Characterization of an antagonist interleukin-6 dimer by stable isotope labeling, cross-linking, and mass spectrometry

Abstract

The homodimeric form of a recombinant cytokine interleukin-6 (IL-6D) is known to antagonize IL-6 signaling. In this study, spatially proximal residues between IL-6 chains in IL-6D were identified using a method for specific recognition of intermolecular cross-linked peptides. Our strategy involved mixing 1:1 15N-labeled and unlabeled (14N) protein to form a mixture of isotopically labeled and unlabeled homodimers, which was chemically cross-linked. This cross-linked IL-6D was subjected to proteolysis by trypsin and the generated peptides were analyzed by electrospray ionization time-of-flight mass spectrometry (MS). Molecular ions from cross-linked peptides of intermolecular origin are label..