Location of disulfide bonds in mature alpha-L-fucosidase from pea

Fuc-9 is the mature form of a vacuolar alpha -L-fucosidase enzyme which see ms to play an important role In plant growth regulation. Fuc-9 is a 202-res idue protein containing five Cys residues located at positions 64, 109, 127 , 162 and 169. In this study. the disulfide structure of Fuc-9 was determin ed by MALDI-TOF mass spectrometry (MS), with minimal clean-up of the sample s and at a nanomolar scale. Two strategies, based on a specific chemical cl eavage (with 2-nitro-5-thiocyanobenzoic acid and alkaline conditions) at th e Cys residues and modification of Cys residues by acrylamide/deuterium lab eled acrylamide alkylation, were used. Using these methods. the disulfide p airings CysB4-Cys109 and Cys162-Cys169 could be established. The advantages and limitations of our experimental approach are discussed. Copyright (C) 2001 European Peptide Society and John Wiley & Sons, Ltd.