Biotinylated peptides/proteins. I. Determination of stoichiometry of derivatization

John S. Smith, Brian T. Miller, Susan L. Knock, Alexander Kurosky

    Research output: Contribution to journalArticle

    27 Citations (Scopus)

    Abstract

    A method is described for the determination of the stoichiometry of biotinylation of peptides and proteins after reaction with an N-hydroxysuccinimide ester of biotin containing the extended spacer arm 6-aminohexanoic acid (NHS-ε{lunate}Ahx-biotin). The method of analysis, based on the quantification of phenylthiocarbamyl derivatives of 6-aminohexanoic acid, is able to measure low picomolar amounts of biotinyl derivative. Analyses were performed using an automated on-line hydrolyzer-derivatizer followed by high-performance liquid chromatography. Compositional analyses determined for known peptides were in excellent agreement with analyses obtained by mass spectrometry. Procedures are also described for the production of biotinylated protein probes that can be labeled reproducibly to contain specific amounts of biotin. The analytical advantage and steric freedom provided by the 6-amino-hexanoic acid spacer arm argue strongly for the NHS-ε{lunate}Ahx-biotin reagent to be a reagent of choice for the biotinylation of peptides and proteins.

    Original languageEnglish (US)
    Pages (from-to)247-253
    Number of pages7
    JournalAnalytical Biochemistry
    Volume197
    Issue number1
    DOIs
    StatePublished - Aug 15 1991

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    Biotin
    Aminocaproic Acid
    Stoichiometry
    Biotinylation
    Peptides
    Proteins
    Derivatives
    High performance liquid chromatography
    Mass spectrometry
    Mass Spectrometry
    Esters
    High Pressure Liquid Chromatography

    ASJC Scopus subject areas

    • Biochemistry
    • Biophysics
    • Molecular Biology

    Cite this

    Biotinylated peptides/proteins. I. Determination of stoichiometry of derivatization. / Smith, John S.; Miller, Brian T.; Knock, Susan L.; Kurosky, Alexander.

    In: Analytical Biochemistry, Vol. 197, No. 1, 15.08.1991, p. 247-253.

    Research output: Contribution to journalArticle

    Smith, John S. ; Miller, Brian T. ; Knock, Susan L. ; Kurosky, Alexander. / Biotinylated peptides/proteins. I. Determination of stoichiometry of derivatization. In: Analytical Biochemistry. 1991 ; Vol. 197, No. 1. pp. 247-253.
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