title: Characterization and de novo sequencing of Atlantic salmon vitellogenin protein by electrospray tandem and matrix-assisted laser desorption/ionization mass spectrometry.
abstract: Vitellogenin (VTG) is a protein produced by the liver of oviparous animals in response to circulating estrogens. In the plasma of males and immature females, VTG is undetectable. VTG has been used as a biomarker for exposure to endocrine disruptors in many species. In the present study, characterization of intact Atlantic salmon VTG was effected using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI ToF MS). Tryptic digest peptides were analyzed by MALDI ToF MS to obtain a peptide mass fingerprint. De novo sequencing of the tryptic peptides used low-energy collisionally-induced dissociation (CID) in an electrospray ionization quadrupole-ToF orthogonal hybrid mass spectrometer (ESI Q-ToF MS/MS). The interpretation of the product-ion spectra obtained from the ESI Q-ToF MS/MS was done by Lutefisk, a computer-based software algorithm. The molecular mass of the intact protein was found to be 187335 Da. A total of 14 tryptic peptides were sequenced and compared with the complete rainbow trout VTG and the partial Atlantic salmon VTG sequences found in the Swiss-Prot database. De novo sequencing by CID MS/MS of 11 Atlantic salmon tryptic digest peptides with selected precursor ions at m/z 788.24, 700.20, 794.75, 834.31, 889.28, 819.79, 865.27, 843.81, 572.20, 573.66 and 561.68 showed high homology with the known sequence of rainbow trout VTG. The last two precursor peptide ions, found at m/z 573.66 and m/z 561.68, also specifically matched the known portion of the Atlantic salmon VTG sequence. Finally, three tryptic precursor peptide ions found at m/z 795.18, 893.28 and 791.05, provided product-ion spectra, which were exclusive to the unsequenced portion of the Atlantic salmon VTG.
authors.affiliation: Special Projects, Oceans Division, Northwest Atlantic Fisheries Center, St John's, Newfoundland A1C 5X1, Canada. banoubjo@dfo-mpo.gc.ca
authors.forename: Joseph
authors.initials: J
authors.lastname: Banoub
authors.forename: Alejandro
authors.initials: A
authors.lastname: Cohen
authors.forename: Atef
authors.initials: A
authors.lastname: Mansour
authors.forename: Pierre
authors.initials: P
authors.lastname: Thibault
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D020317:Salmo salar; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism; D014819:Vitellogenins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D014819:Vitellogenins; D014357:Trypsin