Isotope dilution mass-spectrometry based serum low-abundance protein quantitative detection pretreatment process

An isotope dilution and low-abundance protein technology, which is applied in the field of isotope dilution mass spectrometry quantitative detection of serum low-abundance protein pretreatment process, can solve the problem of poor repeatability and reproducibility, large differences, and affect the accurate quantification of serum low-abundance proteins and other issues to achieve the effect of good repeatability and reproducibility and wide coverage

Active Publication Date: 2016-11-23
AFFILIATED HOSPITAL OF NANTONG UNIV
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Problems solved by technology

[0002] Isotope dilution mass spectrometry has strong specificity and good reproducibility, so it is the best method for absolute quantification of low-abundance proteins in serum, but the accuracy of this method significantly depends on the quality of protein pretreatment
There are many pretreatment processes for low-abundance serum proteins, and there are large differences between different processes. The defects of poor repeatability and reproducibility are common, which seriously affects the accurate quantification of low-abundance proteins in serum by isotope dilution mass spectrometry.

Method used

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Embodiment Construction

[0015] This specific embodiment adopts following technical scheme: its steps are as follows:

[0016] Step 1: Removal of high-abundance proteins: Use 100 μL of serum according to the reagent instructions BlueAlbumin&IgG Depletion Kit (Sigma-Aldrich) removes most of albumin and IgG, take 180 μL of serum after depletion of high-abundance proteins, and mix with 70 μL of 25mM ammonium bicarbonate (ABC) solution;

[0017] Step 2: Denaturation: Heat the mixed sample above for 5 minutes in a water bath at 100°C, then cool it in an ice bath for 5 minutes, then add about 96.1 mg of urea (final concentration 8M) for chemical denaturation, vortex for 30 seconds and let stand for 5 minutes, and finally add 250 μL of 25 mM ABC solution dilution;

[0018] Step 3: Reduction: Add 50 μL of 100 mM dithiothreitol (DTT) (final concentration 10 mM), and bathe in water at 60°C for 30 minutes to keep the opened disulfide bonds of the denatured protein in a reduced state;

[0019] Step 4: Alkylati...

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Abstract

The invention relates to the technical field of medical health and discloses an isotope dilution mass-spectrometry based serum low-abundance protein quantitative detection pretreatment process. The process includes: step one, high-abundance protein removal; step two, denaturation; step three, reduction; step four, alkylation; step five, alkylation termination; step six, trypsinization; step seven, solid-phase extraction; step eight, evaporation to dryness; and step nine, redissolution. The process is high in repeatability and reproducibility, wide in coverage and applicable to pretreatment of various low-abundance proteins in serum detected on the basis of the isotope dilution mass-spectrometry.

Description

technical field [0001] The invention relates to the field of medical and health technologies, in particular to a pretreatment process for the quantitative detection of serum low-abundance proteins by isotope dilution mass spectrometry. Background technique [0002] Isotope dilution mass spectrometry has strong specificity and good reproducibility, so it is the best method for absolute quantification of low-abundance proteins in serum, but the accuracy of this method significantly depends on the quality of protein pretreatment. There are many pretreatment processes for low-abundance serum proteins, and there are large differences between different processes. The defects of poor repeatability and reproducibility are common, which seriously affect the accurate quantification of low-abundance serum proteins by isotope dilution mass spectrometry. Contents of the invention [0003] The purpose of the present invention is to provide isotope dilution mass spectrometry quantitative...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28
CPCG01N1/28
Inventor 季伙燕王建新王惠民鞠少卿丛辉王旭东苏建友
Owner AFFILIATED HOSPITAL OF NANTONG UNIV
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