Optimizing the connectivity in disulfide-rich peptides: alpha-conotoxin SII as a case study.
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.
Bingham
Jon-Paul
JP
Department of Biology, Clarkson University, Potsdam, NY 13699, USA. jbingham@clarkson.edu
Broxton
Natalie M
NM
Livett
Bruce G
BG
Down
John G
JG
Jones
Alun
A
Moczydlowski
Edward G
EG
D000595:Amino Acid Sequence; D000818:Animals; D020916:Conotoxins / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D003964:Diaphragm / Q000187:drug effects; D004220:Disulfides / Q000737:chemistry*; D013058:Mass Spectrometry; D010791:Phrenic Nerve / Q000187:drug effects; D051381:Rats; D011978:Receptors, Nicotinic / Q000187:drug effects
D020916:Conotoxins; D004220:Disulfides; D011978:Receptors, Nicotinic; C077379:alpha-conotoxin SII