Discovery of Novel Cyanotoxins by Detection of Their Native Fluorescence and Unique MS/MS Fragments, and Determination of Their Toxicities

Abstract Cyanopeptides are not easily identifiable and quantified in water during harmful cyanobacterial blooms (cHABs) because they are present as multiple congeners and a few of them are available as pure standards. This prevents their identification and purification as well determination of their toxicities and potential to

cause diseases. The goal of this proposal is to apply modern fluorescence and MS methodologies and enable discovery and purification of novel microcystins (MCs), anabaenopeptins (APs), and cyanopeptolins (CPs) for toxicological studies in liver and kidney cell cultures. To accomplish this goal, we will combine methods

for sample preparation of toxic cyanopeptides with liquid chromatography (LC) coupled to fluorescence and tandem mass spectrometry (MS/MS and MSn) detection. The rationale is that the LC separation and sequential detection of cyanotoxins by native fluorescence detector and a high-resolution mass spectrometer

will provide structural characterization of numerous novel MCs, APs, and CPs in samples collected during cHABs in western Lake Erie. The specific aims are: 1. To optimize HPLC native fluorescence detection with an Orbitrap mass spectrometer for structural analyses of MCs, APs, and CPs, 2. To perform structural

analyses, of these cyanopepides in cHAB samples using unique fragments and novel neutral losses during MS/MS, and 3. To purify novel congeners using preparative LC and size exclusion chromatography and study their toxicities in cell culture. Two important scientific questions will be answered: 1) is it possible

to isolate novel MCs, APs and CPs that will serve as cyanotoxin standards for toxicological studies, and 2) what is the relationship between structure of novel congeners and their ability to intoxicate liver and kidney cells? The results of this proposal will also be useful for the expending the databases containing structures

and toxicities of cyanotoxin congeners.