Identification of tyrosine sulfation in Conus pennaceus conotoxins alpha-PnIA and alpha-PnIB: Further investigation of labile sulfo- and phosphopeptides by electrospray, matrix-assisted laser desorption/ionization (MALDI) and atmospheric pressure MALDI mass spectrometry

Liquid chromatography/electrospray ionization mass spectrometry was used to investigate the peptide composition of the venom of Conus pennaceus, a mol luscivorous cone shell from the Red Sea. Based on observed M(r)s, this veno m contained all known conotoxins previously isolated and identified from th is species. Interestingly, the doubly protonated species of only two of the se conotoxins, alpha-PnIA and alpha-PnIB, showed additional related ions at + 40 m/z (+ 80 Da), indicating the presence of either sulfation or phospho rylation in both components. Highperformance liquid chromatographic (HPLC) fractions containing these two conotoxins were examined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry in both positive and negative ion modes, as well as by MALDI high-energy collision-induced disso ciation. These experiments established the presence of a single sulfated ty rosine residue within both alpha-PnIA and alpha-PnIB, Hence their post-tran slationally modified sequences are GCCSLPPCAANNPDY(S)C-NH2 (alpha-PnIA) and GCCSLPPCALSNPDY(S)C-NH, (alpha-PnIB), This assignment was supported by com parison of their mass spectral behavior with that of known sulfated and pho sphorylated peptides, This data clarified further the distinguishing featur es of the ionization and fragmentation of such modified peptides, Selective disulfide folding of synthetic alpha-PnIB demonstrated that both sulfated and non-sulfated toxins co-elute on reversed-phase HPLC and that alpha-PnIB possesses the same disulfide connectivity as other 'classical' alpha-conot oxins reported previously. Copyright (C) 1999 John Wiley & Sons, Ltd.