Interactome and interface protocol (2IP): A novel strategy for high sensitivity topology mapping of protein complexes
Interactome and interface protocol (2IP): A novel strategy for high sensitivity topology mapping of protein complexes
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DOI：
10.1002/pmic.200700688
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发表时间：
2007-11
期刊：
影响因子：
3.4
通讯作者：
R. Weerasekera;Y. She;Kelly A. Markham;Yu Bai;N. Opalka;S. Orlicky;F. Sicheri;T. Kislinger;G. Schmitt-Ulms
中科院分区：
文献类型：
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作者：
R. Weerasekera;Y. She;Kelly A. Markham;Yu Bai;N. Opalka;S. Orlicky;F. Sicheri;T. Kislinger;G. Schmitt-Ulms
除了一些特征良好的蛋白质组合外，人们对多组分蛋白质复合体的拓扑结构和界面知之甚少。在这里，我们报告了一种新的间接策略，用于蛋白质复合体的低分辨率拓扑映射。在交联后，纯化的蛋白质复合体用溴化氰(CNBr)进行化学切割，得到的片段通过二维电泳法进行分解。这样产生的CNBR片段图与从未交联物获得的2-D CNBR片段图的并排比较揭示了含有交联型CNBR片段的候选凝胶点。对这些信息点的凝胶内胰酶作用和MALDI MS分析确定了基于未经修饰的胰蛋白酶多肽的潜在的交联型CNBR片段。将这些交联型CNBR片段的累积理论分子质量和预测等电点与原始凝胶点坐标进行匹配，才能确定交联度。上述策略被成功地用大肠杆菌RNA聚合酶(RNAP)核心复合体验证，并随后应用于查询酵母Skp1-CDc53/Cullin-F box(SCF)泛素连接酶复合体的四级结构。该方法所需样本量小，可应用于多亚单位蛋白质复合体，且不依赖于专门的数据挖掘软件。
A few well‐characterized protein assemblies aside, little is known about the topology and interfaces of multiconstituent protein complexes. Here we report on a novel indirect strategy for low‐resolution topology mapping of protein complexes. Following crosslinking, purified protein complexes are subjected to chemical cleavage with cyanogen bromide (CNBr) and the resulting fragments are resolved by 2‐D electrophoresis. The side‐by‐side comparison of a thus generated and a 2‐D CNBr fragment map obtained from uncrosslinked material reveals candidate gel spots harboring crosslinked CNBr fragments. In‐gel trypsinization and MALDI MS analysis of these informative spots identify the underlying crosslinked CNBr fragments based on unmodified tryptic peptides. Matching the cumulative theoretical molecular mass and predicted pI of these crosslinked CNBr fragments with original gel spot coordinates is required for confident crosslink assignment. The above strategy was successfully validated with the Escherichia coli RNA polymerase (RNAP) core complex and subsequently applied to query the quaternary structure of components of the yeast Skp1‐Cdc53/Cullin‐F box (SCF) ubiquitin ligase complex. This protocol requires low picomole sample quantities, can be applied to multisubunit protein complexes, and does not rely on specialized data mining software.