Our objective is to develop innovative and sensitive immunoproteomic assays applicable to analysis of estrogen dependent proteins involved in reproductive processes. This technology employs an antibody capture step fottowed by identification of captured proteins by mass spectometry. Immnunoproteomics has the potential to allow rapid (<30 min) assays requiring small volumes of sample and antibody. Key to any immunoproteomic assay is optimizing the buffering systems for tissue extraction, the antibody capture step and washing of the immobilized antigen. This enhances the signal to noise ratio of the mass spectrometric ste and potentially obviate variations in protein-antibody interactions that will plague muItiple assay procedures. Towards this end we will initially develop the immunoproteomic technology for known estrogen markers, followed by measurement of those markers in the complex biological mixtures of cell extracts. Thus, we have two Specific Ams. 1. Develop an immunoproteomic assay for selected estrogen sensitive markers. 2. Demonstrate estrogenization with the technology in Specific Aim 1. Our studies will focus on the progesterone receptor and FKBP52. Cytosolic extracts from a variety of reproductive tissues including rabbit uterine cytosol, Ishakawa cells, and T47D breast cancer cells will be employed. Assorted buffers will be utilized to extract/solubilize proteins, optimize antibody capture and wash immobilized antgen prior to laser desorption. Mass spectral analyses of time-of-flight (TOF) utilized Ciphergen TM SELDI [surface enhanced laser desorption ionization] technology will detect the presence of the estrogen sensitive proteins. Applications include: assessment of action of environmental estrogens, screening for endocrine disruptors, a diagnostic tool for breast and other steroid sensitve carcinomas, study of anti-estrogens, steroid sensitivity profiling, and assessment of postranslatonal modifications.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Small Research Grants (R03)
Project #
5R03HD043171-02
Application #
6699080
Study Section
Pediatrics Subcommittee (CHHD)
Program Officer
Yoshinaga, Koji
Project Start
2003-01-20
Project End
2005-12-31
Budget Start
2004-01-01
Budget End
2005-12-31
Support Year
2
Fiscal Year
2004
Total Cost
$73,500
Indirect Cost
Name
University of Toledo
Department
Physiology
Type
Schools of Medicine
DUNS #
807418939
City
Toledo
State
OH
Country
United States
Zip Code
43614
Pang, Haiyan; Rowan, Brian G; Al-Dhaheri, Mariam et al. (2004) Epidermal growth factor suppresses induction by progestin of the adhesion protein desmoplakin in T47D breast cancer cells. Breast Cancer Res 6:R239-45