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Title: An efficient extraction method to enhance analysis of low abundant proteins from soybean seed

Author
item Natarajan, Savithiry - Savi
item Krishnan, Hari
item Lakshman, Sukla
item Garrett, Wesley

Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/3/2009
Publication Date: 8/3/2009
Citation: Natarajan, S.S., Krishnan, H.B., Lakshman, S., Garrett, W.M. 2009. An efficient extraction method to enhance analysis of low abundant proteins from soybean seed. Analytical Biochemistry. 394(2):259-268.

Interpretive Summary: Genetically modified (GM) soybean is widely grown in the U.S. and abroad and it is likely that soybean cultivars with a variety of modifications to enhance quality and productivity will be developed in the future. It is therefore important to determine if any unintended changes occur in GM soybean seeds. Most allergenic proteins are less abundant and difficult to isolate using conventional extraction methods. In this series of experiments, we determined the best chemical procedure to separate soybean seed proteins. We developed an efficient protein extraction method and identified some of the seed proteins. Protein seed composition of normal soybean is important to know so that we can compare them to GM soybeans. This comparison will allow scientists to identify unintended effects that may occur when new transgenic soybean cultivars are developed and will help to improve consumer acceptance of GM soybeans. This information will be used by scientists who are developing transgenic soybean cultivars.

Technical Abstract: Large amounts of the major seed storage proteins, such as ß-conglycinin and glycinin, in soybean (Glycine max) seeds hinder the isolation and characterization of less abundant seed proteins. We investigated whether isopropanol extraction could facilitate resolution of the less abundant proteins from soybean seeds in one dimensional polyacrylamide gel electrophoresis (1D-PAGE) and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). 1D-PAGE of proteins extracted by different concentrations (10, 20, 30, 40, 50, 60, 70 and 80%) of isopropanol showed that greater than 30% isopropanal was suitable for preferential enrichment of low abundant proteins. Analysis of 2D-PAGE showed that proteins which were less abundant or absent by the conventional extraction procedure were clearly seen in the 40% isopropanol extracts. Increasing above 40% isopropanol concentration resulted in a decrease in the number of less abundant protein spots. We have identified a total of 107 protein spots using Matrix-assisted laser desorption/ionization time of flight mass spectrophotometry (MALDI-TOF-MS) and liquid chromatography mass spectrometry (LC-MS/MS). Our results suggest that extraction of soybean seed powder with 40% isopropanol enriches for lower abundance proteins and is a suitable method for 2D-PAGE separation and identification. This methodology could potentially allow the extraction and characterization of low abundant proteins of other legume seeds containing highly abundant storage proteins.