PEPTIDE REFOLDING DEFINED BY ENZYMATIC DIGESTION

Rockefeller University, New York NY

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Abstract

We have synthesized a cysteine-rich domain in h-factor IX. This EFG-like domain contains 45 amino acids and three disulfide bonds. An extremely difficult point in this work is the definition of the peptide disulfide bond refolding pattern. In order to characterize this peptide and deduce the correct and formation of native disulfide bonds, a series of enzymatic digestions are carried out (under non-reducing conditions) and the disulfide connectivity is established using MS. Typically, V8-protease and trypsin are used to produce the needed proteolytic peptides. During these studies, we have found that matrix-assisted laser desorption ion trap mass spectrometry is particularly useful for this application to S-S mapping (J. Qin & B. T. Chait "Rapid, Accurate Identification and Characterization of Posttranslational Modifications of Proteins by MALDI Ion Trap Mass Spectrometry" Anal. Chem. 69 (1997) 4002 - 4009).