Browsing by Author "Wilkins, A. L."

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Conjugation of microcystins with thiols is reversible: Base-catalyzed deconjugation for chemical analysis
(ACS Publications) Miles, C. O.; Sandvik, M.; Nonga, H. E.; Ballot, A.; Wilkins, A. L.; Rise, F.; Jaabaek, J. A. H.; Loader, J. I.
Conjugation of microcystins with thiols is reversible: Base-catalyzed deconjugation for chemical analysis
(Chemical Research in Toxicology) Miles, C. O.; Sandvik, M.; Nonga, H. E.; Ballot, A.; Wilkins, A. L.; Rise, F.; Jaabaek, J. A. H.; Loader, J. I.
Conjugation of microcystins with thiols is reversible: Base-catalyzed deconjugation for chemical analysis
(ACS Publications, 2016-03-21) Miles, C. O.; Sandvik, M.; Nonga, H. E.; Ballot, A.; Wilkins, A. L.; Rise, F.; Jaabaek, J. A. H.; Loader, J. I.
Microcystins are potent cyclic heptapeptide toxins found in many freshwater cyanobacteria. Most microcystins contain an α,β-unsaturated amide that can react with thiolcontaining amino acids, peptides, and proteins in vivo and in vitro. While soluble conjugates formed from small peptides can be extracted and analyzed directly by LC−MS, microcystins conjugated to proteins are analyzed after oxidative cleavage of their Adda side chains, but information on which microcystin analogues were present is lost. Observations during the development of thiol-derivatization-based LC−MS methods for microcystin analysis indicated that the reaction of thiols with microcystins was reversible. The kinetics of deconjugation was investigated with mercaptoethanol as a model thiol to identify suitable reaction conditions. A range of microcystins conjugated to mercaptoethanol, methanethiol, cysteine, and glutathione were then successfully deconjugated, demonstrating the feasibility of releasing conjugated forms of microcystins for chemical analysis. Reagents for removing the released thiols or for trapping the released microcystins increased the reaction rate. Optimization of methodologies based on this reaction should increase the method’s utility for measuring free and conjugated microcystins. The results also indicate that thiol-conjugated microcystins slowly release free microcystins, even at neutral pH, with consequences for assessment of toxin exposure, metabolism, and trophic transfer. A range of other common natural and environmental toxins, such as deoxynivalenol and acrylamide, also contain α,β-unsaturated carbonyl groups and can be expected to behave in a similar manner.
Identification of microcystins in a Lake Victoria cyanobacterial bloom using LC–MS with thiol derivatization
(Elsevier) Miles, C. O.; Sandvik, H. E.; Rundberget, T.; Wilkins, A. L.; Rise, F.; Ballot, A.
Thiol Derivatization for LC-MS Identification of Microcystins in Complex Matrices
(ACS Publications) Miles, C. O.; Sandvik, M.; Nonga, H. E.; Rundberget, T.; Wilkins, A. L.; Rise, F.; Ballot, A.
Thiol Derivatization for LC-MS Identification of Microcystins in Complex Matrices
(ACS Publications, 2012-07-18) Miles, C. O.; Sandvik, M.; Nonga, H. E.; Rundberget, T.; Wilkins, A. L.; Rise, F.; Ballot, A.
Microcystins are a group of cyclic heptapeptides originating from cyanobacteria. Cyanobacteria also produce a range of peptides and other compounds that can result in complex chromatograms when samples are analyzed by LC-MS. Derivatization with appropriate thiols (e.g., mercaptoethanol) of the olefin in the α,β-unsaturated amide present in most microcystins was shown to simplify analysis of LC-MS chromatograms of sample extracts, making it much easier to identify peaks corresponding to candidate microcystins. Furthermore, interpretation of MS2 spectra was facilitated by addition of the mass associated with the thiol to the α,β-unsaturated amide of microcystins. Cyanotoxins containing Mdha or Dha reacted readily with thiols, whereas Mser, Ser, Mdhb, and thiol-derivatives of Mdha or Dha did not react under the conditions used. This approach therefore provides a convenient LC-MS method to obtain evidence for the presence of Mdha or Dha and can likely be used to differentiate between the isobaric amino acids Mdha and Dhb in candidate cyanotoxin peaks. When O-(2-mercaptoethyl)-O′-methyl-hexa(ethylene glycol) (MEMHEG) (Mwt. 356) was used as the thiol, the resulting derivatives eluted in an LC-MS mass window that was largely free of interferences. This approach simplifies detection of candidate microcystin analogues even in the presence of complex mixtures of coeluting components. The method was used for qualitative analysis of a Microcystis aeruginosa culture from Lake Naivasha, Kenya, and the results were verified using precursor-ion scanning and high-resolution mass spectrometry.