Characterization of wheat gliadin proteins by combined two-dimensional gel electrophoresis and tandem mass spectrometry
Characterization of wheat gliadin proteins by combined two-dimensional gel electrophoresis and tandem mass spectrometry
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DOI：
10.1002/pmic.200401168
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发表时间：
2005-07-01
期刊：
影响因子：
3.4
通讯作者：
Ferranti, P
中科院分区：
文献类型：
--
作者：
Mamone, G;Addeo, F;Ferranti, P
一种基于蛋白质组学的方法被用来表征小麦醇溶蛋白从意大利普通小麦（小麦）品种。通过将70%醇溶性粗蛋白提取物在两个pH梯度范围内的固定化pH梯度条上进行等电聚焦，获得大约40个点的二维凝胶电泳（2-DE）图谱，即，3-10或pH 6-11，并在第二维中进行十二烷基硫酸钠-聚丙烯酰胺电泳。通过基质辅助激光解吸/电离-飞行时间质谱和纳米电喷雾电离-串联质谱（MS/MS）分析表征每个斑点的糜蛋白酶消化物，为每个消化物提供“肽图”。随后在序列数据库中查找测量的质量。为了准确鉴定亲本蛋白，有必要通过对肽的MS/MS实验来确定从头序列。通过部分质量指纹，我们确定了蛋白质分子，如α/β-，γ-，ω-麦醇溶蛋白，和高分子量麦谷蛋白。沿着2-DE图谱上的单个点根据其氨基酸序列特征进行区分。α-醇溶蛋白是数据库中最具代表性的小麦蛋白质，其N-末端相对序列具有高度的保守性，在2-DE图谱中仅含有少量的沉默氨基酸替换。其他密切相关的醇溶蛋白通过测序内部肽链来鉴定。这些结果揭示了醇溶蛋白异质性的复杂本质。该方法为小麦品种间醇溶蛋白的鉴别提供了可靠的参考数据。
A proteomics-based approach was used for characterizing wheat gliadins from an Italian common wheat (Triticum aestivum) cultivar. A two-dimensional gel electrophoresis (2-DE) map of roughly 40 spots was obtained by submitting the 70 % alcohol-soluble crude protein extract to isoelectric focusing on immobilized pH gradient strips across two pH gradient ranges, i.e., 3-10 or pH 6-11, and to sodium dodecyl sulfate-polyacrylamide electrophoresis in the second dimension. The chymotryptic digest of each spot was characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and nano electrospray ionization-tandem mass spectrometry (MS/MS) analysis, providing a "peptide map" for each digest. The measured masses were subsequently sought in databases for sequences. For accurate identification of the parent protein, it was necessary to determine de novo sequences by MS/MS experiments on the peptides. By partial mass fingerprinting, we identified protein molecules such as alpha/beta-, gamma-, omega-gliadin, and high molecular weight-glutenin. The single spots along the 2-DE map were discriminated on the basis of their amino acid sequence traits. a-Gliadin, the most represented wheat protein in databases, was highly conserved as the relative N-terminal sequence of the components from the 2-DE map contained only a few silent amino acid substitutions. The other closely related gliadins were identified by sequencing internal peptide chains. The results gave insight into the complex nature of gliadin heterogeneity. This approach has provided us with sound reference data for differentiating gliadins amongst wheat varieties.