Method for qualitative identification of strawberry stigma protein based on two-dimensional liquid chromatography tandem mass spectrometry

Abstract

The invention discloses a method for qualitative identification of strawberry stigma protein based on the two-dimensional liquid chromatography tandem mass spectrometry platform, comprising the following steps: 1) carrying out extraction and purification of strawberry stigma to obtain protein powder; 2) carrying out protein quantification and reductive alkylation; 3) carrying out proteolysis; 4) carrying out first-dimensional SCX strong cation exchange liquid chromatography separation of a peptide mixture; 5) carrying out second-dimensional reverse C18 liquid phase separation tandem Q Exactivemass spectrometry; and 6) carrying out data analysis: The original files obtained by the mass spectrometer were identified by the Maxquant search library to obtain a protein and peptide information list. The method of the invention can be adopted for qualitative identification of the red-cheek strawberry stigma protein, and provides a basis for building the red-cheek strawberry stigma protein information database.

Description

technical field [0001] The invention relates to the field of plant proteomics, and provides a method for identifying stigma proteins of strawberries (especially red-cheeked strawberries) by using strong cation exchange chromatography and reverse polarity chromatography for two-dimensional separation and connecting Q Exactive mass spectrometers in series. Background technique [0002] Strawberry belongs to Rosaceae (Rosaceae) Fragaria (Fragaria), and is deeply loved by people because of its sweet taste and rich nutritional elements. With the improvement of people's consumption level, the market demand for new varieties is also increasing. Red cheek strawberry is a high-quality large-fruited variety bred in Japan (introduced by Hangzhou Academy of Agricultural Sciences in 2006). It has become the most popular variety in the market because of its large and upright fruit shape, sweet and sour taste, and high fruit hardness. One, but this variety has weak resistance to anthracnos...

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Problems solved by technology

[0004] However, the complexity of the peptides obtained only by C18 one-dimensional liquid phase is still high, the resolution is not enough, and the amount of proteins identified by mass spectrometry is limited. Usually, the number of proteins identified by Q Exactive single needle is only about 2000-3500; SCX-C18 tandem and The separation effect of high pH reverse C18-conventional reverse C18 tandem two-dimensional liquid phase is greatly affected by the liquid phase gradient. The commonly used gradient reported in the literature is not effective for the separation of the stigma protein peptides of red cheek strawberry. In order to improve the identification Separation gradient needs to be optimized

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