Protein Sequencing Tools for Biological Therapeutics

 描述（由申请人提供）：质谱法已成为鉴定和表征复杂混合物中少量蛋白质的首选方法。然而，以高吞吐量方式执行识别的能力取决于可用性 高质量的蛋白质序列数据库。这意味着来自具有未测序或测序差的基因组的生物体的蛋白质（例如肽毒素）和响应于环境而快速改变其一级序列的蛋白质（例如抗体）已被排除在高通量分析之外。下一代测序（NGS）的广泛可用性并没有缓解这个问题，而是NGS导致了低质量、未经策划的蛋白质序列数据库的激增，包括个性化数据库、癌症数据库和具有不确定组装的数据库。数据库搜索蛋白质组学和从头肽测序之间的传统划分不再成立;许多最有趣的生物学问题现在最好通过结合两种技术的最佳数据分析来解决。 在这个第二阶段的STTR项目中，我们建议开发两个商业软件产品的测序生物感兴趣的肽和蛋白质，无论序列数据库的质量。一种产品将针对肽水平，应用于循环抗体、肽毒素和人类白细胞抗原的可变区。另一个产品将针对蛋白质水平，应用于纯化蛋白质的端到端测序，特别是治疗性单克隆抗体。该系统将包括肽级测序仪作为一个组件，以及用于将肽组装成完整序列以及用于可视化和手动验证的工具。 拟议的项目有可能在疫苗开发、治疗性抗体开发和癌症免疫治疗等领域对人类健康产生重大影响。

 DESCRIPTION (provided by applicant): Mass spectrometry has become a method of choice for identifying and characterizing small quantities of proteins in complex mixtures. However, the ability to perform the identification in a high-throughput fashion has depended on the availability of high-quality protein sequence databases. This means that proteins from organisms with unsequenced or poorly sequenced genomes (e.g. peptide toxins) and proteins that modify their primary sequences rapidly in response to the environment (e.g. antibodies) have been excluded from high-throughput analysis. Widespread availability of Next Generation Sequencing (NGS) has not alleviated this problem, but rather NGS has led to a proliferation of lower quality, uncurated protein sequence databases, including personalized databases, cancer databases, and databases with uncertain assembly. The traditional division between database-search proteomics and de novo peptide sequencing no longer holds; many of the most interesting biological questions are now best addressed by data analysis that combines the best of both techniques. In this Phase II STTR project, we propose to develop two commercial software products for sequencing biologically interesting peptides and proteins, regardless of the quality of sequence databases. One product will be aimed at the peptide level, with applications to variable regions of circulating antibodies, peptide toxins, and human leukocyte antigens. The other product will be aimed at the protein level, with applications to end-to-end sequencing of purified proteins, especially therapeutic monoclonal antibodies. This system will include the peptide-level sequencer as a component, as well as tools for assembly of the peptides into the full sequence and for visualization and manual validation. The proposed project has the potential for great impact on human health in areas such as vaccine development, therapeutic antibody development, and cancer immunotherapies.