DETECTION OF SECRETED PEPTIDES USING HYPOTHESIS-DRIVEN MULTISTAGE MS

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。建立了一种快速检测和表征微生物分泌的微量多肽的新方法，包括信息素、毒力因子和群体感应肽。该程序，有针对性的多级质谱（MS）的基础上，利用一种新的MALDI-离子阱质谱仪，以克服目前的MS方法（有限的动态范围，信号抑制效应，化学噪声）的局限性，损害观察低丰度肽从复杂的生物基质。作为概念的证明，假设存在于上清液中，但在单级质谱中没有足够的丰度来观察的分泌肽进行多级MS。高度特异性的片段化特征使得能够明确鉴定感兴趣的肽和区分来自背景的信号。作为例子，我们证明了快速（1分钟）测定交配型的细胞在菌落中的酿酒酵母和阐明自诱导肽（AIP）从致病性葡萄球菌的上清液。我们确认了金黄色葡萄球菌第I、II、IV组agrD编码的环状AIP的一级结构，并提供了直接证据表明天然第III组AIP是七肽（INCDFLL）。我们还表明，从葡萄球菌intermedius同源肽是一个九肽（RIPTSTGFF）与通过缩合的丝氨酸侧链与肽的C-末端形成的内酯环。这是第一次证明葡萄球菌AIP中通过内酯形成发生环化。这些实施例证明了本方法用于表征分泌肽的分析能力及其用于鉴定微生物的潜在效用。描述这项工作的手稿已经出版：M。Kalkum，G. J.里昂和B.T.使用假设驱动的多级质谱法检测分泌肽Sci. USA 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.