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  • Title: Identification of the interface of a large protein-protein complex using H/D exchange and Fourier transform ion cyclotron resonance mass spectrometry.
    Author: Yamada N, Suzuki E, Hirayama K.
    Journal: Rapid Commun Mass Spectrom; 2002; 16(4):293-9. PubMed ID: 11816044.
    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.
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