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Novel serum/plasma proteome analysis method

A proteome and plasma technology, applied in the field of serum/plasma proteome analysis, can solve the problem of limited number of serum/plasma proteins

Active Publication Date: 2021-04-27
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, without removing high-abundance proteins, the number of serum / plasma proteins identified by current mass spectrometry techniques is limited, and 23% of the biomarkers found so far are mainly concentrated in these 300 high-abundance proteins

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  • Novel serum/plasma proteome analysis method
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  • Novel serum/plasma proteome analysis method

Examples

Experimental program
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Effect test

preparation example Construction

[0054] The present invention provides a sample preparation method of serum / plasma proteome, comprising the following steps:

[0055] (1) After the serum sample is centrifuged at low temperature and low speed, the bottom precipitate is removed, and the supernatant is retained;

[0056] Serum is 100 μl to 1000 μl, preferably 200 μl, the low temperature is 2-8°C, preferably 4°C, the centrifugal force is 1200-2000RCF, preferably 1500RCF, and the centrifugation time is 5-10 minutes, preferably 5 minutes;

[0057] (2) Add 4 kinds of mixed nanoparticles modified by different surface groups to the supernatant, and incubate at 37°C for 30-120 minutes. The 4 kinds of nanoparticles modified by different surface groups are SiO modified by surface amino groups. 2 Nanoparticles, surface hydroxyl-modified SiO 2 Nanoparticles, surface zwitterionic modified SiO 2 Nanoparticles, TiO 2 The total amount of nanoparticles is 100 μg, and the mass ratio is 1:1:1:1.

[0058] Among them, the surfac...

Embodiment 1

[0067] Embodiment 1, preparation of serum / plasma proteome

[0068](1) Thaw 200μl serum from the refrigerator at 37°C, centrifuge at 1500RCF for 10 minutes at 4°C, remove the bottom precipitate and transfer the supernatant to a new tube.

[0069] (2) Add 100 μg of mixed nanoparticles (nanoparticles modified with four different surface groups in equal amounts) to the supernatant and incubate at 37° C. for 1 h with rotation.

[0070] (3) After the incubation was completed, centrifuge at 11000 RCF for 90 minutes to remove the supernatant solution. After the pellet was pipetted 5 times with 500 μl of 0.05% CHAPS, centrifuge at 11000 RCF for 90 minutes to remove the supernatant.

[0071] (4) Add 20 μl of 1% SDC, 10 mM TECP, 40 mM CAA and 50 mM TEAB digestion buffer (pH 8.5), react at 95° C. for 10 minutes, add sequencing-grade trypsin 500 ng, and react at 37° C. for 16 hours.

[0072] (5) Add 20% TFA to the system in step (4) so ​​that the final concentration of TFA in the solution...

Embodiment 2

[0073] Example 2, mass spectrometry detection of serum proteome

[0074] The preparation method of the serum proteome sample is the same as that in Example 1.

[0075] Add 20% formic acid to the sample to a final concentration of 1% to terminate the reaction, place on ice for 10 minutes, centrifuge at 15,700 RCF for 10 minutes, collect the substrate precipitate and supernatant, wash the precipitate twice with 0.5% TFA, and collect the supernatant, 3 The secondary supernatants were combined and freeze-dried at low temperature and desalted.

[0076] Then 20 μl of sample buffer (0.1% formic acid) was used to dissolve, and then 2 μl of mass spectrometry was taken for detection, and the data was collected by using ThermoScientific nano-level liquid chromatography tandem high-resolution mass spectrometry (nLC-Easy1000-Q Exactive-HF).

[0077] The specifications of nanoliter liquid chromatography pre-column and analytical column are as follows:

[0078] Pre-column: 3μm particle siz...

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Abstract

The invention discloses a novel serum / plasma proteome analysis method. The mixed nanoparticles modified by different groups can effectively adsorb different types of serum / plasma proteins, selective enrichment of low-abundance proteins in serum / plasma is realized, and interference of high-abundance proteins in serum / plasma on low-abundance protein identification during mass spectrometry identification is effectively avoided. By combining reversed-phase C18 column online separation and a mass spectrometric data independent data acquisition mode, qualitative and quantitative analysis of more than 1500 serum / plasma proteins under 100 min chromatographic gradient can be realized. The method disclosed by the invention has the following advantages that an expensive commercialized serum / plasma high-abundance protein removal device is not needed, and selective enrichment of low-abundance proteins in serum / plasma can be completed only by using cheap and easily available nanoparticles; and according to the method, enrichment of low-abundance protein in serum / plasma can be completed only through four links of incubation, centrifugal cleaning and centrifugation, and errors caused by artificial complex operation are avoided.

Description

technical field [0001] The invention relates to a new method for serum / plasma proteome analysis, which belongs to the field of proteome analysis. Background technique [0002] Serum / plasma is the only liquid that can flow freely between various organs of the body. Serum / plasma not only contains proteins that maintain the functions of serum / plasma itself, but also contains proteins released by other tissues and organs throughout the body. Currently, it is speculated that serum / plasma contains proteins There are more than 10,000 types of proteins, and the types and abundance changes of proteins in serum / plasma contain huge clinical physiological or pathological information. Therefore, serum / plasma has become the main research object of clinical biomarker research. The initiation of the International Human Plasma Proteome Project (HUPO HPPP) is to realize the comprehensive analysis of human plasma protein components, and to determine the differences in plasma proteomes between ...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06G01N30/14G01N30/08G01N30/72G01N30/86G01N30/88
CPCG01N30/02G01N30/06G01N30/14G01N30/08G01N30/72G01N30/8696G01N30/8679G01N30/88G01N2030/067G01N2030/8822G01N2030/8831Y02A50/30
Inventor 张万军付斌刘明伟姜颖任亮亮张养军秦伟捷
Owner ACADEMY OF MILITARY MEDICAL SCI
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