Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.
J Cell Biol. 2010 May 17;189(4):739-54. doi: 10.1083/jcb.200911091.
Protein interactions are involved in all cellular processes. Their efficient and reliable characterization is therefore essential for understanding biological mechanisms. In this study, we show that combining bacterial artificial chromosome (BAC) TransgeneOmics with quantitative interaction proteomics, which we call quantitative BAC-green fluorescent protein interactomics (QUBIC), allows specific and highly sensitive detection of interactions using rapid, generic, and quantitative procedures with minimal material. We applied this approach to identify known and novel components of well-studied complexes such as the anaphase-promoting complex. Furthermore, we demonstrate second generation interaction proteomics by incorporating directed mutational transgene modification and drug perturbation into QUBIC. These methods identified domain/isoform-specific interactors of pericentrin- and phosphorylation-specific interactors of TACC3, which are necessary for its recruitment to mitotic spindles. The scalability, simplicity, cost effectiveness, and sensitivity of this method provide a basis for its general use in small-scale experiments and in mapping the human protein interactome.
蛋白质相互作用涉及所有细胞过程。因此，有效地、可靠地对其进行描述对于理解生物机制至关重要。在这项研究中，我们表明，将细菌人工染色体 (BAC) TransgeneOmics 与定量相互作用蛋白质组学相结合，我们称之为定量 BAC-绿色荧光蛋白相互作用组学 (QUBIC)，可以使用快速、通用且定量的程序以及最小的材料来特异性和高度灵敏地检测相互作用。我们应用这种方法来鉴定已研究清楚的复合物（如有丝分裂促进复合物）的已知和新组件。此外，我们通过将定向突变转基因修饰和药物干扰纳入 QUBIC 来展示第二代相互作用蛋白质组学。这些方法鉴定了中心体蛋白的结构域/同工型特异性相互作用蛋白和 TACC3 的磷酸化特异性相互作用蛋白，这对于其招募到有丝分裂纺锤体是必需的。该方法的可扩展性、简单性、成本效益和敏感性为其在小规模实验中以及在绘制人类蛋白质相互作用组图谱中的广泛应用提供了基础。
