A Proteomic (SELDI-TOF-MS) Approach to Estrogen Agonist Screening

Abstract

A small fish model and surface-enhanced laser desorption/ionization time-of-flight mass spectrometry were used to investigate plasma protein expression as a means to screen chemicals for estrogenic activity. Adult male sheepshead minnows (Cyprinodon variegatus) were placed into aquaria for seawater control, solvent control, and treatments of 17beta-estradiol (E2), methoxychlor (MXC), bisphenol-A (BPA), 4-tert-pentylphenol (TPP), endosulfan (ES), and chlorpyriphos (CP). Fish plasma was applied to weak cation exchange (CM10) ProteinChip arrays, processed, and analyzed. The array produced approximately 42 peaks for E2 plasma and 30 peaks for solvent control plasma. Estrogen-responsive mass spectral biomarker peaks were identified by comparison of E2-treated and control plasma spectra. Thirteen potential protein biomarkers with a range from 1 to 13 kDa were up- or downregulated in E2-treated fish and their performance as estrogenic effects markers was evaluated by comparing spectra from control, estrogen agonist, and nonagonist stressor-treated males and normal female fish plasma. One of the biomarkers, mass-to-charge ratio 3025.5, was identified by high-resolution tandem mass spectrometry as C. variegatus zona radiata protein, fragment 2. The weak environmental estrogens MXC, BPA, and TPP elicited protein expression profiles consistent with the estrogen expression model. Estrogen-responsive peaks were not detected in plasma from fish in the seawater, vehicle, ES, or CP treatments. No difference was found between plasma protein expression of seawater control and solvent control fish. We show that water exposure of fish to estrogen agonists produces distinct plasma protein biomarkers that can be reproducibly detected at low levels using protein chips and mass spectrometry.

Review of recent proteomic applications in aquatic toxicology

“…Similar to research on model species, the most commonly used method of protein separation in non-model species is 2DGE, although LC has been utilized in a few studies [6]. Tandem TOF/TOF is the most frequently used mass spectrometric setup in non-model proteomic research, but TOF analyzers coupled with quadropoles are not uncommon [33]. An FT-ICR mass analyzer coupled with an ion trap was also used in one study [31].…”

“…Surface-enhanced laser desorption ionization (SELDI)-TOF is a technique that exploits the chemical characteristics (e.g., hydrophobicity, charge) of proteins as a means of simplifying complex samples; SELDI-TOF does not enable one to identify differentially expressed proteins, but does yield information that may be helpful in identifying these proteins (peaks) of interest through subsequent tandem MS analysis. This technique has been used in at least two recent non-model studies [33,34], and was highlighted in another [3]. The isobaric tags for relative and absolute quantification (iTRAQ) approach was also used recently to quantitatively profile hepatic fathead minnow proteins involved in androgen receptor signaling [6].…”

“…That study [6] was the first to apply this technique to aquatic toxicology and proved its utility and power. Proteomic research on non-model fish species have examined physiological responses to endocrine disrupting compounds [33,34], Cd [36,37], and microcystin-LR [7,38,39], among other environmental contaminants [30]. Protein profiles have mostly been evaluated in hepatic tissues, but occasionally in gill [37], brain [36], and kidney tissues [40].…”

Toxicogenomics Applied to Ecological Risk Assessment

“…Another study that employed SELDI-TOF MS with a weak cation exchange ProteinChip to analyze protein expression in hemolymph from field collected blue mussels (Mytilus edulis) exposed to crude oil, oil spiked with PAHs, or oil spiked with alkylphenols in the laboratory was able to classify masked samples with 90% to 95% accuracy . Other investigators demonstrated that either SELDI-TOF MS or MALDI-TOF MS could be used in conjunction with ProteinChip arrays to detect anonymous biomarkers of xenoestrogen exposure in plasma samples from adult male sheepshead minnows (Walker et al, 2007;Salinas et al, 2008). These studies represented progress toward developing a laboratory-based assay that could possibly evaluate multiple endocrine modes of action based on a single laboratory exposure.…”

“…By the approach, over 40 proteins differentially expressed following exposure to a strong androgen, an antiandrogen, or a mixture of the two were identified, making it possible to conduct ontology-and pathway-based analyses similar to those commonly utilized in transcriptomic studies (Martyniuk et al, 2008). In addition to studies that are specifically mechanistic in orientation, a variety of proteomics studies aimed at PES and/or biomarker identification have determined the identities of at least some differentially expressed proteins, in some cases identifying sufficient numbers to facilitate ontology-and pathway-based analyses (Rodriguez-Ortega et al, 2003;Apraiz et al, 2006;Amelina et al, 2007;Walker et al, 2007). Finally, a few groups have employed proteomic approaches to examine alterations in protein modification associated with polluted versus reference field sites or laboratory exposures (McDonagh et al, 2005;McDonagh and Sheehan, 2007).…”