A reassessment of the electrophoretic mobility of high molecular weight glutenin subunits of wheat
A reassessment of the electrophoretic mobility of high molecular weight glutenin subunits of wheat
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DOI：
10.1016/j.jcs.2012.08.003
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发表时间：
2012-11-01
影响因子：
3.8
通讯作者：
Koehler, Peter
中科院分区：
文献类型：
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作者：
Lagrain, Bert;Rombouts, Ine;Koehler, Peter
小麦高分子量谷蛋白亚基(HMW-GS)对面包品质有重要影响。它们的组成通常是在谷蛋白二硫键还原后用Tris-甘氨酸SDS-PAGE鉴定的。然而，它们的分子量和一级结构之间的关系，以及它们在Tris-甘氨酸SDS-PAGE中的迁移率之间的关系已经被证明是模糊的。我们演示了一种在凝胶和运行缓冲液中使用中性而不是碱性的pH的Bis-Tris SDS-PAGE程序。在这种方法中，普遍存在的HMW-GS以5>；2顺序迁移到3>；1>；6，近似于2*>；7>；8>；12>；10，这与在Tris-甘氨酸系统中得到的顺序不同。用反相高效液相色谱法分离HMW-GS，N端测序鉴定HMW-GS。将MS数据与蛋白质数据库中的氨基酸序列进行比对，对凝乳酶多肽进行LC-MS/MS分析，进一步证实HMW-GS的蛋白质序列。HMW-GS的氨基酸数目较好地反映了Bis-Tris SOS-PAGE的迁移率顺序。结果表明，Bis-Tris SDS-PAGE不仅可以用来鉴定HMW-GS，而且可以用来估计其多肽链的长度，从而避免了以前观察到的迁移顺序的异常。(C)2012爱思唯尔有限公司。保留所有权利。
The high molecular weight subunits of wheat glutenin (HMW-GS) are important for bread-making quality. Their composition is routinely identified by Tris-glycine SDS-PAGE after reduction of glutenin disulfide bonds. However, the relation between their molecular weight and, hence, their primary structure, and their mobility in Tris-glycine SDS-PAGE has proven to be ambiguous. We demonstrate a Bis-Tris SDS-PAGE procedure with a neutral, instead of alkaline, pH in the gel and running buffers. In this method commonly occurring HMW-GS from wheat migrated in the order 5 > 2 approximate to 3 > 1 > 6 approximate to 2* > 7 > 8 > 9 > 12 > 10, which is different from the order obtained in the Tris-glycine system. HMW-GS were identified by N-terminal sequencing after isolation with RP-HPLC. Protein sequences of HMW-GS were further confirmed by LC-MS/MS analyses of chymotryptic peptides after comparing the MS data to amino acid sequences in protein databases. The numbers of amino acids of HMW-GS reflected well the mobility order in Bis-Tris SOS-PAGE. The results indicate that Bis-Tris SDS-PAGE may not only be used to identify HMW-GS, but also to estimate the length of their polypeptide chain, as such avoiding previously observed anomalies in migration order. (c) 2012 Elsevier Ltd. All rights reserved.