Abstract:
Peptidoglycan is a major and essential component of the bacterial cell envelope that confers cell shape and provides protection against internal osmotic pressure. This complex macromolecule is made of glycan strands cross-linked by short peptides, and its structure is continually modified throughout growth via a process referred to as \"remodeling.\" Peptidoglycan remodeling allows cells to grow, adapt to their environment, and release fragments that can act as signaling molecules during host-pathogen interactions. Preparing peptidoglycan samples for structural analysis first requires purification of the peptidoglycan sacculus, followed by its enzymatic digestion into disaccharide peptides (muropeptides). These muropeptides can then be characterized by liquid chromatography coupled mass spectrometry (LC-MS) and used to infer the structure of intact peptidoglycan sacculi. Due to the presence of unusual crosslinks, noncanonical amino acids, and amino sugars, the analysis of peptidoglycan LC-MS datasets cannot be handled by traditional proteomics software. In this chapter, we describe a protocol to perform the analysis of peptidoglycan LC-MS datasets using the open-source software PGFinder. We provide a step-by-step strategy to deconvolute data from various mass spectrometry instruments, generate muropeptide databases, perform a PGFinder search, and process the data output.
摘要:
肽聚糖是细菌细胞包膜的主要和必要组分,其赋予细胞形状并提供针对内部渗透压的保护。这种复杂的大分子由由短肽交联的聚糖链组成,它的结构在整个生长过程中通过称为“重塑”的过程不断改变。“肽聚糖重塑允许细胞生长,适应他们的环境,并释放在宿主-病原体相互作用期间可以充当信号分子的片段。制备用于结构分析的肽聚糖样品首先需要纯化肽聚糖囊,然后将其酶消化成二糖肽(muropeptides)。然后可以通过液相色谱耦合质谱(LC-MS)表征这些muropeptips,并用于推断完整的肽聚糖囊的结构。由于存在不寻常的交联,非常规氨基酸,和氨基糖,传统的蛋白质组学软件无法处理肽聚糖LC-MS数据集的分析。在这一章中,我们描述了使用开源软件PGFinder进行肽聚糖LC-MS数据集分析的方案。我们提供了一个循序渐进的策略,从各种质谱仪器中去卷积数据,生成muropeptide数据库,执行PGFinder搜索,并处理数据输出。