eXtRactIOn anD anaLYsIs OF sOYBean cYst neMatODe (HETERODERA GLYCINES) PROteIns BY tWO-DIMensIOnaL geL eLectROPhOResIs

Chen, X. , M. H. MacDonald, M. G. Wesley, B. F. Matthews, and S. S. Natarajan. 2011. Extraction and analysis of soybean cyst nematode (Heterodera glycines) proteins by two-dimensional gel electrophoresis. Nematropica 41:240-247. 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 Matrixassisted 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. Chen, X. , M. H. MacDonald, M. G. Wesley, B. F. Matthews, and S. S. Natarajan. 2011. Extraccion y analisis de proteinas del nematodo quiste de la soya (Heterodera glycines) por electroforesis bidimensional. Nematropica 41:240-247. El nematodo quiste de la soya (Heterodera glycines, SCN) es el nematodo mas danino del cultivo de la soya en el mundo. En este estudio, se evaluaron tres metodos de extraccion de proteinas: acetato de fenol/amonia (metodo de fenol) solubilizacon con tiourea/urea (metodo de lisis) y acido tricloroacetico/acetona (metodo TCA), para determinar su eficacia en la separacion de proteinas de Heterodera glycines por electroforesis bidimensional en poliacrilamida (2-DE). Con los tres metodos se separaron bien las proteinas, con variaciones menores en la intensidad. Se obtuvo mayor resolucion de las proteinas y mayor intensidad de estas con el metodo de fenol que con los otros dos metodos. Usando el metodo de fenol tambien se resolvieron mas claramente y con mayor intensidad las proteinas en la region de alto pI. Se analizaron las proteinas obtenidas por el metodo de fenol para evaluar la viabilidad de identificacion por espectrometria de masas. Se seleccionaron 20 puntos de proteinas al azar, se hizo digestion con tripsina, y se analizo con espectrometria de masas (Matrix-assisted laser desorption/ ionization time of flight mass spectrometry, MALDI-TOF-MS) o espectrometria de masas de cromatografia liquida (liquid chromatography mass spectrometry, LC-MS/MS). Estos resultados sugieren que los metodos de fenol y de lisis directa son confiables y eficientes para la separacion bidimensional de las proteinas de Heterodera glycines, siendo el metodo de fenol superior para las proteinas alcalinas.