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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Mycotoxin Prevention and Applied Microbiology Research » Research » Publications at this Location » Publication #315924
Title: Analysis of carbohydrates in Fusarium verticillioides using size-exclusion HPLC – DRI and direct analysis in real time ionization – time-of-flight – mass spectrometry (DART-MS)

Author
item ROBERTS, ETHAN - Bradley University
item BOUDREAU, BETH - Bradley University
item Brown, Daren
item MCQUADE, KRISTI - Bradley University
item REMSEN, EDWARD - Bradley University

Submitted to: Analytical Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/3/2015
Publication Date: 1/21/2016
Publication URL: http://handle.nal.usda.gov/10113/62354
Citation: Roberts, E.S., Boudreau, B.A., Brown, D.W., McQuade, K.L., Remsen, E.E. 2016. Analysis of carbohydrates in Fusarium verticillioides using size-exclusion HPLC – DRI and direct analysis in real time ionization – time-of-flight – mass spectrometry (DART-MS). Analytical Methods. 8(3):673-681.

Interpretive Summary: A technique was developed to determine levels of different sugars from Fusarium verticillioides.Sugars enhance tolerance to osmotic, heat and cold stresses, and desiccation, in part, by increasing the stability of proteins and membranes. Often, multiple assays are used to characterize different sugars present in the same extract. The developed analytical technique is based on direct analysis in real time ionization in conjunction with time-of-flight mass spectrometry. The procedure allows for the qualitative and quantitative analysis of multiple sugars in a complex biological matrix with minimal sample preparation. This study verified that the trehalose synthase mutant strain of F. verticillioides does not accumulate any trehalose. This development will aid plant pathologist, mycologists, and other scientists in the further characterization of the role of the sugar trehalose in the F. verticillioides response to stress events and in the development of new strategies to limit mycotoxin contamination in crop plants.

Technical Abstract: Direct analysis in real time ionization – time-of-flight – mass spectrometry (DART-MS) and size-exclusion HPLC – DRI are used, respectively, to qualitatively and quantitatively determine the carbohydrates extracted from the corn rot fungus Fusarium verticillioides. In situ permethylation in the DART beam forms tri-methylammonium adducts of the carbohydrates, which enables positive ion MS detection and analysis of these compounds in either whole fungal extracts or size-exclusion separated HPLC fractions of the extracts. This method detects the disaccharide trehalose at concentrations greater than 3.0 ppm. Additionally, this procedure allows for analysis of glucose as well as the sugar alcohols mannitol, arabitol, and glycerol. The reliability of the DART-MS method is confirmed by the absence of trehalose in a mutant strain of F. verticillioides lacking the gene for trehalose-6-phosphate synthase. The capabilities demonstrated here suggest that the DART-MS method, in conjunction with size-exclusion HPLC, is an effective approach for the analysis of saccharide biomarkers in fungi.