Protein Sequencing Tools for Biological Therapeutics

 描述(申请人提供)：质谱学已经成为识别和表征复杂混合物中少量蛋白质的一种选择方法。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，来自基因组未测序或测序不佳的生物的蛋白质(例如多肽毒素)和对环境做出反应而快速修改其初级序列的蛋白质(例如抗体)已被排除在高通量分析之外。下一代测序技术(NGS)的广泛应用并没有缓解这一问题，相反，NGS导致了质量较低的、未精选的蛋白质序列数据库的激增，包括个性化数据库、癌症数据库和组装不确定的数据库。数据库搜索蛋白质组学和从头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地通过结合这两种技术的数据分析来解决。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，用于对具有生物学意义的多肽和蛋白质进行测序，而不考虑序列数据库的质量。一种产品将针对多肽水平，应用于循环抗体、多肽毒素和人类白细胞抗原的可变区。另一种产品将针对蛋白质水平，应用于纯化蛋白质的端到端测序，特别是治疗性单抗。该系统将包括多肽水平测序仪作为一个组件，以及将多肽组装成全序列以及可视化和手动验证的工具。拟议的项目可能在疫苗开发、治疗性抗体开发和癌症免疫疗法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.