Sulfide Oxidations for LC-MS Analysis of Methionine-Containing Microcystins in Dolichospermum flos-aquae NIVA-CYA 656

Abstract

Microcystins are cyclic heptapeptides produced by a range of cyanobacteria. More than 150 microcystin analogues have been reported from cultures, algal blooms, or other contaminated samples. Relatively few analytical standards are available, making identification and quantitation of these toxins a challenge, even with LC-MS technology. We developed a two-step oxidative procedure that allows LC-MS identification of microcystins containing methionine and methionine sulfoxide, and reveals the oxidation state of the methionyl sulfur atom. The procedure was used in parallel with mercaptoethanol derivatization and LC-MS(2) analysis to demonstrate the presence of [Asp(3)]MC-MR (12) and MC-MR (17) in a culture of Dolichospermum flos-aquae, together with low levels of [Asp(3)]MC-M(O)R (5) and MC-M(O)R (7), as well as 20 other microcystins. Fresh culture contained only traces of sulfoxides 5 and 7, but these increased during storage or sample extraction and preparation. This suggests that microcystins containing methionine sulfoxide are primarily postextraction oxidation artifacts, rather than being produced by biosynthesis in cyanobacteria. A simple, rapid extraction under inert gas followed promptly by LC-MS analysis minimized oxidation artifacts for D. flos-aquae.

Glutathione-Conjugates of Deoxynivalenol in Naturally Contaminated Grain Are Primarily Linked via the Epoxide Group

“…Oxidation with hydrogen peroxide followed by LC-MS analysis may thus be applied as a simple technique to verify putative DON–thiol conjugates in complex mixtures, an approach analogous to that developed for identifying Met-containing peptides in cyanobacterial blooms [12]. Addition of hydrogen peroxide to an extract from Norwegian oats oxidized approximately 90% of the DON-13-GSH to the corresponding sulfoxides (DON-13-GS(O)H) within 90 min (Figure 4).…”

Preparation and Characterization of Cysteine Adducts of Deoxynivalenol

“…Oxidation of the sulfide to a sulfoxide followed by LC−MS/MS could be used to help to confirm the presence of thiol adducts in samples, in a manner similar to that reported for identifying the presence of methionine residues in cyclic peptides. 20 Autoxidation of sulfides to their sulfoxides is a common process occurring in nature, 21 and such compounds could potentially also occur via metabolism.…”

Appendix 3: Tables of Microcystins and Nodularins

“…In some cases, especially with MC-RR and related congeners, the dominant ion observed in mass spectrometry is the doubly protonated ion [M + 2H] 2+ . Acyclo-LR: [28,42,54] 105 [2,3,9] …”