334
M. Vincenti . A. Irico
As the most refined analysis of dinophysistoxins took advantage of the great selectivity allowed by MS-MS, the same is occurring for recent ciguatoxin determinations.
Good linearity of response was observed in MS-MS quantitative determinations, and
detection limits in the range of hundreds of picograms (40-200 ppt in fish flesh) were
obtained (Jones et al. 1997). As for dinophysistoxins, the specific MS-MS experiment
adopted was SRM, in which multiple loss of water molecules was followed.
17.6
Polypeptidic Toxins: Conotoxins
Conotoxins are biochemically active pep tides present in the venom of some tropical
marine snails. These toxins are active on sodium and calcium channels as well as on
nicotinic acetylcholine receptors. Although relatively small, quite specific receptor
properties are imparted by the constrained and rigid tridimensional conformation of
conotoxins. This is mainly due to the presence of several cysteine residues in the
amino acidic sequence, forming multiple disulfide bonds. Also I"carboxyglutaric residues contribute significantly to the tridimensional conformation. The structural elucidation of conotoxins is complicated by the presence of several post-translational
modifications, including C-terminal amidation, proline hydroxylation, tryptophan
halogenation and glutaric acid carboxylation.
Mass spectrometric methods have been extensively used for protein sequence determination, and proved particularly useful in the identification of post-translational
modifications, which are not feasible by Edman degradation techniques (Hunt et al.
1986). In the general procedure, the protonated molecular ion of the polypeptide, either produced by ESI or by other soft-ionization techniques, is mass-selected and subsequently fragmented by cm in an MS-MS apparatus. The secondary product ions
are detected by scanning the second mass analyser. The resulting daughter-ion spectrum is interpreted on the basis of regular and rather predictable sequences of bond
cleavages in the peptide backbone, with charge retention either on the N-terminal (sequences an' bn> en) or on the C-terminal (sequences xn>Yn>zn) fragment (Hunt et al.1986).
Each amino acid (unmodified or post-translationally modified) is identified by the
mass gap between consecutive fragments in each sequence, possibly with the support
of interpreting software programs. Single amino acids can also be mass-analysed after peptide degradation, and disulfide bonds can be detected by comparing ESI -MS
and ESI-MS-MS spectra before and after cysteine derivatization.
The whole set of procedures mentioned and the availability of an extremely wide
set of literature on peptide analysis by MS methods, make the structural identification of an unknown polypeptide a more standardized task than that of a cyclic
polyether. Nevertheless, investigation of conotoxins is rather complicated, since the
large number of disulfide bonds, post-translational modifications and individual variations (interspecies, intraspecies and within-individual) put the interpreting procedure
out of traditional schemes. A general overview of such problems and ESI -MS tools to
solve them has been recently published (Bingham 1996). The same group, in a research
paper (Jones et al.1996), used an LC-ESI-MS approach to identify several conopeptides
and to demonstrate the large compositional variability of venom mixtures. Disulfide
bonds were determined by stepwise reduction and alkylation of an isolated conotoxin.
The derivatization mixture was subsequently analysed by LC-ESI-MS (see Fig. 17.10a)
M. Vincenti . A. Irico
As the most refined analysis of dinophysistoxins took advantage of the great selectivity allowed by MS-MS, the same is occurring for recent ciguatoxin determinations.
Good linearity of response was observed in MS-MS quantitative determinations, and
detection limits in the range of hundreds of picograms (40-200 ppt in fish flesh) were
obtained (Jones et al. 1997). As for dinophysistoxins, the specific MS-MS experiment
adopted was SRM, in which multiple loss of water molecules was followed.
17.6
Polypeptidic Toxins: Conotoxins
Conotoxins are biochemically active pep tides present in the venom of some tropical
marine snails. These toxins are active on sodium and calcium channels as well as on
nicotinic acetylcholine receptors. Although relatively small, quite specific receptor
properties are imparted by the constrained and rigid tridimensional conformation of
conotoxins. This is mainly due to the presence of several cysteine residues in the
amino acidic sequence, forming multiple disulfide bonds. Also I"carboxyglutaric residues contribute significantly to the tridimensional conformation. The structural elucidation of conotoxins is complicated by the presence of several post-translational
modifications, including C-terminal amidation, proline hydroxylation, tryptophan
halogenation and glutaric acid carboxylation.
Mass spectrometric methods have been extensively used for protein sequence determination, and proved particularly useful in the identification of post-translational
modifications, which are not feasible by Edman degradation techniques (Hunt et al.
1986). In the general procedure, the protonated molecular ion of the polypeptide, either produced by ESI or by other soft-ionization techniques, is mass-selected and subsequently fragmented by cm in an MS-MS apparatus. The secondary product ions
are detected by scanning the second mass analyser. The resulting daughter-ion spectrum is interpreted on the basis of regular and rather predictable sequences of bond
cleavages in the peptide backbone, with charge retention either on the N-terminal (sequences an' bn> en) or on the C-terminal (sequences xn>Yn>zn) fragment (Hunt et al.1986).
Each amino acid (unmodified or post-translationally modified) is identified by the
mass gap between consecutive fragments in each sequence, possibly with the support
of interpreting software programs. Single amino acids can also be mass-analysed after peptide degradation, and disulfide bonds can be detected by comparing ESI -MS
and ESI-MS-MS spectra before and after cysteine derivatization.
The whole set of procedures mentioned and the availability of an extremely wide
set of literature on peptide analysis by MS methods, make the structural identification of an unknown polypeptide a more standardized task than that of a cyclic
polyether. Nevertheless, investigation of conotoxins is rather complicated, since the
large number of disulfide bonds, post-translational modifications and individual variations (interspecies, intraspecies and within-individual) put the interpreting procedure
out of traditional schemes. A general overview of such problems and ESI -MS tools to
solve them has been recently published (Bingham 1996). The same group, in a research
paper (Jones et al.1996), used an LC-ESI-MS approach to identify several conopeptides
and to demonstrate the large compositional variability of venom mixtures. Disulfide
bonds were determined by stepwise reduction and alkylation of an isolated conotoxin.
The derivatization mixture was subsequently analysed by LC-ESI-MS (see Fig. 17.10a)