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

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.