Structural characterization of monomeric folding intermediates of recombinant human macrophage-colony stimulating factor β (rhM-CSFβ) by chemical trapping, chromatographic separation and mass spectrometric peptide mapping

We have developed a strategy for the characterization of protein folding intermediates that combines selective modification of bis-cysteinyl thiol groups with melarsen oxide (MEL), chromatographic separation and mass spectrometric characterization of the resulting protein derivatives. In the unfolding reaction of recombinant human macrophage-colony stimulating-factor β (rhM-CSFβ) we observed monomeric M·4MEL and dimeric D·2MEL intermediates. The major locations of the MEL groups in D·2MEL were at C157 and C159. In M·4MEL, MEL groups were predominantly located at C31 and C102. These results indicate the presence of highly structured dimeric and monomeric intermediates. In the completely reduced R·4MEL derivative, MEL groups were distributed such that the smallest ring structures resulted.