Tran John C, Wall Mark J, Doucette Alan A
Department of Chemistry, Dalhousie University, 6274 Coburg Road, Halifax, Nova Scotia, Canada B3H 4J3.
J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Mar 15;877(8-9):807-13. doi: 10.1016/j.jchromb.2009.02.024. Epub 2009 Feb 13.
Solution isoelectric focusing (sIEF) is evaluated relative to ion exchange chromatography (IEC) as a preferred charge-based prefractionation tool for proteome mixtures. While IEC is extensively employed for proteome prefractionation prior to MS analysis, we demonstrate here that conventional salt gradient IEC has significant shortcomings compared to sIEF. Here, we critically evaluated a custom eight-channel sIEF device for intact protein separation, relative to strong cation exchange (SCX) and strong anion exchange (SAX) chromatography. The resolution, recovery, and uniformity of separation obtained with our sIEF device were comparable or superior to that of optimized IEC separations. Most importantly for intact proteins, sIEF separations strongly correlate with the proteins' isoelectric point, which contrasts with IEC where no correlation was observed. To validate the sIEF platform for proteome analysis, prefractionation through sIEF resulted in the confident identification of a greater number of proteins from yeast (211) following LC-MS/MS, relative to those obtained through SAX (173) or SCX (148).
将溶液等电聚焦（sIEF）作为蛋白质组混合物基于电荷的首选预分级工具，与离子交换色谱法（IEC）进行了比较评估。虽然IEC在质谱分析之前广泛用于蛋白质组预分级，但我们在此证明，与sIEF相比，传统的盐梯度IEC存在明显缺点。在这里，相对于强阳离子交换（SCX）和强阴离子交换（SAX）色谱法，我们严格评估了一种用于完整蛋白质分离的定制八通道sIEF装置。我们的sIEF装置获得的分离分辨率、回收率和均匀性与优化的IEC分离相当或更优。对于完整蛋白质而言，最重要的是，sIEF分离与蛋白质的等电点密切相关，这与未观察到相关性的IEC形成对比。为了验证sIEF平台用于蛋白质组分析的效果，通过sIEF进行预分级后，与通过SAX（173种）或SCX（148种）获得的蛋白质相比，在液相色谱-串联质谱（LC-MS/MS）分析后，能够更可靠地鉴定出更多来自酵母的蛋白质（211种）。
