title: Rapid quantification of clostridial epsilon toxin in complex food and biological matrixes by immunopurification and ultraperformance liquid chromatography-tandem mass spectrometry.
abstract: 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·mL(-1) for ETX, making this approach complementary to enzyme linked immunosorbent assay (ELISA) techniques.
authors.affiliation: CEA, iBiTec-S, Service de Pharmacologie et d'Immunoanalyse, 91191 Gif-sur-Yvette, France.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Seyer
authors.forename: François
authors.initials: F
authors.lastname: Fenaille
authors.forename: Cecile
authors.initials: C
authors.lastname: Féraudet-Tarisse
authors.forename: Hervé
authors.initials: H
authors.lastname: Volland
authors.forename: Michel R
authors.initials: MR
authors.lastname: Popoff
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Tabet
authors.forename: Christophe
authors.initials: C
authors.lastname: Junot
authors.forename: François
authors.initials: F
authors.lastname: Becher
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001905:Botulinum Toxins / Q000302:isolation & purification*; D002138:Calibration; D002853:Chromatography, Liquid; D003013:Clostridium / Q000737:chemistry*; D045424:Complex Mixtures / Q000737:chemistry*; D005504:Food Analysis / Q000379:methods*; D007118:Immunoassay; D008892:Milk / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis; D059748:Proteolysis; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014357:Trypsin
chemical_list: D045424:Complex Mixtures; D010455:Peptides; D014357:Trypsin; D001905:Botulinum Toxins