Attempt to Detect Natural Anticancer Compounds—Protein Binding through Precursor Ion Scan and MS/MS Measurements

DOI Web Site Web Site 41 References
  • NEGISHI Jun
    Department of Life Science and Biotechnology, Faculty of Chemistry, Material and Bioengineering
  • SHIRAHAMA Tatsuya
    Department of Life Science and Biotechnology, Faculty of Chemistry, Material and Bioengineering
  • NAGAOKA Yasuo
    Department of Life Science and Biotechnology, Faculty of Chemistry, Material and Bioengineering High Technology Research Center, Kansai University
  • TAKEMOTO Yoshiki
    Department of Life Science and Biotechnology, Faculty of Chemistry, Material and Bioengineering
  • UESATO Shinichi
    Department of Life Science and Biotechnology, Faculty of Chemistry, Material and Bioengineering High Technology Research Center, Kansai University

Bibliographic Information

Other Title
  • Attempt to detect natural anticancer compounds: protein binding through precursor ion scan and MS/MS measurements

Search this article

Abstract

  A 2′-succinyltaxol-bovine serum albumin (BSA) conjugate was prepared as an antigen to produce an anti-taxol monoclonal antibody by immunizing mice. Formation of a linkage between hapten and protein is usually confirmed by the UV or fluorescamine method. However, it was difficult to confirm the binding of 2′-succinyltaxol to BSA by these methods owing to the similar UV absorption maxima of 2′-succinyltaxol (273 nm) and BSA (280 nm). In the present study, we therefore conducted a mass spectrometric analysis using the precursor ion scan and MS/MS techniques to confirm the formulation of the 2′-succinyltaxol-BSA conjugate in the following way: The conjugate was subjected to thermal denaturalization, dithiothreitol (DTT)-reduction, iodoacetamide-alkylation and trypsin-digestion, affording a peptide fragment mixture. This was then analyzed by electrospray ionization (ESI)-MS in the positive mode by scanning the peaks containing a mass of 854 corresponding to taxol. The detected peaks were in turn subjected to MS/MS measurements. Among them, a peak at m/z 1247.4 was found to be a peptide fragment containing Lys (ε-2′-succinyltaxol), demonstrating the formulation of the 2′-succinyltaxol-BSA conjugate. In order to confirm the feasibility of this analytical method, the deacetylvinblastine (deacetylVLB)-BSA antigen which produced the anti-VLB monoclonal antibody (MAb-10-A9), was subjected to the same analytical treatment as above, giving a peak at m/z 851.3 originating from a Lys (ε-deacetylVLB). Thus, this new method could serve as an additional tool for confirmation of the formation of hapten-protein conjugates which are difficult to detect by the above spectrophotometric methods.<br>

Journal

  • YAKUGAKU ZASSHI

    YAKUGAKU ZASSHI 128 (9), 1317-1323, 2008-09-01

    The Pharmaceutical Society of Japan

References(41)*help

See more

Keywords

Details 詳細情報について

Report a problem

Back to top