Expanding CyanoHAB Monitoring: New Micropeptins and Generalizable MS/MS Workflows for the Annotation of Cyanopeptide Classes

ABSTRACT Cyanobacterial harmful algal blooms (cyanoHABs) are a major ecological and public health concern, commonly monitored for hepatotoxic microcystins and cylindrospermopsins and neurotoxic anatoxins and saxitoxins. However, the broader suite of bioactive metabolites produced during blooms remains under characterized. Here, we interrogated a chromatography fraction library generated from a cyanoHAB in Muskegon, Michigan. From this library, we isolated two new micropeptins ( 1 and 2 ), including an analog bearing a bishomologated tyrosine residue, and we confirmed the structure of ferintoic acid C ( 3 ). Structures were established using complementary spectrometric and spectroscopic methods. To expand chemical space coverage beyond isolated compounds, we analyzed LC-MS/MS data using the GNPS2 Analysis Hub query language for product ion searching, enabling annotation of cyanopeptide classes and class-specific modifications across the fraction set, which provided a practical and user-friendly approach for identifying cyanopeptide classes. One of the new micropeptins ( 1 ) exhibited moderate inhibition of neutrophil elastase, consistent with roles in ecological interactions and potential relevance to human exposure. Analysis of field samples from ongoing Lake Erie blooms showed recurring micropeptins but no evidence of microcystins. Together, these results challenge microcystin-centric assessments of bloom hazard and support expanded monitoring of non-microcystin cyanopeptides. SYNOPSIS Routine cyanoHAB monitoring targets few regulated toxins; we reveal abundant, under characterized cyanopeptides and enable rapid class-level annotation across datasets with a new LC-MS/MS analysis pipeline. GRAPHICAL ABSTRACT