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Oak Ridge Conference |
1 The Charles B. Stout Neuroscience Mass Spectrometry Laboratory and the Departments of
2 Neurology and
3 Molecular Sciences, The University of Tennessee Health Science Center, Memphis, TN 38163.
aAddress correspondence to this author at: The University of Tennessee Health Science Center, 847 Monroe Ave., Room 117, Memphis, TN 38163. Fax 901-448-7842; e-mail ddesiderio{at}utmem.edu.
Abstract
Background: A human proteome is relatively dynamic compared with its corresponding genome. Our aim was to study the heterogeneity of a human pituitary proteome as a function of gender, age, and race.
Methods: Pituitary control tissues (n = 8) were used to extract proteins; each control tissue was analyzed (n = 3–5) with two-dimensional gel electrophoresis (2DGE) and PDQuest software. We obtained 30 high-resolution 2DGE gels and conducted a comparative analysis as a function of gender, age, and race. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography-electrospray ionization-quadrupole-ion trap tandem mass spectrometry were used to characterize the protein in each differential spot.
Results: We detected 
1000 protein spots in each 2DGE map, and 51 differential spots (7 differing with gender, 17 with age, 15 with race, and 12 with the coeffect of age and race). Among those 51, we characterized 28 proteins [5 differing with gender, 8 with age, 6 with race, 8 with the coeffect of age and race, and 1 (somatotropin chain 1) with all of these]. Somatotropin was related to gender, age, and race, and prolactin was higher in females than males. The differentially expressed proteins that were related to age were mainly those proteins associated with cell growth, proliferation, differentiation, apoptosis, and death; those proteins showed no difference with gender and race. Age and race affected some proteins associated with hormone regulation (e.g., follistatin, thyroid hormone receptor ß-2, adenylate cyclase-inhibiting G
protein).
Conclusions: A heterogeneity exists in the human pituitary proteome as a function of gender, age, and race. These findings will serve as a basis for our comparative proteomics studies of human pituitary adenomas.
Reference maps of a human pituitary adenoma proteome (1) and of a healthy (control) human pituitary proteome (2)(3) have been established in our laboratory. The next goal of our research program is to locate, detect, and characterize all of the proteins expressed differentially between human pituitary adenomas and controls. Those data are needed for our long-term goal: to clarify the molecular mechanisms involved in human pituitary adenoma formation.
The human pituitary is a key endocrine regulation organ. Human endocrinology differs according to gender, age, and race. When comparing a pituitary adenoma and control proteomes, one obtains adenoma tissue from a neurosurgical operation and controls from postmortem sources. Because of the special neuroanatomical location of the human pituitary (sella turcica), its key role in human physiologic function, and its small volume (0.5–1.0 cm3 in the adult human), one cannot obtain any control pituitary tissue from a patient with a pituitary adenoma, whereas for other cancer studies [e.g., lung cancer (4) and breast cancer], control tissue can be obtained during surgery from the patient with the tumor. Pituitary control tissues can be obtained only from postmortem sources (5), e.g., a death from another disease or an accident. Furthermore, other uncontrolled experimental factors (gender, age, and race) are involved when a comparative proteomics study is performed between pituitary adenomas and controls.
In addition, although a human genome is relatively stable across gender, age, and race, the functional expression profiles (transcriptome and proteome) of a genome are dynamic (6) and do reflect differences across gender, age, and race. The proteome is the ultimate functional expression of a genome, and a difference (although perhaps not very large) must exist among the different control human pituitary proteomes. A pituitary adenoma is the most common change of the pituitary (7)(8)(9)(10)(11). To accurately locate and to detect differentially expressed proteins in a human pituitary adenoma, a detailed comparative analysis is needed of a set of control human pituitary proteomes across gender, age, and race. In this study, we present experimental data that will clarify the heterogeneity among control human pituitary proteomes and that will provide key data for our comparative proteomics study of human pituitary adenomas.
For the above reasons, we performed a large-scale comparative analysis in this study. Eight human pituitary control tissues were used to extract proteins, and each control tissue was analyzed (three to five times) by two-dimensional gel electrophoresis (2DGE).
1
We obtained 30 2DGE gels for comparative analyses across gender, age, and race. The differentially expressed proteins were characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and liquid chromatography-electrospray ionization-quadrupole-ion trap tandem mass spectrometry (LC-ESI-Q-IT MS/MS). Scheme 1
contains the overall experimental strategy that was used to determine the heterogeneity of the proteomes in a set of control human pituitary samples.
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Materials and Methods
pituitary tissues
We obtained eight control pituitary tissues: seven from the Memphis Regional Medical Center and one from the National Disease Research Interchange (Philadelphia, PA). The whole pituitary tissue was removed during autopsy, frozen immediately in liquid nitrogen, and stored (-80 °C) until processed. Table 1
contains the information for each pituitary specimen.
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protein extraction
The whole pituitary tissue (0.45–0.70 g; n = 8) was homogenized (4 °C) in 10 mL of 2 mol/L acetic acid containing 1 mL/L mercaptoethanol. The homogenate was sonicated (20 s) and lyophilized. The lyophilisate was stored (-80 °C) until analysis. The protein content in the lyophilisate was measured with a bicinchoninic acid protein assay (Pierce). For an 18-cm IPG strip, pH 3–10 NL (Amersham Pharmacia Biotech), we used a total of 70 µg of pituitary protein. The protein was mixed with 250 µL of the dissolving solution [7 mol/L urea, 2 mol/L thiourea, 40 g/L CHAPS, 100 mmol/L dithiothreitol (DTT), 20 mL/L pharmalyte, and a trace of bromphenol blue]. The mixture was vortex-mixed for 5 min, sonicated for 5 min, and allowed to stand for 40 min. We then added 110 µL of rehydration solution (7 mol/L urea, 2 mol/L thiourea, 40 g/L CHAPS, 60 mmol/L DTT, 5 mL/L IPG buffer pH 3–10 NL, and a trace of bromphenol blue) to the above mixture. The mixture was sonicated for 5 min and then allowed to stand for 40 min, after which it was vortex-mixed for 5 min and centrifuged for 20 min at 13 000g and room temperature. The supernatant is called the "protein sample solution".
2dge
First dimension.
Isoelectric focusing (IEF) was performed on a Multiphor II instrument (Amersham Pharmacia Biotech) with precast IPG strips (pH 3–10 NL; 180 x 3 x 0.5 mm). A 350-µL aliquot of the protein sample solution was loaded on an IPG strip. The IPG strip was rehydrated overnight (18 h). IEF was performed at 20 °C with the following conditions: a 1-min gradient at 100 V, 2 mA, and 5 W; a 2-h gradient at 100 V, 2 mA, and 5 W; a 1-min gradient at 500 V, 2 mA, and 5 W; a 1.5-h gradient at 3500 V, 2 mA, and 5 W; and an 8-h gradient at 3500 V, 2 mA, and 5 W. After IEF, the IPG strip was stored (-80 °C) until the second-dimension electrophoresis was performed.
Second dimension.
The second-dimension electrophoresis involved sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on a PROTEAN plus DodecaTM Cell (Bio-Rad) that can analyze up to 12 gels at a time. The 12% PAGE resolving gel (190 x 205 x 1.0 mm) was cast with a PROTEAN plus Multicasting Chamber (Bio-Rad). The resolving-gel solution for 12 gels was made by mixing 180 mL of 400 g/L acrylamide/bis-acrylamide (29:1 by weight; cross-linking ratio = 3.3%); 150 mL of 1.5 mol/L Tris-HCl, pH 8.8; 270 mL of distilled, deionized water, 3 mL of 100 g/L ammonia persulfate; and 150 µL of TEMED. The IPG strip with the protein sample was equilibrated for 10 min in 4 mL of reducing equilibrium buffer (375 mmol/L Tris-HCl, pH 8.8; 6 mol/L urea; 200 mL/L glycerol; 20 g/L SDS; a trace of bromphenol blue; and 20 g/L DTT). The IPG strip was equilibrated for 10 min in an alkylation equilibrium solution (4 mL) that contained 25 g/L iodoacetamide instead of 20 g/L DTT. A 10 g/L solution of low-molecular-weight agarose was used to seal the equilibrated IPG strip to the top of the resolving gel. Second-dimension electrophoresis was performed in 25 L of Tris-glycine-SDS electrophoresis buffer (25 mmol/L Tris base, 192 mmol/L glycine, 1 g/L SDS) with a constant voltage (200 V for 370 min). The 2DGE-separated protein spots were visualized with a modified silver-staining method (1).
image analysis
The silver-stained two-dimensional gels were digitized with SilverFast scan software (Epson Corporate) and an EPSON Expression 800 scanner (Model G710U). The same scanning conditions were used for each two-dimensional gel in a matched set (reflective, positive, 36-bit color, 200 dpi, same output size, RGB scan). The scan data (tiff file) were input to The Discovery SeriesTM PDQuest 2DGE Gel Analysis software for a PC computer (Ver. 7.1.0; Bio-Rad). For each two-dimensional gel, three image types were created: the original unaltered scan (two-dimensional scan), the filtered and processed scan (filtered image), and the synthetic image (gaussian image) that contained the gaussian spots with a defined volume and quality. All subsequent spot-matching and analysis steps in the PDQuest software were performed on the gaussian spots. For a between-gel comparison, a set of spot-generation conditions (weakest spot, smallest spot, size of the largest spot, a selected region of the background) was used. The total density in a gel image was used to normalize each spot volume in the gel image to minimize the effect of experimental factors on spot volume.
A matched set was created from the gaussian images, which contained 30 gels from the eight pituitary specimens; Gel C4-1 was used as the master gel for comparisons. The spatial reproducibility (the percentage of matched-spot and spot-positional deviation) and the quantitative reproducibility (volume and quality) were analyzed. These gel images in the matched set were grouped by gender, age, and race (Table 2
). The comparison analyses were performed with the mean normalized volume across gender, age, and race (M vs F; B-30 vs W-40; W-40 vs W-50; B-30 vs W-50). The "cutoff point" value for a significant difference for a differential spot was a threefold difference. Each differential spot must also exist in each gel of, for example, 17 male gels, but it may not exist in any female gel.
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calculation of experimental PI and molecular weight
Protein calibrators (cat. no. M3411; Sigma) were used as internal markers (Table 2
) to calculate the experimental pI and mass values on a two-dimensional gel. Those proteins were amyloglucosidase from Aspergillus niger (89/70 kDa; pI 3.8), ovalbumin (45 kDa; pI 5.1), carbonic anhydrase from human erythrocytes (29 kDa; pI 7.0), and myoglobin from horse heart (17 kDa; pI 7.6).
ms
The differential spots were excised from the two-dimensional gels, and the proteins were subjected to in-gel trypsin digestion (2)(12). The tryptic peptide mixture was purified with a ZipTipC18 microcolumn (prod. no, ZTC18S096; Millipore), according to the methods recommended by the manufacturer. For MALDI-TOF analysis, the purified tryptic peptides were eluted directly from the microcolumn onto the MALDI plate with 2 µL of a -cyano-4-hydroxycinnamic acid solution (12.5 g/L in 500 mL/L acetonitrile–1 mL/L trifluoroacetic acid; 7 cycles), and the matrix was dried in ambient air. For LC-ESI-Q-IT analysis, the purified tryptic peptide mixture was eluted with 6 µL of 850 mL/L acetonitrile–1 mL/L trifluoroacetic acid (10 cycles), and the elute was air-dried. Before analysis, the dried tryptic peptide mixture was redissolved in 5 µL of 20 mL/L acetonitrile–5 mL/L acetic acid.
For MALDI-TOF-MS analysis, the peptide-mass fingerprinting (PMF) data for each protein were generated on a Perseptive Biosytems MALDI-TOF Voyager DE-RP mass spectrometer. The instrument settings included the delayed extraction, reflective, and positive-ion modes. A protonated molecule ion, [M+H]+, is generally produced by MALDI. The mass spectra are internally mass-calibrated with two fragment-ion masses of the trypsin autodigestion products: amino acid sequences 108–115 ([M+H]+ = 842.509 Da) and 58–77 ([M+H]+ = 2211.104 Da). The blank-gel experiments were performed in parallel to remove masses from trypsin, matrix, known contaminants (typically keratins), and other unknown contaminants. The corrected mass list is the PMF data that are used to search databases.
For LC-ESI-Q-IT analysis, the MS/MS data were generated on an LCQDeca mass spectrometer equipped with a standard electrospray source (ThermoFinnigan) to obtain the amino acid sequences of the LC-separated tryptic peptides. The LCQDeca instrument settings were as follows: ESI voltage, 2.0 kV; capillary probe temperature, 110 °C; and electron multiplier, -900 V. Samples were injected manually (5-µL aliquot), and the tryptic peptide mixture was separated on a capillary column, the New Objective PicoFrit [360 µm (o.d.) x 75 µm (i.d.); 15-µm (i.d.) tip pores; packing length, 8 cm] packed with Magic C18AQ material (5-µm beads; 200 Å pores; Michrom Bioresources). Peptides were eluted (flow rate, 0.4 µL/min) with the following gradient made by mixing solution A (water containing 1 mL/L formic acid) and solution B [900 mL/L acetonitrile containing 1 mL/L formic acid): (a) 0% solution B for 5 min; (b) linear gradient up to 65% solution B within 30 min; (c) 65% solution B for 15 min; (d) linear gradient down to 0% solution B within 5 min; and (e) maintain 0% solution B for 30 min. The MS/MS data were acquired in the "triple-play" data-dependent scan mode, which included four scan events: a full-range MS scan and three MS/MS scans that depended on the scan mode. Data were managed with the Xcalibur software provided by ThermoFinnigan.
database analysis
We used the MALDI-TOF MS PMF data to characterize the protein by searching the SWISS-PROT/TrEMBL database with PeptIdent software (http://us.expasy.org/tools/peptident.html) and the LC-ESI-Q-IT MS/MS data to characterize the protein by searching the SWISS-PROT or NCBInr databases with the SEQUEST software that is part of the LCQDeca software package. The parameters for searching the databases and the criteria for evaluating the results of the database search have been described (1).
Results and Discussion
analysis of the 2dge pattern of human pituitary proteomes
High-resolution, 2DGE separation of proteins is a key step in performing a comparison between human pituitary proteomes. The same experimental conditions, including the amount of protein, sample-dissolving solution, IEF, IPG strip equilibration solution, SDS-PAGE, gel scan, and PDQuest process, were used to conduct two-dimensional polyacrylamide gel electrophoresis experiments and image analysis for all pituitary samples. Analysis of up to 12 gels at a time minimized any between-gel variability. Each pituitary sample was analyzed three to five times by 2DGE. The results indicated that a good 2DGE pattern was attained (Fig. 1
). Approximately 1000 protein spots were detected in the two-dimensional map. Most of the 2DGE-separated spots were distributed within the pH 4–9 area and the mass range 15–100 kDa.
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Use of a commercial IPG strip (18-cm IPGstrip pH 3–10 NL) for the first-dimension IEF, a vertical multigel electrophoresis system (can analyze up to 12 gels at a time) for second-dimension SDS-PAGE, and an appropriate amount of protein loaded on the gel (70 µg) (13)(14) allowed us to obtain a high-resolution, high-reproducibility 2DGE pattern of a human pituitary proteome. For each pituitary sample, the mean between-gel, matched percentage was 85–99%. We obtained the highest mean (SD) between-gel matched percentage [98.9 (0.5)%] for sample C4 because one gel from sample C4 was used as the master gel. The positional deviation of the matched-spots among the different matched gels was 1.61 (0.58) mm in the IEF direction and 1.36 (0.55) mm in the SDS-PAGE direction. Moreover, we observed a high correlation for the volume of the matched spots among the different replicate two-dimensional gels for each pituitary sample. For each sample, the correlation coefficient (r) of the normalized volumes for between-gel matched-spots was >0.75 (range, 0.768–0.905). According to statistical principles, if r was >0.7, then a strong correlation relationship existed between gels. For example, a representative scatter-plot (not shown) from a correlation analysis of the normalized spot volume between gels C4-1 and C4-2 had a best-fit line of: y = 0.909x + 0.051 (r = 0.907; n = 1256). A high correlation for spot volume is an important requirement for accurate comparisons between gels because a differential spot is actually determined by the difference between a spot volume on two gels.
Because of its high resolution and high reproducibility, the 2DGE-PDQuest system can accurately locate and detect differential protein spots. For example, to determine a differential spot between gels A and B, this system can establish that the spot exists in gel A but not in gel B or in gel B but not in gel A, that the spot volume in gel A is greater than that in gel B, or that the spot volume in gel B greater than that in gel A.
comparative analysis between pituitary proteomes
The differential spots were determined with use of silver-stained two-dimensional gels and the PDQuest analysis system. Seven differential spots were found between the male and female groups: two were increased in the male pituitary (vs female), and five were increased in the female pituitary (vs male; Table 3
and Fig. 1
). Seventeen differential spots were found between groups W-40 and W-50: 5 were increased in the W-40 group vs W-50, and 12 were increased in the W-50 group vs W-40 (Table 3
and Fig. 1
). Seventeen differential spots were found between the B-30 and W-40 groups: 13 were increased in the B-30 group vs W-40, and 4 were increased in the W-40 group vs B-30 (Table 3
and Fig. 1
). Twenty-seven differential spots were found between the B-30 and W-50 groups: 10 were increased in the B-30 group vs W-50, and 17 were increased in the W-50 group vs B-30 (Table 3
and Fig. 1
).
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The experimental pI values and molecular weights of each differential spot in Table 3
were obtained by inputting the standard 2DGE markers (Fig. 1
). Shown in Fig. 2
is a representative differential spot that was found between the W-40 (n = 8) and W-50 (n = 5) groups (spot 4001). Those data show that a differential spot exists in each gel of the W-50 group and that the mean normalized spot volume in the W-50 group is significantly higher than that in the W-40 group (P <0.01) with a volume ratio of 5.7 (W-50/W-40).
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We further analyzed the results from the comparison between the three compared groups: B-30 vs W-40, W-40 vs W-50, and B-30 vs W-50. The B-30 group contained gels C2 and C7, W-40 contained gels C3 and C5, and W-50 contained gels C9 and C10 (Table 2
). Differences between two factors, race and age, existed within those three groups. When we compared those three groups, we found differential spots between different paired compared groups (Table 3
and Fig. 3
). Only an age difference existed between groups W-40 and W-50, the two groups that contained samples from white individuals. Therefore, the differential spots 6714, 1322, 0103, 0121, 1319, 3512, and 6313 (see Fig. 3
) resulted from the age factor. Spots 6714 and 1322 should not relate to race. For group B-30 vs group W-40, the age difference was only 8 years; therefore, the differential spots 0120, 0203, 0325, 2303, 2311, 5504, 6315, 0712, 2615, and 2622 resulted primarily from differences in race. Therefore, 17 differential spots resulted from the age difference, 15 differential spots from the race difference, and 12 differential spots from the coeffect of age and race factors. Fig. 3
illustrates these results.
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characterization of differentially expressed proteins by ms
The differential spots were excised from two-dimensional gels, in-gel trypsin digestion was performed, and MS was used to characterize the protein (1). The interfering masses from trypsin, -cyano-4-hydroxycinnamic acid matrix, keratins, and other unknown contaminants were removed to obtain a corrected mass list for the PMF data before database analysis. Fig. 4
shows the corrected MALDI-TOF mass spectrum for spot 2504. Sixteen tryptic peptide masses were obtained, labeled (see Fig. 4
), and used to search the SWISS-PROT database. Spot 2504 was identified as somatotropin chain 1 (SWISS-PROT No. P01241; Table 3
); 12 peptide fragments were matched, and the sequence coverage was 57%. A total of 33 differentially expressed proteins (6 proteins related to gender, 9 to age, 8 to race, and 10 to the coeffect of age and race) were characterized by MALDI-TOF PMF data (Fig. 3
and Table 3
). Table 3
contains the protein names and corresponding SWISS-PROT access numbers. The theoretical pI and molecular mass values were obtained from the SWISS-PROT database.
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Two differentially expressed proteins (spots 0712 and 2504) were characterized by both MALDI-TOF PMF and LC-ESI-Q-IT MS/MS analysis (Table 3
). The LC-separated tryptic peptides were analyzed by ESI-Q-IT MS, which produced the MS/MS data. The MS/MS data were then used to search the database with SEQUEST software. As shown in Fig. 5
, the protein in spot 2504 was characterized as somatotropin chain 1 (SWISS-PROT no. P01241) based on the LC-ESI MS/MS data. Six tryptic peptides were sequenced. The number of amino acids in the tryptic peptides that were sequenced ranged from 6 to 19, and the sequence coverage was 23%. Both methods (PMF and MS/MS) yielded the same protein.
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functional analysis of the characterized proteins
In this study, we found that most of the differential spots found across gender, age, and race were low-intensity 2DGE spots. Eighteen differential spots were not identified by MS (15 spots with PMF; 3 spots with MS/MS). The reasons that those differentially expressed proteins could not be characterized may be as follows: (a) those spots were very weak, i.e., they contained too little protein; (b) although protein loss (e.g., from adsorption) during experiments was minimized by the use of low-adsorption materials (e.g., siliconized tips and tubes), loss of protein and peptide still occurred to some degree; (c) the commonly used purification method for tryptic peptides, a Millipore ZipTipC18 column, will lose some biological samples; and (d) although MALDI-TOF and LC-ESI-MS/MS are very sensitive, there are still limits to their detection sensitivities.
For the 33 differentially expressed proteins, we further analyzed their physiologic functions in the human pituitary according to their functional groups, which are summarized in Table 4
. Functional analysis of the proteins is a key step to determine the relationship of these proteins across gender, age, and race because some unavoidable factors (e.g., blood contamination of pituitary tissue) might affect the comparison results obtained with the 2DGE-PDQuest analysis system. Table 4
shows the functional characteristics of each protein. A main protein in pituitary tissue, somatotropin, correlated with gender, age, and race. The expression of prolactin was higher in females than in males. An interesting phenomenon was that some proteins that are associated with cell growth, proliferation, differentiation, apoptosis, and death {somatotropin, developmentally regulated GTP-binding protein 2, insulin-like growth factor binding, ATP-binding protein associated with cell differentiation, and interleukin-1ß-converting enzyme (ICE)-like apoptotic protease 5 [Fas-associating protein with Death Domain (MORT1)-associated Caenorhabditis elegans cell death gene (CED-3) homolog]} were correlated with age. Follistatin demonstrated a significant decrease with an increase in age. Some proteins associated with the regulation of a hormone, e.g., thyroid hormone receptor ß-2 and adenylate cyclase-inhibiting G protein, were also related to the coeffect of age and race. Some proteins (e.g., hemoglobin, vascular anticoagulant-, fibrinogen, serum albumin, hemopexin, and serum amyloid P-component) possibly resulted from blood contamination of the pituitary tissue during the postmortem because these proteins are located in red blood cells, platelets, and serum or are secreted in the liver. For the other proteins, we have not yet discovered why they are differentially expressed in this set of pituitary proteomes.
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In summary, the functional characteristics of these differentially expressed proteins provide a preliminary heterogeneous profile of human pituitary proteomes. Such a heterogeneity database is needed for our comparative proteomics studies of the human pituitary and for our studies of the basic molecular mechanisms in human pituitary adenoma formation.
Conclusion
The human pituitary is the critical regulatory center of the endocrine system. The pituitary gland consists of three lobes: the anterior, neurointermediate (only a few cell layers thick in humans), and posterior lobes. The anterior lobe consists of several different cell types (somatotrophs, mammotrophs, corticotrophs, thyrotrophs, and gonadotrophs), which are involved in the biosynthesis and secretion of key hormones (growth hormone, prolactin, adenocorticotropic hormone, thyroid-stimulating hormone, and follicle-stimulating hormone/luteinizing hormone, respectively) that regulate multiple, diverse, and important physiologic and metabolic functions in the human body (2). The endocrine status of an individual differs according to gender, age, and race. A pituitary adenoma is the most common pathologic change in the pituitary, and can produce serious physiologic and metabolic responses (8)(9)(11). A goal of our research program is to detect significantly differentially expressed proteins in a human pituitary adenoma to clarify the basic molecular mechanisms of adenoma formation. However, because control pituitary tissue varies according to gender, age, and race, it is essential to evaluate the heterogeneity among human pituitary proteomes as a function of those three variables.
Comparative proteomics based on 2DGE, PDQuest analysis, and MS (15)(16)(17)(18) provides a powerful tool to accurately analyze a human pituitary proteome across gender, age, and race. The first heterogeneity profile among human pituitary proteomes has been described here. A high-resolution, high-reproducibility 2DGE map, which contains 1000 proteins of a human pituitary proteome, was obtained. We found 51 differential spots (7 spots related to gender, 17 spots to age, 15 spots to race, and 12 spots to the coeffect of age and race) when we compared pituitary proteomes across gender, age, and race, and characterized 33 of these spots by use of in-gel trypsin digestion, MS (MALDI-TOF and LC-ESI-Q-IT), and database analysis. These proteins have different functional roles, and they reveal a preliminary heterogeneity profile of human pituitary proteomes across gender, age, and race. These heterogeneity data may help clarify the dynamic change of human pituitary proteomes with gender, age, and race and could serve as a basis for comparative proteomics studies of pituitary adenomas. If these differentially expressed proteins are also obtained when we perform a comparative proteomic study between pituitary adenoma and control tissues, then one should very carefully evaluate whether they are really differentially expressed proteins related to the pituitary adenoma or whether they result from the inherent heterogeneity of the human pituitary proteome. These heterogeneity data will also be collected on our proteome database of the human pituitary (http://www.utmem.edu/proteomics).
Acknowledgments
We gratefully acknowledge financial assistance (to D.M.D.) from NIH (NS 42843). The MALDI-TOF mass spectrometer was purchased with grants from NIH (RR-10522) and the National Science Foundation (DBI 9604633), and the LCQ was purchased with a grant from NIH (RR-14593). The pituitary tissues were provided by the University of Tennessee MED Hospital and the National Disease Research Interchange (Philadelphia, PA).
Footnotes
1 Nonstandard abbreviations: 2DGE, two-dimensional gel electrophoresis; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; LC-ESI-Q-IT MS/MS, liquid chromatography-electrospray ionization-quadrupole-ion trap tandem mass spectrometry; DTT, dithiothreitol; IEF, isoelectric focusing; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; and PMF, peptide-mass fingerprinting. 
References
The following articles in journals at HighWire Press have cited this article:
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