McCarthy Fiona M, Burgess Shane C, van den Berg Bart H J, Koter Marek D, Pharr G Todd
College of Veterinary Medicine, PO Box 6100, Mississippi State University, Mississippi 39762, USA.
J Proteome Res. 2005 Mar-Apr;4(2):316-24. doi: 10.1021/pr049842d.
Differential detergent fractionation (DDF), which relies on detergents to sequentially extract proteins from eukaryotic cells, has been used to increase proteome coverage of 2D-PAGE. Here, we used DDF extraction in conjunction with the nonelectrophoretic proteomics method of liquid chromatography and electrospray ionization tandem mass spectrometry. We demonstrate that DDF can be used with 2D-LC ESI MS2 for comprehensive cellular proteomics, including a large proportion of membrane proteins. Compared to some published methods designed to isolate membrane proteins specifically, DDF extraction yields comprehensive proteomes which include twice as many membrane proteins. Two-thirds of these membrane proteins have more than one trans-membrane domain. Since DDF separates proteins based upon their physicochemistry and subcellular localization, this method also provides data useful for functional genome annotation. As more genome sequences are completed, methods which can aid in functional annotation will become increasingly important.
差异去污剂分级分离（DDF）依赖去污剂从真核细胞中顺序提取蛋白质，已被用于增加二维聚丙烯酰胺凝胶电泳（2D-PAGE）的蛋白质组覆盖率。在此，我们将DDF提取与液相色谱和电喷雾电离串联质谱的非电泳蛋白质组学方法结合使用。我们证明DDF可与二维液相色谱电喷雾电离串联质谱（2D-LC ESI MS2）一起用于全面的细胞蛋白质组学研究，包括很大比例的膜蛋白。与一些专门设计用于分离膜蛋白的已发表方法相比，DDF提取产生的综合蛋白质组包含的膜蛋白数量是其两倍。这些膜蛋白中有三分之二具有不止一个跨膜结构域。由于DDF根据蛋白质的物理化学性质和亚细胞定位进行分离，该方法还提供了有助于功能基因组注释的数据。随着越来越多的基因组序列完成，有助于功能注释的方法将变得越来越重要。
