Characterization of nodularin variants in Nodularia spumigena from the Baltic Sea using liquid chromatography/mass spectrometry/mass spectrometry

Nodularin is a potent hepatotoxic cyclic pentapeptide produced by planktonic cyanobacterium Nodularia spumigena. Bloom and culture samples of the cyanobacterium collected and isolated from the Gulf of Gdańsk, southern Baltic Sea, were analyzed. Hybrid quadrupole-time-of-flight liquid chromatography/mass spectrometry/mass spectrometry (TOF-LC/MS/MS) with ionspray (ISP) and collision-induced dissociation (CID) were used to characterize nodularin and its analogues. The identification process was based on the comparison of recorded product ion spectra with the previously reported FAB-MS/CID (high-energy) mass spectra of the corresponding nodularin variants. Amino acid structures and sequences were derived from the fragmentation pattern of the [M+H]⁺ ions. Apart from unmodified nodularin with an arginine residue (NOD-R), three demethylated variants have been found. The sites of demethylation were located on aspartic acid [Asp¹]NOD, the Adda residue [DMAdda³]NOD, and dehydrobutyric acid [dhb⁵]NOD. In two other nodularin variants an additional methyl group is located in the Adda [MeAdda]NOD and Glu [Glu⁴(OMe)]NOD residues. The linear NOD and the geometrical isomer of NOD-R, reported earlier in N. spumigena from New Zealand, have also been detected. Two of the total eight nodularin variants characterized in the present study, [dhb⁵]NOD and [MeAdda]NOD, have not been described earlier.