Combined LC-MS/MS and Molecular Networking Approach Reveals New Cyanotoxins from the 2014 Cyanobacterial Bloom in Green Lake, Seattle.

Cyanotoxins obtained from a freshwater cyanobacterial collection at Green Lake, Seattle during a cyanobacterial harmful algal bloom in the summer of 2014 were studied using a new approach based on molecular networking analysis of liquid chromatography tandem mass spectrometry (LC-MS/MS) data. This MS networking approach is particularly well-suited for the detection of new cyanotoxin variants and resulted in the discovery of three new cyclic peptides, namely microcystin-MhtyR (6), which comprised about half of the total microcystin content in the bloom, and ferintoic acids C (12) and D (13). Structure elucidation of 6 was aided by a new microscale methylation procedure. Metagenomic analysis of the bloom using the 16S-ITS rRNA region identified Microcystis aeruginosa as the predominant cyanobacterium in the sample. Fragments of the putative biosynthetic genes for the new cyanotoxins were also identified, and their sequences correlated to the structure of the isolated cyanotoxins.