Structural characterization and location of disulphide linkages of a potent vasodilatory peptide, recombinant maxadilan, by a multiple mass spectrometric approach.
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.
Yoshida
S
S
Safety & Analytical Research Center, Shiseido Research Center, Yokohama, Japan.
Takamatsu
T
T
Denda
S
S
Ohnuma
M
M
Tajima
M
M
Lerner
E A
EA
Kanda
F
F
D000595:Amino Acid Sequence; D002918:Chymotrypsin; D004220:Disulfides / Q000032:analysis*; D006868:Hydrolysis; D007301:Insect Hormones / Q000032:analysis*; D019476:Insect Proteins; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000032:analysis; D016339:Spectrometry, Mass, Fast Atom Bombardment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin; D014665:Vasodilator Agents / Q000032:analysis*
D004220:Disulfides; D007301:Insect Hormones; D019476:Insect Proteins; D011994:Recombinant Proteins; D014665:Vasodilator Agents; C069147:maxadilan protein, insect; D002918:Chymotrypsin; D014357:Trypsin