Analysis of soybean-proteins (Glycine max) by two-dimensional electrophoresis and MALDI-TOF :

University essay from SLU/Dept. of Biomedical Sciences and Veterinary Public Health

Abstract: Food allergy is a growing problem in the world today, which makes it important to find out which proteins in food that causes allergic reactions. Soybeans are one of the most common food allergens in many countries and little is known about it allergenic properties. There are about 15 known soybean-proteins that have been shown to be allergenic. Most of these are seed proteins and the most common ones are Globulines called Glycinin and β-conglycinin. Other proteins known to be allergenic are Gly m Bd 28, soybean agglutinin (SBA), Kunitz trypsin inhibitor (TI), Gly m 4 and Gly m Bd30. The main aim of this study was to set up a two-dimensional (2-D) electrophoresis method for separation of proteins in soybean extract. The 2-D technique was used to identify soybean components and especially allergenic components in the soybean extract. The 2-D electrophoresis method was very useful for separation of proteins. First the proteins were separated during isoelectric focusing according to the proteins isoelectric point. In a second step they were separated in an electrophoresis according to their molecular weight. The gels were then stained with Coomassie brilliant blue (CBB) and proteins could be seen as blue spots on a clear background. To identify the proteins in the soybean extract known soybean components were run separately on 2-D electrophoresis and compared to the gel over the whole soybean extract. To further confirm the identity of allergic components in the soybean extract matrix-assisted laser desorption ionization (MALDI) was used. The soybean extract was also gel filtrated through a Sephadex gel column and collected in 20 different fractions. From these fractions some were chosen for 2-D electrophoresis, these were also compared to the previously gels over the extract. Results from 2-D electrophoresis showed that glycinin was a very common protein in the soybean extract. The Trypsin inhibitor was also relatively common. SBA, β-conglycinin and Gly m 4 gave rise to fewer spots. In the gel filtrated fractions several of the Glycinin and β-conglycinin were successfully separated. All proteins identified through 2-D electrophoresis were summarized on a gel with the whole soybean extract. To confirm the identity of some of the proteins MALDI-TOF was used. Several Glycinin and β-conglycinin spots could be confirmed and also three other proteins with no known allergenic properties. The 2-D electrophoresis method in combination with MALDI-TOF was shown to be a very useful combination for identification of proteins.

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