Table_3_Investigation of Protein and Epitope Characteristics of Oats and Its Implications for Celiac Disease.XLSX

The use of pure oats (oats cultivated with special care to avoid gluten contamination from wheat, rye, and barley) in the gluten-free diet (GFD) represents important nutritional benefits for the celiac consumer. However, emerging evidence suggests that some oat cultivars may contain wheat gliadin analog polypeptides. Consequently, it is necessary to screen oats in terms of protein and epitope composition to be able to select safe varieties for gluten-free applications. The overall aim of our study is to investigate the variability of oat protein composition directly related to health-related and techno-functional properties. Elements of an oat sample population representing 162 cultivated varieties from 20 countries and the protein composition of resulting samples have been characterized. Size distribution of the total protein extracts has been analyzed by size exclusion-high performance liquid chromatography (SE-HPLC) while the 70% ethanol-extracted proteins were analyzed by RP-HPLC. Protein extracts separated into three main groups of fractions on the SE-HPLC column: polymeric proteins, avenins (both containing three subgroups based on their size), and soluble proteins, representing respectively 68.79–86.60, 8.86–27.72, and 2.89–11.85% of the total protein content. The ratio of polymeric to monomeric proteins varied between 1.37 and 3.73. Seventy-six reversed phase-HPLC-separated peaks have been differentiated from the ethanol extractable proteins of the entire population. Their distribution among the cultivars varied significantly, 6–23 peaks per cultivar. The number of appearances of peaks also showed large variation: one peak has been found in 107 samples, while 15 peaks have been identified, which appeared in less than five cultivars. An estimation method for ranking the avenin-epitope content of the samples has been developed by using MS spectrometric data of collected RP-HPLC peaks and bioinformatics methods. Using ELISA methodology with the R5 antibody, a high number of the investigated samples were found to be contaminated with wheat, barley, or rye.

在无麸质饮食（gluten-free diet, GFD）中使用纯燕麦——即经特殊种植管控以规避小麦、黑麦与大麦带来的麸质交叉污染的燕麦——可为乳糜泻患者带来重要的营养益处。然而，新近研究证据表明，部分燕麦品种可能含有小麦麦醇溶蛋白类似多肽。因此，有必要从蛋白质与表位组成维度对燕麦进行筛选，以筛选出适用于无麸质应用的安全品种。
本研究的总体目标是探究与健康相关及技术功能特性直接相关的燕麦蛋白质组成的变异性。研究对来自20个国家的162个栽培品种组成的燕麦样本群体，以及对应样本的蛋白质组成进行了表征。采用体积排阻高效液相色谱（size exclusion-high performance liquid chromatography, SE-HPLC）分析总蛋白质提取物的粒径分布，同时采用反相高效液相色谱（reverse phase-high performance liquid chromatography, RP-HPLC）分析70%乙醇提取的蛋白质组分。经SE-HPLC色谱柱分离后，蛋白质提取物可分为三大主要组分：聚合蛋白、燕麦醇溶蛋白（按粒径可划分为三个亚组）以及可溶性蛋白，三者分别占总蛋白质含量的68.79%~86.60%、8.86%~27.72%与2.89%~11.85%。聚合蛋白与单体蛋白的比值范围为1.37~3.73。
从全部样本群体的乙醇可提取蛋白质中，共分辨出76个反相高效液相色谱峰。这些峰在不同燕麦品种间的分布差异显著，每个品种可检测到6~23个峰。各峰的出现频次同样存在较大差异：其中1个峰在107个样本中被检出，另有15个峰仅在不足5个品种中被发现。通过采集RP-HPLC峰的质谱（Mass Spectrometry, MS）数据并结合生物信息学方法，开发了一种用于评估样品燕麦醇溶蛋白表位含量的排序方法。采用结合R5抗体的酶联免疫吸附测定（enzyme-linked immunosorbent assay, ELISA）方法对大量受试样本进行检测后发现，诸多样本存在小麦、黑麦或大麦污染情况。