【作者】 朱莉思；

【导师】 高友鹤；

【作者基本信息】 北京协和医学院 ， 病理生理学， 2014， 博士

【摘要】 蛋白质几乎参与所有的生物学过程。他们通过与其他蛋白质或者其他分子发生相互作用行使功能。本论文建立了两个研究蛋白质相互作用的蛋白质组学新策略：4Facts策略用于鉴定实时的蛋白质-配体相互作用；4S策略用于整体分析蛋白质-细胞相互作用。4Facts策略——快速固定(Fast Fix)、亲和捕获(Fish)、分子量筛选(Filter),可以在不对生物学样品进行预先干预的条件下全面、灵敏而准确地研究实时原位的蛋白质-配体相互作用。在本策略中,SDS-PAGE (十二烷基硫酸钠聚丙烯酰胺凝胶电泳)被用于打断非共价结合以去除未被共价交联的复合物,以及提供分子量信息用于筛选配体蛋白。候选蛋白被认定为靶蛋白的配体蛋白,需要同时满足以下三个条件：第一,必须与靶蛋白在同一胶条中被鉴定；第二,必须在实验组胶条中被鉴定,而不出现在相同、相邻或更高分子量范围的对照组胶条中；第三,所在胶条的分子量范围包含或大于它与靶蛋白的理论分子量之和。我们将4Facts策略应用于人血液中的蛋白质-配体相互作用,以血浆白蛋白以及转铁蛋白为例展示该策略的可行性。人血液不经任何处理被直接加入10%的甲醛溶液中进行5秒的快速交联反应。运用该策略,我们鉴定到35个白蛋白的配体以及52个转铁蛋白的配体。与此前三个主要的白蛋白相互作用蛋白质组学研究进行比较,本研究鉴定到的35个白蛋白配体中的68.57%在其他研究中也被鉴定到。该策略实现了5秒的短时间交联,未来可能被应用于研究动态的蛋白质-蛋白质相互作用。4S策略——表面酶切(Surface Shaving)结合种属特异性(Species Specificity),可以被用于整体分析不同物种间的蛋白质-细胞相互作用。我们以牛血清-人Ca Ski细胞相互作用为例来说明4S策略。牛血清培养的Ca Ski细胞在经过三次PBS洗涤之后与胰酶孵育。表面酶切上清以及作为对照的牛血清和细胞裂解液被收集,用于质谱分析。一些蛋白质因为共享多肽而无法被定量。我们把共享多肽的谱图数按照各蛋白所含种属特异的多肽的谱图数的比例,分配给各个蛋白,然后通过NSAF值对牛源蛋白(包含牛种属特异的肽)以及人源蛋白(包含人种属特异的肽)进行定量。运用该策略,30个牛源蛋白相对于牛血清被富集,我们认为这些牛血清蛋白与人Ca Ski细胞发生相互作用,可能在细胞培养中起着重要作用。基因注释显示它们之中的大部分具有结合功能。另外,216个人源蛋白相对于细胞裂解液被富集,它们可能是细胞膜蛋白或者分泌蛋白。该策略实现了对不同物种间蛋白质-细胞相互作用的整体分析,未来可能被应用于病原-宿主微环境相互作用。更多还原

【Abstract】 Proteins mediate virtually all biological process. They function through interactions with other proteins as well as other molecules. In this dissertation, studies have been made on setting up new proteomic strategies for identification of real-time protein-ligand interactions and for global analyses of protein-cell interactions.The4Facts strategy-Fast Fix, Fish, and Filter, can be used to comprehensively, sensitively and precisely identify real-time protein-protein interactions without prior intervention. In this strategy, SDS-PAGE is used to disrupt non-covalent bonds, thereby eliminating uncross-linked complexes and simultaneously providing molecular weight information for identification. Only proteins that simultaneously satisfy the following three criteria can be considered true ligands:1. the target protein must be identified in the same slice as its ligands;2. the ligands must be identified in slices for the experimental group but not in the corresponding, neighboring or higher control slices;3. the ligands appear in the range of molecular weights equal to or greater than the sum of the theoretical molecular weights of the target and the ligand. Using albumin and transferrin as examples, the4Facts strategy was applied to identify protein-ligand interactions in human blood. Human blood was directly added to10%formaldehyde for5s of cross-linking. The use of this strategy identified35ligands for albumin and52ligands for transferrin. Comparison with three major previous studies of the albuminome revealed that68.57%of the35albumin ligands identified in our study were also identified in these other studies. Since fast fixing of5s was achieved, this strategy can be potentially used in identification of dynamic protein-protein interactions, and become a new standard in future.The4S strategy-Surface Shaving and Species Specificity, can be used to achieve global analyses of protein-cell interactions between different species. Bovine serum-human Ca Ski cell interaction was used as an example to illustrate this strategy. Cells cultured in bovine serum were washed three times with PBS and then incubated with trypsin. The supernatant after cell surface shaving were send to MS analysis. Bovine serum and Ca Ski cell lysate were analyzed as control. Many proteins were reported in protein groups and cannot be quantified because of shared peptides. We assigned SpC (spectral counts) of shared peptides to different proteins according to the SpC ratio of species specific peptides in those proteins. Bovine proteins (proteins with bovine specific peptides) and human proteins (proteins with human specific peptides) were quantified by spectral counting using NSAF values. Using this strategy,30bovine proteins were identified as proteins that interact with Ca Ski cells as they were enriched comparing with bovine serum. They might play important roles in cell culture. Most of them have binding function with GO annotation. In addition,216human proteins were enriched comparing with cell lysate. They might be cell membrane proteins or secreted proteins. This strategy achieved global analyses of protein-cell interactions between different species, and can be potentially used in identification of pathogen-host microenvironment interactions, etc.更多还原