Differential Mass Spectrometry of Rat Plasma Reveals Proteins That Are Responsive to 17β-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 β. To identify potential ER biomarkers, we profiled the rat plasma glycoproteome after treatment with vehicle or 17β-estradiol (E2) or an ERR-selective agonist PPT by differential mass spectrometry. Our comparative proteomic experiment identifies novel E2- and PPTresponsive proteins, such as serine protease inhibitor family members. Keywords: 17β-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 β, which have different gene expression patterns in vivo.1-3 Estrogens bind to the ligand-binding domain of ERR/ ERβ 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 ERβ 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 ERβ 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 ERβ 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. * 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) 5943371; e-mail,
[email protected]. † 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
17β-Estradiol (E2) is the most potent member of the class of estrogens and binds to both ERR and ERβ.4 The amino acid sequences in the ligand-binding domains of ERR and ERβ 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 ERβ differentially regulate gene expression of targeted genes and consequently have different effects on different tissues. Because of the diverse functions of ERR and ERβ, 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 ERβ.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 ERβ. 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 estrogenresponsive genes exhibit tissue- and temporal-specific expression 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 Journal of Proteome Research 2008, 7, 4373–4383 4373 Published on Web 09/12/2008
research articles markers, in particular, acute protein markers, in animal plasma that are responsive to estrogen or SERMs treatment. In this study, we utilized high-resolution Fourier transform mass spectrometry (FTMS) and differential mass spectrometry (dMS) to identify proteins in plasma that respond acutely to E2 or PPT treatment in rats. Differential mass spectrometry is a general proteomics workflow that provides relative quantitation and identifies statistically significant changes in full scan mass spectrometry data.33,34 Because protein glycosylation is a very common post-translational modification and more than half of all proteins are thought to be glycosylated,35,36 we focused on the N-linked glyco-proteome, which contains glycosylated proteins with carbohydrates linked to asparagine residues.37 To look for acute E2- and PPT-responsive proteins, we biochemically isolated formerly N-linked glycosylated peptides from plasma of vehicle-, E2-, or PPT-treated rats using a method that was described previously.38 Isolated peptides were profiled using micro capillary liquid chromatography mass spectrometry (LC-MS) and statistically significant differences between these complex mass spectrometric profiles were detected using dMS analysis.33,34 Peptides that were responsive to E2 or PPT treatment were identified by targeted tandem mass spectrometry (MS/MS). In addition to confirming several estradiol-responsive proteins in rat plasma, our proteomic profiling studies discovered a number of novel plasma proteins that are responsive to E2 or PPT treatment in rat. Furthermore, for one identified PPT-responsive protein, alpha-1-acid glycoprotein, we validated our proteomics quantification by a quantitative ELISA analysis.
Materials and Methods In Vivo Procedures and Tissue Collection. Adult, female Sprague-Dawley rats (175-225 g; Taconic Farms, Germantown, NY) were purchased ovariectomized and allowed at least 2 weeks to acclimate following arrival. Animals had free access to water and a phytoestrogen-reduced diet (TD96155, Harlan Teklad). All animal care and handling procedures were approved by the Institutional Animal Care and Use Committee at Merck & Co., Inc., and performed in accordance with NIH guidelines. Animals were assigned to one of three groups in this study: vehicle (V, sesame oil), 17β-estradiol (E2, 0.05 mg/kg, solubilized in sesame oil, Sigma, Catalog no. E1024), or 4,4′,4′′(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT, 3 mg/kg, solubilized in sesame oil ERR agonist, Tocris Cookson, Inc., Catalog no. 1426). Each treatment group had 9 rats for a total of 27 rats. Treatments were injected subcutaneously in the dorsal cervical region once daily for 4 days. The dose and duration of E2 and PPT treatment were based on published39 and unpublished (Laura S. Lubbers) data. Four hours after the final dose, animals were euthanized with CO2. Uterine tissue was excised and weighed for all animals. The change in uterine weight among groups was compared using one way analysis of variance followed by Student-Newman-Keuls test. Blood was obtained via cardiac puncture using prechilled syringes containing K3-EDTA (1 mg/mL blood, final concentration). Blood then was placed into chilled tubes containing aprotinin (500 KIU/mL blood; TRASYLOL VLE, Serological Proteins, Inc.; Code 96-072-3) and a Dipeptidyl Peptidase IV; EC 3.4.14.5 inhibitor (10 µL/mL blood; LINCO Catalog no. DPP4-010) and mixed. Blood was centrifuged at 1500g and 4 °C within 20 min of collection. Plasma was harvested, aliquoted, and immediately frozen on dry ice. Order of necropsy was 4374
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Zhao et al. interwoven among three treatment groups. Time from cardiocentesis to final freezing of plasma on dry ice was minimized and kept constant for all samples. Biochemical Isolation of Formerly N-Linked Glycosylated Peptides from Rat Plasma. Biochemical isolation of formerly N-linked glycosylated peptides from rat plasma was performed using the method developed by Zhang et al.38 with minor modifications. In this study, two aliquots of samples from vehicle-treated rats, V-1 and V-2, as well as one set of samples from E2 and PPT treated rats, were analyzed in a single block design with samples interwoven using an order that alternated between V-1, E2, V-2, and PPT across individual animals in each treatment group. A mixture of three purified human N-linked glycoproteins was added into each sample at the beginning of the biochemical isolation experiment to serve as a spike-in/ recovery internal control. The final concentrations of the three human proteins in 25 µL of rat plasma sample were 50 µg/mL of R2-HS-Glycoprotein (Calbiochem, no. 362199), 50 µg/mL of R1-Antitrypsin (Calbiochem, no. 178251), and 10 µg/mL of R1Antichymotrypsin (Calbiochem, no. 178196). Twenty-five microliters of rat plasma sample with three human proteins spiked-in was added to 75 µL of coupling buffer (100 mM NaOAc and 150 mM NaCl, pH 5.5; Bio-Rad, no. 153-6054) with 15 mM NaIO4 (Bio-Rad, no. 153-6055) and incubated at room temperature for 1 h. After desalting the oxidation mixture by a protein desalting spin column (Pierce, no. 89862), the oxidized sample was conjugated to 200 µL of packed hydrazide resin (Bio-Rad, no. 153-6047) in a mini-spin column (Pierce, no. 69705) at room temperature for 18 h. The hydrazide resin was then washed six times with 300 µL of 8 M urea/0.4 M NH4HCO3 (Sigma, A-6141) pH 8.3 to remove nonglycoproteins. Each wash cycle included 5 min of rotating incubation at room temperature and 3 min of centrifugation at 3000 rpm. The hydrazide resin-conjugated proteins were reduced in 300 µL of 8 mM TECP (Pierce, no. 20490)/50 mM NH4HCO3 at room temperature for 30 min, alkylated in 300 µL of 10 mM iodoacetamide (Sigma, I-6125)/50 mM NH4HCO3 at room temperature for 30 min, and digested with 25 µg of trypsin (Promega, no. V5280) in 300 µL of 50 mM NH4HCO3 at 37 °C with shaking at 400 rpm for 18 h. To remove nonglycosylated peptides, the hydrazide resin was washed three times with 300 µL of 1.5 M NaCl, three times with 300 µL of 80% acetonitrile (Fisher, A9981)/0.1% trifluoroacetic acid (Pierce, no. 28904) (vol/vol), three times with 300 µL of methanol (Fisher, A454-1), and six times with 300 µL of 50 mM NH4HCO3 at room temperature. Formerly N-linked glycopeptides were released from the hydrazide resin by treatment with 3 µL (1500 U) of PNGase F (NEB, P0705L) in 300 µL of 50 mM NH4HCO3 at 37 °C with shaking at 400 rpm for 18 h. The released peptides were dried and resuspended in 25 µL of 0.1 M HOAc. The resuspended peptides were further diluted 5-fold in 0.1 M HOAc. One microliter of diluted sample was used for LC-MS profiling analysis. All isolated peptides were stored at -20 °C. LC-MS Data Acquisition. Each sample was profiled once using a microcapillary HPLC system coupled with an LTQ-FT mass spectrometer (Thermo Electro Corp., Madison WI) via a nanoelecrospray ionization source (Proxeon, Odense Denmark). The microcapillary HPLC apparatus consisted of a FAMOS micro autosampler (Dionex Corp., Sunnyvale CA) and an 1100 series capillary pump (Agilent Technologies, Palo Alto, CA). An HPLC solvent system composed of solvent A (0.1 M HOAc) and solvent B (90% MeCN) was used to produce a linear gradient that increased organic composition from 0% B to 30%
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E2- and PPT-Responsive Plasma Proteins B at a rate of 0.7% B/µL. The micro liter pickup mode was used to inject 1.0 µL of each sample onto the head of a 100 µm × 2.5 cm ProteoPep sample trap (New Objective Inc., Woburn MA) and a 9 µL volume of solvent A was used to elute salts and other hydrophilic components of the sample to waste. Following sample desalting, the LTQ-FT divert valve was switched to direct the HPLC flow to a 75 µm × 5 cm PicoFrit column (New Objective, Inc., Woburn MA) packed with Poros R2. A flow rate of 1.0 µL/min was used to elute peptides from the column and introduce them into the nanoelectrospray source. The primary instrument parameters that were used to ionize the peptides and transmit the resulting gas phase ions into the LTQ-FT were 3.5 kV source voltage, 240 °C capillary temperature, 25.0 V capillary potential, 85 V tube lens potential, and no sheath or auxiliary gas flow. AGC was used to limit the maximum ion current introduced into the mass spectrometer to 1.0 × 106 (arbitrary units) and 1.5 × 104 (arbitrary units) for Fourier Transform (FT) and Linear Ion Trap (IT) full scan spectra, respectively. An AGC target value of 5.0 × 103 was used for IT MSn spectra. Full scan FT spectra were recorded from m/z 300 to 2000 at a resolution setting of 50,000 and mass accuracy
