STRUCTURAL CHARACTERIZATION AND LOCATION OF DISULFIDE LINKAGES OF A POTENT VASODILATORY PEPTIDE, RECOMBINANT MAXADILAN, BY A MULTIPLE MASS-SPECTROMETRIC APPROACH

A multiple mass spectrometric strategy using fast-atom bombardment (FA B) and matrix-assisted laser desorption/ionization (MALDI) has been us ed to confirm the sequence and to locate the disulfide linkages of rec ombinant maxadilan (r-maxadilan) (average molecular mass 7422.5 Da), a potent vasodilatory peptide from Lutzomyia longipalpis, MALDI measure ments of intact r-maxadilan, its reduced form and its pyridylethylated form (p-maxadilan) indicated the presence of four Cys residues withou t major post-translational modifications. FAB and FAB-tandem mass spec trometry measurements of chymotryptic digests of p-maxadilan were suff icient to map the primary structure of p-maxadilan, though the complem entary use of MALDI was necessary for complete mapping using Asp-N dig estion due to a strong suppression observed in FAB. Assignment of the Cys-5-Cys-9 linkage was achieved by comparison of FAB mass spectra bef ore and after reduction of tryptic digests of r-maxadilan, Since the m olecular weight of the peptide fragment containing the Cys-18-Cys-55 l inkage is more than 4000, MALDI measurement was indispensable for assi gnment of this linkage, The results fully support the value of the mul tiple mass spectrometric strategy in the structural characterization o f peptides and proteins.