Novel Microcystins from Planktothrix prolifica NIVA-CYA 544 Identified by LC-MS/MS, Functional Group Derivatization and 15N-labeling

Abstract

Microcystins are cyclic heptapeptides from cyanobacteria that are potent inhibitors of protein phosphatases and are toxic to animals and humans. At present, more than 250 microcystin variants are known, with variants reported for all seven peptide moieties. While d-glutamic acid (d-Glu) is highly-conserved at position-6 of microcystins, there has been only one report of a cyanobacterium (Anabaena) producing microcystins containing l-Glu at the variable 2- and 4-positions. Liquid chromatography–mass spectrometry analyses of extracts from Planktothrix prolifica NIVA-CYA 544 led to the tentative identification of two new Glu-containing microcystins, [d-Asp3]MC-ER (12) and [d-Asp3]MC-EE (13). Structure determination was aided by thiol derivatization of the Mdha7-moiety and esterification of the carboxylic acid groups, while 15N-labeling of the culture and isotopic profile analysis assisted the determination of the number of nitrogen atoms present and the elemental composition of molecular and product-ions. The major microcystin analog in the extracts was [d-Asp3]MC-RR (1). A microcystin with an unprecedented high-molecular-mass (2116 Da) was also detected and tentatively identified as a sulfide-linked conjugate of [d-Asp3]MC-RR (15) by LC–HRMS/MS and sulfide oxidation, together with its sulfoxide (16) produced via autoxidation. Low levels of [d-Asp3]MC-RW (14), [d-Asp3]MC-LR (4), [d-Asp3,Mser7]MC-RR (11), [d-Asp3]MC-RY (17), [d-Asp3]MC-RF (18), [d-Asp3]MC-RR–glutathione conjugate (19), and [d-Asp3]MC-RCit (20), the first reported microcystin containing citrulline, were also identified in the extract, and an oxidized derivative of [d-Asp3]MC-RR and the cysteine conjugate of 1 were partially characterized.

Structural Diversity, Characterization and Toxicology of Microcystins

“…), but when the possible elemental composition and ring or double bond equivalents (RDBE) are limited to a reasonable range similar to known MCs, in most cases a candidate formula can be determined. Careful evaluation of isotopic patterns in HRMS can further help narrow down possible molecular formulae, relying less on pre-determined limits in elements, as reported recently in Mallia et al [99]. Known parent (unconjugated) MCs have between 44 and 57 C atoms, 63 and 84 H atoms, 7 and 13 N atoms, 12 and 17 O atoms, and up to one S atom in methionine-containing MCs [53].…”

“…However, when combined with modern LC-MS, similar approaches are even more powerful due to the ease with which multiple products of reactions with multiple reactants can be selectively detected. The reactivity of the double bond in Dha 7 and Mdha 7 of MCs towards thiols has emerged as being particularly useful for characterizing MCs congeners [81,82,99]. In particular, mercaptoethanol derivatization has been exploited in such a manner both to identify new MCs from complex samples [77,108] or to quickly differentiate Mdha 7 -from Dhb 7 -containing MCs [109], which is not possible using LC-MS alone unless authentic standards are available.…”

“…In particular, mercaptoethanol derivatization has been exploited in such a manner both to identify new MCs from complex samples [77,108] or to quickly differentiate Mdha 7 -from Dhb 7 -containing MCs [109], which is not possible using LC-MS alone unless authentic standards are available. Another useful reaction is methyl esterification that, together with LC-MS, can be used to count the number of reactive carboxylic acid (the CO 2 H group of Glu 6 is reactive, but not that of Masp 3 /Asp 3 ) and phenolic groups in MCs [96,99]. Selective oxidation with mild oxidants can also be combined with LC-MS to identify MCs containing sulfide and sulfoxides, such as methionine (e.g., MC-MR) [81,93].…”

“…However, cyanobacteria are known to produce both Cys and GSH [198], and low levels of GSH conjugates of MCs have been detected in several cyanobacterial cultures using untargeted LC-HRMS methods (D. G. Beach and C. O. Miles, unpublished observations; [99]). GSH conjugates can be further metabolized to γ-GluCys and Cys conjugates [199], so it is possible that the γ-GluCys and Cys conjugates observed from cyanobacteria are produced via this route or by direct conjugation of MCs to these smaller peptides.…”

Cylindrospermopsin- and Deoxycylindrospermopsin-Producing Raphidiopsis raciborskii and Microcystin-Producing Microcystis spp. in Meiktila Lake, Myanmar

“…Underivatized samples were analysed by LC-HRMS/MS in positive and negative ionisation modes as previously described [12,13] and then after reaction with mercaptoethanol (targeting Mdha 7 /Dha 7 moieties in MCs) [14] and Oxone/DMSO (targets sulfide groups in methionine and Cys/GSH conjugates of MCs) [15]. Results of these analyses are summarised in Figure 5, Table 4 and Table S1.…”