USDA-ARS Western Regional Research Center, U.S. Department of Agriculture-Agricultural Research Service, 800 Buchanan Street, Albany, CA 94710, USA.
BMC Plant Biol. 2010 Jan 11;10:7. doi: 10.1186/1471-2229-10-7.
The gamma gliadins are a complex group of proteins that together with other gluten proteins determine the functional properties of wheat flour. The proteins have unusually high levels of glutamine and proline and contain large regions of repetitive sequences. While most gamma gliadins are monomeric proteins containing eight conserved cysteine residues, some contain an additional cysteine residue that enables them to be linked with other gluten proteins into large polymers that are critical for flour quality. The ability to differentiate among the gamma gliadins is important for studies of wheat flour quality because proteins with similar sequences can have different effects on functional properties.
The complement of gamma gliadin genes expressed in the wheat cultivar Butte 86 was evaluated by analyzing publicly available expressed sequence tag (EST) data. Eleven contigs were assembled from 153 Butte 86 ESTs. Nine of the contigs encoded full-length proteins and four of the proteins contained nine cysteine residues. Only one of the encoded proteins was a perfect match with a sequence reported in NCBI. Contigs from four different publicly available EST assemblies encoded proteins that were perfect matches with some, but not all, of the Butte 86 gamma gliadins and the complement of identical proteins was different for each assembly. A specialized database that included the sequences of Butte 86 gamma gliadins was constructed for identification of flour proteins by tandem mass spectrometry (MS/MS). In a pilot experiment, proteins corresponding to six Butte 86 gamma gliadin contigs were distinguished by MS/MS, including one containing the extra cysteine residue. Two other proteins were identified as one of two closely related Butte 86 proteins but could not be distinguished unequivocally. Unique peptide tags specific for Butte 86 gamma gliadins are reported.
Inclusion of cultivar-specific gamma gliadin sequences in databases maximizes the number and quality of peptide identifications and increases sequence coverage of these gamma gliadins by MS/MS. This approach makes it possible to distinguish closely related proteins, to associate individual proteins with sequences of specific genes, and to evaluate proteomic data in a biological context to better address questions about wheat flour quality.
γ-麦谷蛋白是一组复杂的蛋白质，它们与其他面筋蛋白一起决定了小麦粉的功能特性。这些蛋白质含有异常高的谷氨酰胺和脯氨酸水平，并含有大量重复序列的区域。虽然大多数 γ-麦谷蛋白是含有 8 个保守半胱氨酸残基的单体蛋白，但有些蛋白含有额外的半胱氨酸残基，使其能够与其他面筋蛋白连接成大聚合物，这些聚合物对面粉质量至关重要。区分 γ-麦谷蛋白的能力对于研究小麦粉质量非常重要，因为具有相似序列的蛋白质可能对面筋功能特性有不同的影响。
通过分析公共表达序列标签 (EST) 数据，评估了小麦品种 Butte 86 中表达的 γ-麦谷蛋白基因的组成。从 153 个 Butte 86 EST 中组装了 11 个 contigs。9 个 contigs 编码全长蛋白，其中 4 个蛋白含有 9 个半胱氨酸残基。只有一个编码的蛋白与 NCBI 中报道的序列完全匹配。来自四个不同公共 EST 组装的 contigs 编码的蛋白与 Butte 86 γ-麦谷蛋白的一些而非全部完全匹配，并且每个组装的相同蛋白的互补物都不同。为了通过串联质谱 (MS/MS) 鉴定面粉蛋白，构建了一个包含 Butte 86 γ-麦谷蛋白序列的专用数据库。在一个试点实验中，通过 MS/MS 区分了六个 Butte 86 γ-麦谷蛋白 contigs 对应的蛋白，包括一个含有额外半胱氨酸残基的蛋白。另外两个蛋白被鉴定为两个密切相关的 Butte 86 蛋白之一，但不能明确区分。报道了特定于 Butte 86 γ-麦谷蛋白的独特肽标签。
在数据库中包含品种特异性 γ-麦谷蛋白序列可最大限度地增加肽鉴定的数量和质量，并通过 MS/MS 增加这些 γ-麦谷蛋白的序列覆盖率。这种方法使得区分密切相关的蛋白、将单个蛋白与特定基因的序列相关联以及在生物学背景下评估蛋白质组学数据成为可能，从而更好地解决关于小麦粉质量的问题。
