Rapid Quantification of Clostridial Epsilon Toxin in Complex Food and Biological Matrixes by Immunopurification and Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry

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• 作者：Alexandre Seyer ; Fran莽ois Fenaille ; Cecile F茅raudet-Tarisse ; Herv茅 Volland ; Michel R. Popoff ; Jean-Claude Tabet ; Christophe Junot ; Fran莽ois Becher
• 刊名：Analytical Chemistry
• 出版年：2012
• 出版时间：June 5, 2012
• 年：2012
• 卷：84
• 期：11
• 页码：5103-5109
• 全文大小：286K
• 年卷期：v.84,no.11(June 5, 2012)
• ISSN：1520-6882

文摘

Epsilon toxin (ETX) is one of the most lethal toxins produced by Clostridium species and is considered as a potential bioterrorist weapon. Here, we present a rapid mass spectrometry-based method for ETX quantification in complex matrixes. As a prerequisite, naturally occurring prototoxin and toxin species were first structurally characterized by top-down and bottom-up experiments, to identify the most pertinent peptides for quantification. Following selective ETX immunoextraction and trypsin digestion, two proteotypic peptides shared by all the toxin forms were separated by ultraperformance liquid chromatography (UPLC) and monitored by ESI-MS (electrospray ionization-mass spectrometry) operating in the multiple reaction monitoring mode (MRM) with collision-induced dissociation. Thorough protocol optimization, i.e., a 15 min immunocapture, a 2 h enzymatic digestion, and an UPLC-MS/MS detection, allowed the whole quantification process including the calibration curve to be performed in less than 4 h, without compromising assay robustness and sensitivity. The assay sensitivity in milk and serum was estimated at 5 ng路mL^鈥? for ETX, making this approach complementary to enzyme linked immunosorbent assay (ELISA) techniques.