Characterization and quantification of Atlantic salmon, rainbow trout and Atlantic cod vitellogenin applying combined mass spectrometric techniques

Cohen, Alejandro Martin (2006) Characterization and quantification of Atlantic salmon, rainbow trout and Atlantic cod vitellogenin applying combined mass spectrometric techniques. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Vitellogenin is a complex phosphoglycolipoprotein that is secreted into the bloodstream of sexually mature, female, oviparous animals in response to circulating estrogens. It is then incorporated into the ovaries by receptor mediated endocytosis, where it is further cleaved into smaller peptides which are constituents of the egg yolk proteins. It is generally accepted that these peptides serve as the main nutritional reserve for the developing embryo. -- Quantification of vitellogenin in blood is useful for different purposes. The reproductive status and degree of sexual maturation of oviparous animals can be assessed according to the levels of vitellogenin in plasma. The expression of this protein can also be induced in males under the effect of estrogenic compounds. Relying on this observation, some studies have used vitellogenin as a biomarker of environmental endocrine disruption in many species. -- The objective in this work was to develop a novel quantitative technique for measuring plasma levels of Atlantic salmon and rainbow trout vitellogenin using high performance liquid chromatography coupled to tandem mass spectrometry. These two species are of vital importance for the sustainable commercial fisheries and aquaculture activities in Atlantic Canada. -- To achieve this goal, the first step of this project consisted of a comprehensive characterization and partial sequencing of Atlantic salmon and rainbow trout vitellogenin. The initial approach employed both chemical and enzymatic digestions of vitellogenin coupled with tandem mass spectrometry, one of the most powerful techniques available for sequencing, identification and characterization of proteins. The information from these studies was then used to select a 'signature peptide' characteristic for both species. This peptide was used as a surrogate of the precursor protein for quantification purposes. A detailed account is given on all the technical features of the quantitative technique. Special attention was placed on the tuning and testing of this system, focusing on the recovery, specificity and sensitivity of the method. Additional characterization and sequencing was also performed for Atlantic cod vitellogenin. The results obtained in this work allow this method to be considered as an alternative to existing immunological assays.

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