Jang You-Ran, Cho Kyoungwon, Kim Sewon, Sim Jae-Ryeong, Lee Su-Bin, Kim Beom-Gi, Gu Yong Q, Altenbach Susan B, Lim Sun-Hyung, Goo Tae-Won, Lee Jong-Yeol
National Institute of Agricultural Sciences, RDA, Jeonju, South Korea.
Department of Biotechnology, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, South Korea.
Front Plant Sci. 2020 Dec 4;11:600489. doi: 10.3389/fpls.2020.600489. eCollection 2020.
The wheat gliadins are a complex group of flour proteins that can trigger celiac disease and serious food allergies. As a result, mutation breeding and biotechnology approaches are being used to develop new wheat lines with reduced immunogenic potential. Key to these efforts is the development of rapid, high-throughput methods that can be used as a first step in selecting lines with altered gliadin contents. In this paper, we optimized matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and reversed-phase high-performance liquid chromatography (RP-HPLC) methods for the separation of gliadins from cv. Chinese Spring (CS). We evaluated the quality of the resulting profiles using the complete set of gliadin gene sequences recently obtained from this cultivar as well as a set of aneuploid lines in CS. The gliadins were resolved into 13 peaks by MALDI-TOF-MS. α- or γ-gliadins that contain abundant celiac disease epitopes and are likely targets for efforts to reduce the immunogenicity of flour were found in several peaks. However, other peaks contained multiple α- and γ-gliadins, including one peak with as many as 12 different gliadins. In comparison, separation of proteins by RP-HPLC yielded 28 gliadin peaks, including 13 peaks containing α-gliadins and eight peaks containing γ-gliadins. While the separation of α- and γ-gliadins gliadins achieved by RP-HPLC was better than that achieved by MALDI-TOF-MS, it was not possible to link peaks with individual protein sequences. Both MALDI-TOF-MS and RP-HPLC provided adequate separation of ω-gliadins. While MALDI-TOF-MS is faster and could prove useful in studies that target specific gliadins, RP-HPLC is an effective method that can be applied more broadly to detect changes in gliadin composition.
小麦醇溶蛋白是一类复杂的面粉蛋白，可引发乳糜泻和严重的食物过敏。因此，人们正在采用诱变育种和生物技术方法来培育具有较低免疫原性潜力的新型小麦品系。这些努力的关键在于开发快速、高通量的方法，可作为筛选醇溶蛋白含量改变品系的第一步。在本文中，我们优化了基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF-MS）和反相高效液相色谱（RP-HPLC）方法，用于从中国春（CS）品种中分离醇溶蛋白。我们使用最近从该品种以及CS的一组非整倍体系中获得的完整醇溶蛋白基因序列，评估了所得图谱的质量。通过MALDI-TOF-MS将醇溶蛋白解析为13个峰。在几个峰中发现了含有丰富乳糜泻表位且可能是降低面粉免疫原性努力目标的α-或γ-醇溶蛋白。然而，其他峰包含多种α-和γ-醇溶蛋白，包括一个含有多达12种不同醇溶蛋白的峰。相比之下，通过RP-HPLC分离蛋白质产生了28个醇溶蛋白峰，包括13个含有α-醇溶蛋白的峰和8个含有γ-醇溶蛋白的峰。虽然RP-HPLC实现的α-和γ-醇溶蛋白的分离优于MALDI-TOF-MS，但无法将峰与单个蛋白质序列联系起来。MALDI-TOF-MS和RP-HPLC都能充分分离ω-醇溶蛋白。虽然MALDI-TOF-MS速度更快，可能在针对特定醇溶蛋白的研究中有用，但RP-HPLC是一种有效的方法，可更广泛地应用于检测醇溶蛋白组成的变化。
