Optimizing the connectivity in disulfide-rich peptides: ?-conotoxin SII as a case study
Analytical Biochemistry(2005)
摘要
We describe a strategy for the efficient, unambiguous assignment of disulfide connectivities in α-conotoxin SII, of which ∼30% of its mass is cysteine, as an example of a generalizable technique for investigation of cysteine-rich peptides. α-Conotoxin SII was shown to possess 3–8, 2–18, and 4–14 disulfide bond connectivity. Sequential disulfide bond connectivity analysis was performed by partial reduction with Tris(2-carboxyethyl)phosphine and real-time mass monitoring by direct-infusion electrospray mass spectrometry (ESMS). This method achieved high yields of the differentially reduced disulfide bonded intermediates and economic use of reduced peptide. Intermediates were alkylated with either N-phenylmaleimide or 4-vinylpyridine. The resulting alkyl products were assigned by ESMS and their alkyl positions sequentially identified via conventional Edman degradation. The methodology described allows a more efficient, rapid, and reliable assignment of disulfide bond connectivity in synthetic and native cysteine-rich peptides.
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关键词
Disulfide connectivity,Mass spectrometry,MS/MS,Conotoxin,N-Phenylmaleimide
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