Lexhaller Barbara, Colgrave Michelle L, Scherf Katharina A
Leibniz-Institute for Food Systems Biology at the Technical University of Munich, Freising, Germany.
CSIRO Agriculture and Food, St Lucia, QLD, Australia.
Front Plant Sci. 2019 Dec 13;10:1530. doi: 10.3389/fpls.2019.01530. eCollection 2019.
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 α-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): α-gliadins, γ-gliadins, ω5-gliadins, ω1,2-gliadins, high- and low-molecular-weight glutenin subunits of wheat, ω-secalins, high-molecular-weight secalins, γ-75k-secalins and γ-40k-secalins of rye, and C-hordeins, γ-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.
食用小麦、黑麦和大麦可能会引发对小麦的不良反应，如乳糜泻、非乳糜泻性麸质/小麦敏感或小麦过敏。储存蛋白（麸质）被认为是主要诱因，但其他功能性蛋白组，如α-淀粉酶/胰蛋白酶抑制剂或酶，对患有小麦不良反应的人也可能有害。麸质被广泛用作小麦、黑麦或大麦复杂蛋白质混合物的统称，可细分为以下麸质蛋白类型（GPTs）：α-醇溶蛋白、γ-醇溶蛋白、ω5-醇溶蛋白、ω1,2-醇溶蛋白、小麦的高分子量和低分子量谷蛋白亚基、ω-黑麦碱、高分子量黑麦碱、黑麦的γ-75k-黑麦碱和γ-40k-黑麦碱，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。从面粉中分离出的GPTs可作为临床研究、诊断或食品分析的参考材料，并有助于阐明疾病机制。采用先根据溶解度进行蛋白质分离，再进行制备型反相高效液相色谱的联合策略，根据疏水性纯化GPTs。由于麸质蛋白的异质性及其部分聚合性质，仅获得单一蛋白组的高度纯化GPTs具有挑战性。因此，表征和鉴定每种GPT中的蛋白质及其比例至关重要。在本研究中，通过采用涉及GPTs胰蛋白酶和糜蛋白酶水解产物的液相色谱-串联质谱的无导向蛋白质组学策略鉴定单个蛋白质，证明了小麦、黑麦和大麦麸质的复杂性。获得了不同的蛋白组，并揭示了GPTs的相对组成。采用多反应监测液相色谱-串联质谱对最丰富的麸质蛋白进行相对定量。这些分析还能够鉴定已知的小麦过敏原和乳糜泻活性肽。结合功能测定，这些发现可能有助于阐明麸质/小麦相关疾病的机制，并可能有助于更精确地表征分析或诊断测定的参考材料。
