Combined use of ESI-MS and UV diode-array detection for localization of disulfide bonds in proteins: application to an alpha-L-fucosidase of pea

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 det ection (HPLC diode array). The denatured protein is subjected to proteolysi s and the peptide mixture is divided into three fractions: (i) underivatize d peptides, (ii) ethylpyridylated peptides, and (iii) reduced and ethylpyri dylated peptides. The three peptide ensembles are then subjected to chromat ographic 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 d isulfide bonds were located between residues Cys(64) and Cys(109) and betwe en Cys(162) and Cys(169), while Cys(127) was free.