DETECTION OF SECRETED PEPTIDES USING HYPOTHESIS-DRIVEN MULTISTAGE MS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。建立了一种快速检测和鉴定微生物分泌的微量肽的新方法，包括信息素、毒力因子和群体感应肽。该程序基于靶向多级质谱（MS），利用新型maldi -离子阱质谱仪克服了当前MS方法的局限性（有限的动态范围，信号抑制效应，化学噪声），这些局限性影响了对复杂生物基质中低丰度肽的观察。作为概念的证明，假设分泌肽存在于上清中，但在单级质谱中没有足够的丰度来观察到，因此需要进行多阶段质谱分析。高度特异性的片段特征可以明确识别感兴趣的肽，并从背景中区分信号。作为例子，我们展示了在酿酒酵母菌落中快速（1分钟）确定交配类型的细胞，并从致病性葡萄球菌的上清液中阐明了自诱导肽（AIPs）。我们确认了agrD编码的金黄色葡萄球菌I、II、IV群环状AIP的初级结构，并提供了iii群原生AIP为七肽（incdll）的直接证据。我们还发现，来自中间葡萄球菌的同源肽是一种非肽（RIPTSTGFF），其丝氨酸侧链与肽的c端缩合形成内酯环。这是葡萄球菌AIP通过内酯形成发生环化的第一个证明。这些实例证明了本方法表征分泌肽的分析能力及其在鉴定微生物方面的潜在效用。一份描述这项工作的手稿已经发表：M。Kalkum， G.J. Lyon和B.T. Chait -使用假设驱动的多级质谱法检测分泌肽-自然科学学报。美国100(2003)2795-2800。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A new method for the rapid detection and characterization of trace amounts of peptides secreted from microorganisms, including pheromones, virulence factors, and quorum sensing peptides was developed. The procedure, based on targeted multi-stage mass spectrometry (MS), utilizes a novel MALDI-ion trap mass spectrometer to overcome limitations of current MS methods (limited dynamic range, signal suppression effects, chemical noise) that impair observation of low abundance peptides from complex biological matrixes. As a proof of concept, secreted peptides that are hypothesized to be present in the supernatant, but that are not be sufficiently abundant to be observed in single-stage mass spectra, are subjected to multi-stage MS. Highly specific fragmentation signatures enable unambiguous identification of the peptides of interest and differentiation of the signals from the background. As examples, we demonstrate the rapid (1 min) determination of the mating type of cells in colonies of Saccharomyces cerevisiae and the elucidation of autoinducing peptides (AIPs) from supernatants of pathogenic Staphylococci. We confirm the primary structures of the agrD encoded cyclic AIPs of Staphylococcus aureus for group I, II, IV and provide direct evidence that the native group-III AIP is a heptapeptide (INCDFLL). We also show that the homologous peptide from Staphylococcus intermedius is a nonapeptide (RIPTSTGFF) with a lactone ring formed through condensation of the serine side chain with the peptide's C-terminus. This is the first demonstration of cyclization in a staphylococcal AIP that occurs via lactone formation. These examples demonstrate the analytical power of the present procedure for characterizing secreted peptides and its potential utility for identifying microorganisms. A manuscript describing this work has been published:M. Kalkum, G.J. Lyon and B.T. Chait ¿ Detection of Secreted Peptides using Hypothesis-driven Multistage Mass Spectrometry¿ Proc Natl Acad. Sci. USA 100 (2003) 2795-2800.