Micro-heterogeneities in Native and Recombinant Proteins Studied by Electrospray Ionization and Fast Atom Bombardment Mass Spectrometry Coupled with Micro-column Liquid Chromatography.

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  • ISHIKAWA Keiichiro
    Dept. of Advanced Chemical Technology, National Institute of Materials and Chemical Research
  • TUOMINEN Jari
    VTT Chemical Technology
  • MURAKI Michio
    Molecular Biology Dept., National Institute of Bioscience and Human-technology
  • NAGAHORA Hitoshi
    Molecular Biology Dept., National Institute of Bioscience and Human-technology
  • JIGAMI Yoshifumi
    Molecular Biology Dept., National Institute of Bioscience and Human-technology
  • KOGA Yoshinori
    Dept. of Advanced Chemical Technology, National Institute of Materials and Chemical Research
  • NIWA Yoshio
    Dept. of Inorganic Materials, National Institute of Materials and Chemical Research

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Other Title
  • ミクロカラム液体クロマトグラフィー／エレクトロスプレーおよび高速原子衝撃イオン化質量分析法による天然および遺伝子組み換えタンパク質の微小構造変異の解析
  • ミクロ カラム エキタイ クロマトグラフィー エレクトロ スプレー オヨビ コ

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A home-made electrospray ionization (ESI) source, which generated multiply charged gas-phase ions of proteins under atmospheric pressure, was coupled with a tandem quadrupole mass spectrometer. A detection limit of 18 fmol (1.8 × 10-14 mol) and a precision of 0.01% in the molecular weight measurement were attained using hen-egg lysozyme as a typical protein.<br>ESI mass spectrometry (MS) was applied to the prompt identification of the amino acid substitutions in recombinant human lysozymes (HLY). However, the direct analysis of HLY was prevented by the co-existing inorganic salts added to retain its biological activities. On-line coupling of ESI-MS with micro-column liquid chromatography (ESI-LC/MS) was developed to perform direct molecular weight measurements of salt-containing proteins at the picomole level. LC/MS interfacing by ESI required to alleviate the sensitivity losses brought about by the high electric conductivity and the high surface tension of the LC eluent.<br>ESI-MS and ESI-LC/MS were successfully applied to the characterization of the post-translational modifications found in recombinant wheat germ agglutinin (WGA2). These methods along with fast atom bombardment (FAB) MS and FAB LC/MS were also applied to the characterization of natural variants of β-casein (β-CA). The possibility and limitation of the present methods for the analysis of complex proteins were discussed.

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