Proteome identification method and applications thereof

A proteome and identification method technology, applied in measurement devices, material analysis by electromagnetic means, instruments, etc., can solve the problems of reducing the number of identifications, increasing the difficulty of wheat protein mass spectrometry identification, etc., achieving good protein quality, major application value, high purity effect

Inactive Publication Date: 2017-07-04
INST OF BOTANY CHINESE ACAD OF SCI
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Problems solved by technology

During iTRAQ's nano-liter mass spectrometry identification, high-abundance glutenin will cover the mass spectrometry signals of low-abundance albumin and globulin, thereby reducing the number of iTRAQ identifications
In addition, due to the incomplete sequencing of the whole wheat genome, the number of wheat protein entries in the database is far less than that of Arabidopsis, rice and other species, which also increases the difficulty of mass spectrometry identification of wheat proteins
[0008] At present, there are few reports on the identification of

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  • Proteome identification method and applications thereof

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Embodiment 1

[0075] Embodiment 1, wheat seed proteome identification method

[0076] 1. Extraction of total protein from wheat seeds

[0077] Grind the mature seeds of dehulled wheat Jimai No. 19 into powder, take 0.05g of the powder and 400μL of solution S1 into a centrifuge tube, then add 400μL of Tris saturated phenol (pH=8.0), and mix thoroughly for 30 minutes at 4°C with a shaker For the above, centrifuge at 4°C and 20000g for 20 minutes, the liquid in the centrifuge tube is separated, that is, the upper phenol phase and the lower water phase; absorb the upper phenol phase into a new centrifuge tube to obtain the extract 1, and pour it into the remaining Add 400 μL of Tris saturated phenol extraction water phase to the lower water phase, shake at 4°C for 20 minutes, then centrifuge at 20,000g for 20 minutes at 4°C, the liquid in the centrifuge tube will be separated, that is, the upper phenol phase and the lower water phase; The upper layer of phenol was absorbed into a new centrifug...

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Abstract

The present invention discloses a proteome identification method and applications thereof. The proteome identification method comprises: 1) extracting the total protein of an organism, and dissolving the total protein by using a protein lysate to obtain a protein solution; 2) sequentially carrying out reducing and alkylation on the protein in the protein solution to obtain a reduced alkylated product; 3) carrying out enzymolysis on the protein in the reduced alkylated product by using a protease to obtain an enzymolysis product; 4) labeling the protein in the enzymolysis product by using an iTRAQ reagent to obtain a labeled product; 5) pre-grading the labeled product by using UPLC-Q-TOF-MS to obtain different small proteome samples, carrying out primary identification on each small proteome samples by using MALDI-TOF-TOF-MS, and combining the group having the absorption peak being not covered to obtain different large proteome samples; and 6) carrying out proteome identification on different large groups of the samples by using NanoLC-Q-TOF-MS to obtain the proteome identification data.

Description

technical field [0001] The invention relates to a proteome identification method and its application in the field of biotechnology. Background technique [0002] Protein is the product of gene expression, and it is also the main executor and embodiment of gene function. The study of protein is of great significance to reveal the function of gene and the essence of life phenomenon. Proteomics is an emerging discipline that emerged under the background of breakthroughs in genomics research and high-throughput protein analysis technology. research to elucidate its biological function. Currently, proteomics technologies include two-dimensional gel electrophoresis, two-dimensional fluorescent differential gel electrophoresis, isotope affinity labeling technology, and isotope-labeled protein quantitative technology, including various protein separation and identification technologies. [0003] Isotopic tags for relative and absolute quantitation (iTRAQ) technology is a new and p...

Claims

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Application Information

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IPC IPC(8): G01N27/62
CPCG01N33/6851G01N2333/415
Inventor 曲乐庆荣立伟田丽宏
Owner INST OF BOTANY CHINESE ACAD OF SCI
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