Total chemical synthesis and NMR characterization of the glycopeptide tx5a, a heavily post-translationally modified conotoxin, reveals that the glycan structure is alpha-D-Gal-(1-->3)-alpha-D-GalNAc.

The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* = 6-bromotryptophan and Thr* = Gal-GalNAc-threonine), isolated from Conus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post-translationally modified residues: four cysteine residues, two gamma-carboxyglutamic acid residues, and one residue each of 6-bromotryptophan, 4-trans-hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue. Previous investigations have demonstrated that tx5a contains a disaccharide composed of N-acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized. We hypothesized that tx5a contained the T-antigen, beta-D-Gal-(1-->3)-alpha-D-GalNAc, one of the most common O-linked glycan structures, identified previously in another Conus glycopeptide, contalukin-G. We therefore utilized the peracetylated form of this glycan attached to Fmoc-threonine in an attempted synthesis. While the result-ing synthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* =6-bromotryptophan and Thr* = beta-D-Gal-(1-->3)-alpha-D-GalNAc-threonine) and the native peptide had almost identical mass spectra, a comparison of their RP-HPLC chromatograms suggested that the two forms were not identical. Two-dimensional 1H homonuclear and 13C-1H heteronuclear NMR spectroscopy of native tx5a isolated from Conus textile was then used to determine that the glycan present on tx5a indeed is not the aforementioned T-antigen, but rather alpha-D-Gal-(1-->3)-alpha-D-GalNAc.

Fragmentation of tunichrome Sp-1 is dominated by an unusual gas-phase intramolecular rearrangement.

Tunichrome Sp-1 is a modified pentapeptide from the ascidian Styela plicata, having the structure H-DOPA-DOPA-Gly-Pro-dcDeltaDOPA (where DOPA = 3,4-dihydroxyphenylalanine and dcDeltaDOPA = decarboxy-(E)-alpha,beta-dehydro-DOPA). The tandem mass spectrum of the peptide is dominated by a number of abundant fragment ions that involve a gas-phase rearrangement where the dcDeltaDOPA group becomes covalently attached to the N-terminus. The high degree of rearrangement in Sp-1 compared with a related octapeptide, plicatamide, allowed for detailed multiple mass spectrometric (MS(n)) (up to n = 6) experiments, and hence permitted a detailed assessment of the origin and routes to the formation of the various rearrangement ions. Analyses on both a triple-quadrupole and a quadrupole time-of-flight mass spectrometer were made to ascertain whether the gas-phase rearrangements observed for tunichrome Sp-1 were unique to an ion trap mass spectrometer (i.e. the hypothesis being that perhaps the extended trapping times were required to facilitate this unusual gas-phase rearrangement). Interestingly, analyses on both the triple-quadrupole and quadruple time-of-flight mass spectrometers revealed an identical phenomenon, with the rearrangement fragment ions present at approximately the same abundance as the non-rearranged a-, b- and y-type sequence ions. We suggest that the smaller size of tunichrome Sp-1 compared with plicatamide facilitates the transfer of the dcDeltaDOPA group in this gas-phase rearrangement. This rearrangement was not observed for peptide analogs of tunichrome Sp-1 that did not contain the dcDeltaDOPA at the C-terminus, confirming that the presence of dcDeltaDOPA is critical for the rearrangement.