Differential Mass Spectrometry of Rat Plasma Reveals Proteins That Are Responsive to 17-Estradiol and a Selective Estrogen Receptor Modulator PPT Xuemei Zhao, † Ekaterina G. Deyanova, † Laura S. Lubbers, ‡ Pete Zafian, ‡ Jenny J. Li, † Andy Liaw, § Qinghua Song, § Yi Du, † Robert E. Settlage, † Gerry J. Hickey, ‡ Nathan A. Yates, † and Ronald C. Hendrickson* ,† Department of Proteomics, Department of Pharmacology, and Biometrics Research, Merck Research Laboratories, Rahway, New Jersey 07065 Received April 23, 2008 Estrogens are a class of steroid hormones that interact with two related but distinct nuclear receptors, estrogen receptor (ER) R and . To identify potential ER biomarkers, we profiled the rat plasma glycoproteome after treatment with vehicle or 17-estradiol (E2) or an ERR-selective agonist PPT by differential mass spectrometry. Our comparative proteomic experiment identifies novel E2- and PPT- responsive proteins, such as serine protease inhibitor family members. Keywords: 17-estradiol • estrogen receptor • mass spectrometry • PPT • proteomics • plasma protein • N-linked glycoproteins Introduction Estrogens are a class of steroid hormones that have profound influences on reproductive tissues, such as breast, ovary, and uterus in females, as well as testes and prostate in males, and nonreproductive tissues, such as bone, the cardiovascular system, and the central nervous system. Estrogens exert their diverse effects on different tissues through interaction with two related but distinct nuclear receptors, estrogen receptor (ER) R and , which have different gene expression patterns in vivo. 1-3 Estrogens bind to the ligand-binding domain of ERR/ ER to induce a conformational change of the receptor(s). The ligand-receptor complexes regulate transcription of their targeted genes through distinct pathways. For example, both ERR and ER function as ligand-dependent transcriptional activators that interact directly with an estrogen-response element (ERE) on promoter DNA. In addition, ERR functions as a transcriptional activator, whereas ER functions as a transcriptional repressor, by interaction of the ligand-receptor complexes with other transcription factors, such as AP-1 and Sp1, without direct ERR and ER binding to DNA. In these pathways, ligand-receptor complexes regulate transcription of targeted genes through recruitment of different coregulators and components of the transcriptional machinery on different promoters. To date, considerable effort has been invested in mRNA profiling of estrogen targeted genes; however, little is known about alteration of the proteome in response to estrogens. 17-Estradiol (E2) is the most potent member of the class of estrogens and binds to both ERR and ER. 4 The amino acid sequences in the ligand-binding domains of ERR and ER share 56% identity in humans and 55% homology in rats. 5,6 E2 binds with high and relatively similar affinity to both ER subtypes. 5 ERR and ER differentially regulate gene expression of targeted genes and consequently have different effects on different tissues. Because of the diverse functions of ERR and ER, great effort has been invested in the study of selective estrogen receptor modulators (SERMs), which are ligands that have the capacity to selectively bind to and activate ERR or ER. 7-11 For example, the nonsteroidal compound propylpyrazole triol (PPT) is an ERR-selective agonist based on its transactivation effects on gene constructs containing consensus EREs. 12,13 Its binding affinity to ERR is about 50% that of E2, and has a 410-fold binding affinity preference for ERR over ER. Compared with estrogens, SERMs exert similar and distinct tissue-specific effects, 3,10,14-17 and thus will assist in the development of estrogen pharmaceuticals with improved tissue selectivity and specificity. To understand the broad physiological function of estrogens as well as the effect of SERMs, multiple mRNA profiling studies on ER targeted genes have been performed in different cell lines and animal tissues and have revealed rich information on changes in mRNA expression profiles induced by ER ligands in different biological systems. 17-26 Information on global profiling of ER targeted genes at the protein level, however, is very limited. 27 In addition, it is well-known that estrogen- responsive genes exhibit tissue- and temporal-specific expres- sion patterns in adult animals. 28-32 Therefore, to monitor the engagement of estrogens or SERMs to their targets in clinical trials, we are interested in searching for pharmacodynamic * To whom correspondence should be addressed. Mailing address: Ronald C. Hendrickson, Merck & Co., Inc., RY800-B301, 126 E. Lincoln Ave, P.O. Box 2000, Rahway, NJ 07065-0900. Phone, (732) 594-5940; fax, (732) 594- 3371; e-mail, ronald_hendrickson@merck.com. † Department of Proteomics, Merck Research Laboratories. ‡ Department of Pharmacology, Merck Research Laboratories. § Biometrics Research, Merck Research Laboratories. 10.1021/pr800309z CCC: $40.75  2008 American Chemical Society Journal of Proteome Research 2008, 7, 4373–4383 4373 Published on Web 09/12/2008