Characterization and Relative Quantitation of Wheat, Rye, and Barley Gluten Protein Types by Liquid Chromatography-Tandem Mass Spectrometry
Characterization and Relative Quantitation of Wheat, Rye, and Barley Gluten Protein Types by Liquid Chromatography-Tandem Mass Spectrometry
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DOI：
10.3389/fpls.2019.01530
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发表时间：
2019-12-13
影响因子：
5.6
通讯作者：
Scherf, Katharina A.
中科院分区：
文献类型：
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作者：
Lexhaller, Barbara;Colgrave, Michelle L.;Scherf, Katharina A.
小麦、黑麦和大麦的消费可能导致对小麦的不良反应，如乳糜泻、非乳糜泻面筋/小麦敏感性或小麦过敏。储藏蛋白质（麸质）被认为是主要的触发因素，但其他功能性蛋白质组，如α-淀粉酶/胰蛋白酶抑制剂或酶可能对遭受小麦不良反应的人有害。面筋被广泛用作小麦、黑麦或大麦的复杂蛋白质混合物的集合术语，并且可以细分为以下面筋蛋白类型（GPT）：α-麦醇溶蛋白、γ-麦醇溶蛋白、ω-5-麦醇溶蛋白、ω-1，2-麦醇溶蛋白、小麦的高分子量和低分子量麦谷蛋白亚基、黑麦的ω-黑麦蛋白、高分子量黑麦蛋白、γ-75k-黑麦蛋白和γ-40k-黑麦蛋白，以及大麦的C-大麦醇溶蛋白、γ-大麦醇溶蛋白、B-大麦醇溶蛋白和D-大麦醇溶蛋白。从面粉中分离的GPT可用作临床研究、诊断或食品分析的参考材料，并可用于阐明疾病机制。一个组合的策略，蛋白质分离，根据溶解度，然后制备反相高效液相色谱纯化的GPT根据疏水性。由于谷蛋白的异质性和它们的部分聚合性质，获得仅具有一个蛋白质基团的高度纯化的GPT是一个挑战。因此，必须表征和鉴定蛋白质及其在每个GPT中的比例。在这项研究中，从小麦，黑麦和大麦面筋的复杂性，证明了通过识别的个别蛋白质，采用一种无方向的蛋白质组学策略，涉及液相色谱-串联质谱的胰蛋白酶和胰凝乳蛋白酶水解产物的GPT。获得不同的蛋白质组，并揭示了GPT的相对组成。多反应监测液相色谱-串联质谱法用于相对定量的最丰富的面筋蛋白。这些分析还允许识别已知的小麦过敏原和乳糜泻活性肽。结合功能测定，这些研究结果可能揭示面筋/小麦相关疾病的机制，并可能是有用的，更精确地表征分析或诊断测定的参考材料。
The consumption of wheat, rye, and barley may cause adverse reactions to wheat such as celiac disease, non-celiac gluten/wheat sensitivity, or wheat allergy. The storage proteins (gluten) are known as major triggers, but also other functional protein groups such as a-amylase/trypsin-inhibitors or enzymes are possibly harmful for people suffering of adverse reactions to wheat. Gluten is widely used as a collective term for the complex protein mixture of wheat, rye or barley and can be subdivided into the following gluten protein types (GPTs): alpha-gliadins, gamma-gliadins, omega 5-gliadins, omega 1,2-gliadins, high- and low-molecular-weight glutenin subunits of wheat, omega-secalins, high-molecular-weight secalins, gamma-75k-secalins and gamma-40k-secalins of rye, and C-hordeins, gamma-hordeins, B-hordeins, and D-hordeins of barley. GPTs isolated from the flours are useful as reference materials for clinical studies, diagnostics or in food analyses and to elucidate disease mechanisms. A combined strategy of protein separation according to solubility followed by preparative reversed-phase high-performance liquid chromatography was employed to purify the GPTs according to hydrophobicity. Due to the heterogeneity of gluten proteins and their partly polymeric nature, it is a challenge to obtain highly purified GPTs with only one protein group. Therefore, it is essential to characterize and identify the proteins and their proportions in each GPT. In this study, the complexity of gluten from wheat, rye, and barley was demonstrated by identification of the individual proteins employing an undirected proteomics strategy involving liquid chromatography-tandem mass spectrometry of tryptic and chymotryptic hydrolysates of the GPTs. Different protein groups were obtained and the relative composition of the GPTs was revealed. Multiple reaction monitoring liquid chromatography-tandem mass spectrometry was used for the relative quantitation of the most abundant gluten proteins. These analyses also allowed the identification of known wheat allergens and celiac disease-active peptides. Combined with functional assays, these findings may shed light on the mechanisms of gluten/wheat-related disorders and may be useful to characterize reference materials for analytical or diagnostic assays more precisely.