| 摘要 |
The present invention is directed to a simple method for absolute quantification of plasma vitellogenin from two or more different fish species such as Rainbow trout and Atlantic salmon, or Atlantic cod and haddock. In the case of Rainbow tro ut and Atlantic salmon, plasma samples obtained from control and P-estradiol induced fish were digested with trypsin. A characteristic 'signature peptide ' was selected and analyzed by high performance liquid chromatography coupled to a n electrospray quadrupole-time-of-flight tandem mass spectrometer, using a deuterated homologue peptide as an internal standard. The hybrid tandem mass spectrometer was operated in a 'pseudo' selected reaction monitoring mode by which three diagnostic product ions were monitored for identification and quantification purposes. The reproducibility (coefficient of variation -5%) and sensitivity (limit of quantification of 0.009 mg/mL) achieved by this simple assay allow it to be considered as an alternative to immunological assays. In the case of Atlantic cod and haddock, the amino acid sequence of the vitellogenin protein has not yet been determined, but, the Atlantic cod vitellogenin has been characterized using a'bottom-up' mass spectrometric approach. Vitellogenin synthesis was induced 'in vivo' with P-Estradiol,and subjected to trypsin digestion for characterization by matrix-assisted laser desorption/ionizatio n- Quadrupole-Time-of-flight tandem mass spectrometry. A peptide mass fingerpri nt was obtained and 'de novo' sequencing of the most abundant tryptic peptides was performed by low energy collision induced dissociation-tandem mass spectrometry. Thus, the sequences of various tryptic peptides have been elucidated. It has also been determined that Atlantic cod vitellogenin share s a series of common peptides with the two different known vitellogenin sequence s of Haddock, a closely related species. There are also disclosed novel isolat ed signature peptides, namely Thr-Tyr-Phe-Ala-Gly-Ala-Ala-Ala-Asp-Val-Leu-Glu- Val-Gly-Val-Arg and Asp Leu Gly Leu Ala Tyr Thr Glu Lys.
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