Combined use of ESI‐MS and UV diode‐array detection for localization of disulfide bonds in proteins: application to an α‐l‐fucosidase of pea

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 α‐l ‐fucosidase from pea. The two disulfide bonds were located between residues Cys⁶⁴ and Cys¹⁰⁹ and between Cys¹⁶² and Cys¹⁶⁹, while Cys¹²⁷ was free.