Probing the folding pathways of long R3 insulin-like growth factor-I (LR3IGF-I) and IGF-I via capture and identification of disulfide intermediates by cyanylation methodology and mass spectrometry

Abstract

This report describes an integrated investigation of the refolding and reductive unfolding of insulin-like growth factor (IGF-I) and its variant, long R3 IGF-I (LR3IGF-I), which has a Glu3 to Arg3 substitution and a hydrophobic 13-amino acid N-terminal extension. The refolding performed in glutathione redox buffer was quenched at different time points by adjusting the pH to 2.0-3.0 with a 1 N HCl solution of 1-cyano-4-dimethyl- aminopyridinium tetrafluoroborate, which trapped intermediates via cyanylation of free sulfhydryl groups. The disulfide structure of the intermediates was determined by chemical cleavage followed by mass mapping with mass spectrometry. Six refolding intermediates of IGF-I and three refolding intermediates of LR3IGF-I were isolated and characterized. Folding pathways of IGF-I and LR3IGF-I are proposed based on the time-dependent distribution and disulfide structure of the corresponding trapped intermediates. Similarities and differences in the refolding behavior of IGF- I and LR3IGF-I are discussed.