Location of disulphide bonds in human insulin-like growth factors (IGFs) synthesized by recombinant DNA technology.
Natural human insulin-like growth factor (IGF) I is a relatively large single chain peptide (mol. wt 7649) with a known sequence of 70 amino acids. C6----C48, C47----C52 and C18----C61 assignments have been previously proposed for the three disulphide bonds linking six cysteine residues (C6, C18, C47, C48, C52 and C61), on the basis of analogy (and homology) with proinsulin. In this work, IGF I synthesized by recombinant DNA technology (r-IGF I, with identical biological activity and chromatographic behaviour) was subjected to a three-step mass spectrometric analysis in combination with degradation methods for structural verification. Firstly, the correct molecular weight of the intact peptide was determined by high-mass fast atom bombardment (FAB) analysis. Secondly, twofold enzymatic degradation (chymotrypsin followed by V8 protease, 'FAB mapping' of the cleavage products) was employed in order that fragments with 'isolated' S-S bonds would be produced which allow an unambiguous assignment. This immediately established the C18----C61 linkage as it was contained in a singly bridged two-chain peptide. The two other S-S bonds, which cross-link C6 and the 'tight' C47 to C52 segment, remained 'unresolved' within a more complex, doubly bridged triple-chain peptide. Thirdly, further degradation of this structural block, in which cleavage of the C47-C48 bond was required to discern these bonds, was carried out by using FAB tandem mass spectrometry and (for additional corroboration) manual Edman degradation. Both procedures confirmed the original C6----C48/C47----C52 prediction.(ABSTRACT TRUNCATED AT 250 WORDS)
Raschdorf
F
F
Ciba-Geigy Ltd, Basel, Switzerland.
Dahinden
R
R
Maerki
W
W
Richter
W J
WJ
Merryweather
J P
JP
D055598:Chemical Phenomena; D002621:Chemistry; D004274:DNA, Recombinant; D004220:Disulfides / Q000032:analysis*; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000032:analysis* / Q000138:chemical synthesis; D013058:Mass Spectrometry; D011994:Recombinant Proteins / Q000032:analysis; D013002:Somatomedins / Q000032:analysis*
D004274:DNA, Recombinant; D004220:Disulfides; D011994:Recombinant Proteins; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I