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United States Department of Agriculture

Agricultural Research Service

Research Project: EVALUATION OF THE QUALITY AND SAFETY OF TRANSGENIC SOYBEANS Title: Evaluation of protein extraction methods suitable for two-dimensional gel electrophoresis of the soybean cyst nematode (Heterodera glycines)

Authors
item Chen, XI -
item Macdonald, Margaret
item Garrett, Wesley
item Matthews, Benjamin
item Natarajan, Savithiry

Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 17, 2011
Publication Date: December 5, 2011
Citation: Chen, X., Macdonald, M.H., Garrett, W.M., Matthews, B.F., Natarajan, S.S. 2011. Evaluation of protein extraction methods suitable for two-dimensional gel electrophoresis of the soybean cyst nematode (Heterodera glycines). Analytical Biochemistry. 41(2):240-247.

Interpretive Summary: The soybean cyst nematode (SCN) is the major pest of soybean in the U.S. Some soybean cultivars are resistant to one race and susceptible to another. To improve soybean yields by increasing the level of plant resistance to targeted pests, it is important to understand the protein composition in nematode. Therefore, we have evaluated, standardized and applied protein analysis technology to determine and quantify the spectrum of proteins present in nematode. To do this we are using a “proteomics” approach in which nematode proteins are separated, identified and quantified using a device called a mass spectrometer. We evaluated an efficient extraction method to solubilize more nematode proteins. This approach will be important in providing a way to identify nematode proteins and to develop a reference map of nematode proteins. This standardized methodology for defining and quantifying the spectrum of protein in nematode will be useful to scientists who wish to develop nematode resistant soybeans.

Technical Abstract: Soybean cyst nematode (Heterodera glycines, SCN) is the most destructive pathogen of soybean (Glycine max (L.) Merr.) worldwide. In this study, three different protein extraction methods including phenol/ammonium acetate (phenol method), thiourea/urea solublization (lysis method) and trichloroacetic acid/acetone (TCA method) were evaluated to determine their efficacy in separating Heterodera glycines proteins by two-dimensional polyacrylamide gel electrophoresis (2-DE). In all three methods, nematode proteins were well separated with minor variations in the intensity of the protein spots. The phenol method showed higher protein resolution and spot intensity of all proteins compared with the other two methods. In addition, in the high-pI region, proteins were clearly resolved and strongly detected using the phenol method. Protein spots obtained from the phenol method were subjected to further analysis to test their suitability for identification by mass spectrometry. Twenty protein spots were randomly selected, digested with trypsin, and analyzed using Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) or liquid chromatography mass spectrometry (LC-MS/MS). While these results suggest that phenol method and the direct lysis method are more efficient and reliable for the 2D separation of Heterodera glycines proteins, the phenol extraction procedure is superior for the alkaline proteins. This information will be useful to scientists interested in high quality extraction of proteins from nematodes and it will advance research targeted to developing new modes of resistance in plants to nematodes.

Last Modified: 10/22/2014
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