Designer Proteases for Complete Proteomics

智力优势：该项目的目标是从α-裂解蛋白酶（aLP）设计具有新的和不同的底物特异性的蛋白酶。该项目将开发新的方法和工具，以确定蛋白质的一级结构，特别是确定哪些氨基酸是后修饰的。目前几乎所有的大规模蛋白质测序实验都是使用质谱法进行的，以分析用蛋白酶胰蛋白酶消化的蛋白质片段，胰蛋白酶在赖氨酸（K）和精氨酸（R）残基后切割。胰蛋白酶切割产生足够的肽以提供足够的独特蛋白质序列用于蛋白质鉴定，但翻译后修饰（PTM）定位需要更完整的蛋白质序列覆盖。初步结果表明，aLP是理想的蛋白质组学，因为一些活性位点突变改变其对小的脂肪族残基或大的疏水残基的底物特异性。这些“正交”类型的氨基酸（与带电荷的K和R相比）允许访问蛋白质序列的更多和不同的部分。在aLP平台的基础上，将设计具有不同底物切割特异性的工程蛋白酶家族，以覆盖整个蛋白质序列。 一个应用程序，将被调查的是确定哪些网站的蛋白质被修改的小泛素样修饰（SUMO）。所产生的突变体为检测S.粟酒酵母蛋白质组 使用突变体aLP切割SUMO化的蛋白质产生可用现有方法检测的GG标记的赖氨酸。因此，aLP将提供第一个全面调查哪些蛋白质是SUMO化的酵母。更广泛的影响：该项目的更广泛的影响包括开发新的蛋白酶，这将是广泛有用的蛋白质组学社区。对从事蛋白质组学研究的研究人员的调查揭示了对这种强活性的“正交”蛋白酶的迫切需求。该项目将产生大型蛋白质组学数据集，有助于预测修饰位点，并为细胞生物学和蛋白质结构/功能的基础研究提供信息。 还将制定数据分析方案，通过网络广泛传播。两名本科研究人员将在整个项目期间参与该项目。PI在指导本科生方面有着悠久的历史，这些本科生已经进入了科学领域，特别注重鼓励多样性。PI在UCSD创立了研究学者计划，为来自不同背景的高中生提供研究培训。参与者由指定的教师和研究生对指导。指导一直持续到参与者选择大学和专业。在前两年，超过90%的学生完成了该计划，决定从事科学研究。一名或两名来自研究学者计划的学生将从事NSF项目的各个方面。

Intellectual Merit: The goal of the project is to design proteases from alpha-lytic protease (aLP) that have new and different substrate specificities. The project will develop new methods and tools to determine the primary structure of proteins, in particular to determine which amino acids are post-translationally modified. Nearly all large-scale protein sequencing experiments are currently performed using mass spectrometry to analyze pieces of proteins that were digested with the protease, trypsin, which cleaves after lysine (K) and arginine (R) residues. Trypsin cleavage produces enough peptides to provide sufficient unique protein sequence for protein identification, but more complete coverage of protein sequences is required for post-translational modification (PTM) localization. Preliminary results show that aLP is ideal for proteomics because a few active site mutations alter its substrate specificity towards either small aliphatic residues or large hydrophobic residues. These "orthogonal" types of amino acids (compared to K and R, which are charged) allow access to more and different parts of the protein sequence. Building on the aLP platform, a family of engineered proteases will be designed with varying substrate cleavage specificities to enable coverage of entire protein sequences. An application that will be investigated is the determination of which sites in proteins are modified with the small ubiquitin-like modification (SUMO). The generated mutants provide ideal substrate specificity for detection of SUMOylation in the S. pombe yeast proteome. Cleavage of SUMOylated proteins using a mutant aLP generates GG-tagged lysines that can be detected with existing approaches. Thus aLP will provide the first comprehensive survey of which proteins are SUMOylated in yeast.Broader Impacts: The broader impacts of the project include development of novel proteases that will be widely useful to the proteomics community. A survey of researchers engaged in proteomics studies reveals the dire need for such robustly active "orthogonal" proteases. The project will result in large proteomic data sets that will aid in the prediction of modification sites and provide information for basic research into cell biology and protein structure/function. Data analysis programs will also be developed that will be widely disseminated via the web. Two undergraduate researchers will be involved in the project throughout the project period. The PI has a long history of mentoring undergraduates, who have entered careers in science, with a particular focus on encouraging diversity. The PI founded at UCSD the Research Scholars program that provides research training for high school students from diverse backgrounds. The participants are mentored by assigned pairs of faculty and graduate students. Mentoring is continued up to the point where participants choose colleges and majors. In the first two years, more than 90% of the students completed the program having decided on a career in scientific research. One and possibly 2 students from the Research Scholars Program will work on aspects of this NSF project.