Abstract
A simplified strategy is described for the assignment of disulfide bonds in proteins of medium to high molecular mass (10-30 kDa). The method combines the use of high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) and HPLC with UV diode-array detection (HPLC diode array). The denatured protein is subjected to proteolysis and the peptide mixture is divided into three fractions: (i) underivatized peptides, (ii) ethylpyridylated peptides, and (iii) reduced and ethylpyridylated peptides. The three peptide ensembles are then subjected to chromatographic and spectroscopic analysis. A systematic methodology is described to analyze the large amount of data obtained. The method was applied to the localization of disulfide bonds in alpha-L-fucosidase from pea. The two disulfide bonds were located between residues Cys64 and Cys109 and between Cys162 and Cys169, while Cys127 was free.
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Schedule a callMore From: The journal of peptide research : official journal of the American Peptide Society
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