Analysis of Soybean Seed Proteins Using Proteomics

Abstract

In order to determine the variation of seed proteins that may occur in the crop as a result of genetic modification, accurate and replicable methodology for protein extraction, isolation, and characterization is essential. Modern proteomic tools are being used to study the expression of proteins to examine alterations in protein profiles caused by genetic mutations and environmental stress [2-10]. Proteome analysis is performed using a variety of methods including structural proteomics such as high throughput (HT) X-ray crystallography and HT nuclear magnetic resonance (NMR) spectroscopy; expressional or analytical proteomics such as gel based electrophoresis (IDE, 2DE, 2DIGE), gel-free (LC-MS/MS or multidimensional protein identification technology (Mud PIT), protein chips, DNA chips, mass spectrometry (MS), micro sequencing; and functional or interaction proteomics such as HT functional assays, ligand chips, yeast 2-hybrid, deletion analysis, and motif analysis [11-14]. In this mini review, we discuss some of the expression analysis methodology that we’ve applied to study soybean seed proteins. Because the extraction of soybean seed proteins for accurate 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is challenging, we initially optimized protein extraction techniques by comparing different methods. Extraction of protein suitable for 2D-PAGE is sample-dependent and is achieved by optimizing the concentration of chaotropic agents, detergents, reducing agents, buffers, enzymes and ampholytes. Also helpful in this regard are advances in immobile pH gradient (IPG) technology and the development of electrophoretic instruments have improved the reproducibility of protein separation. The availability of commercial IPG strips in linear and nonlinear gradients with multiple narrow pH ranges [15], allows effective protein separation for various downstream analyses. We compared four different solubilization methods (urea/thiourea, urea, modified trichloroacetic acid (TCA)/ acetone and phenol) for extraction of proteins from soybean seeds for subsequent analysis by 2D-PAGE [16]. Our study demonstrated that the modified TCA/acetone method with urea/thiourea solubilization resolved more protein spots than the urea or phenol extractions. Using the phenol/urea method, resolution of proteins was generally poor and spots diffuse in the high molecular weight region of the gel, particularly when separating the proteins at pH 4.0 to 7.0. Moreover, while overall protein separation was similar in the TCA/acetone and urea/thiourea methods, low molecular weight proteins were more consistently resolved with the TCA/acetone method. In addition, characterization of low abundant proteins is a challenge because they are often masked by highly abundant proteins. Recently, Boschetti and Righetti [17] published a review of plant proteomic methods to isolate