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.
