Multidimensional liquid chromatography-mass spectrometry method for identifying proteins and proteomes in tobacco

A multi-dimensional liquid chromatography, liquid chromatography technology, applied in the direction of measurement devices, instruments, scientific instruments, etc., can solve the problems of lack of detection throughput, large amount of proteome, etc.

Active Publication Date: 2018-01-19
SHANGHAI TOBACCO GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a multi-dimensional liquid chromatography-mass spectrometry method for identifying proteins and p

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  • Multidimensional liquid chromatography-mass spectrometry method for identifying proteins and proteomes in tobacco

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Embodiment 1

[0095] 1. Protein extraction

[0096] Take samples of fresh tobacco leaves, using "Sample extraction techniques for enhanced proteomic analysis of plant tissues" (Isaacson T; Damasceno CM; Saravanan RS; HeY; CataláC; SaladiéM; Rose JK, Nature Protocols, 2006, 1, 769-774). The protein was extracted by TCA / acetone precipitation method and phenol method respectively, and then the extracted proteins were combined and re-dissolved by adding 4-8M urea aqueous solution to obtain protein sample solution. In the protein sample solution, the ratio of the volume (μl) of the solvent added for the reconstitution to the mass (mg) of the protein is the volume ratio of 55-65:1.

[0097] The protein sample solution is quantified by ultraviolet spectrophotometry. First, take the protein sample solution, add the precipitant for rotary suspension and then centrifuge, take the precipitate and add the dissolving agent to suspend and dissolve, then add the color reagent to mix, and then perform the incub...

Embodiment 2

[0122] 1. Protein extraction

[0123] Take samples of fresh tobacco leaves, using "Sample extraction techniques for enhanced proteomic analysis of plant tissues" (Isaacson T; Damasceno CM; Saravanan RS; HeY; CataláC; SaladiéM; Rose JK, Nature Protocols, 2006, 1, 769-774). The protein was extracted by TCA / acetone precipitation method and phenol method, and then the extracted protein was combined and reconstituted with 6M urea aqueous solution to obtain a protein sample solution. In the protein sample solution, the ratio of the volume (μl) of the solvent added for the reconstitution to the mass (mg) of the protein is 60:1.

[0124] The protein sample solution was quantified by ultraviolet spectrophotometry. First, take 10μL of protein sample solution, add 500μL of precipitant, rotate and suspend at room temperature for 2-3 minutes, then add 500μL of coprecipitant, suspend evenly, centrifuge, and precipitate into protein. Discard Remove the supernatant, centrifuge again for 5 minutes...

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Abstract

The invention provides a multidimensional liquid chromatography-mass spectrometry method for identifying proteins and proteomes in tobacco, and the multidimensional liquid chromatography-mass spectrometry method specifically comprises the following steps: 1) taking a tobacco leaf sample, using a trichloroacetic acid-acetone precipitation method and a phenol method respectively to extract proteins,and then merging the proteins and re-dissolving to obtain a protein sample solution; 2) subjecting the protein sample solution to enzymolysis, desalting and drying to obtain dry peptide powder as a sample; and 3) sequentially passing the prepared sample through a first-dimensional liquid chromatography and a second-dimensional liquid chromatography for gathering, performing elution separation, and detecting by mass spectrometry. The method provided by the invention reserves the proteins in the sample to the maximum extent, greatly improves the peak capacity of the chromatogram, and has the advantages of high detection flux, high sensitivity and large amount of identified proteomes.

Description

Technical field [0001] The invention belongs to the technical field of tobacco protein and proteomics research, and relates to a multi-dimensional liquid chromatography mass spectrometry method for identifying proteins and proteomes in tobacco, and specifically relates to a multidimensional liquid chromatography for extracting, enzymatically and determining the proteins and proteomes in fresh tobacco leaves. GC / MS usage. Background technique [0002] With the development of various detection technologies, the concept of proteome was proposed in 1994. It is an emerging subject and hotspot in functional genomics research in the post-genome era. Proteomics is the study of proteins at the overall, dynamic, and network level. The study of proteomics is a requirement for studying and understanding complex life activities. [0003] Tobacco (Nicotiana tabacum) is an important economic crop. At the same time, as a complex model plant, tobacco is representative in the fields of plant geneti...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06G01N30/46G01N30/72G01N30/89
Inventor 陈敏董惠忠吴达
Owner SHANGHAI TOBACCO GRP CO LTD
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