Detection of anatoxin-a and three analogs in Anabaena spp. cultures: new fluorescence polarization assay and toxin profile by LC-MS/MS.
Anatoxin-a (ATX) is a potent neurotoxin produced by several species of Anabaena spp. Cyanobacteria blooms around the world have been increasing in recent years; therefore, it is urgent to develop sensitive techniques that unequivocally confirm the presence of these toxins in fresh water and cyanobacterial samples. In addition, the identification of different ATX analogues is essential to later determine its toxicity. In this paper we designed a fluorescent polarization (FP) method to detect ATXs in water samples. A nicotinic acetylcholine receptor (nAChR) labeled with a fluorescein derivative was used to develop this assay. Data showed a direct relationship between the amount of toxin in a sample and the changes in the polarization degree of the emitted light by the labeled nAChR, indicating an interaction between the two molecules. This method was used to measure the amount of ATX in three Anabaena spp. cultures. Results indicate that it is a good method to show ATXs presence in algal samples. In order to check the toxin profile of Anabaena cultures a LC-MS/MS method was also developed. Within this new method, ATX-a, retention time (RT) 5 min, and three other molecules with a mass m/z 180.1 eluting at 4.14 min, 5.90 min and 7.14 min with MS/MS spectra characteristic of ATX toxin group not previously identified were detected in the Anabaena spp. cultures. These ATX analogues may have an important role in the toxicity of the sample.
Department of Pharmacology, Veterinary School, University of Santiago de Compostela, Lugo 27002, Spain. jonandoni.sanchez@rai.usc.es.
Jon A
JA
Sanchez
Department of Pharmacology, Veterinary School, University of Santiago de Compostela, Lugo 27002, Spain. mariapaz.otero@rai.usc.es.
Paz
P
Otero
Department of Pharmacology, Veterinary School, University of Santiago de Compostela, Lugo 27002, Spain. amparo.alfonso@usc.es.
Amparo
A
Alfonso
Department of Biology, Faculty of Sciences, University of Porto, Rua do Campo Alegre, Porto 4619-007, Portugal. vtr.rms@gmail.com.
Vitor
V
Ramos
Department of Biology, Faculty of Sciences, University of Porto, Rua do Campo Alegre, Porto 4619-007, Portugal. vmvascon@fc.up.pt.
Vitor
V
Vasconcelos
CNRS, Institut de Neurobiologie Alfred Fessard-FRC2118, Laboratoire de Neurobiologie et Développement-UPR3294, 1 Avenue de la Terrasse, Gif sur Yvette Cedex 91198, France. araoz@inaf.cnrs-gif.fr.
Romulo
R
Aráoz
CNRS, Institut de Neurobiologie Alfred Fessard-FRC2118, Laboratoire de Neurobiologie et Développement-UPR3294, 1 Avenue de la Terrasse, Gif sur Yvette Cedex 91198, France. Jordi.Molgo@inaf.cnrs-gif.fr.
Jordi
J
Molgó
Department of Physiology, Veterinary School, University of Santiago de Compostela, Lugo 27002, Spain. mmercedes.rodriguez@usc.es.
Mercedes R
MR
Vieytes
Department of Pharmacology, Veterinary School, University of Santiago de Compostela, Lugo 27002, Spain. luis.botana@usc.es.
Luis M
LM
Botana
D017033:Anabaena; D000818:Animals; D001681:Biological Assay; D002853:Chromatography, Liquid; D000087522:Cyanobacteria Toxins; D005454:Fluorescence Polarization; D009498:Neurotoxins / Q000032:analysis* / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014101:Torpedo; D014326:Tropanes / Q000032:analysis* / Q000378:metabolism
D000087522:Cyanobacteria Toxins; D009498:Neurotoxins; D011978:Receptors, Nicotinic; D014326:Tropanes; C509783:anatoxin a