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* Department of Cell and Developmental Biology and Anatomy, University of South Carolina School of Medicine, Columbia, South Carolina 29208; and Department of Medicine, Division of Endocrinology, Diabetes and Metabolism, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado 80045
1To whom requests for reprints should be addressed at Department of Cell and Developmental Biology and Anatomy, University of South Carolina School of Medicine, Columbia, South Carolina 29208. E-mail: blake{at}med.sc.edu
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Abstract |
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 Top Abstract The Anterior Pituitary GlandThe AP Proteome and...Factors to Consider When...LC-MS/MS Study of the...DIGE and MALDI-ToF MS...Matching Proteomic Results to...References |
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Key Words: anterior pituitary gland • DIGE • Golden Syrian hamster pituitary • mass spectrometry • mouse pituitary • proteome • rat pituitary
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The Anterior Pituitary Gland |
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TopAbstractThe Anterior Pituitary GlandThe AP Proteome and...Factors to Consider When...LC-MS/MS Study of the...DIGE and MALDI-ToF MS...Matching Proteomic Results to...References |
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The effects and interactions of hypothalamic factors and target organ hormones on the secretion of the AP hormones and their PTMs are not fully understood. In addition, little is known about the regulation of non-hormonal AP proteins, many of which mediate the synthesis and release of the AP hormones. When mass spectrometry (MS) is used to analyze the AP in appropriate animal models (e.g., Ref. 14), it is now possible to determine the response of the AP proteome to exogenously administered hypothalamic factors or target organ secretions, alone or in combination. Furthermore, such investigations open the door for discovery of novel AP secretions and intracellular proteins involved in regulating AP function. In spite of the potential of proteomics to contribute to this important area, there has been a paucity of such studies conducted on the entire pituitary gland (e.g., Refs. 15, 16) and specifically on the AP (17, 18).
Here we present, summarize, and compare some of the proteomic studies we have conducted on the AP of three rodent species. We discuss important decisions that needed to be made as first steps before employing high-performance liquid chromatography (LC) in line with tandem MS (MS/MS) to analyze subcellular fractions of the AP of Golden Syrian hamsters and mice. We then describe how difference gel electrophoresis (DIGE) technology was used in conjunction with matrix-assisted laser desorption ionization–time of flight MS (MALDI-ToF MS) and peptide mass fingerprinting (PMF) to quantify the effects of estrogen on the AP proteome in rats. Subsequently, we discuss the matching of proteomic results to specific cell types within the gland. Although the present overview focuses on the AP, these studies have the potential to serve as a model for investigation of other heterogeneous and multifunctional organs.
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The AP Proteome and Proteomic Strategies |
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TopAbstractThe Anterior Pituitary GlandThe AP Proteome and...Factors to Consider When...LC-MS/MS Study of the...DIGE and MALDI-ToF MS...Matching Proteomic Results to...References |
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We have employed two different proteomic strategies to study the AP. These are illustrated in the flow diagrams in Figure 1
. Procedures for subcellular fractionation of the AP do not necessarily differ between the two strategies, but they are distinguished by the timing of when the digestion is performed (i.e., trypsin digestion followed by peptide separation or protein separation followed by trypsin digestion). In the trypsin digestion and peptide separation strategy, peptides were separated and analyzed by LC-MS/MS using the database search software SEQUEST (19, 20). Because a mixture of proteins is digested in these studies, a peptide containing a PTM cannot be traced back to a specific protein form (i.e., a splice isoform or some other modified form of a protein). Although we did not quantify proteins in these studies, relative quantification of the proteins in two samples can be performed by using isotopic labeling strategies (e.g., Ref. 21). In the protein separation and trypsin digestion method, proteins are minimally labeled with cyanine dyes (CyDyes) and separated by 2D gel electrophoresis. Fluorescent gel images are acquired with a laser scanner and the gel spots quantified using DeCyder software (22). In our studies the gels were then poststained with Deep Purple and the spots excised robotically. The proteins in the gel spots were then digested with trypsin, and the mixture of proteolytic peptides was analyzed by MALDI-ToF MS. Proteins were identified by PMF using the database search software MASCOT.
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Factors to Consider When Conducting Proteomics on the AP |
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TopAbstractThe Anterior Pituitary GlandThe AP Proteome and...Factors to Consider When...LC-MS/MS Study of the...DIGE and MALDI-ToF MS...Matching Proteomic Results to...References |
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The decision was made to conduct initial proteomic analysis of the AP in Golden Syrian hamster despite the fact that its genome was only partially sequenced. It was selected because one of us (C.A.B.) has used this animal model extensively for neuroendocrine investigations. Subsequent studies were conducted on mouse and rat, species in which the genome has been sequenced. In our initial studies of the AP of the young adult male, we adopted hamster and mouse models. Thereafter, ovariectomized rats were used to assess the effects of estrogen on the AP proteome.
Rodents were kept in a room with controlled lighting conditions (12:12-hr light:dark cycle). They were killed during a specified time interval during the first half of the light period. This avoided any changes in the AP proteome that might occur in response to circadian fluctuations (23). In addition, this time interval preceded the time of the surge of gonadotropins in blood that starts during the second half of the light period in proestrous animals or ovariectomized rodents administered estrogen (24, 25).
Stress and anesthetics can influence proteomic analysis by affecting AP hormone release (e.g., Refs. 26–28) and possibly by altering the levels of nonsecreted AP proteins. Thus, it is of interest to avoid stress and to select an anesthetic agent that has minimal effects on AP hormone release. Two different methods of sacrifice were used in the hamster studies. Some hamsters were decapitated; others were anesthetized with sodium pentobarbital before perfusion (17). Decapitation minimizes stress but has the disadvantage of leaving substantial amounts of blood in the AP. Within minutes of injection, pentobarbital suppresses LH release (29) and causes only a small 2- to 4-fold increase in circulating PRL concentrations (30). The subsequent perfusion procedure removes large amounts of blood proteins that can interfere with and mask the analysis of AP proteins. Pentobarbital anesthesia and perfusion were employed in the mouse and rat studies.
Ideally, one would like to be able to expose, remove, and snap freeze or immediately use the AP or any other organ or tissue for proteomic analysis within a few seconds of touching the animal. This would minimize any proteolysis that might occur during the collection procedure. Any proteolysis compromises protein identification, especially when a gel-based, protein separation strategy is employed. Unfortunately, there is poor accessibility to the pituitary gland because of its location inside the skull. In addition, it takes time to separate the AP from the posterior lobe (pars intermedia [PI] and pars nervosa) along the line of Rathke’s cleft (Fig. 2A). In rat, most all of the cells in the PI stain immunohistochemically for ACTH, but only selective cells in the AP stain for ACTH (31). After removal of the posterior lobe, staining for ACTH on horizontal sections of the AP can be used to assess contamination of the tissue with cells of the PI (Fig. 2B). A major concern when analyzing the AP proteome is that it is free of pars nervosa tissue. Analysis of entire pituitary glands and the posterior lobe of mouse by LC-MS/MS has demonstrated that oxytocin and vasopressin neurophysins are excellent indicators of pars nervosa tissue in AP preparations. Others have reported vasopressin-neurophysin 2-copeptin precursor in postmortem human pituitary glands analyzed by LC-MS/MS (16).
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The weight of the AP in a young adult male is approximately 4 mg in hamster, 2 mg in mouse, and 7 mg in rat. Based on analyses of ovariectomized rat APs, the protein recovered in reaction buffer relative to tissue wet weight was approximately 8%. The distribution of protein in our subcellular fractions was approximately 50% soluble, 30% membrane, and 20% cytoskeleton/chromatin. Proteomic analysis can be performed on a single subcellular fraction of AP tissue by LC-MS/MS using approximately 10 µg of protein. The analyses are sensitive and can detect picomolar amounts of a protein. However, we combine APs to form a pool for two reasons. First, it enables us to prepare multiple protein fractions for replicate analyses, and, second, it ensures that the results we obtain are more representative of the proteome of the population in general rather than a single individual. A single hamster, mouse, or rat AP does not provide enough protein to allow direct analysis by DIGE. We therefore combined several glands, prepared subcellular fractions, used 500 µg of protein per gel, and performed analytical replicates.
To increase the total number of proteins identified, AP homogenates were separated into different fractions, and we treated and analyzed each fraction separately. We generated three subcellular fractions in our initial studies of the hamster and mouse AP: nuclear and nonnuclear 100,000 g and cytosolic fractions. In these studies, the trypsin digestion and peptide separation strategy was employed. In rat, the proteomes in two groups were compared. We prepared soluble protein fractions that were similar but not identical to the cytosolic fractions of hamster and mouse. We employed the protein separation and trypsin digestion strategy in these investigations.
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LC-MS/MS Study of the AP of Young Adult Male Golden Syrian Hamster and Mouse |
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TopAbstractThe Anterior Pituitary GlandThe AP Proteome and...Factors to Consider When...LC-MS/MS Study of the...DIGE and MALDI-ToF MS...Matching Proteomic Results to...References |
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In hamster we identified the most proteins in specific fractions when perfusion was employed. Although these comparisons were not conducted under controlled conditions, we made the decision to adopt perfusion for collection of the glands in the subsequent studies of mouse and rat. However, perfusion did not totally eliminate albumin or hemoglobin chains from all the fractions in any of the three species, indicating that blood proteins and red blood cells were not completely washed out of the AP even when this step was incorporated. In hamster, mouse, and rat studies, there was no indication of any contamination of the APs with neurohypophysial tissue as evidenced by failure to detect neurophysins or any of a large number of proteins that have been described as specific to neural tissue.
The protein extraction and subcellular fractionation procedures employed to investigate the hamster (17) and mouse (18) AP proteomes were very similar, and the LC-MS/MS and SEQUEST analyses in the two studies were virtually identical. For these reasons and the fact that young adult males were used in both studies, it was possible to make some interesting comparisons and observations of the AP proteome in both species (18).
The proteins identified in the basic subcellular fractions of hamster and mouse are likely the more abundant proteins in the AP. Changes in these could be investigated in different treatment groups in future quantitative studies using isotope-coded affinity tags (21) and LC-MS/MS. It is also important to note that many of these same proteins were characterized in the AP of the ovariectomized rat using 2D-DIGE and MALDI-ToF MS (32). Proteins identified in the AP of hamster, mouse, and rat included some previously not reported in this tissue and that may be of interest for further study. A brief report of one such protein identified in the hamster AP is summarized at the end of this article.
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DIGE and MALDI-ToF MS Study of the AP of the Estrogen-Treated Ovariectomized Rat |
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TopAbstractThe Anterior Pituitary GlandThe AP Proteome and...Factors to Consider When...LC-MS/MS Study of the...DIGE and MALDI-ToF MS...Matching Proteomic Results to...References |
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Estrogen treatment increased the levels of acidic isoforms of several proteins including GH and PRL; several proteins involved in protein synthesis, folding, and secretion; and several metabolic enzymes. The majority of the downregulated proteins were involved in RNA or DNA interactions, including five heterogeneous nuclear ribonucleoproteins. Detailed lists of the regulated proteins have been reported (32). These experiments demonstrate the efficacy of proteomics to identify AP proteins and perform relative quantifications on them. The results clearly indicated that estrogen acts in vivo to modulate the AP proteome (32), but the data do not enable us to determine whether estrogen acted directly on the AP or indirectly via the hypothalamus or by some other means. Further, because the homogenized AP tissue is a mixture of different hormone-secreting cells, these results do not determine the localization for changes in content of an individual protein.
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Matching Proteomic Results to Specific Cell Types Within the AP and Ancillary Techniques |
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TopAbstractThe Anterior Pituitary GlandThe AP Proteome and...Factors to Consider When...LC-MS/MS Study of the...DIGE and MALDI-ToF MS...Matching Proteomic Results to...References |
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Utilizing LC-MS/MS, we identified SP22 in cytosolic fractions of perfused hamster AP (17). This protein was of interest because it had not been localized specifically to the AP and it had been implicated in apoptosis, androgen receptor function, fertility, and ontogeny of early-onset Parkinson’s disease.
As a first step, we cloned the cDNA for full-length SP22 from the AP in individual Golden Syrian hamsters and rats to confirm the ability of cells within the AP to synthesize SP22 and to eliminate the possibility that all the SP22 was synthesized elsewhere and stored in the AP. Individual tissue samples were placed into nuclease-free microfuge tubes containing Trizol reagent and homogenized. The RNA was extracted and used for reverse transcriptase–polymerase chain reaction (RT-PCR). Total RNA (1 µg) from AP, posterior lobe, and testis (positive control) was reverse transcribed using MuLV reverse transcriptase and amplified using Taq DNA polymerase (34). Primers were made using known homologous sequences for hamster and rat SP22 available in GenBank. Amplified cDNAs (570 base pairs) were obtained from all APs, posterior lobes, and testes. Amplicons were subcloned into plasmids and sequenced to confirm the identity of the SP22 cDNA. DNA sequencing confirmed the presence of the SP22 mRNA amplified in the AP and posterior lobe of the hamster and rat (34).
We then used standard peroxidase and fluorescence immunohistochemical techniques on Bouin’s fixed tissue to determine which cell types contained SP22. Serial sections of the pituitary gland were mounted on slides in a paired flip-flopped orientation as previously described (31), resulting in the exposed surfaces of each pair of sections being mirror images of one another. Mirror image sections were labeled with SP22 and LHß, TSHß, PRL, ACTH, or GH using peroxidase conjugated secondary antibody. Additional sections were colabeled with SP22 and one of the AP hormones using fluorescent secondary antibodies. Immunostaining for SP22 revealed its presence in both species in numerous cells throughout the AP and the pars nervosa but not in the PI. The SP22 colocalized in somatotropes and thyrotropes in hamster and rat. We identified SP22 in a small percentage of corticotropes, gonadotropes, and lactotropes. This was the first report that SP22 was present specifically in the AP and localized primarily in somatotropes and thyrotropes, suggesting that SP22 may help regulate AP function and be particularly important for the control of GH and TSH secretion (34).
Other techniques in addition to RT-PCR and standard peroxidase and fluorescent immunohistochemistry can be used to compliment proteomic findings. Relative quantitative real-time RT-PCR can be used to determine or confirm that upregulation or downregulation of a specific protein under treatment conditions is accompanied by alterations in mRNA accumulation. However, changes in mRNA do not necessarily reflect alterations in protein accumulation, and they do not determine PTMs. For this reason, we emphasize measurement of protein rather than mRNA in functional studies. Western blot analyses enable comparison of protein concentration in multiple animals comprising different groups at one time. The membrane also can be probed multiple times to study additional proteins. In this manner it is possible to compare relative changes in multiple proteins between individual AP samples. In studies using RT-PCR or Western blot analyses, data represent changes in the entire AP.
One can use immunohistochemical-stained sections of AP to determine cell volume density (35) and percent of different AP cell types (36). This information, coupled with AP weight, may help determine whether altered regulation of a protein is due to changes occurring within individual cells or a result of an increase in number of cells that express that protein. In cases where we have had difficulty in staining for nuclear proteins by standard peroxidase and fluorescent immunohistochemistry, we have used heat-induced antigen retrieval successfully (37). Although antisera can be specific with regard to binding to a particular protein, they often are of insufficient specificity to identify isoforms of the protein. The six established AP hormones and other AP proteins have been found to be located in select cell populations or subpopulations. It should be noted that species variations and variations due to physiological state have been observed (38–40).
The previously mentioned proteomic and accessory techniques address analysis of AP tissue and not dispersed AP cells or cell lines. It is important to note that normal function of the AP is often not maintained when the cytoarchitectural relationships within the AP are disrupted (41), and it is highly unlikely that dispersed AP cells or any of the limited number of AP cell lines that are available express the normal complement of proteins.
In summary, recent technological advances provide the opportunity to identify the AP proteome and large-scale changes in the proteome in response to changes in physiological state and experimental or disease conditions. Characterization of these changes, coupled with ancillary techniques, can elucidate changes in the AP that result in normal and altered secretion by this master gland. The procedures and techniques we employed to study the AP could be employed to investigate the proteome of other heterogeneous and multifunctional organs.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractThe Anterior Pituitary GlandThe AP Proteome and...Factors to Consider When...LC-MS/MS Study of the...DIGE and MALDI-ToF MS...Matching Proteomic Results to...References |
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- and ß-subunits in hormone action. Endocrinology 131:82–88, 1992.[Abstract]This article has been cited by other articles:
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  C. A. Blake, L. M. Brown, M. W. Duncan, S. W. Hunsucker, and S. M. Helmke Estrogen Regulation of the Rat Anterior Pituitary Gland Proteome Experimental Biology and Medicine, December 1, 2005; 230(11): 800 - 807. [Abstract] [Full Text] [PDF]
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