Supporting data for "Chemical Approaches to Investigate Histone H3.3-specific Binding Proteins and Microtubule Inner Proteins"

In my first project, a potential specific binder of histone named POI was found through in vivo crosslink-assisted and SILAC-based protein identification. After the development of biotinylated peptide or nucleosome probe for pull-down experiment, POI was validated to specifically recognize hsitone in cell lysate. The SDS-PAGE data (file Gel(1)-(30)) was used to validate the identity and purity of the obtained POI protein, and was further applied in the study of protein-protein interactions between POI and nucleosome probe. Another important part is the validation of the interaction in the nuclear extract, the enriched proteins can be resolved on the gel, tranferred to the PVDF membrane. After the incubation with with the primary antibody followed by secondary antibody, the signal of POI can be visualized after treated with the peroxide-luminol mixture, and the data was shown in the Western blot (1) to (38).
In the second project, an investigation of microtubule inner proteins (MIPs) was attempted utilizing two different in vivo crosslink-assisted and SILAC-based protein identification methods based on the incorporation of the photo-affinity amino acid or on the chemical probes derived from microtubule stabilizing agents (MSA). For the first method, photo-affinity amino acid was incorporated into α or β-tubulin by Amber suppression through transient transfection. For the second method, I synthesized several diazirine-containing photo-affinity probes modified from Paclitaxel, which is a well-known drug that targets microtubule structure. Then the development and optimization of the photo-catalytic crosslinking method specially designed for the microtubule system gradually solves the problem that the UV irradiation and photo-induced cytotoxicity of the iridium catalyst easily trigger the microtubule disassembly. Taking advantage of the chemical probes located in the lumen, the proteins can be captured after photo-catalytic crosslinking and further identified based on proteomics techniques. The data (NMR (1)-(57)) show the identification result of the synthesized compound in the development of chemical probes.

在本研究的第一个项目中，通过体内交联辅助结合基于细胞培养稳定同位素标记（SILAC）的蛋白质组鉴定方法，我们发现了一种名为POI的组蛋白潜在特异性结合蛋白。在开发出用于下拉实验的生物素标记肽或核小体探针后，我们验证了POI可特异性识别细胞裂解液中的组蛋白。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）数据（文件Gel(1)-(30)）被用于验证所获取的POI蛋白的身份与纯度，并进一步应用于POI与核小体探针之间的蛋白质相互作用研究。另一项核心内容是在核提取物中验证该相互作用：富集得到的蛋白经凝胶分离后转印至聚偏氟乙烯（PVDF）膜，先后与一抗、二抗孵育，再用过氧化氢-鲁米诺混合液处理后即可可视化POI的信号，相关数据展示于免疫印迹（Western Blot）图谱(1)-(38)中。
在本研究的第二个项目中，我们针对微管内部蛋白（MIPs）开展研究，采用了两种不同的体内交联辅助结合SILAC的蛋白质组鉴定方法：一种基于光亲和氨基酸掺入策略，另一种基于源自微管稳定剂（MSA）的化学探针策略。第一种方法中，通过琥珀突变抑制（Amber suppression）技术并经瞬时转染，将光亲和氨基酸掺入至α或β微管蛋白中。第二种方法中，我们合成了数种经紫杉醇（Paclitaxel，一种经典的靶向微管结构的药物）修饰得到的含二氮杂环丙烷基团的光亲和探针。针对微管系统定制开发的光催化交联方法的开发与优化，逐步解决了紫外照射及铱催化剂的光诱导细胞毒性易引发微管解聚的问题。借助定位于微管管腔内的化学探针，可在光催化交联后捕获靶蛋白，并通过蛋白质组学技术完成后续鉴定。核磁共振波谱（NMR (1)-(57)）数据展示了化学探针开发过程中合成化合物的鉴定结果。