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Method for detecting medium-low abundance protein in body fluid sample based on mass spectrometry

A low-abundance protein and protein technology, applied in the field of biological detection, can solve the problems of increasing cost, limiting the detection efficiency and application range of blood proteome, and cumbersome operation, so as to achieve the effect of assisting diagnosis and improving detection coverage

Pending Publication Date: 2022-06-07
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is cumbersome to operate and increases the cost, which greatly limits the detection efficiency and application range of blood proteome.

Method used

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  • Method for detecting medium-low abundance protein in body fluid sample based on mass spectrometry
  • Method for detecting medium-low abundance protein in body fluid sample based on mass spectrometry
  • Method for detecting medium-low abundance protein in body fluid sample based on mass spectrometry

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Experimental program
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Effect test

Embodiment 1

[0037] By the method of the present invention, normal human serum samples are detected, and the specific method is as follows:

[0038] 1. Preparation of Enhanced Channel Samples

[0039] Take 10 μL of serum samples from clinical healthy volunteers, use High-Select TM Top14 High Abundant Protein Removal Medium Spin Column (Thermo Scientific #A36371) removes high abundance proteins. 10 μL of serum sample was added directly to the resin slurry in the column, mixed by gentle inversion at room temperature, and the filtrate was collected by centrifugation after 10 min incubation.

[0040] 2. Preparation of Samples for Mass Spectrometry Detection

[0041] Take 1 μL of the same serum sample as in step 1 and dissolve it in 200 μL of urea lysis buffer (9M urea, 20 mM 4-hydroxyethylpiperazineethanesulfonic acid). Take the sample filtrate from step 1 and add 2 mL of urea lysis solution. The protein concentration of all samples was determined by Pierce BCA kit, then an appropriate amo...

Embodiment 2

[0059] By the method of the present invention, 7 cases of liver cancer patients and 7 cases of normal human serum samples are detected, and the specific method is as follows:

[0060] 1. Preparation of Enhanced Channel Samples

[0061] Serum samples from 14 patients and healthy individuals were mixed in equal proportions. Take 10 μL of mixed serum samples, use High-Select TM Top14 High Abundant Protein Removal Medium Spin Column (Thermo Scientific #A36371) removes high abundance proteins. 10 μL of serum sample was added directly to the resin slurry in the column, mixed by gentle inversion at room temperature, and the filtrate was collected by centrifugation after 10 minutes of incubation.

[0062] 2. Preparation of Samples for Mass Spectrometry Detection

[0063] 1 μL of serum samples from 7 patients with liver cancer and 7 normal people were respectively taken and dissolved in 200 μL of urea lysis buffer (9M urea, 20mM 4-hydroxyethylpiperazineethanesulfonic acid). Take th...

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Abstract

The invention discloses a method for detecting low-abundance proteins in samples based on mass spectrometry, which comprises the following steps: 1) removing high-abundance proteins in only one group of samples to realize the enrichment of the low-abundance proteins, and taking the low-abundance proteins as enhanced channel samples for subsequent detection; and 2) carrying out mass spectrometry on the enhanced channel sample and the common channel sample by adopting a high-throughput labeling method to obtain quantitative results of various proteins in the body fluid. On the basis of a high-throughput labeling method and on the premise of retaining high-abundance protein of a sample, the coverage rate of blood low-abundance protein spectrum detection is greatly increased by utilizing an enhanced channel. According to the method, more researchers can be helped to find blood protein markers really related to diseases, and diagnosis of various diseases is assisted.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a method for detecting or assisting the detection of medium and low abundance proteins in a body fluid sample. Background technique [0002] Early diagnosis of disease is crucial for controlling disease development and prognosis. In actual clinical testing, non-invasive body fluid detection methods are used to diagnose various diseases early. Blood is the most commonly used body fluid test sample, and the proteins in it are commonly used disease diagnostic markers. At present, more than one hundred in vitro diagnostic methods based on blood protein markers have been clinically applied, including the diagnosis of infectious diseases, neurological diseases, cancer, diabetes, cardiovascular diseases and prenatal diagnosis. In recent years, the rapid development of mass spectrometry technology and the demand for medical precision have made the research on the detection ...

Claims

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Application Information

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IPC IPC(8): G01N1/34G01N27/62G01N30/02G01N30/14G01N30/72
CPCG01N1/34G01N27/62G01N30/02G01N30/14G01N30/72
Inventor 朱奕颖石文昊
Owner TSINGHUA UNIV