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Protein quantitative method utilizing equiponderance dimethylation marking

A heavy dimethylation and protein technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of large sample loss, poor labeling efficiency, poor labeling efficiency and labeling selectivity, and achieve low labeling price, simple labeling, The effect of high labeling efficiency

Active Publication Date: 2014-10-01
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

IPTL requires multi-step reactions, resulting in poor labeling efficiency and labeling selectivity, while IVTAL is only suitable for cell samples, and QITL requires multi-step reactions, resulting in large sample loss and poor labeling efficiency, especially 18 O mark

Method used

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  • Protein quantitative method utilizing equiponderance dimethylation marking
  • Protein quantitative method utilizing equiponderance dimethylation marking
  • Protein quantitative method utilizing equiponderance dimethylation marking

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Embodiment Construction

[0022] 1. Protein extraction process: first wash the cells with 1×PBS until there is no blood. Use 2 mL of 8M urea plus commercial protease inhibitor cocktail (purchased from sigma) with a final concentration of 0.1% (v / v) as the lysate to suspend about 500 mg of mouse ascites-type liver cancer lymphatic high metastasis cell line (Hca -F) and about 500 mg of a low-metastatic cell line (Hca-P), the cell line was crushed with a tissue disruptor at a speed of 10,000 rpm. Then sonicate for 100 s. After ultrasonication, the protein extract was centrifuged at 25000g for 30min, precipitated with acetone at -80°C, discarded the supernatant by centrifugation, evaporated to dryness, reconstituted with 1mL 8M urea, and then determined the protein concentration by Bradford method. Store at -20°C until use.

[0023] 2. Protein sample processing process: 100 μL of 1 μg / μL Hca-P protein dissolved in 8M urea was added with dithiothreitol to a final concentration of 10 mM. After denaturation...

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Abstract

The invention relates to a new protein quantitative method, in particular to a quantitative method utilizing equiponderance dimethylation marking. The method utilizes the organic combination of methanal and isotope of the methanal, and sodium cyanoborohydride and isotope of the sodium cyanoborohydride for marking peptide fragments, so that the peptide fragments have the similar mass-to-charge ratio on the primary spectrum of the mass spectrum and have the different mass-to-charge ratios on the secondary spectrum of the mass spectrum, and the protein quantitative analysis is achieved on the secondary spectrum. Compared with other quantitative methods, the method has the advantages of being high in quantitative accuracy and precision, and wide in dynamic range.

Description

technical field [0001] The present invention designs a quantitative method of isobaric labeling, which can realize large-scale, high-accuracy and high-precision quantification of protein samples. In addition, this method has other advantages such as up to six-fold labeling at the same time, simple method labeling, and high labeling efficiency. High, no side effects, low price, etc. The method can be used for quantitative analysis of proteomics of biological samples. Background technique [0002] Quantitative proteomics is a hot topic in systems biology research. It provides extremely important data for discovering disease biomarkers, discovering protein-protein interactions, drug-protein interactions, explaining the mechanism of disease, explaining biological metabolic processes, etc. support. Protein is the embodiment and bearer of life activities. It is extremely rich in organisms and participates in all metabolic processes of living organisms. Therefore, changes in prot...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
Inventor 张丽华周愿单亦初杨开广吴琪张珅张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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