for mass spec calibration.

[0048] Test samples: Hela cell supernatant and plasma from human venous blood.

[0049] 2.2 Separation and purification process of exosomes:

[0050] 0.1ml of human plasma sample is diluted 10 times with deionized water, and 1ml solution sample diluted 10 times is used for injection to be tested. (2) The sample volume of Hela cell supernatant is 10ml, adopt 3000r / min*10min to discard the pre...

Embodiment 3

[0072] On the basis of the exosome mass spectrometry analysis method using continuous filtration described in Examples 1 and 2, the liquid sample to be tested in the present invention can also be cell culture fluid. This embodiment provides a continuous filtration mass spectrometry method for cell culture fluid. The experimental data, the step that adopts is described in embodiment 2 in detail, so repeat it no longer;

[0073] This embodiment provides the mass spectrometry analysis spectrum data of the cell culture fluid adopting the method of the present invention, such as image 3 As shown in the figure, the mass spectrometry detection and analysis data of 5 cell lines are shown. The characteristic peaks of the mass spectrometry images of different cell lines are different. A2 represents the 143B cell line, B1 represents the MG63 cell line, B2 represents the HOS cell line, and B3 represents the U2OS cell line, and C1 represents the 3T3E1 cell line;

[0074] by the above i...

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Abstract

The invention relates to an exosome mass spectrum analysis method adopting continuous filtration. The method comprises the following steps: (1) preparing a to-be-detected liquid, and extracting a to-be-detected liquid sample; (2) filtering the to-be-detected liquid sample through a first-stage filter membrane, a second-stage filter membrane and a third-stage filter membrane, and obtaining an exosome sample on the third-stage filter membrane; and (3) directly taking out the third-stage filter membrane, treating the third-stage filter membrane with a mixed solvent prepared from acetonitrile, trifluoroacetic acid, deionized water and sinapic acid, and carrying out mass spectrometric detection on the third-stage filter membrane to obtain a detection result. The method is convenient and stablein analysis, and the detection time is less than 1 hour. The main signals of a graph obtained are consistent with those of a mass spectrum detection graph obtained after exosome cyst separation and purification through an existing standard ultracentrifugation method. The method has the advantages of being low in cost, small in amount of samples needed, rapid, convenient to use, accurate, stable, capable of avoiding the defects that time and resources are consumed in the ultracentrifugation technology and the like and wide in biological and medical application prospect.

Description

technical field [0001] The invention relates to an exosome mass spectrometry analysis method using continuous filtration, which belongs to a medical experiment analysis method. Background technique [0002] Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (Matrix-assisted laserdesorption / ionization time-of-flight mass spectrometry, MALDI-TOF MS) has been developed since the 1980s, and has ultra-high sensitivity and specificity. The molecular weight of biomolecules can be accurately determined to realize the identification and analysis of biomacromolecules; by analyzing the fingerprints of different bacteria, MALDI-TOF MS can realize the rapid identification of medical experiments; in addition, MALDI-TOF MS technology is also widely used in proteomics research. [0003] Exosomes are a kind of extracellular vesicles (Extracellular Vesicles, EV, the size of extracellular vesicles is about 30-1000nm), and its diameter is about 30-150nm. Rich in membr...

Claims

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Application Information

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IPC IPC(8): G01N27/64G01N1/34
CPCG01N1/34G01N27/64
Inventor 沈宇辉
Owner 苏州先觉生物科技有限公司
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