Assignment of the complete disulphide bridge pattern in the human recombinant follitropin beta-chain

The chemical assessment of the complete disulphide bridge pattern in the be ta -chain of human recombinant follicotropin (beta FSH) was accomplished by integrating classical biochemical methodologies with mass spectrometric pr ocedures. A proteolytic strategy consisting of a double digestion of native beta FSH using the broad-specificity protease subtilisin first, followed b y trypsin, was employed. The resulting peptide mixture was directly analyse d by FAB-MS, leading to the assignment of the first three disulphide bridge s. The remaining S-S bridges were determined by HPLC fractionation of the p roteolytic digest followed by ESMS analysis of the individual fractions. Th e pattern of cysteine couplings in beta FSH was determined as: Cys3-Cys51, Cys17-Cys66, Cys20-Cys104, Cys28-Cys82, Cys32-Cys84 and Cys87-Cys94, confir ming the arrangement inferred from the crystal structure of the homologous beta CG. A subset of the S-S bridge pattern comprising Cys3-Cys51, Cys28-Cy s82 and Cys32-Cys84 constitutes a cysteine knot motif similar to that found in the growth factor super-family.