Gas-phase Microsequencing of Peptides and Proteins with a Fluorescent Edman-type Reagent, Fluorescein Isothiocyanate

Koji Muramoto, Kiyoshi Nokihara, Akira Ueda, Kiyoshi Nokihara, Hisao Kamiya

    Research output: Contribution to journalArticlepeer-review

    15 Citations (Scopus)

    Abstract

    A fluorescent Edman-type reagent, fluorescein isothiocyanate (FITC), was adapted to a gas-phase sequencer fitted with a miniature reaction chamber. Fluorescein thiohydantoin amino acids were identified by on-line gradient HPLC with fluorescence monitoring. The optimized protocol for the coupling reaction using FITC and phenylisothiocyanate, and the degree of washing to remove the excess reagents were established. Myoglobin (5pmol) and lysozyme (5pmol) were analyzed by the sequencer to obtain a 35-51% initial yield (I.Y.) and 82-88% repetitive yield (R.Y.) for the former, and 51-66% I.Y. and 91-92% R.Y. for the latter. These figures permitted 20-25 amino acid residues to be identified from the N-terminus of 5 pmol samples applied to the sequencer.

    Original languageEnglish
    Pages (from-to)300-304
    Number of pages5
    JournalBioscience, Biotechnology, and Biochemistry
    Volume58
    Issue number2
    DOIs
    Publication statusPublished - 1994 Jan 1

    ASJC Scopus subject areas

    • Biotechnology
    • Analytical Chemistry
    • Biochemistry
    • Applied Microbiology and Biotechnology
    • Molecular Biology
    • Organic Chemistry

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