Optimizing the connectivity in disulfide-rich peptides: alpha-conotoxin SII as a case study |
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Authors: | Bingham Jon-Paul Broxton Natalie M Livett Bruce G Down John G Jones Alun Moczydlowski Edward G |
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Institution: | Department of Biology, Clarkson University, Potsdam, NY 13699, USA. jbingham@clarkson.edu |
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Abstract: | We describe a strategy for the efficient, unambiguous assignment of disulfide connectivities in alpha-conotoxin SII, of which approximately 30% of its mass is cysteine, as an example of a generalizable technique for investigation of cysteine-rich peptides. alpha-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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Keywords: | Disulfide connectivity Mass spectrometry MS/MS Conotoxin N-Phenylmaleimide |
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