Three-layer sandwich gel electrophoresis: a method of salt removal and protein concentration in proteome analysis.
Three-layer sandwich gel electrophoresis: a method of salt removal and protein concentration in proteome analysis.
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DOI：
10.1021/pr800182b
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发表时间：
2008-09
影响因子：
4.4
通讯作者：
Ting Liu;Angela M Martin;A. Sinai;B. Lynn
中科院分区：
文献类型：
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作者：
Ting Liu;Angela M Martin;A. Sinai;B. Lynn
样品制备在成功的蛋白质组学应用中起着关键作用。电喷雾质谱法的特征对可用于样品制备的缓冲液、洗涤剂和其他试剂的类型施加了限制。不幸的是，许多这些质谱不相容的试剂显着提高蛋白质从复杂的矩阵回收。这个问题促使我们寻找更好的清除协议。我们的数据表明，三层夹心凝胶电泳（TSGE）协议可以解决这个问题，并提供从苛刻的解决方案，一个功能，从其他清洁协议不可用的极低浓度的蛋白质的近定量回收。TSGE方案的标志是琼脂糖凝胶（用作基质以包埋感兴趣的蛋白质）与低百分比和高百分比聚丙烯酰胺凝胶（分别用作浓缩层和密封层）的性质的组合。通过将目标蛋白质从琼脂糖基质中电泳驱动到浓缩层中，TSGE方案同时将样品浓缩在浓缩层中，并提供适合下游缓冲液交换和蛋白水解消化的环境。结合2D-LC-MS/MS，TSGE协议进行了评估，在分析从原生动物寄生虫弓形虫的全细胞提取物。我们的实验蛋白质组学结果与基因数据的计算机预测的比较表明，TSGE没有偏见的蛋白质鉴定。
Sample preparation plays a critical role in successful proteomic applications. Features of electrospray mass spectrometry impose limits on the types of buffers, detergents and other reagents that can be used in sample preparation. Unfortunately, many of these mass spectrometry incompatible reagents significantly enhance protein recoveries from complex matrices. This problem prompted our search for a better cleanup protocol. Our data suggest that the Three-layer Sandwich Gel Electrophoresis (TSGE) protocol can solve this problem and provide near quantitative recovery of extremely low concentration proteins from harsh solutions, a feature not available from other cleanup protocols. The hallmark of the TSGE protocol is the combination of the properties of agarose gels (that serve as the matrix to immobilize the proteins of interest) with low- and high-percentage polyacrylamide gels (that serve as the concentration and sealing layers, respectively). By electrophoretically driving the proteins of interest from the agarose matrix into the concentration layer, the TSGE protocol simultaneously concentrates the sample in the concentration layer and provides an environment amenable to downstream buffer exchange and proteolytic digestion. In combination with 2D-LC-MS/MS, the TSGE protocol was evaluated in the analysis of a whole cell extract from the protozoan parasite Toxoplasma gondii. Comparison of our experimental proteomic results with in silico predictions from gene data indicated that TSGE did not bias the protein identification.