DNA/RNA QUADRUPLEXES: STRUCTURE, STABILITY AND INTERACTIONS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 蛋白质水解在调节多种生物过程中起着重要作用。 一个这样的过程是细胞凋亡，其中已知大约300种蛋白质经历蛋白水解加工作为导致程序性细胞死亡的生化事件的一部分。 不幸的是，目前用于监测复杂样品中的蛋白水解事件的方法受到严重的限制。 我们正在开发一种新的方法，用于在复杂的生化混合物中蛋白质水解的全局分析，该方法基于使用工程肽连接酶来选择性地标记α胺蛋白N-末端。 该标记允许亲和纯化和富集N-末端肽，用于随后通过串联质谱法进行测序。 这种方法不仅能够监测由于细胞或血清中蛋白质水解而新形成的N-末端，而且还降低了样品复杂性以获得更大的蛋白质组覆盖率。 我们正在应用这种方法来描述细胞凋亡过程中发生的蛋白水解，以及细胞凋亡后血清蛋白水平的变化。 我们的目标是调查不同刺激和不同细胞类型引起的细胞凋亡中蛋白水解模式的多样性，以确定细胞凋亡中蛋白水解的新靶点，并探讨血清蛋白水平在癌症和化疗的不同阶段如何变化。 这项工作将为细胞凋亡的生物学以及如何在化疗中最好地利用细胞凋亡提供新的线索。 这项工作还将建立一种新的蛋白质组学方法，该方法将在生物学的其他领域得到应用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Proteolysis plays an important role in the regulation of diverse biological processes. One such process is apoptosis, in which approximately 300 proteins are known to undergo proteolytic processing as part of the biochemical events leading to programmed cell death. Unfortunately, current methods for monitoring proteolytic events in complex samples suffer from serious limitations. We are developing a novel method for global profiling of proteolysis in complex biochemical mixtures that is based on the use of an engineered peptide ligase to selectively label the alpha amines protein N-termini. The label permits affinity purification and enrichment of N-terminal peptides for subsequent sequencing by tandem mass spectrometry. This approach not only enables monitoring of newly formed N-termini as a result of proteolysis is cells or serum, but also decreases sample complexity for greater proteomic coverage. We are applying this method to profile the proteolysis that occurs during apoptosis in cells, and to profile changes in serum protein levels following apoptosis. Our goal is to survey the diversity of proteolysis patterns in apoptosis elicited by different stimuli and in different cell types, to identify new targets of proteolysis in apoptosis, and to explore how levels of serum proteins change in different stages of cancer and chemotherapy. This work will shed new light on the biology of apoptosis and how apoptosis can be best exploited in chemotherapy. The work will also establish a novel proteomic method that will find application in other areas of biology.