Semiquantitative Proteomic Analysis of the Human Spliceosome via a Novel Two-Dimensional Gel Electrophoresis Method
Semiquantitative Proteomic Analysis of the Human Spliceosome via a Novel Two-Dimensional Gel Electrophoresis Method
复制标题
DOI：
10.1128/mcb.05266-11
复制
发表时间：
2011-07-01
影响因子：
5.3
通讯作者：
Luehrmann, Reinhard
中科院分区：
文献类型：
--
作者：
Agafonov, Dmitry E.;Deckert, Jochen;Luehrmann, Reinhard
超过200种蛋白质与人类剪接体相关，但对它们在特定剪接体复合物中的相对丰度知之甚少。在这里，我们描述了一种新的二维（2D）电泳方法，允许分离高分子量的蛋白质，而没有凝胶内沉淀，因此没有蛋白质的损失。使用这个系统加上质谱，我们确定了171个蛋白质共2D地图上的阶段特异性spliceosomal复合物。通过用具有宽线性强度范围的荧光染料染色，我们可以将蛋白质定量和分类为高、中或低丰度。亲和纯化的人B、B-act和C复合物分别含有69、63和72种高/中丰度蛋白质。剪接体蛋白的募集和释放是根据它们在A、B、B-act和C剪接体复合物中的丰度来进行的。用磷酸特异性染料染色显示，通过2D凝胶分析在人剪接体复合物中检测到的蛋白质中约有三分之一被磷酸化。这里描述的2D凝胶电泳系统允许第一次的相对丰度的蛋白质存在于一个特定的spliceosomal复合物的客观看法，也揭示了额外的光spliceosomal蛋白质的组成动态和磷酸化状态在特定阶段的剪接。
More than 200 proteins associate with human spliceosomes, but little is known about their relative abundances in a given spliceosomal complex. Here we describe a novel two-dimensional (2D) electrophoresis method that allows separation of high-molecular-mass proteins without in-gel precipitation and thus without loss of protein. Using this system coupled with mass spectrometry, we identified 171 proteins altogether on 2D maps of stage-specific spliceosomal complexes. By staining with a fluorescent dye with a wide linear intensity range, we could quantitate and categorize proteins as present in high, moderate, or low abundance. Affinity-purified human B, B-act, and C complexes contained 69, 63, and 72 highly/moderately abundant proteins, respectively. The recruitment and release of spliceosomal proteins were followed based on their abundances in A, B, B-act, and C spliceosomal complexes. Staining with a phospho-specific dye revealed that approximately one-third of the proteins detected in human spliceosomal complexes by 2D gel analyses are phosphorylated. The 2D gel electrophoresis system described here allows for the first time an objective view of the relative abundances of proteins present in a particular spliceosomal complex and also sheds additional light on the spliceosome's compositional dynamics and the phosphorylation status of spliceosomal proteins at specific stages of splicing.