A Method for Extracting Rice Leaf Plasma Membrane Phosphorylated Proteins Suitable for Two-dimensional Electrophoresis

Abstract

The invention belongs to the technical field of proteomics, specifically discloses for the first time a method for extracting rice leaf plasma membrane phosphorylated proteins suitable for two-dimensional electrophoresis, including extraction of rice leaf rough plasma membranes, purification of rice leaf plasma membranes, and rice leaf plasma membrane extraction methods. Enrichment of membrane phosphorylated proteins and other steps. The extraction method of the present invention is simple in operation, high in extraction efficiency and low in cost, the purity of the obtained rice leaf plasma membrane can reach more than 93%, and the yield of plasma membrane phosphorylated protein enriched from plasma membrane protein is 3.33-3.89% . This method is suitable for plant materials that are difficult to extract plasma membrane phosphorylated proteins and have many interfering impurities; at the same time, the obtained plasma membrane phosphorylated proteins have fewer impurities and interfering substances, which can meet the requirements of two-dimensional electrophoresis. Uniform distribution, less longitudinal tailing and horizontal stripes, and good repeatability; this method is suitable for the preparation and analysis of monocot plasma membrane phosphorylated proteomes.

Description

technical field [0001] The invention relates to the technical field of proteomics, in particular to a method for extracting rice leaf plasma membrane phosphorylated proteins suitable for two-dimensional electrophoresis. Background technique [0002] Protein phosphorylation refers to protein phosphorylation under the action of protein kinase and dephosphorylation under the action of phosphatase. As one of the most important forms of protein post-translational modification, protein phosphorylation regulates almost all life activities in cells, especially plays an extremely important role in regulating cell signal transduction. Phosphoproteomics refers to the use of proteomics analysis methods to observe the modification status and changes of phosphorylated proteins in cells as a whole, and then analyze the regulation and molecular mechanism of specific phosphorylation modifications on life processes; its content It mainly includes the enrichment, detection, identification and...

AI Technical Summary

Problems solved by technology

In the existing studies on the phosphorylated proteome of plant plasma membranes, most of the strategies adopted are: firstly isolate and purify plant plasma membranes, then enzymatically hydrolyze and enrich phosphorylated peptides of plasma membrane proteins, and then analyze by chromatography and mass spectrometry. Carry out protein phosphorylation modification analysis; its advantages are good peptide solubility and high enrichment specificity, but the disadvantage is that protein identification only relies on a single peptide to cause confusion, it is difficult to directly determine the molecular weight and isoelectric point of the protein, and non-specific adsorption Still exist

[0004] At present, there are no related reports on the rice plasma membrane phosphorylated proteome. The main reason is that the monocot plasma membrane is difficult to purify, and the abundance of plasma membrane phosphorylated proteins is very low, and the hydrophobicity of the plasma membrane protein itself easily leads to protein loss, etc.

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