Quantification of vitellogenin

Details Quantification of vitellogenin Quantification of vitellogenin

C - Chemistry, Metallurgy

07

K

C07K 7/08 (2006.01) C07K 7/06 (2006.01) G01N 30/72 (2006.01)

Patent

CA 2588344

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 trout and Atlantic salmon, plasma samples obtained from control and .beta.-estradiol induced fish were digested with trypsin. A characteristic 'signature peptide' was selected and analyzed by high performance liquid chromatography coupled to an 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 .beta.-Estradiol, and subjected to trypsin digestion for characterization by matrix-assisted laser desorption/ionization- Quadrupole-Time-of-flight tandem mass spectrometry. A peptide mass fingerprint 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 shares a series of common peptides with the two different known vitellogenin sequences of Haddock, a closely related species. There are also disclosed novel isolated signature peptides, namely Thr-Tyr-Phe-Ala-Gly-Ala-Ala-Ala-Asp-Val-Leu-Glu- Val-Gly-Val-Arg, Asp Leu Gly Leu Ala Tyr Thr Glu Lys, Phe Phe Gly Gln Glu Ile Ala Asn Ile Asp Lys, Glu Ile Val Leu Leu Gly Tyr Gly Thr Met Ile Ser Lys and Tyr Glu Ser Phe Ala Val Ala Arg.

Affiliated with

Also associated with

Rating

Quantification of vitellogenin does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Quantification of vitellogenin, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Quantification of vitellogenin will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFCA-PAI-O-2084795