Determination of the disulfide structure of sillucin, a highly knotted, cysteine-rich peptide, by cyanylation/cleavage mass mapping.
The disulfide structure of sillucin, a highly knotted, cysteine-rich, antimicrobial peptide, isolated from Rhizomucor pusillus, has been determined to be Cys2--Cys7, Cys12--Cys24, Cys13--Cys30, and Cys14--Cys21 by disulfide mass mapping based on partial reduction and CN-induced cleavage enabled by cyanylation. The denatured 30-residue peptide was subjected to partial reduction by tris(2-carboxyethyl)phosphine hydrochloride at pH 3 to produce a mixture of partially reduced sillucin species; the nascent sulfhydryl groups were immediately cyanylated by 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate. The cyanylated species, separated and collected during reversed phase high-performance liquid chromatography, were treated with aqueous ammonia, which cleaved the peptide chain on the N-terminal side of cyanylated cysteine residues. The CN-induced cleavage mixture was analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry before and after complete reduction of residual disulfide bonds in partially reduced and cyanylated species to mass map the truncated peptides to the sequence. Because the masses of the CN-induced cleavage fragments of both singly and doubly reduced and cyanylated sillucin are related to the linkages of the disulfide bonds in the original molecule, the presence of certain truncated peptide(s) can be used to positively identify the linkage of a specific disulfide bond or exclude the presence of other possible linkages.
Qi
J
J
Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Wu
J
J
Somkuti
G A
GA
Watson
J T
JT
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D006868:Hydrolysis; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D009570:Nitriles / Q000737:chemistry; D010084:Oxidation-Reduction; D010449:Peptide Mapping / Q000379:methods; D010455:Peptides; D010720:Phosphines / Q000737:chemistry; D020033:Protein Isoforms / Q000737:chemistry; D011726:Pyridinium Compounds / Q000737:chemistry; D020103:Rhizomucor / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013329:Structure-Activity Relationship
D000900:Anti-Bacterial Agents; D003545:Cysteine; D004220:Disulfides; D007202:Indicators and Reagents; D009570:Nitriles; D010455:Peptides; D010720:Phosphines; D020033:Protein Isoforms; D011726:Pyridinium Compounds; C080938:tris(2-carboxyethyl)phosphine; C067292:1-cyano-4-dimethylaminopyridinium; C018662:sillucin