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

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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 (273nm) and BSA (280nm). 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 hap-ten-protein conjugates which are difficult to detect by the above spectrophotometric methods.
2008-09-01

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

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