EXTRACTION AND ANALYSIS OF SOYBEAN CYST NEMATODE (HETERODERA GLYCINES) PROTEINS BY TWO-DIMENSIONAL GEL ELECTROPHORESIS

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 efficient and reliable for the 2D separation of Heterodera glycines proteins, the phenol extraction procedure is superior for the alkaline proteins.