In-depth investigation of low-abundant proteins in matured and filling stages seeds of Glycine max by a combination of protamine sulfate precipitation and TMT-based quantitative proteomic analysis

The presence of seed storage proteins is a major bottleneck in soybean seed proteome analysis despite the significant technical advancements in mass spectrometry and data analysis software. Our previous studies have shown that effective ways to improve the detection of low-abundance proteins in soybean seeds using protamine sulfate (PS) with 2-DGE analysis. Besides, verification of the PS precipitation method revealed broad application to other leguminous plants like pea, broad bean, and peanut. Nonetheless, it is difficult to overcome the poor reproducibility and limitation of the dynamic range of identification during 2-DGE analysis. Thus, to elucidate the suitability and efficacy of protamine sulfate precipitation method for shotgun proteome approach, we performed TMT-based quantitative analysis using matured and filling stages seeds by application of two-way pre-fractionation using PS and chromatographic methods. Interestingly, this approach led to the identification of 2,200 and 924 differential proteins in the matured and filling stage of soybean seeds, respectively. These results revealed enrichment of various proteins related to major metabolism in soybean seeds including amino acid, major carbohydrate, lipid metabolism, and among others. Taken together, results provided the new insight into current complexity and limitation of soybean seed proteomics through two-way pre-fractionation combined with TMT-based quantitative analysis.