Assignment of the disulfide bonds in napin, a seed storage protein from Brassica napus, using matrix-assisted laser desorption ionization mass spectrometry.
The sites of the disulfide bonds in a napin protein isolated from Brassica napus have been identified by proteolytic cleavage and subsequent peptide mapping by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Napins consist of two polypeptide chains containing two and six cysteine residues, respectively, that are held together by disulfide bonds. Upon initial cleavage of native napin by Endo-Lys-C, a disulfide-linked core complex of four peptides was obtained. This core peptide was isolated by reversed-phase HPLC and further digested by thermolysin, and the resulting fragments were identified by MALDI-MS. In a separate set of experiments, intact napin was subjected to proteolysis by thermolysin, and an isolated disulfide-linked peptide of interest was subdigested again using thermolysin. The combined data resulting from these experiments allowed the assignment of the disulfide linkages in a relatively abundant napin isoform, BngNAP1, apart from an ambiguity concerning the adjacent cysteines at positions 14' and 15' of the long chain. Two intermolecular disulfide bonds link Cys10 (short chain) with Cys25' (long chain) and Cys23 with Cys14' (or Cys15'), respectively. The long chain of napin contains two intramolecular disulfide bonds connecting Cys27' with Cys80' and Cys14' (or Cys15') with Cys72'.
Gehrig
P M
PM
Massachusetts Institute of Technology, Cambridge, USA.
Biemann
K
K
D055516:2S Albumins, Plant; D000595:Amino Acid Sequence; D001937:Brassica / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D003553:Cystine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010940:Plant Proteins / Q000737:chemistry*; D011487:Protein Conformation; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013820:Thermolysin / Q000378:metabolism
D055516:2S Albumins, Plant; D004220:Disulfides; D010446:Peptide Fragments; D010940:Plant Proteins; D003553:Cystine; C050363:napin protein, Brassica napus; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D013820:Thermolysin; D003545:Cysteine