Identification of six new Alternaria sulfoconjugated metabolites by high-resolution neutral loss filtering.
BACKGROUND
Many species of Alternaria damage important agricultural crops, including small grains and tomatoes. These fungi can produce a variety of secondary metabolites, some of which are toxic to humans and animals. Interest in screening for conjugated or 'modified' mycotoxins has increased because of their tendency to evade traditional analytical screening methods. Two sulfoconjugated Alternaria toxins have been reported and the potential exists for many more.


METHODS
One hundred and forty-eight Canadian strains of Alternaria spp., about half of them isolated from grain, were grown on Potato Dextrose Agar in Petri dishes for 7 days. Plugs of each strain were removed, extracted and screened by a rapid liquid chromatography (LC)/data-dependent tandem mass spectrometry (MS(2)) method in negative electrospray ionization mode. Data generated on an Orbitrap Q-Exactive mass spectrometer was processed by post-acquisition neutral loss filtering (NLF). Seven isolates that produced sulfoconjugates of known Alternaria toxins were selected for growth on three additional types of fermentation media.


RESULTS
Collision-induced dissociation of sulfoconjugated ions displayed a distinctive neutral loss of SO3 (79.957 Da) that was detected in the MS(2) datasets using post-acquisition NLF. A total of 108 of the 148 isolates screened produced sulfoconjugated metabolites on agar plates. Analysis of the seven isolates grown in liquid culture, on rice and Cheerios, led to the discovery of six new, two previously reported and 30 unidentified sulfoconjugated compounds.


CONCLUSIONS
NLF of HRMS(2) data from an Orbitrap Q-Exactive is a powerful tool for the rapid discovery of sulfoconjugated fungal metabolites. This technique could also be applied to the detection of other important conjugated mycotoxins such as glucosides. The majority of the Canadian isolates of Alternaria spp. studied produced sulfoconjugated metabolites, some of which had no known 'free' Alternaria precursor metabolite, indicating that they are possibly new metabolites. The advantage of sulfoconjugation to Alternaria spp. is unknown, and warrants further study into the mechanisms behind the sulfur assimilatory pathways.
Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, ON, N5V 4T3, Canada.|Department of Chemistry, University of Western Ontario, London, ON, N6A 5B7, Canada.
Megan J
MJ
Kelman
Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, ON, N5V 4T3, Canada.
Justin B
JB
Renaud
Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, Ottawa, ON, K1A 0C6, Canada.
Keith A
KA
Seifert
Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, Ottawa, ON, K1A 0C6, Canada.
Jonathan
J
Mack
Canadian Grain Commission, Grain Research Laboratory, Winnipeg, MB, R3C 3G8, Canada.
Kumaran
K
Sivagnanam
Department of Chemistry, University of Western Ontario, London, ON, N6A 5B7, Canada.|Department of Biochemistry, University of Western Ontario, London, ON, N6A 5C1, Canada.
Ken K-C
KK
Yeung
Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, ON, N5V 4T3, Canada.|Department of Chemistry, University of Western Ontario, London, ON, N6A 5B7, Canada.
Mark W
MW
Sumarah
D000528:Alternaria / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002853:Chromatography, Liquid; D002523:Edible Grain / Q000382:microbiology*; D055442:Metabolome; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013457:Sulfur Compounds / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D013457:Sulfur Compounds