发展高效蛋白质膜上酶切新方法及细菌感染中宿主蛋白质组学研究

Pre-fraction by gel electrophoresis is often combined with liquid chromatography-mass spectrometry (LC-MS) for large-scale profiling of complex protein mixtures. An essential component of this widely applied proteomic platform is in-gel protein digestion, whose effectiveness however is often compromised by the limited accessibility of the enzyme to its gel-embedded protein substrates. In addition, this digestion approach is also suffered from the low recovery rate of large and/or hydrophobic peptides. To overcome these limitations, we aim to develop a highly efficient approach that combines electroblotting gel-fractionated proteins onto a membrane support and then performing digestion on the membrane. Traditionally, on-membrane digestion methods are severely challenged by the poor peptide recovery due to their strong binding to the nitrocellulose or PVDF membranes often used in Western blotting. Herein novel membrane materials with moderate protein affinity will be explored and expected to substantially improve peptide recovery as well as the overall performance of on-membrane digestion. The newly developed protein digestion approach, if incorporated into the gel electrophoresis-LC-MS proteomic platform, will greatly improve the proteome coverage as well as the analytical depth. Next we will apply this novel proteomic strategy to profile in large-scale protein expression of host cells during the course of Salmonella infection. The dynamic alterations in host protein levels will allow us gain insights into important cellular pathways that are activated by the host to combat bacterial infection. Such molecular mechanisms will be helpful to develop novel anti-microbial strategies.

在蛋白质组学研究中液质联用常与凝胶电泳分离相结合以提高分析复杂蛋白质混合物的能力。蛋白质胶中酶切是其中的一个重要环节，其效率却受到酶与底物接触有限及部分肽段回收率低的制约。为克服以上不足，申请人拟结合蛋白印迹中将胶中蛋白质电转移到膜载体这一过程，发展高效的膜上酶切新方法。研究将以探索新型膜材料为突破口，大幅度提高酶解后肽段产物的回收率，从而真正发挥凝胶电泳-液质联用技术结合了蛋白质和多肽两个层次分离手段的巨大优势，进而显著提升该技术平台分析复杂蛋白质组的深度和广度。同时，申请人拟运用所发展的蛋白质组学新方法大规模研究沙门氏菌感染中宿主细胞蛋白质组的动态变化，以此揭示宿主为拮抗病原细菌感染所启动或运用的分子机制，从而为设计抗感染新药提供重要的理论基础。

细菌感染仍然是威胁人类健康的一大杀手。在系统或整体层次上研究病原细菌和宿主细胞相互作用的分子机制通常带给我们一些全新的发现。前期我们以沙门氏菌感染宿主上皮细胞为模式体系，大规模定量分析了感染过程中胞内沙门氏菌的蛋白质动态表达，揭示了一系列病原细菌适应宿主胞内环境的分子机制。为进一步探索宿主应对细菌感染的防御机制，我们运用稳定同位素标记的定量蛋白质组学研究了感染过程中宿主细胞的蛋白质动态表达。在检测到的5000多个宿主蛋白质中，我们发现其中194个蛋白质在细菌感染过程中的表达水平发生了显著变化。高通量蛋白质组学研究表明宿主整合素蛋白和糖酵解相关蛋白显著上调，其中糖酵解通路蛋白变化趋势和前期胞内细菌组学数据一致。同时，我们进一步利用质谱选择反应监测、蛋白印迹和免疫荧光的方法证实了宿主细胞核内的PARP1蛋白在感染过程中发生了明显上调，且该上调为沙门氏菌感染所特有，并不依赖于与胞内细菌增殖相关的三型分泌系统。此外，我们的研究发现也表明高通量蛋白质组学是研究病原细菌与宿主相互作用机制的一种强有力手段。