336
M. Vincenti . A. Irico
and the daughter-ion MS-MS spectra of both original and alkylated conopeptides revealed the position of disulfide-bound cysteines. Figure 17.10b shows the ESI-MS-MS
spectrum of alkylated conotoxin and the corresponding interpretation scheme. A different group also used ESI-MS-MS spectra of singly- and multiply-charged conotoxin
molecular ions to characterize the amino acidic sequence of Conus venom components,
without LC separation (Krishnamurthy et al. 1996).
In a more recent study (Eluan Kalume et al. 1998), eleven conotoxins have been investigated in detail, some of which show a large number of post-translationally modified amino acids. Among these, the presence of glycosylic groups has been detected
for the first time in conotoxins. From the isotopic pattern of both conotoxin molecular ions obtained by ESI-MS, and ESI-MS-MS fragment ions obtained upon cm in an
ion-trap mass spectrometer, it has been possible to distinguish the number and position of brominated triptophane residues in six conotoxins. In general, mass spectrometric data were used to complement the gaps present in amino acid sequence data
obtained by Edman degradation, and arising from post-translational modifications.
17.7
Conclusions
The examples of LC-ESI-MS characterization of marine toxins reported so far clearly
indicate the rapidly increasing involvement of such instrumentation in a field previously dominated by biological and biochemical assays. ESI-MS is extremely sensitive
and selective in the same time, so that its on-line combination with a powerful separation method such as LC allows one to undertake the complex task of attributing
the toxicological properties of a toxin mixture to each individual component. From
this analysis, revealing the complex nature and variability of most seafood intoxications, it is possible to set the basis for understanding and preventing these syndromes.
However, the potentiality of chromatographic and MS techniques does not appear
to be fully exploited in toxin analysis at the moment. For example, the combination of
capillary electrophoresis with ESI-MS is likely to be more extensively utilized in the
future, especially for polar and intrinsically-charged toxins. More striking is the relatively infrequent application of MS-MS methods on a routine basis, i.e. to achieve a
higher degree of selectivity in quantitative determinations. Tandem mass spectrometric
techniques are likely to have an even higher impact in the structural characterization
of unknown toxins. The availability of LC-ESI-ion-trap mass spectrometers from at
least two suppliers will favor the diffusion of these instruments capable of performing multiple MS (MS H ). Thus, the molecular ion of an unknown toxin could be massselected and then fragmented, producing a secondary spectrum. If this secondary spectrum is still not sufficient for complete structural elucidation, then the fragment ions
could be mass-selected and fragmented further until structural features and isomeric
distinctions are singled out.
In conclusion, it can be foreseen that LC-ESI-MS and LC-ESI-MS-MS will increasingly complement NMR techniques in the structural elucidation of marine toxins and
will substitute them in those cases when limited pure toxin availability and presence
of impurities prevents the application of NMR. For the routine determination of known
toxins in raw seafood materials, LC-ESI-MS will probably not replace the fast and sen-
M. Vincenti . A. Irico
and the daughter-ion MS-MS spectra of both original and alkylated conopeptides revealed the position of disulfide-bound cysteines. Figure 17.10b shows the ESI-MS-MS
spectrum of alkylated conotoxin and the corresponding interpretation scheme. A different group also used ESI-MS-MS spectra of singly- and multiply-charged conotoxin
molecular ions to characterize the amino acidic sequence of Conus venom components,
without LC separation (Krishnamurthy et al. 1996).
In a more recent study (Eluan Kalume et al. 1998), eleven conotoxins have been investigated in detail, some of which show a large number of post-translationally modified amino acids. Among these, the presence of glycosylic groups has been detected
for the first time in conotoxins. From the isotopic pattern of both conotoxin molecular ions obtained by ESI-MS, and ESI-MS-MS fragment ions obtained upon cm in an
ion-trap mass spectrometer, it has been possible to distinguish the number and position of brominated triptophane residues in six conotoxins. In general, mass spectrometric data were used to complement the gaps present in amino acid sequence data
obtained by Edman degradation, and arising from post-translational modifications.
17.7
Conclusions
The examples of LC-ESI-MS characterization of marine toxins reported so far clearly
indicate the rapidly increasing involvement of such instrumentation in a field previously dominated by biological and biochemical assays. ESI-MS is extremely sensitive
and selective in the same time, so that its on-line combination with a powerful separation method such as LC allows one to undertake the complex task of attributing
the toxicological properties of a toxin mixture to each individual component. From
this analysis, revealing the complex nature and variability of most seafood intoxications, it is possible to set the basis for understanding and preventing these syndromes.
However, the potentiality of chromatographic and MS techniques does not appear
to be fully exploited in toxin analysis at the moment. For example, the combination of
capillary electrophoresis with ESI-MS is likely to be more extensively utilized in the
future, especially for polar and intrinsically-charged toxins. More striking is the relatively infrequent application of MS-MS methods on a routine basis, i.e. to achieve a
higher degree of selectivity in quantitative determinations. Tandem mass spectrometric
techniques are likely to have an even higher impact in the structural characterization
of unknown toxins. The availability of LC-ESI-ion-trap mass spectrometers from at
least two suppliers will favor the diffusion of these instruments capable of performing multiple MS (MS H ). Thus, the molecular ion of an unknown toxin could be massselected and then fragmented, producing a secondary spectrum. If this secondary spectrum is still not sufficient for complete structural elucidation, then the fragment ions
could be mass-selected and fragmented further until structural features and isomeric
distinctions are singled out.
In conclusion, it can be foreseen that LC-ESI-MS and LC-ESI-MS-MS will increasingly complement NMR techniques in the structural elucidation of marine toxins and
will substitute them in those cases when limited pure toxin availability and presence
of impurities prevents the application of NMR. For the routine determination of known
toxins in raw seafood materials, LC-ESI-MS will probably not replace the fast and sen-