Department of Crop & Soil Sciences, Washington State University, Pullman, WA 99164, USA.
Department of Agricultural and Biological Engineering, University of Florida, Gainesville, FL 32611, USA.
Nutrients. 2014 Apr 15;6(4):1578-97. doi: 10.3390/nu6041578.
Wheat prolamins, commonly known as "gluten", are a complex mixture of 71-78 proteins, which constitute ~80% of the proteins in the wheat grains and supply 50% of the global dietary protein demand. Prolamins are also responsible for numerous gluten-induced disorders and determine the unique visco-elastic properties of the wheat dough. These properties necessitate the reliable determination of the prolamin composition in wheat grains and their derived products. Therefore, this study examined the impact of HPLC conditions, including column type, column temperature, flow rate, and the gradient of polar and non-polar solvents in the mobile phase, to improve the analytical resolution of prolamins. The following conditions were found optimal for analyses: column temperature 60 °C, flow rate 1.0 mL/min and an elution gradient of 20%-60% of 0.1% trifluoroacetic acid + acetonitrile in 60 min. For further improvement of resolution, gliadin and glutenin extracts were analyzed using MALDI-TOF-MS in combination with HPLC fractionation. Two semi-quantitative methods, densitometry of stained polyacrylamide gels and HPLC, were used to determine relative prolamin quantities and the correspondence between the methods was established. The combinatorial gluten analyses approach developed during the present study was used to analyze prolamin profiles of wheat transformants expressing DEMETER silencing artificial microRNA, and the results are discussed.
小麦醇溶蛋白，通常被称为“面筋”，是一种由 71-78 种蛋白质组成的复杂混合物，占小麦籽粒中蛋白质的 80%左右，提供了全球饮食蛋白质需求的 50%。醇溶蛋白还与许多由面筋引起的疾病有关，并决定了小麦面团独特的黏弹性。这些特性要求可靠地测定小麦籽粒及其衍生产品中的醇溶蛋白组成。因此，本研究考察了 HPLC 条件（包括柱类型、柱温、流速以及流动相的极性和非极性溶剂梯度）对提高醇溶蛋白分析分辨率的影响。结果发现以下条件最适合分析：柱温 60°C，流速 1.0mL/min，洗脱梯度为 60 分钟内 0.1%三氟乙酸+乙腈的 20%-60%。为了进一步提高分辨率，使用 MALDI-TOF-MS 结合 HPLC 分级分析对麦醇溶蛋白和麦谷蛋白提取物进行分析。使用染色聚丙烯酰胺凝胶的密度测定法和 HPLC 两种半定量方法来确定相对醇溶蛋白的量，并建立了方法之间的对应关系。本研究开发的组合面筋分析方法用于分析表达 DEMETER 沉默人工 microRNA 的小麦转化体的醇溶蛋白图谱，并对结果进行了讨论。
