UV-MALDI mass spectrometry analysis of AAL toxin TA

PATRIARCA, ANDREA; ERRA-BALSELLS, R.; GIUDICESSI, S.; TERMINIELLO, L.; VAQUERA, S.; FERNÁNDEZ PINTO, V.

Mendoza

Conferencia; MycoRed ISM conference; 2011

International Society for Mycotoxicology

AAL toxins belong to the sphinganine-analog mycotoxins (SAMT) due to their structural similarity to sphinganine. SAMT execute their toxicity through the competitive inhibiton of sphinganine N-acetyltransferase. The inhibition of this enzyme leads to various diseases in animals and humans.  AAL toxins are produced by Alternaria arborescens and are the causal agent of stem-canker disease in tomatos. Assessment of toxicological properties and health risks posed by AAL toxins as well as detailed investigations into their biosynthesis require appropriate methods for screening and structural confirmation. The ultraviolet matrix-assisted laser/desorption ionization time of flight mass spectrometry (UV-MALDI-TOF MS) instrumentation is a technique that enables a highly sensitive and rapid analysis of complex native mixtures obtained from cells and tissues. The objective of this work is to determine if UV-MALDI-TOF MS is an appropriate method for screening and confirmation of AAL toxins in fungal cultures. A representative culture of A. arborescens EGS 39-128 and two native A. arborescens strains isolated from tomato ITEM 8162 and ITEM 8134, were used. For mycotoxin production, the  strains were inoculated in autoclaved rice and incubated at 25ºC for 21 days. The extraction and quantification of AAL toxins were made according to Solfrizzo et al 2005. Samples of AAL TA standard and products extracted from the fungal cultures were analyzed by UV-MALDI-MS performed on the Ultraflex II TOF/TOF Bruker Daltonics mass spectrometer. Matrix selection was performed and the best results were obtained with 9H-[3,4-b]piridoindole (nHo; norharmane) in negative ion  mode and  2,5-dihydroxibenzoic acid (DHB) in a  positive ion mode. ESI-MS analyses were performed using a Bruker micrOTOF-Q II mass spectrometer.The UV-MALDI-TOF spectrum of the AAL TA standard revealed a molecular ion [M + H]+ at m/z 522, whereas the adducts [M + Na]+ at m/z 544 and [M + K]+ at m/z 560 were also observed with high signal intensity. The same peaks were observed in ESI spectrum. The main fragments of AAL TA showed high intensity and a reproducible pattern: [M + H – H2O]+ at m/z 504, [M + H – 2 H2O]+ at m/z 486, [M + H – TCA]+ at m/z 346, [M + H – TCA – H2O]+ at m/z 328, [M + H – TCA – 2 H2O]+ at m/z 310, [M + H – TCA – 3 H2O]+ at m/z 292, [M + H – TCA – 4 H2O]+ at m/z 274.  EGS 39-128 and ITEM 8162  cultures of A. arborescens yielded a MALDI-TOF and ESI spectra similar to that observed for the AAL TA standard. For the native culture ITEM 8134 no one of the characteristic ions was observed on both the UV-MALDI-TOF and ESI spectra. It could be concluded from the present results that UV-MALDI-TOF is an adequate method for screening and confirmation of AAL toxins from fungal culture extracts. It represents a better option to chromatographic techniques that require complex and laborious derivatization of the toxins and it would be a good complement to HPLC-ESI-MS/MS methods.