Westbrook J A, Yan J X, Wait R, Welson S Y, Dunn M J
National Heart & Lung Institute, Imperial College School of Medicine, Heart Science Centre, Harefield Hospital, Middlesex, UK.
Electrophoresis. 2001 Aug;22(14):2865-71. doi: 10.1002/1522-2683(200108)22:14<2865::AID-ELPS2865>3.0.CO;2-Y.
Two-dimensional gel electrophoresis (2-DE) enables separation of complex mixtures of proteins on a single polyacrylamide gel according to isoelectric point, molecular weight, solubility, and relative abundance. For this reason, 2-DE together with mass spectrometry (MS) has become a key technology in proteome analysis. The introduction of immobilised pH gradients (IPGs) for isoelectric focusing of proteins affords improved reproducibility and permits full-scale proteome analyses to be undertaken. Whilst broad-range IPGs are useful for investigating simple proteomes (e.g. Mycoplasma genitalium) it is becoming clear that additional resolving power is needed for separating the more complex proteomes of eukaryotic organisms. The use of narrow-range and very narrow-range IPGs provides the means with which to dissect a complex proteome. We have compared very narrow-range IPGs (3.5-4.5L, 4-5L, 4.5-5.5L, 5-6L, and 5.5-6.7L) with broad- (3-10NL) and narrow-range IPGs (4-7L and 6-9L) for the visualisation of the human heart proteome. The superior ability of very narrow-range IPGs to separate different protein species and isoforms, compared with 3-10NL and 4-7L 2-D gels is demonstrated. The results are supported by MS identifications which further show that reduction of the number of comigrating protein species results in less ambiguous and more reliable database search results.
二维凝胶电泳（2-DE）能够根据等电点、分子量、溶解度和相对丰度，在单一聚丙烯酰胺凝胶上分离复杂的蛋白质混合物。因此，2-DE与质谱（MS）一起已成为蛋白质组分析中的关键技术。用于蛋白质等电聚焦的固定化pH梯度（IPG）的引入提高了重现性，并允许进行全面的蛋白质组分析。虽然宽范围的IPG对于研究简单蛋白质组（如生殖支原体）很有用，但越来越明显的是，分离真核生物更复杂的蛋白质组需要额外的分辨率。使用窄范围和极窄范围的IPG提供了剖析复杂蛋白质组的方法。我们将极窄范围的IPG（3.5-4.5L、4-5L、4.5-5.5L、5-6L和5.5-6.7L）与宽范围（3-10NL）和窄范围的IPG（4-7L和6-9L）进行了比较，以可视化人类心脏蛋白质组。结果表明，与3-10NL和4-7L二维凝胶相比，极窄范围的IPG在分离不同蛋白质种类和同工型方面具有更强的能力。质谱鉴定结果支持了这些结果，进一步表明共迁移蛋白质种类数量的减少导致数据库搜索结果的歧义性降低且更可靠。
