Dziuba Jerzy, Szerszunowicz Iwona, Nałęcz Dorota, Dziuba Marta
Department of Food Biochemistry, University of Warmia and Mazury in Olsztyn, Poland.
Acta Sci Pol Technol Aliment. 2014 Apr-Jun;13(2):181-90. doi: 10.17306/j.afs.2014.2.7.
Proteomic analysis is emerging as a highly useful tool in food research, including studies of food allergies. Two-dimensional gel electrophoresis involving isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis is the most effective method of separating hundreds or even thousands of proteins. In this study, albumin and globulin tractions of pea seeds cv. Ramrod were subjected to proteomic analysis. Selected potentially alergenic proteins were identified based on their molecular weights and isoelectric points.
Pea seeds (Pisum sativum L.) cv. Ramrod harvested over a period of two years (Plant Breeding Station in Piaski-Szelejewo) were used in the experiment. The isolated albumins, globulins and legumin and vicilin fractions of globulins were separated by two-dimensional gel electrophoresis. Proteomic images were analysed in the ImageMaster 2D Platinum program with the use of algorithms from the Melanie application. The relative content, isoelectric points and molecular weights were computed for all identified proteins. Electrophoregrams were analysed by matching spot positions from three independent replications.
The proteomes of albumins, globulins and legumin and vicilin fractions of globulins produced up to several hundred spots (proteins). Spots most characteristic of a given fraction were identified by computer analysis and spot matching. The albumin proteome accumulated spots of relatively high intensity over a broad range of pi values of ~4.2-8.1 in 3 molecular weight (MW) ranges: I - high molecular-weight albumins with MW of ~50-110 kDa, II - average molecular-weight albumins with MW of ~20-35 kDa, and III - low molecular-weight albumins with MW of ~13-17 kDa. 2D gel electrophoregrams revealed the presence of 81 characteristic spots, including 24 characteristic of legumin and 14 - of vicilin.
Two-dimensional gel electrophoresis proved to be a useful tool for identifying pea proteins. Patterns of spots with similar isoelectric points and different molecular weights or spots with different isoelectric points and similar molecular weights play an important role in proteome analysis. The regions characteristic of albumin, globulin and legumin and vicilin fractions of globulin with typical MW and pi values were identified as the results of performed 2D electrophoretic separations of pea proteins. 2D gel electrophoresis of albumins and the vicilin fraction of globulins revealed the presence of 4 and 2 spots, respectively, representing potentially allergenic proteins. They probably corresponded to vicilin fragments synthesized during post-translational modification of the analysed protein.
蛋白质组学分析正在成为食品研究（包括食物过敏研究）中一种非常有用的工具。二维凝胶电泳，包括等电聚焦和十二烷基硫酸钠聚丙烯酰胺凝胶电泳，是分离数百甚至数千种蛋白质最有效的方法。在本研究中，对豌豆品种Ramrod种子的白蛋白和球蛋白组分进行了蛋白质组学分析。根据其分子量和等电点鉴定出选定的潜在致敏蛋白。
实验使用了在两年时间内（皮亚斯基 - 谢莱耶沃植物育种站）收获的豌豆（Pisum sativum L.）品种Ramrod种子。通过二维凝胶电泳分离分离得到的白蛋白、球蛋白以及球蛋白的豆球蛋白和豌豆球蛋白组分。使用Melanie应用程序中的算法在ImageMaster 2D Platinum程序中分析蛋白质组图像。计算所有鉴定出的蛋白质的相对含量、等电点和分子量。通过匹配三个独立重复实验的斑点位置来分析电泳图谱。
白蛋白、球蛋白以及球蛋白的豆球蛋白和豌豆球蛋白组分的蛋白质组产生了多达数百个斑点（蛋白质）。通过计算机分析和斑点匹配鉴定出了给定组分最具特征的斑点。白蛋白蛋白质组在约4.2 - 8.1的广泛等电点值范围内，在3个分子量（MW）范围内积累了相对高强度的斑点：I - 分子量约为50 - 110 kDa的高分子量白蛋白，II - 分子量约为20 - 35 kDa的中等分子量白蛋白，III - 分子量约为13 - 17 kDa的低分子量白蛋白。二维凝胶电泳图谱显示存在81个特征斑点，包括24个豆球蛋白特征斑点和14个豌豆球蛋白特征斑点。
二维凝胶电泳被证明是鉴定豌豆蛋白的有用工具。具有相似等电点和不同分子量的斑点模式或具有不同等电点和相似分子量的斑点模式在蛋白质组分析中起重要作用。通过对豌豆蛋白进行二维电泳分离，鉴定出了白蛋白、球蛋白以及球蛋白的豆球蛋白和豌豆球蛋白组分具有典型分子量和等电点值的特征区域。白蛋白和球蛋白的豌豆球蛋白组分的二维凝胶电泳分别显示存在4个和2个代表潜在致敏蛋白的斑点。它们可能对应于分析蛋白质在翻译后修饰过程中合成的豌豆球蛋白片段。
