New approaches to sequencing of complex peptides.

描述（由申请人提供）：非核糖体肽（NRP），如青霉素、万古霉素和从微生物来源分离的相关分子，几十年来一直是药物发现的主要成分。我们建议采用多级质谱（MSn）从头测序的NRP，包括环状NRP。环状肽的MSn谱的分析导致从同一谱解释多个线性肽的困难的组合问题。该建议开发了新的组合算法来解决这个问题。由于基于MSn的NPRs质谱分析是快速和廉价的，并且需要最少量的材料（<1.5g），因此该方法开启了在大生物活性海洋蓝藻筛选程序中积累的许多未知NRP的高通量测序的可能性。在NRP测序工作自动化的同时，我们将从海洋放线菌中收获一组孤儿基因簇，以生成环肽文库。在这个建议中开发的算法将被用来充分表征这个环状亚胺库。这项工作不仅为NRP的自动化表征奠定了基础，而且也将适用于其他肽类天然产物如肽类、肽衍生毒素或羊毛硫抗生素的表征。公共卫生相关性：该项目描述了一种新的基于质谱的方法和相应算法的开发和应用，该方法和算法允许对非核糖体衍生的复杂治疗剂进行从头测序。

DESCRIPTION (provided by applicant): Nonribosomal peptides (NRPs) such as penicillin, vancomycin and related molecules isolated from microbial sources have been a staple for drug discovery for many decades. We propose to employ multi-stage mass-spectrometry (MSn) for de novo sequencing of NRPs, including cyclic NRPs. Analysis of MSn spectra of a cyclic peptide results in the difficult combinatorial problem of interpreting multiple linear peptides from the same spectrum. This proposal develops new combinatorial algorithms for solving this issue. Since the MSn based mass spectrometry analysis of NPRs is fast and inexpensive and requires minimal amounts of material (<1 5g), this approach opens a possibility of high-throughput sequencing of many unknown NRPs accumulated in large bioactivity marine cyanobacterial screening programs. In parallel to the automation of the NRPs sequencing efforts, we will harvest a set of orphan gene clusters from marine actinomycetes to generate a library of cyclic peptides. The algorithms developed in this proposal will be used to fully characterize this cyclic imine library. This work not only sets the stage for the automated characterization of NRPs but will also be applicable to the characterization of other peptidic natural products such as peptaibols, peptide derived toxins or lantibiotics. PUBLIC HEALTH RELEVANCE: This project describes the development and application of a novel mass spectrometry based method and corresponding algorithms that allow the de novo sequencing of complex therapeutic agents that are non-ribosomally derived.