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Abstract：
The in-depth analysis of complex proteome samples requires fractionation of the sample into subsamples prior to LC-MS/MS in shotgun proteomics experiments. We have established a 3D workflow for shotgun proteomics that relies on protein separation by 1D PAGE, gel fractionation, trypsin digestion, and peptide separation by in-gel IEF, prior to RP-HPLC-MS/MS. Our results show that applying peptide IEF can significantly increase the number of proteins identified from PAGE subfractionation. This method delivers deeper proteome coverage and provides a large degree of flexibility in experimentally approaching highly complex mixtures by still relying on protein separation according to molecular weight in the first dimension.
摘要：
复杂蛋白质组样品的深入分析需要在shot弹枪蛋白质组学实验中的LC-MS/MS之前将样品分级分离为子样品。我们已经建立了shot弹枪蛋白质组学的3D工作流程，该流程依赖于通过1DPAGE进行蛋白质分离，凝胶分馏,胰蛋白酶消化，和通过凝胶内IEF分离肽，在RP-HPLC-MS/MS之前我们的结果表明，应用IEF肽可以显着增加从PAGE亚分级分离鉴定的蛋白质数量。该方法提供了更深的蛋白质组覆盖，并通过仍然依赖于根据第一维分子量的蛋白质分离，在实验上接近高度复杂的混合物时提供了很大程度的灵活性。
