Method for monitoring and qualification of 15N metabolic labeled protein in combination with mass spectrum parallel reaction

A parallel reaction and protein technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of differences in protein enzyme digestion process, expensive synthetic peptides, synthetic peptides can not accurately reflect the absolute amount of endogenous proteins, etc., to improve efficiency effect

Pending Publication Date: 2019-03-08
北京蛋白世界生物科技有限公司
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Problems solved by technology

First, this method requires preliminary experiments and extensive data analysis to determine the target peptide for synthesis
Second, the protease digestion process of different batches of samples can produce significant differences
The results showed that the synthetic peptides could not accurately reflect the ab

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  • Method for monitoring and qualification of 15N metabolic labeled protein in combination with mass spectrum parallel reaction
  • Method for monitoring and qualification of 15N metabolic labeled protein in combination with mass spectrum parallel reaction
  • Method for monitoring and qualification of 15N metabolic labeled protein in combination with mass spectrum parallel reaction

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Embodiment

[0040] 15 A quantitative method for monitoring and quantifying N metabolically labeled proteins in combination with mass spectrometry, comprising the following steps:

[0041] a) to 15 N metabolically labeled internal reference protein, obtained 15 N-labeled protein;

[0042] b) Add the lysate obtained in step a) to the concentration of 0.25 μg / μL cell lysate 15 N-labeled protein, and performed SDS-PAGE electrophoresis to obtain a protein gel;

[0043] in cell lysates 15 The concentration of N-labeled protein ranges from 0 fmol / μL to 100 fmol / μL, and the specific concentration is shown in Table 1. Samples 1 to 4 were obtained, and samples 1 to 4 were subjected to SDS-PAGE electrophoresis in turn, and the following in-gel enzymatic hydrolysis, mass spectrometry detection, PRM quantitative analysis steps

[0044] Table 1 Sample data

[0045]

[0046] Electrophoresis conditions: After 10 minutes of electrophoresis at 80V, electrophoresis at 120V until bromophenol blue i...

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Abstract

The invention discloses a method for monitoring and qualification of 15N metabolic labeled protein in combination with mass spectrum parallel reaction. 15N metabolic labeled internal reference proteincan accurately reflect the structure and the state of endogenous protein to be detected, has the same enzymolysis efficiency with the protein to be detected, solves the problem that internal reference peptide ion strength signals are affected by protease digestion, co-elute matrix, sample composition and instrument status during mass spectrum selection, tracking and quantification to cause nonreproducibility, and meanwhile improves the efficiency of monitoring and qualification of mass spectrum parallel reaction; in addition, the method can produce various quantitative polypeptide used for parallel reaction monitoring, improves the qualification accuracy, reduces the cost for synthesizing polypeptide, can be applied to quantitative proteomic research and detection of protein markers, andhas great help in medical diagnosis, environmental health, food safety inspection and judicial expertise.

Description

technical field [0001] The invention relates to the analytical field of mass spectrometry detection, especially a 15 A method for the quantification of N metabolically labeled proteins combined with mass spectrometry for parallel reaction monitoring. Background technique [0002] Mass spectrometry parallel reaction monitoring (Parallel reaction monitoring, PRM) technology, as a mass spectrometry detection method, has the advantages of strong specificity, high sensitivity, high accuracy, good reproducibility, wide linear dynamic range, automation and high throughput. Advantages, these qualities can meet the urgent needs of many research fields today. Real-time quantitative monitoring through PRM technology can be used for pharmacokinetics, clinical diagnosis, illicit drug inspection, food and cosmetics and other industrial quality control, environmental testing, agricultural botany research, and the discovery of metabolomics and proteomics biomarkers, etc. Related research....

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06G01N30/72G01N30/88
CPCG01N30/02G01N30/06G01N30/72G01N30/88G01N2030/067G01N2030/8831
Inventor 陆天聪
Owner 北京蛋白世界生物科技有限公司
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