Quantitative measurement of fathead minnow vitellogenin by liquid chromatography combined with tandem mass spectrometry using a signature peptide of vitellogenin

Vitellogenin (VTG) has been proposed as a sensitive biomarker of exposure to environmental estrogenic contaminants that induce VTG production in oviparous species. Enzyme-linked immunosorbent assay (ELISA) methods are currently widely used to measure the VTG levels. In this paper, a new liquid chromatography combined with tandem mass spectrometry (LC/ESI-MS/MS) method for the quantitative analysis of VTG in the plasma of fathead minnows exposed to 17alpha-ethinylestradiol (EE2) has been developed. This method includes, first, the selection of the signature peptide, which involves sodium dodecyl sulfate-polyarylamide gel electrophoresis separation, in-gel digestion, LC/ESI-MS/MS analysis with an ion trap mass spectrometer, and sequence determination with the TurboSEQUEST MS/MS database application; second, optimization of the selected signature peptide in multireaction monitor (MRM) mode with a triple quadrupole mass spectrometer; and third, trypsin digestion of plasma and VTG quantitation via MRM-mode LC/ESI-MS/MS. A series of plasma samples from fathead minnows following exposure to EE2 was assayed. A good correlation was found when EE2-induced plasma samples from fathead minnows were analyzed with ELISA and the described new method. Although used here with fathead minnow, the new LC/ESI-MS/MS method could be easily applied to the analysis of VTG expressed in any other fish species. Quantitation of VTG by this method was found to be highly specific and linear. The absence of potential artifactual measurements of VTG at low exposure levels could also be critical in future studies that evaluate weakly estrogenic compounds in aquatic species.

[1]  J. Giesy,et al.  Quantification of rainbow trout (Oncorhynchus mykiss) zona radiata and vitellogenin mRNA levels using real-time PCR after in vivo treatment with estradiol-17β or α-zearalenol , 2000, The Journal of Steroid Biochemistry and Molecular Biology.

[2]  D. Burks,et al.  Vitellogenin gene transcription: A relative quantitative exposure indicator of environmental estrogens , 2001, Environmental toxicology and chemistry.

[3]  N. Hiramatsu,et al.  Transition of serum vitellogenin cycle in Sakhalin taimen (Hucho perryi). , 1997, Comparative biochemistry and physiology. Part C, Pharmacology, toxicology & endocrinology.

[4]  A. Shevchenko,et al.  Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. , 1996, Analytical chemistry.

[5]  K. Pedersen,et al.  Vitellogenin in Zoarces viviparus: purification, quantification by ELISA and induction by estradiol-17 beta and 4-nonylphenol. , 1998, Comparative biochemistry and physiology. Part C, Pharmacology, toxicology & endocrinology.

[6]  T. Yan,et al.  A zebrafish vitellogenin gene (vg3) encodes a novel vitellogenin without a phosvitin domain and may represent a primitive vertebrate vitellogenin gene. , 2000, Gene.

[7]  J. Yates,et al.  An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database , 1994, Journal of the American Society for Mass Spectrometry.

[8]  A. Soto,et al.  Developmental effects of endocrine-disrupting chemicals in wildlife and humans. , 1993, Environmental health perspectives.

[9]  N. Spooner,et al.  Reduction of signal suppression effects in ESI-MS using a nanosplitting device. , 2001, Analytical chemistry.

[10]  J. Rinchard,et al.  Comparative study of reproductive biology in single and multiple‐spawner cyprinid fish. II. Sex steroid and plasma protein phosphorus concentrations , 1997 .

[11]  Ronald J. Moore,et al.  Analysis of α2u-Globulin in Rat Urine and Kidneys by Liquid Chromatography−Electrospray Ionization Mass Spectrometry , 1998 .

[12]  Erin M. Snyder,et al.  Effects of nonylphenol ethoxylate exposure on reproductive output and bioindicators of environmental estrogen exposure in fathead minnows, Pimephales promelas , 2001, Environmental toxicology and chemistry.

[13]  A. Goksøyr,et al.  Monoclonal antibody enzyme‐linked immunosorbent assay to quantify vitellogenin for studies on environmental estrogens in the rainbow trout (Oncorhynchus mykiss) , 2002, Environmental toxicology and chemistry.

[14]  H Segner,et al.  Development and validation of a homologous zebrafish (Danio rerio Hamilton-Buchanan) vitellogenin enzyme-linked immunosorbent assay (ELISA) and its application for studies on estrogenic chemicals. , 2001, Comparative biochemistry and physiology. Toxicology & pharmacology : CBP.

[15]  T. Braunbeck,et al.  Measurement of vitellogenin gene expression by RT-PCR as a tool to identify endocrine disruption in Japanese medaka (Oryzias latipes) , 2002, Biomarkers : biochemical indicators of exposure, response, and susceptibility to chemicals.

[16]  E. Tamiya,et al.  Development and application of a monoclonal antibody-based sandwich ELISA for quantification of Japanese medaka (Oryzias latipes) vitellogenin. , 2002, Comparative biochemistry and physiology. Toxicology & pharmacology : CBP.

[17]  Edward A. Dratz,et al.  Absolute quantification of the G protein-coupled receptor rhodopsin by LC/MS/MS using proteolysis product peptides and synthetic peptide standards. , 2003 .

[18]  E. Bradbury,et al.  Precise peptide sequencing and protein quantification in the human proteome through in vivo lysine-specific mass tagging , 2003, Journal of the American Society for Mass Spectrometry.

[19]  P. Bjerregaard,et al.  In vivo estrogenic activity of branched and linear alkylphenols in rainbow trout (Oncorhynchus mykiss). , 1999, The Science of the total environment.

[20]  J. Sumpter,et al.  Vitellogenesis as a biomarker for estrogenic contamination of the aquatic environment. , 1995, Environmental health perspectives.

[21]  E. Oberdörster,et al.  Vitellogenin induction in painted turtle, Chrysemys picta, as a biomarker of exposure to environmental levels of estradiol. , 2001, Aquatic toxicology.

[22]  F. Regnier,et al.  Strategy for qualitative and quantitative analysis in proteomics based on signature peptides. , 2000, Journal of chromatography. B, Biomedical sciences and applications.

[23]  F. Regnier,et al.  Signature-peptide approach to detecting proteins in complex mixtures. , 2000, Journal of chromatography. A.

[24]  Salvatore Sechi,et al.  A method to identify and simultaneously determine the relative quantities of proteins isolated by gel electrophoresis. , 2002, Rapid communications in mass spectrometry : RCM.

[25]  R. Robinson,et al.  Œstrogenic Activity of Certain Synthetic Compounds , 1938, Nature.

[26]  J. Specker,et al.  Induction by beta-estradiol of vitellogenin in striped bass (Morone saxatilis): characterization and quantification in plasma and mucus. , 1992, General and comparative endocrinology.

[27]  L. Christensen,et al.  Monitoring and risk assessment for endocrine disruptors in the aquatic environment: a biomarker approach. , 1998, Archives of toxicology. Supplement. = Archiv fur Toxikologie. Supplement.

[28]  F. Regnier,et al.  Automated signature peptide approach for proteomics. , 2001, Journal of chromatography. A.

[29]  K. Yamauchi,et al.  Changes in hepatic vitellogenin mRNA levels during oocyte development in the Japanese eel, Anguilla japonica. , 2002, General and comparative endocrinology.

[30]  S. B. Jones,et al.  An approach for assessment of water quality using semipermeable membrane devices (SPMDs) and bioindicator tests. , 2000, Chemosphere.

[31]  G. Ankley,et al.  Fathead minnow vitellogenin: Complementary DNA sequence and messenger RNA and protein expression after 17β‐estradiol treatment , 2000 .

[32]  D. Chelius,et al.  Identification and relative quantitation of protein mixtures by enzymatic digestion followed by capillary reversed-phase liquid chromatography-tandem mass spectrometry. , 2002, Analytical chemistry.

[33]  P. Walsh,et al.  5 Vitellogenesis and Oocyte Assembly , 1988 .

[34]  Gerald T Ankley,et al.  Comparison of ELISAs for detecting vitellogenin in the fathead minnow (Pimephales promelas). , 2003, Comparative biochemistry and physiology. Toxicology & pharmacology : CBP.

[35]  O. Carnevali,et al.  Gilthead seabream (Sparus aurata) vitellogenin: purification, partial characterization, and validation of an enzyme-linked immunosorbent assay (ELISA). , 1998, General and comparative endocrinology.

[36]  Richard D. Smith,et al.  Stable isotope-coded proteomic mass spectrometry. , 2003, Current opinion in biotechnology.