In-Depth Investigation of Low-Abundance Proteins in Matured and Filling Stages Seeds of Glycine max Employing a Combination of Protamine Sulfate Precipitation and TMT-Based Quantitative Proteomic Analysis.

Cheol Woo Min,Youngsoo Kim,Joonho Park,Ganesh Kumar Agrawal,Randeep Rakwal,Pingfang Yang,Sun Tae Kim,Ravi Gupta,Jin Woo Bae

doi:10.3390/cells9061517

Abstract

Despite the significant technical advancements in mass spectrometry-based proteomics and bioinformatics resources, dynamic resolution of soybean seed proteome is still limited because of the high abundance of seed storage proteins (SSPs). These SSPs occupy a large proportion of the total seed protein and hinder the identification of low-abundance proteins. Here, we report a TMT-based quantitative proteome analysis of matured and filling stages seeds of high-protein (Saedanbaek) and low-protein (Daewon) soybean cultivars by application of a two-way pre-fractionation both at the levels of proteins (by PS) and peptides (by basic pH reverse phase chromatography). Interestingly, this approach led to the identification of more than 5900 proteins which is the highest number of proteins reported to date from soybean seeds. Comparative protein profiles of Saedanbaek and Daewon led to the identification of 2200 and 924 differential proteins in mature and filling stages seeds, respectively. Functional annotation of the differential proteins revealed enrichment of proteins related to major metabolism including amino acid, major carbohydrate, and lipid metabolism. In parallel, analysis of free amino acids and fatty acids in the filling stages showed higher contents of all the amino acids in the Saedanbaek while the fatty acids contents were found to be higher in the Daewon. Taken together, these results provide new insights into proteome changes during filling stages in soybean seeds. Moreover, results reported here also provide a framework for systemic and large-scale dissection of seed proteome for the seeds rich in SSPs by two-way pre-fractionation combined with TMT-based quantitative proteome analysis.

Highlights

Soybean seeds are considered as one of the most important crops because of their large-scale use as a dietary supplement to humans and livestock
For tandem mass tags (TMTs)-based proteome analysis, isolated proteins from all the fractions were subjected to in-solution trypsin digestion by the filter-aided sample preparation (FASP) method [20] and digested peptides from 3 biological replicates of 6 samples were labeled with TMT 6-plex kit (Figure 1A)
Peptide pre-fractionation techniques such as basic pH reversed-phase (BPRP) or SCX reduces the complexity of pooled peptides and increases the protein coverage in the shotgun proteomic analysis [2], high-abundance proteins (HAPs) lead to masking or suppression of low abundance proteins (LAPs) during 2-DGE and shotgun proteomic analyses [38]

Summary

Introduction

Soybean seeds are considered as one of the most important crops because of their large-scale use as a dietary supplement to humans and livestock. Calcium [6] have been developed that facilitate the identification of low-abundance proteins on two-dimensional gel electrophoresis (2-DGE). These HAPs depletion methods seem to be effective and are broadly applied for the depletion of HAPs from different plants including broad bean, pea, wild soybean, and peanut, which contain their own specific HAPs [5]. Development of shotgun proteomic analysis techniques allows comparison of tandem mass spectra derived from peptide fragmentation that facilitates the relative (or absolute) quantification using the ion intensities or spectral counting and peptide labeling approaches [10]. The tandem mass tags (TMTs) were presented as an alternative approach having the same features of other isotope labeling techniques, with mass tags of identical nominal mass and chemical structure, and that allowed up to 16 samples per MS run [9,12]

Methods

Results

Discussion

Conclusion