Timperio Anna Maria, D'Amici Gian Maria, Zolla Lello
Department of Environmental Sciences, University of Tuscia, Largo dell'Università snc, Viterbo, Italy.
Methods Mol Biol. 2012;909:43-62. doi: 10.1007/978-1-61779-959-4_4.
The present chapter describes methods for the separation and identification of proteins in liver metabolism through a comparison of the protein expression profiles of the two breeds taken into account as a model: Holstein Friesian and Chianina cattle. The liver has received special attention, containing as it does, enzymes involved in energy generation, carbohydrate, lipid, amino acid, and xenobiotic metabolism, as well as proteins involved in polypeptide synthesis, folding, and cell structure. The first step in the procedure is the preparation of purified protein fractions from liver tissues, followed by sample preparation for 2-DE analysis in order to identify proteins which could be differentially expressed in the livers of the two breeds and relate them to different liver functions. Data can be then statistically elaborated with cluster analysis, which stressed the up-/on-regulation trend of these proteins. Quantitative data can be used to perform a two-way hierarchical cluster analysis of the 39 differentially expressed protein spots, either up- or on-regulated in Chianina versus Holstein Friesian liver samples. Thus, spots from 2-DE maps can be carefully excised from the gel and subjected to in-gel trypsin digestion and analyzed by tandem mass spectrometry in their contents.
本章介绍了通过比较荷斯坦弗里生牛和契安尼纳牛这两个作为模型的品种的蛋白质表达谱，来分离和鉴定肝脏代谢中蛋白质的方法。肝脏受到了特别关注，因为它含有参与能量生成、碳水化合物、脂质、氨基酸和外源性物质代谢的酶，以及参与多肽合成、折叠和细胞结构的蛋白质。该程序的第一步是从肝脏组织中制备纯化的蛋白质组分，然后进行二维电泳（2-DE）分析的样品制备，以鉴定可能在两个品种的肝脏中差异表达的蛋白质，并将它们与不同的肝脏功能联系起来。然后可以用聚类分析对数据进行统计处理，聚类分析强调了这些蛋白质的上调/下调趋势。定量数据可用于对契安尼纳牛与荷斯坦弗里生牛肝脏样品中上调或下调的39个差异表达蛋白点进行双向层次聚类分析。因此，可以从二维电泳图谱中小心地切下斑点，进行胶内胰蛋白酶消化，并对其内容物进行串联质谱分析。
