通过完整细胞免疫捕获和靶向质谱法检测复杂基质中的鼠疫耶尔森菌

Detection of Yersinia pestis in Complex Matrices by Intact Cell Immunocapture and Targeted Mass Spectrometry.

作者信息

Chenau Jérôme, Fenaille François, Simon Stéphanie, Filali Sofia, Volland Hervé, Junot Christophe, Carniel Elisabeth, Becher François

机构信息

CEA, iBiTec-S, Service de Pharmacologie et d'Immunoanalyse, Bât. 136, 91191, Gif-sur-Yvette, France.

Institut Pasteur, Unité de Recherche Yersinia, Paris, France.

出版信息

Methods Mol Biol. 2017;1600:69-83. doi: 10.1007/978-1-4939-6958-6_7.

DOI:10.1007/978-1-4939-6958-6_7

PMID:28478558

Abstract

We describe an immunoaffinity-liquid chromatography-tandem mass spectrometry (immuno-LC-MS/MS) protocol for the direct (i.e., without prior culture), sensitive and specific detection of Yersinia pestis in complex matrices. Immunoaffinity enables isolation and concentration of intact bacterial cells from food and environmental samples. After protein extraction and digestion, suitable proteotypic peptides corresponding to three Y. pestis-specific protein markers (murine toxine, plasminogen activator and pesticin) are monitored by targeted LC-MS/MS using the selected reaction monitoring (SRM) mode. This immuno-LC-MS/MS assay has a limit of detection of 2 × 10 CFU/mL in milk or tap water, and 4.5 × 10 CFU in 10 mg of soil.

摘要

我们描述了一种免疫亲和-液相色谱-串联质谱（immuno-LC-MS/MS）方法，用于在复杂基质中直接（即无需预先培养）、灵敏且特异检测鼠疫耶尔森菌。免疫亲和能够从食品和环境样本中分离并浓缩完整的细菌细胞。蛋白质提取和消化后，使用选择反应监测（SRM）模式通过靶向LC-MS/MS监测对应于三种鼠疫耶尔森菌特异性蛋白质标志物（鼠毒素、纤溶酶原激活剂和鼠疫菌素）的合适的蛋白型肽段。这种免疫-LC-MS/MS检测方法在牛奶或自来水中的检测限为2×10 CFU/mL，在10 mg土壤中的检测限为4.5×10 CFU。