Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Feb 8;22(4):1686.
doi: 10.3390/ijms22041686.

Proteomics Complementation of the Rat Uterotrophic Assay for Estrogenic Endocrine Disruptors: A Roadmap of Advancing High Resolution Mass Spectrometry-Based Shotgun Survey to Targeted Biomarker Quantifications

Laszlo Prokai  1 , Fatima Rahlouni  1 , Khadiza Zaman  1 , Vien Nguyen  1 , Katalin Prokai-Tatrai  1
Affiliations

Proteomics Complementation of the Rat Uterotrophic Assay for Estrogenic Endocrine Disruptors: A Roadmap of Advancing High Resolution Mass Spectrometry-Based Shotgun Survey to Targeted Biomarker Quantifications

Laszlo Prokai et al. Int J Mol Sci. .

Abstract

The widely used rat uterotrophic assay to assess known and potential estrogenic compounds only considers uterine weight gain as endpoint measurement. To complement this method with an advanced technology that reveals molecular targets, we analyzed changes in protein expression using label-free quantitative proteomics by nanoflow liquid chromatography coupled with high-resolution mass spectrometry and tandem mass spectrometry from uterine protein extracts of ovariectomized rats after daily 17β-estradiol exposure for five days in comparison with those of vehicle-treated control animals. Our discovery-driven study revealed 165 uterine proteins significantly regulated by estrogen treatment and mapped by pathway analyses. Estrogen-regulated proteins represented cell death, survival and development, cellular growth and proliferation, and protein synthesis as top molecular and cellular functions, and a network found with the presence of nuclear estrogen receptor(s) as a prominent molecular node confirmed the relevance of our findings to hormone-associated events. An exploratory application of targeted proteomics to bisphenol A as a well-known example of an estrogenic endocrine disruptor is also presented. Overall, the results of this study have demonstrated the power of combining untargeted and targeted quantitative proteomic strategies to identify and verify candidate molecular markers for the evaluation of endocrine-disrupting chemicals to complement a conventional bioassay.

Keywords: 17β-estradiol; bisphenol A; endocrine disruption; estrogen-regulated proteins; high resolution mass spectrometry; label-free proteomics; liquid chromatography–mass spectrometry; protein networks; rat uterus; targeted proteomics.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Screening from acquired high mass resolution (Orbitrap) mass spectra during the untargeted shotgun acquisitions for transglutaminase 2 (TGM2) as a prioritized uterine protein marker for targeted assay development. (a) Locating a proteotypic tryptic peptide of TGM2 by accurate mass of its doubly-charged ion [M+2H]2+ (TIC: total-ion chromatogram, brown and green traces: uterus samples from control and E2-treated rat, respectively); (b) Verifying accurate masses and isotope peak distribution of the doubly-charged ions (R: mass resolution, inset: theoretical m/z and isotope peak distribution); (c) Confirming the peptide sequence SEGTYC@C@GPVSVR by the acquired ion-trap tandem mass spectrometry (MS/MS) scan (b and y sequence ions marked according to nomenclature by Roepstorff and Fohlman [41]; C@ is carbamidomethylated cysteine).
Figure 2
Figure 2
Molecular interaction Ingenuity Pathway Analysis (IPA®) network associated with connective tissue development and function, organ morphology, reproductive system development, and function assembled from the E2-regulated uterine proteins TGM2, elongation factor 2 (EEF2), selenium binding protein 1 (SELBP1), and lumican (LUM) in the rat. The shapes (see legend in the blue box) represent molecular classes of the regulated proteins. In the network, red and green colors denote upregulation and downregulation in response to E2 treatment, respectively. The intensity of the color indicates the relative magnitude of fold change in protein expression pattern based on spectral counts. Solid and dashed lines represent direct and indirect interactions, respectively. Abbreviations: ABLIM1, actin binding LIM protein 1; ABCC9, adenosine triphosphate (ATP) binding cassette subfamily C member 9; ACTC1, actin alpha cardiac muscle 1; BLK, proto-oncogene, Src family tyrosine kinase; CDCA7, cell division cycle associated 7; CDK18, cyclin dependent kinase 18; CNBP, CCHC-type zinc finger nucleic acid binding protein; COPE, COPI coat complex epsilon; ESR1, estrogen receptor 1; F7, coagulation factor 7; FARSB, phenylalanyl–tRNA synthetase subunit beta; GDP, guanosine diphosphate; GM2A, ganglioside activator; GRM5, glutamate metabotropic receptor 5; HSPB2, heat shock protein family B (small) member 2; KCNQ5, potassium voltage-gated channel subfamily Q member 5; MCM4, minichromosome maintenance complex component 4; mir, microRNA; MYO9A, myosin IXa; NDRG1, N-myc downstream regulated 1; PADI4, peptidyl arginine deiminase 4; PI3, peptidase inhibitor 3; PLAZR1, phospholipase A2 receptor 1; PNRC1, proline rich nuclear receptor coactivator; PPPP5C, protein phosphatase 5 catalytic subunit; SLC25A15, solute carrier family 25 member 15; TMOD2, tropomodulin 2; WEE1, G2 checkpoint kinase.
Figure 3
Figure 3
Uterus wet weights of E2-treated and BPA-treated OVX Sprague-Dawley rats compared to vehicle-treated controls in the reported experiment. Data are given as average ± standard error, and asterisks (*) indicate statistically significant differences from OVX controls with ANOVA followed by post hoc Dunnett��s tests (p < 0.05).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Colborn T., vom Saal F.S., Soto A.M. Developmental effects of endocrine-disrupting chemicals in wildlife and humans. Environ. Health Perspect. 1993;101:378–384. doi: 10.1289/ehp.93101378. - DOI - PMC - PubMed
    1. Ankley G., Francis E., Gray E., Kavlock R., McMaster S., Reese D., Sayles G., Sergeant A., Vallero D. Research Plan for Endocrine Disruptors. Office of Research and Development, United States Environmental Protection Agency; Washington, DC, USA: 1998. pp. 1–47.
    1. United States Environmental Protection Agency Endocrine Disruptor Screening Program (EDSP) [(accessed on 27 October 2018)]; Available online: http://www.epa.gov/endo/
    1. United States Environmental Protection Agency . Next Generation Risk Assessment: Incorporation of Recent Advances in Molecular, Computational, and Systems Biology. National Center for Environmental Assessment Office of Research and Development, United States Environmental Protection Agency; Washington, DC, USA: 2014. pp. 1–12.
    1. Baker V.A. Endocrine disrupters—Testing strategies to assess human hazard. Toxicol. Vitr. 2001;15:413–419. doi: 10.1016/S0887-2333(01)00045-5. - DOI - PubMed

LinkOut - more resources