title: Identification of the interface of a large protein-protein complex using H/D exchange and Fourier transform ion cyclotron resonance mass spectrometry.
abstract: An infrared multiphoton dissociation (IRMPD) spectrum, obtained by Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS), was used to dissociate and to identify fragment ions from recombinant human interleukin-6 (IL-6; 21 KDa). The entire sequence was assigned by a single IRMPD experiment, and the observed fragment ions reflected the IL-6 secondary structure. This method was combined with H/D off-exchange to identify IL-6 and anti-human IL-6 mouse monoclonal antibody MH166 (150-kDa) binding sites in the IL-6 molecule. To facilitate the data analysis, the protein complex formation and the hydrogen exchange were performed with an immobilized antibody. Quenching of the hydrogen exchange reaction and collection of the deuterated IL-6 were performed by elution under acidic conditions to measure the mass spectrum directly. IL-6 was dissociated by using IRMPD, and the interface of IL-6 bound to anti-IL-6 antibody MH166 was determined to analyze the deuterium incorporation level of each fragment ion. Thus, two discontinuous regions, Leu 126-Lys 131 and Asp 160-Met 184, were identified as the antibody binding sites. These regions are adjacent to each other on the tertiary structures determined by NMR and X-ray analyses.
authors.affiliation: Central Research Laboratories, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan. naoyuki_yamada@ajinomoto.com
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Yamada
authors.forename: Ei-ichiro
authors.initials: E
authors.lastname: Suzuki
authors.forename: Kazuo
authors.initials: K
authors.lastname: Hirayama
mesh_terms: D000818:Animals; D019138:Antibodies, Blocking / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry; D017357:Cyclotrons; D003903:Deuterium / Q000737:chemistry; D005583:Fourier Analysis; D006801:Humans; D015850:Interleukin-6 / Q000737:chemistry; D013058:Mass Spectrometry; D051379:Mice; D011506:Proteins / Q000737:chemistry*
chemical_list: D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D015850:Interleukin-6; D011506:Proteins; D003903:Deuterium