Structural comparison of recombinant human macrophage colony stimulating factor β and a partially reduced derivative using hydrogen deuterium exchange and electrospray ionization mass spectrometry

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Abstract
Hydrogen deuterium exchange, monitored by electrospray ionization mass spectrometry, has been employed to characterize structural features of a derivative of recombinant human macrophage colony stimulating factor beta (rhm-CSFβ) in which two of the nine disulfide bridges (Cys157/Cys159–Cys′157/Cys′159) were selectively reduced and alkylated. Removal of these two disulfide bridges did not affect the biological activity of the protein. Similarities between CD and fluorescence spectra for rhm-CSFβ and its derivative indicate that removing the disulfide bonds did not strongly alter the overall three-dimensional structure of rhm-CSFβ. However, differences between deuterium exchange data of the intact proteins indicate that more NHs underwent fast deuterium exchange in the derivative than in rhm-CSFβ. Regions located near the disulfide bond removal site were shown to exhibit faster deuterium exchange behavior in the derivative than in rhm-CSFβ.