Identification of the interface of a large protein-protein complex using H/D exchange and Fourier transform ion cyclotron resonance mass spectrometry.
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.
Yamada
Naoyuki
N
Central Research Laboratories, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan. naoyuki_yamada@ajinomoto.com
Suzuki
Ei-ichiro
E
Hirayama
Kazuo
K
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*
D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D015850:Interleukin-6; D011506:Proteins; D003903:Deuterium