稳定同位素磷酰化标记蛋白质定量及其在HIV潜伏激活机制研究中的应用

Mass spectrometry-based proteomics as a central life science technology plays key roles in diverse areas such as molecular biology, new drug discovery and clinical diagnostic. In this project, organic phosphorus chemistry will be introduced for the quantitative proteomics. A novel stable isotope N-phosphorylation labeling strategy based on organic phosphorus chemistry will be developed for peptide de novo sequencing and protein identification and quantification. Meanwhile, stable isotope N-phosphorylation labeling strategy will be extensively apllied to investigate the global protein expression level changes in cells as induced by chemical activators of HIV latency, which may provide new sights for understanding the molecular mechanisms of super elongation complex (SEC) in controlling HIV and cellular gene expression.In addition, the dynamic changes of phosphorylated proteins will also be identified and quantified by using this novel method. A series of organic phosphite labeling reagents with different chemical structures and multi-functions will be synthesized and characterized for peptide sequencing and protein identification and quantification using mass spectrometry. Stable isotope N-phosphorylation labeling strategy will be systematically studied using model synthetic peptide and standard proteins.In combination with the experimental data of mass spectrometry,theoretical calculations using density functional theory (DFT) will also be applied to investigate the related mechanisms of N-phosphorylation labeling, such as the chemical mechanism of phosphorylation labeling reaction with high selectivity, enhancement of the intensities of b ions and reducing the complexity of the tandem mass spectrometry, and significant improvement of ionization efficiency, which may favor the understanding of the chemical and biological intrinsic properties of phosphoryl group. In summary, stable isotope N-phosphorylation labeling as a novel and original strategy for mass spectrometry-based protein quantification may play an important role in biological science such as the protein deep coverage and quantification, protein-protein interactions and protein post-translational modifications.

基于质谱的蛋白质组学在分子生物学、新药物研发及临床诊断等领域中发挥着重要作用。本项目拟把有机磷化学引入定量蛋白质组学研究，建立一种基于有机磷化学的稳定同位素标记多肽从头测序和蛋白质定量质谱分析新策略―稳定同位素磷酰化标记；同时，把该策略应用于HIV潜伏激活剂处理细胞后的蛋白质表达差异组学分析，特别是磷酸化蛋白质组定量分析，对超级转录延伸复合体SECs在细胞中及HIV基因转录调控中的机制进行系统研究。本项目合成表征一系列结构新颖功能多样的磷酸酯标记试剂，应用于多肽从头测序、蛋白质鉴定和定量质谱分析，并运用量子化学密度泛函理论对磷酰化标记相关反应机理如磷酰基增敏机制等进行系统理论计算研究，深入理解磷酰基的化学生物学性质。总之，稳定同位素磷酰化标记作为一种原创性的蛋白质定量分析新策略，有望在蛋白质组的深度覆盖与定量以及蛋白质翻译后修饰等生命科学研究中发挥重要作用。

基于质谱的多标稳定同位素标记技术在定量蛋白质组学和代谢组学研究中发挥着重要作用。基于前期稳定同位素标记策略，项目首先开发了三标稳定同位素磷酰化标记策略-3-SIPL。3-SIPL基于有机磷化学，运用简单稳定同位素合成试剂合成并表征了 13C4- and 13C418O2-标记的氢亚磷酸酯。建立了半微量稳定同位素标记和合成方法。运用标准合成肽段及标准蛋白样品对方法的重复性、准确性、线性范围等进行了考查。并运用3-SIPL对药物处理条件下HIV-1基因转录调控关键蛋白复合物的蛋白-蛋白相互作用进行了定量分析，共鉴定145个可能的相互作用蛋白，其中115个具有三标定量信息。研究发现发现蛋白复合物7SK snRNP包含的蛋白在药物处理条件下下调10倍左右，如LARP7 和HEXIM2等，同时发现HSP90AB1 及PRMT5与CDK9的相互作用也发生的较大的变化；其他蛋白复合物并没有发生明显变化，如SEC（包含 AFF4, AFF1, ELL1, ENL, 和AF9)和BRD4/P-TEFb (包含 BRD4 and Cyclin T)等蛋白复合物。随后运用传统生物化学方法对差异蛋白进行验证，结果与3-SIPL定量结果一致。蛋白复合物7SK snRNP中与CDK9相互作用蛋白的减少说明再药物处理条件下，需要7SK snRNP释放更多的CDK9来补充激酶活性的抑制。.另外，项目成功开发了稳定同位素N-磷酰化氨基酸标记两标定量策略-SIPAL，对含有氨基的小分子手性代谢物进行成功分离和定量分析。2-SIPAL基于N-二异丙基磷酰化L-丙氨酸，开发16O/18O-DIPP-L-Ala-NHS试剂。该策略对20种L-氨基酸，10种D-氨基酸和小肽进行了标记和LC-MS分析。SIPAL具有高选择性和标记效率，15分钟水相条件下即可完成标记。磷酰基的引入不但提高了质谱分析灵敏度，同时有利于色谱分离，特别是针对水溶性高极性代谢物分子。同时磷酰基团的引入，可以利用核磁共振磷谱对手性异构体比例进行分析；标记后形成的酰胺键为HPLC-UV分析提供可能。该方法成功对尿样中氨基代谢物进行了分析，共发现313个可能的氨基代谢物。最后，基于MALDI-TOF MS，对标准合成肽段进行2-SIPAL标记，显示该策略在蛋白质组学定量分析中的潜在应用。