Exosome mass spectrum analysis method adopting continuous filtration

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...

AI Technical Summary

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 for later use; b, body fluid sample purification: filter the body fluid sample in step a through a filter membrane, and further remove cell debris and other impurities in the body fluid , let it stand for 10-15 minutes, and keep the precipitate for later use; c, exosome extraction: suspend the precipitate obtained in step b with PBS buffer, suspend the exosome in the upper layer of the liquid, and then use a sterile needle Absorb the liquid containing exosomes in the upper layer and store it at -80°C for future use.” Although this patent discloses that the body fluid sample is filtered and extracted through a filter membrane, the accuracy of the mass spectrometry detection results after filtration cannot meet the requirements. Filtration and treatment methods are also not up to the requirements, and cannot meet the requirements of replacing ultracentrifugation for experimental detection

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