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Cell exosome lipidomics analysis method based on LC-MS

An analysis method, exosome technology, applied in the field of biotechnology detection, can solve the problems of poor quantitative accuracy, poor linear response of mass spectrometry, and inability to analyze absolute quantitative analysis of analytes, so as to achieve the effect of improving accuracy and good stability

Pending Publication Date: 2020-06-30
上海中科新生命生物科技有限公司
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Problems solved by technology

This method has certain limitations: due to too many ions detected at the same time and severe ion suppression, the linear response of the mass spectrometer is poor, and the absolute quantitative analysis of the analyte cannot be performed by the conventional standard curve method. The mass spectrum response intensity gives a relative quantitative result, the accuracy of the quantification is poor, and the wrong result is often obtained

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Embodiment Construction

[0023] The technical solutions of the present invention will be described below in conjunction with the accompanying drawings and embodiments.

[0024] A method for analyzing cellular exosome lipidomics based on LC-MS. This embodiment detects human 293T cells, including the following steps:

[0025] Step S1, take 100ml of human 293T cell supernatant sample, and centrifuge at 800g, 4°C for 15min; take the supernatant again at 2,000g, and centrifuge at 4°C for 30min; take the supernatant again and centrifuge at 7,500g, 4°C for 30min ;Take the supernatant through a 0.22μm filter membrane, take the filtrate and put it in an ultra-high-speed centrifuge at 100,000g, 4°C, centrifuge for 2h, remove the supernatant, pay attention to the tiny white precipitate at the bottom of the tube, do not completely remove all the supernatant, be careful Mix well, transfer the bottom solution sample to a new centrifuge tube, add PBS solution to resuspend, and balance, then centrifuge at 100,000g fo...

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Abstract

The invention provides a cell exosome lipidomics analysis method based on LC-MS. A differential centrifugation enrichment exosome is matched with an internal standard method for quantitative measurement; optimized analysis parameters of liquid chromatography and mass spectrometry are combined; various stable isotope internal standard substances are added into a to-be-detected substance, so that the quantitative influence caused by an ion inhibition effect can be effectively reduced, high-coverage lipidomics is carried out on a cell exosome sample to obtain absolute quantitative analysis, the stability is better, and the quantitative accuracy can be greatly improved.

Description

technical field [0001] The invention relates to the field of biotechnology detection, in particular to an LC-MS-based method for analyzing cellular exosome lipidomics. Background technique [0002] Exosomes are double-layered vesicles actively secreted by cells, which can carry a variety of proteins, nucleic acids, and lipids to circulate in body fluids. Carry out material transport and information exchange between cells, and thus participate in a variety of physiological and pathological processes, such as presenting antigens, promoting tumor growth and migration, repairing damaged tissues, etc. Lipid is an important part of cell membrane and plays an important role in cell signal transduction, material transportation, energy storage and so on. Abnormal lipid metabolism is inseparable from the process of exosomes carrying and transporting lipids. Therefore, the establishment of a reliable, stable, and high-coverage exosome lipid metabolism analysis method has positive gui...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/14G01N30/34G01N30/72
CPCG01N30/02G01N30/06G01N30/14G01N30/34G01N30/72G01N2030/045
Inventor 张惠萍刘泰驿
Owner 上海中科新生命生物科技有限公司
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