Mass spectrometric mapping of disulfide bonds in recombinant human interleukin-13

Interleukin 13 (IL-13), a member of the alpha-helical family of cytokines, has similar to 30% primary sequence homology Kith IL-4 and shares a common receptor component. The biologically active rhIL-13 is monomeric and non-gl ycosylated, and contains two disulfide bonds as determined by comparative e lectrospray mass spectrometric (RIS) analysis of the protein before and aft er reduction with dithiothreitol-dithioerythritol. A trypsin-resistant core peptide of rhIL-13 was isolated and analyzed by plasma desorption (PD) MS, identifying a disulfide-linked core peptide. Subsequent digestion of this core peptide by pepsin, followed by PDMS analysis of the resulting cystine- containing peptic fragments, provided rapid determination of the existing d isulfide bonds between cysteine residues 28-56 and 44-70, This disulfide ar rangement is similar to that observed for the analogous four internal cyste ine residues in hIL-4. The conservation of disulfide bend arrangements betw een hIL-13 and hIL-4, coupled with their alpha-helical structure and sequen ce homologies, confirms that IL-13 and IL-I are structural homologues, It i s also consistent with their reported similarities in biological function a nd receptor binding kinetics. Copyright (C) 2000 John Wiley & Sons, Ltd.