Quantification of Greenland halibut serum vitellogenin: a trip from the deep sea to the mass spectrometer.
This paper focuses on the sequential steps involved in developing a technique for quantifying Greenland halibut vitellogenin, a serum protein biomarker, using a comprehensive mass spectrometric approach. In the first phase of this study, in-gel trypsin digestions of serum proteins separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). A characteristic band around a molecular mass of 185 kDa, present in the mature female specimens, but absent in the male samples, was identified as vitellognin according to the peptide mass fingerprint obtained by MALDI-MS. Subsequently, MALDI and electrospray ionization tandem mass spectrometry (ESI-MS/MS) analyses were performed on the digest of the vitellogenin band for de novo sequencing. From these studies, a characteristic 'signature' peptide (sequence: FFGQEIAFANIDK) was selected from a list of candidate peptides as a surrogate analytical standard used for quantification purposes. Sample preparation for vitellogenin quantification consisted of a simple one-step overnight trypsin digestion. Samples were spiked with an isotopologue signature peptide standard and analyzed by high-performance liquid chromatography (HPLC) coupled in-line to an electrospray quadrupole-hexapole-quadrupole tandem mass spectrometer, operated in selective reaction monitoring mode. Transitions [(m/z 750.0 --> 1020.4 and 750.0 --> 1205.4) and (754.8 --> 1028.6 and 754.8 --> 1213.2)] were monitored for the signature peptide and the internal standard, respectively. Samples obtained from the field showed that vitellogenin levels were in accordance with fish maturity determined by macroscopic examination of the gonad, proving this technique suitable for measuring vitellogenin as a serum protein biomarker for reproductive maturity in female fish.
Cohen
Alejandro M
AM
Fisheries and Oceans Canada, Science Branch, St John's, Newfoundland, A1C 5X1, Canada.
Jahouh
Farid
F
Sioud
Salim
S
Rideout
Rick M
RM
Morgan
M Joanne
MJ
Banoub
Joseph H
JH
D000595:Amino Acid Sequence; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005432:Flounder / Q000097:blood*; D006066:Gonads / Q000737:chemistry; D006116:Greenland; D008297:Male; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014819:Vitellogenins / Q000032:analysis* / Q000737:chemistry*
D014819:Vitellogenins