Method optimization for proteomic analysis of soybean leaf: improvements in identification of new and low-abundance proteins

Abstract

The most critical step in any proteomic study is protein extraction and sample preparation. Better solubilization increases the separation and resolution of gels, allowing identification of a higher number of proteins and more accurate quantitation of differences in gene expression. Despite the existence of published results for the optimization of proteomic analyses of soybean seeds, no comparable data are available for proteomic studies of soybean leaf tissue. In this work we have tested the effects of modification of a TCA-acetone method on the resolution of 2-DE gels of leaves and roots of soybean. Better focusing was obtained when both mercaptoethanol and dithiothreitol were used in the extraction buffer simultaneously. Increasing the number of washes of TCA precipitated protein with acetone, using a final wash with 80% ethanol and using sonication to ressuspend the pellet increased the number of detected proteins as well the resolution of the 2-DE gels. Using this approach we have constructed a soybean protein map. The major group of identified proteins corresponded to genes of unknown function. The second and third most abundant groups of proteins were composed of photosynthesis and metabolism related genes. The resulting protocol improved protein solubility and gel resolution allowing the identification of 122 soybean leaf proteins, 72 of which were not detected in other published soybean leaf 2-DE gel datasets, including a transcription factor and several signaling proteins.

Research Article Differential proteomics in contrasting cowpea genotypes submitted to different water regimes

“…The most expressed proteins in the tolerant genotype were those directly involved in protein metabolism, followed by proteins responsible for photosynthesis, both under full irrigation and water deficit. According to Mesquita et al (2012), such differences are important mechanisms for tolerance and identification of potential candidate molecules to manipulation for use in genetics in order to select drought-tolerant genotypes. Changes in gene expression and adaptations to tolerance are directly involved with change in protein metabolism, either synthesis or degradation since they play crucial roles in stress tolerance (Kosová et al, 2013).…”

“…Total protein extraction was carried out in five biological replications. The leaves were macerated in liquid nitrogen, and the extraction performed based on the method described by Mesquita et al, (2012). Protein concentration was determined according to Bradford (1976).…”

Arabidopsis AtPARK13, Which Confers Thermotolerance, Targets Misfolded Proteins

“…Two-dimensional Gel Electrophoresis and Mass Spectrometry-Total Arabidopsis protein extracts were subjected to twodimensional PAGE, and protein spots were analyzed using ImageMaster 2D Platinum version 7 (GE Healthcare) followed by peptide mass fingerprinting and data-dependent sequencing, as described previously (23).…”

A theoretical approach for discriminating accurately intrinsic pattern of biological systems and recognizing three kind soybean proteomes

“…Currently, the major researches on soybean proteomics included expression differences of proteins in leaves, roots, seeds and seed linage [13,14,15,16], the expression differences, in root, leaves, flower buds, seeds under different growth and stress conditions [17,18,19,20,21], the researches on low abundant proteins [22,23,24], the allergen protein studies [25,26,27], the analysis on proteins of transgenosis and normal soybeans [28], the quantitative analysis on proteomic profiles [29,30]. In all these works, the mainly used techniques are some separation technologies, such as 2-DE [1,13,15,19~23,25,28,30,31,32,33], SDS-PAGE [14,24,29,31], HPLC-MS [1,14,20,24], by which the overall expression spectra of proteome could be obtained.…”