Designer Proteases for Complete Proteomics

智力价值：该项目的目标是从α-裂解酶(ALP)中设计具有新的和不同底物特异性的蛋白酶。该项目将开发新的方法和工具来确定蛋白质的一级结构，特别是确定哪些氨基酸是翻译后修饰的。目前几乎所有的大规模蛋白质测序实验都是使用质谱仪来分析被胰酶消化的蛋白质片段，胰酶在赖氨酸(K)和精氨酸(R)残基之后裂解。胰酶裂解产生的多肽足以为蛋白质鉴定提供足够的唯一蛋白质序列，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是蛋白质组学的理想选择，因为少数活性位点突变会改变其对小脂肪残基或大疏水残基的底物专一性。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中更多和不同的部分。在ALP平台的基础上，将设计一系列具有不同底物切割特性的工程蛋白酶，以实现对整个蛋白质序列的覆盖。一个将被研究的应用是确定蛋白质中的哪些位置被小泛素样修饰(SUMO)修饰。所产生的突变体为检测S.pombe酵母蛋白质组中的SUMO化提供了理想的底物特异性。用突变的ALP裂解SuMOylated蛋白质产生GG标记的赖氨酸，可以用现有的方法检测到。因此，ALP将提供关于酵母中哪些蛋白质被SUMO化的第一次全面调查。广泛的影响：该项目的更广泛的影响包括开发对蛋白质组学社区将广泛有用的新型蛋白酶。对从事蛋白质组学研究的研究人员进行的一项调查显示，人们迫切需要这种活性极强的“正交”蛋白酶。该项目将产生大量蛋白质组数据集，这些数据集将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。还将开发数据分析程序，通过网络广泛传播。两名本科生研究人员将在整个项目期间参与该项目。PI有很长的指导本科生的历史，这些本科生已经进入了科学职业生涯，特别关注鼓励多样性。加州大学圣迭戈分校设立了研究学者项目，为不同背景的高中生提供研究培训。参与者由指定的教职员工和研究生对进行指导。辅导一直持续到参与者选择大学和专业。在最初的两年里，超过90%的学生完成了这个项目，决定了从事科学研究。来自研究学者计划的一名或可能两名学生将致力于这个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.