A Data-Independent Methodology for the Structural Characterization of Microcystins and Anabaenopeptins Leading to the Identification of Four New Congeners

Abstract

Toxin-producing cyanobacteria are responsible for the presence of hundreds of bioactive compounds in aquatic environments undergoing increasing eutrophication. The identification of cyanotoxins is still emerging, due to the great diversity of potential congeners, yet high-resolution mass spectrometry (HRMS) has the potential to deepen this knowledge in aquatic environments. In this study, high-throughput and sensitive on-line solid-phase extraction ultra-high performance liquid chromatography (SPE-UHPLC) coupled to HRMS was applied to a data-independent acquisition (DIA) workflow for the suspect screening of cyanopeptides, including microcystin and anabaenopeptin toxin classes. The unambiguous characterization of 11 uncommon cyanopeptides was possible using a characterization workflow through extensive analysis of fragmentation patterns. This method also allowed the characterization of four unknown cyanotoxins ([Leu1, Ser7] MC-HtyR, [Asp3]MC-RHar, AP731, and AP803). The quantification of 17 common cyanotoxins along with the semi-quantification of the characterized uncommon cyanopeptides resulted with the identification of 23 different cyanotoxins in 12 lakes in Canada, United Kingdom and France. The concentrations of the compounds varied between 39 and 41,000 ng L−1. To our knowledge, this is the first DIA method applied for the suspect screening of two families of cyanopeptides simultaneously. Moreover, this study shows the great diversity of cyanotoxins in lake water cyanobacterial blooms, a growing concern in aquatic systems.

Insight into Unprecedented Diversity of Cyanopeptides in Eutrophic Ponds Using an MS/MS Networking Approach

“…Similarly, numerous reports have been published, where molecular networking is employed to track changes in secondary metabolic profiles, including MCs and other peptides [ 46 , 47 ]. Fragmentation spectra of unknown variants discovered for APTs and MCs using molecular networking have been further manually curated for the identification of diagnostic ion peak [ 45 , 48 , 49 , 50 , 51 , 52 ].…”

“…However, we reported here their putative presence based on dereplication. Further, mass spectra of these putative new variants were evaluated for the presence of characteristic ion peak at m/z 84.081 (an immonium fragment ion of lysine) together with other fragment ions of amino acids ( Figures S2 and S3 ) [ 45 , 49 , 50 ]. The recent increased reports of APTs co-occurrence with MCs raise the attention to this class of CNPs since their impact on the cohabiting aquatic organisms remains unclear, and their ecological role is uncertain [ 46 ].…”

Absence of Cyanotoxins in Llayta, Edible Nostocaceae Colonies from the Andes Highlands

“…DMAdda (m/z 121.06) and ADMAdda (m/z 163.08) specific fragment ions were also absent. The absence of all those reported [ 15 , 16 ] characteristic fragments confirms the safety of Llayta as a food ingredient with respect to cyanobacteria toxins.…”

Ultra-Trace Analysis of Cyanotoxins by Liquid Chromatography Coupled to High-Resolution Mass Spectrometry

“…DIA is useful for non-targeted screening or suspect screening with a long list of suspects. DIA mode was recently applied by Roy-Lachapelle et al [25] for the determination of microcystins. In our case, we focused on 10 commonly found cyanotoxins.…”

“…High and ultra-high performance liquid chromatography coupled with mass spectrometry (HPLC-MS, UHPLC-MS) and tandem mass spectrometry (MS/MS) are the techniques of choice, in particular with triple quadrupole (QqQ) analyzers [11,[15][16][17][18]. However, due to the advances in identification and sensitivity, several HRMS methods have been applied for the determination of cyclic peptides [19][20][21] and multi-class cyanotoxins [22][23][24][25] in freshwater. These methods present limits of detection from 0.3 ng/L to 3900 ng/L, being 0.3-5.6 ng/L the lowest ones reported by Greer et al [18] by UHPLC-MS/MS.…”