Session: Parallel session 7 - Club jeune FPS

Proteins in pea seed starch at the scale of the single granule by high resolution mass spectrometry

Starch is a main storage form of carbon for plants and is composed of glucose residues linked together by α-1,4 O-glycosidic linkages and branched through α-1,6 bonds. The two polymers composing starch are amylose and amylopectin which is the most abundant. A wide range of granule morphologies exists regarding to the plant species and their localization such as leaves, roots or tubers. Starch granules contain a limited proteome constituted of around 100 proteins. The aim of our work is to miniaturize a proteomics analysis of a single granule to correlate the protein distribution with the morphology and the amylose, amylopectin ratio.

In a preliminary step we analyzed proteins extracted from 300, 150, 75, 40, 20 10, 1, 0.5 down to 0.1 µg of starch. As the protein content of starch is 0.6% we obtained 1.8 µg down to 0.6 ng of proteins. Taking in account the pea starch granule size this represents 16,300, 8170, 4080, 2130, 1090, 544, 54, 27, 5 granules respectively. Then, we counted almost exactly 10,000, 1,000 and 100 granules. For protein extraction from starch granules, starch was firstly gelatinized within a 10 kDa Amicon® filter overnight at 70 °C. Then eFASP digestion with trypsin enzyme were was performed followed by nanoLC-MSMS on a Q-Exactive+ Orbitrap. The bioinformatics data treatment was performed with Proteome Discoverer.

Granule Bond Starch Synthase (GBSS) is the most abundant protein in starch. In the first experiment from 300 to 0.1 µg of starch, GBSS was identified with 49 to 8 unique peptides, respectively. In counted granules, GBSS was identified in 10,000, 1,000 and 100 starch granules with 31; 20 and 2 unique peptides respectively, which is less than expected from the previous results. In order to improve the sensitivity of the analysis we are currently optimizing the protein extraction from granules, performing DIA analysis and we are planning to perform SCope experiment with TMT labeling.