Diagnostic Fragmentation Filtering for Cyanopeptolin Detection

Abstract

Cyanobacteria are ubiquitous photosynthetic prokaryotes that produce structurally diverse bioactive metabolites. Although microcystins are extensively studied, other cyanopeptides produced by common bloom-forming species have received little attention. Cyanopeptolins are a large cyanopeptide group that contain a characteristic 3-amino-6-hydroxy-2piperidone (Ahp) moiety. In the present study we used diagnostic fragmentation filtering (DFF), a semitargeted liquid chromatography-tandem mass spectrometry (MS/MS) product ion filtering approach, to investigate cyanopeptolin diversity from 5 Microcystis strains and 4 bloom samples collected from lakes in Ontario and Quebec, Canada. Data processing by DFF was used to search MS/MS data sets for pairs of diagnostic product ions corresponding to cyanopeptolin partial sequences. For example, diagnostic product ions at m/z 150.0912 and 215.1183 identified cyanopeptolins with the NMe-Tyr-Phe-Ahp partial sequence. Forty-eight different cyanopeptolins, including 35 new variants, were detected from studied strains and bloom samples. Different cyanopeptolin profiles were identified from each sample. We detected a new compound, cyanopeptolin 1143, from a bloom and elucidated its planar structure from subsequent targeted MS/MS experiments. Diagnostic fragmentation filtering is a rapid, easy-to-perform postacquisition metabolomics strategy for inferring structural features and prioritizing new compounds for further study and dereplication. More work on cyanopeptolin occurrence and toxicity is needed because their concentrations in freshwater lakes after blooms can be similar to those of microcystins.

Metabolomics Reveals Strain-Specific Cyanopeptide Profiles and Their Production Dynamics in Microcystis aeruginosa and M. flos-aquae

“…Microbial natural products, including cyanopeptides, are typically biosynthesized as mixtures of structurally related compounds [26]. The tandem mass spectrometry (MS/MS) spectra of chemicals are highly dependent on their molecular structure, meaning groups of structurally related compounds often generate common product ions [27,28]. Post-acquisition data processing approaches using MS/MS datasets acquired with modern high-resolution instruments, such as quadrupole time-of-flight and Orbitrap mass analyzers, can exploit these features of the natural products [28,29].…”

Application of Metabolomics for the Detection and Evaluation of Cyanopeptide Mixtures in Ontario and Quebec Lakes

“…16) subclassifies cyanopeptolin congeners 142 . This substitution remains fairly consistent within the group, making subclassification advantageous in characterizing the vast structural diversity for CPs 52,54,141 . Determination of shared partial amino acid sequences with diagnostic product ions provides further structural subclassification.…”

“…Cyanopeptolins are a group (>230 compounds) of cyclic oligopeptides containing a pentapeptide ring and a diagnostic 3-amino-6-hydroxy-2-piperidone (Ahp) moiety at position 3 (Fig. 7) 141 . Position 2 is commonly occupied by an NMe-aromatic residue such as Phe and Tyr, with possible halogenation of Tyr (Fig.…”

“…Screening and analysis of LC-(HR)MS/MS raw data for characteristic features for chemical groups such as retention time ranges, precursor ion m/z ranges, and diagnostic product ions can effectively filter massive datasets for all metabolites with a particular chemical feature relating to a chemical group or compound.Diagnostic fragmentation filtering (DFF) is one of such techniques, filtering large chemical data sets for identification of multiple diagnostic product ions. DFF is a feature of the open-source mass spectrometry data analysis software, mzMine2 and allows for the identification of diagnostic product ion pairs or combinations which may be tailored to a specific chemical class141 . DFF advantageously uses structural similarities in product ion spectra to indicate structurally similar chemicals141 .…”

“…DFF is a feature of the open-source mass spectrometry data analysis software, mzMine2 and allows for the identification of diagnostic product ion pairs or combinations which may be tailored to a specific chemical class141 . DFF advantageously uses structural similarities in product ion spectra to indicate structurally similar chemicals141 . DFF searches output all metabolites within the large data set that contain specified product ions within their MS/MS.…”