Rapid sequencing and disulfide mapping of peptides containing disulfide bonds by using 1,5-diaminonaphthalene as a reductive matrix.
MS/MS is indispensable for the amino acid sequencing of peptides. However, its use is limited for peptides containing disulfide bonds. We have applied the reducing properties of 1,5-diaminonaphthalene (1,5-DAN) as a MALDI matrix to amino acid sequencing and disulfide bond mapping of human urotensin II possessing one disulfide bond, and human guanylin possessing two disulfide bonds. 1,5-DAN was used in the same manner as the usual MALDI matrices without any pre-treatment of the peptide, and MS/MS was performed using a matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometer (MALDI QIT TOFMS). The results demonstrated that MS/MS of the molecular ions reduced by 1,5-DAN provided a series of significant b-/y-product ions. All 11 amino acid residues of urotensin II were identified using 1,5-DAN, while only 5 out of 11 residues were identified using 2,5-dihydroxybenzoic acid (DHB); similarly 11 out of 15 amino acid residues of guanylin were identified using 1,5-DAN, while only three were identified using DHB. In addition, comparison of the theoretical and measured values of the mass differences between corresponding MS/MS product ions using 1,5-DAN and DHB narrowed down the possible disulfide bond arrangement candidates. Consequently, 1,5-DAN as a reductive matrix facilitates rapid amino acid sequencing and disulfide mapping for peptides containing disulfide bonds.
Fukuyama
Yuko
Y
Koichi Tanaka Mass Spectrometry Research Laboratory, Shimadzu Corporation, 1, Nishinokyo-Kuwabaracho, Nakagyo-ku, Kyoto 604-8511, Japan. yukof@shimadzu.co.jp
Iwamoto
Shinichi
S
Tanaka
Koichi
K
D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004220:Disulfides / Q000032:analysis* / Q000737:chemistry*; D005768:Gastrointestinal Hormones / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D015394:Molecular Structure; D045265:Natriuretic Peptides / Q000737:chemistry; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014579:Urotensins / Q000737:chemistry
D004220:Disulfides; D005768:Gastrointestinal Hormones; D045265:Natriuretic Peptides; D010446:Peptide Fragments; D010455:Peptides; D014579:Urotensins; C510889:1,5-diaminonaphthalene; C072685:guanylin; C029677:urotensin II; D015081:2-Naphthylamine