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Serum protein preparation method and serum proteome mass spectrometric detection method

A serum protein and proteome technology, which is applied in the preparation of test samples, measurement devices, and material analysis by electromagnetic means, can solve the problems of increasing the scanning time of mass spectrometry detection, reducing the dynamic range of serum samples, and reducing quantitative accuracy.

Pending Publication Date: 2022-03-01
北京青莲百奥生物科技有限公司
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, when the high-abundance protein in serum is removed, many low-abundance proteins combined with it will also be lost at the same time, affecting the authenticity and accuracy of protein quantification results in serum.
The use of chromatographic multi-stage splitting can reduce the complexity of serum samples and reduce the dynamic range of serum samples, but it will also significantly increase the scanning time of mass spectrometry detection. More importantly, multi-stage splitting leads to increased sample variability and reduced quantitative accuracy, which cannot be applied For large-scale, high-throughput clinical proteome analysis

Method used

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  • Serum protein preparation method and serum proteome mass spectrometric detection method
  • Serum protein preparation method and serum proteome mass spectrometric detection method
  • Serum protein preparation method and serum proteome mass spectrometric detection method

Examples

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preparation example Construction

[0075] The invention provides a sample preparation method of serum proteome, comprising the following steps:

[0076] (1) After the serum sample is centrifuged at low temperature and low speed, the bottom particles and insoluble matter are removed, and the supernatant is retained;

[0077]The serum is 25µl to 1000µl, the low temperature is 2~8°C, preferably 4°C, the centrifugal force is 500~2000 RCF, preferably 1000RCF, and the centrifugation time is 10~20 minutes, preferably 15 minutes;

[0078] (2) Dilute the supernatant with equal volume of PBS buffer, add the mixed magnetic nanoparticles with hydrophilic and hydrophobic surface modification, and incubate at 37°C for 5-10 minutes, the mixed magnetic nanoparticles with hydrophilic and hydrophobic surface modification are Surface dextran-modified hydrophilic magnetic nanoparticles and surface polystyrene-modified hydrophobic magnetic nanoparticles total 1.25 mg, and the mass ratio is 10:1;

[0079] (3) After the serum mixed ...

Embodiment 1

[0084] Embodiment 1, the preparation of serum proteome

[0085] (1) Thaw 25 μl of serum from the refrigerator at room temperature, centrifuge at 1500 RCF for 10 minutes at 4°C, remove the bottom cells and pellets, and transfer the supernatant to a new tube.

[0086] (2) Add 25 μl PBS buffer to the supernatant, then add 1.25 mg mixed nanoparticles (mixed hydrophilic and hydrophobic magnetic nanomaterials at a mass ratio of 10:1) and incubate at 37°C for 10 minutes with rotation.

[0087] (3) After the incubation was completed, magnetic separation was performed for 5 minutes to remove the supernatant solution, the precipitate was washed with 200 μl PBS vortex shaking for 2 minutes, and the supernatant solution was removed by magnetic separation for 5 minutes.

[0088] (4) Add 20 μl of 1% DDM, 10 mM TECP, 40 mM CAA and 100 mM TEAB digestion buffer (pH8.5), react at 95°C for 10 minutes, add sequencing grade trypsin 1ug, and react at 37°C for 16 hours.

[0089] (5) Add 20% TFA (vo...

Embodiment 2

[0090] Example 2, mass spectrometry detection of serum proteome

[0091] The preparation method of the serum proteome sample is the same as that in Example 1.

[0092] 10 μl of loading buffer (0.1% (v / v) formic acid) was used to dissolve, and then 2 μl of mass spectrometry was taken for detection, and the data was collected using ThermoScientific nanoliter liquid chromatography tandem high-resolution mass spectrometry (nLC-Easy1200-OrbitrapExploris 480).

[0093] The specifications of nanoliter liquid chromatography pre-column and analytical column are as follows:

[0094] Pre-column: 3µm particle size C 18 Filler, 2cm×150µm inner diameter (filler is Dr.Maisch GmbH company).

[0095] Analytical column (separation column): 1.9µm particle size C 18 Filler, inner diameter 25cm×75µm (filler is Dr.Maisch GmbH company).

[0096] Mobile phase A is 0.1% (v / v) volume ratio (1000ul of water is added to 1ul of formic acid) formic acid (formic acid, FA) aqueous solution, mobile phase ...

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Abstract

The invention discloses a serum protein preparation method and a serum proteome mass spectrometry detection method. Aiming at the limitation of an existing serum proteome sample preparation method, a magnetic nano material based on mixing of a surface hydrophilic material and a surface hydrophobic material is established, and non-concentration-dependent enrichment of serum protein is realized by utilizing a'protein crown 'formed by contact of nano particle-protein in a solution; in combination with a high-field asymmetric waveform ion mobility spectrometry (FAIMS) and an optimized established data independent mass spectrum scanning mode, deep coverage and accurate quantitative detection of serum proteome are realized.

Description

technical field [0001] The invention relates to a serum protein preparation method and a serum proteome mass spectrometry detection method, belonging to the field of proteome analysis. Background technique [0002] Serum samples are important clinical samples with stable sources and rich biological information. Proteins are the direct executors of life activities or biological functions. Therefore, the abundance changes of macromolecular proteins in serum samples can indirectly and sensitively reflect the disease state of internal organs. Currently, serum samples are the preferred biomarkers in clinical practice. one of the sources. However, serum samples are also the most difficult samples to analyze. The content of albumin in serum accounts for about 50% of the total mass of all serum proteins, and the abundance of the 22 most abundant proteins in serum accounts for 99% of the total abundance of all serum proteins. It is predicted that there are more than 10,000 protein ...

Claims

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Application Information

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IPC IPC(8): G01N27/624G01N30/89G01N1/28
CPCG01N27/624G01N30/89G01N1/28
Inventor 孙龙钦李京丽
Owner 北京青莲百奥生物科技有限公司
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