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 beta-chain of human recombinant follicotropin (betaFSH) was accomplished by integrating classical biochemical methodologies with mass spectrometric procedures. A proteolytic strategy consisting of a double digestion of native betaFSH using the broad-specificity protease subtilisin first, followed by trypsin, was employed. The resulting peptide mixture was directly analysed by FAB-MS, leading to the assignment of the first three disulphide bridges. The remaining S-S bridges were determined by HPLC fractionation of the proteolytic digest followed by ESMS analysis of the individual fractions. The pattern of cysteine couplings in betaFSH was determined as: Cys3-Cys5l, Cys17-Cys66, Cys20-Cys104, Cys28-Cys82, Cys32-Cys84 and Cys87-Cys94, confirming the arrangement inferred from the crystal structure of the homologous betaCG. A subset of the S-S bridge pattern comprising Cys3-Cys51, Cys28-Cys82 and Cys32-Cys84 constitutes a cysteine knot motif similar to that found in the growth factor superfamily.
Amoresano
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A
Centro Internazionale di Servizi di Spettrometria di Massa, CNR-Università di Napoli Federico II, Napoli, Italy
Orrù
S
S
Siciliano
R A
RA
De Luca
E
E
Napoleoni
R
R
Sirna
A
A
Pucci
P
P
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry*; D005640:Follicle Stimulating Hormone / Q000737:chemistry*; D037201:Follicle Stimulating Hormone, beta Subunit; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry; D020539:Sequence Analysis, Protein / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization
D004220:Disulfides; D037201:Follicle Stimulating Hormone, beta Subunit; D011994:Recombinant Proteins; D005640:Follicle Stimulating Hormone