Structural Characterization and Location of Disulphide Linkages of a Potent Vasodilatory Peptide, Recombinant Maxadilan, by a Multiple Mass Spectrometric Approach

Abstract

A multiple mass spectrometric strategy using fast-atom bombardment (FAB) and matrix-assisted laser desorption/ionization (MALDI) has been used to confirm the sequence and to locate the disulfide linkages of recombinant maxadilan (r-maxadilan) (average molecular mass 7422.5 Da), a potent vasodilatory peptide from Lutzomyia longipalpis. MALDI measurements of intact r-maxadilan, its reduced form and its pyridylethylated form (p-maxadilan) indicated the presence of four Cys residues without major post-translational modifications. FAB and FAB-tandem mass spectrometry measurements of chymotryptic digests of p-maxadilan were sufficient to map the primary structure of p-maxadilan, though the complementary use of MALDI was necessary for complete mapping using Asp-N digestion due to a strong suppression observed in FAB. Assignment of the Cys-5-Cys-9 linkage was achieved by comparison of FAB mass spectra before and after reduction of tryptic digests of r-maxadilan. Since the molecular weight of the peptide fragment containing the Cys-18-Cys 55 linkage is more than 4000, MALDI measurement was indispensable for assignment of this linkage. The results fully support the value of the multiple mass spectrometric strategy in the structural characterization of peptides and proteins.

Maxadilan Is a Specific Agonist and Its Deleted Peptide (M65) Is a Specific Antagonist for PACAP Type 1 Receptor

“…1,2 It contains four cysteine residues that participate in the formation of disulfide bonds between positions 1‐5 and 14‐51. 3 The amino acid sequences of maxadilan, M65, and PACAP are shown in Figure 1. PACAP shows a 68% amino acid sequence homology with VIP.…”

“…1,2 It contains four cysteine residues that participate in the formation of disulfide bonds between positions 1‐5 and 14‐51. 3 Although maxadilan possesses both biological activity and specific binding sites, it is not present in mammals. As flies are evolutionarily distant from vertebrates and maxadilan does not share sequence homology with other known peptides, it was postulated that this peptide would act either at an orphan receptor whose endogenous ligand was unknown or at a receptor whose ligand had a structure distinct from maxadilan.…”

Functional Characterization of Structural Alterations in the Sequence of the Vasodilatory Peptide Maxadilan Yields a Pituitary Adenylate Cyclase-activating Peptide Type 1 Receptor-specific Antagonist

“…It contains four cysteine residues that participate in the formation of two disulfide bonds between positions 1 and 5 and positions 14 and 51 ( Fig. 1) (6). Initial studies had suggested that maxadilan shared properties with CGRP, but binding of maxadilan to CGRP receptors could not be demonstrated.…”

CysMap and CysJoin: Database and tools for protein disulphides localisation

“…Anyhow, no software dedicated to disulphide bridges assignment is available at the moment. Since 1985 [5] and up to recent years [6–22], mass spectrometry has been widely used for the assignment of disulphide bridges due to the advantage that the separation of the peptide mixtures generated by proteolytic digestion of native proteins is often unnecessary. The technique allows the direct identification of cluster peptides linked by disulphide bonds in the mass spectra of digestion mixtures by their unique mass values.…”