|Molecular Vision 2006;
Received 19 August 2005 | Accepted 12 April 2006 | Published 18 April 2006
Protein expression in a transformed trabecular meshwork cell line: proteome analysis
H. Thomas Steely,1
Glen W. Dillow,1
Terry A. Braun,2
Thomas L. Casavant,2
Mitchell D. McCartney,1
Abbot F. Clark1
1Alcon Research, Ltd., Fort Worth, TX; 2The University of Iowa Coordinated Laboratory for Computational Genomics, Iowa City, IA
Correspondence to: H. Thomas Steely, Alcon Research, Ltd., 6201 South Freeway, Fort Worth, TX, 76134; Phone: (817) 551-4522; email: email@example.com
Purpose: Characterization of the human trabecular meshwork (TM) proteome is hindered by the small mass of intact tissue and the slow growth of cultured cell strains. We have previously characterized a transformed TM cell strain (GTM3) that demonstrates many of the same protein expression and cell signaling systems of nontransformed cell strains. The aim of this study was to initiate a proteomic survey of GTM3 cells as the initial step toward characterization of the complete human TM proteome.
Methods: GTM3 cells were cultured to confluence, harvested and solubilized in urea/Nonidet. The protein extract (600 μg) was focused in immobilized isoelectric focusing (IEF) strips, separated by 10% SDS PAGE, and visualized with colloidal Coomassie Blue. Spots of interest were excised, destained, and the contained proteins subjected to in-gel reduction, derivatization, and tryptic digestion. Tryptic peptides were extracted and analyzed by electrospray LC/MS/MS. Protein identification was made using the TurboSequest search algorithm and a recent version of the nonredundant human protein database downloaded from the National Center for Biotechnology Information (NCBI).
Results: Eighty-seven (87) primary proteins and 93 variants of these proteins were identified. A website was created (TM proteome) that combines data such as graphic spot location within the gel, peptide sequence, apparent and calculated pI, apparent and calculated mass, percentage of coverage, and protein informatic website links.
Conclusions: Proteomic analysis of a transformed human TM cell line has been initiated combining preparative two-dimensional PAGE separation, LC/MS/MS analysis of major proteins, and bioinformatic cataloging of the data. Further investigation of data from the transformed cell strain will be used in a comparative fashion for spot identification of analytical proteomic gels of human TM tissue and cultured normal cells. These initial data will form the base from which the characterization of protein expression in the normal and glaucomatous TM can be accomplished.
Glaucoma is a leading cause of irreversible blindness and affects approximately 2-4% of the human population over the age of 40 [1-3]. The disease represents a heterogeneous group of optic neuropathies with multifactorial etiologies. A family history of glaucoma is an important risk factor for the development of glaucoma. A number of glaucoma loci have been mapped and several glaucoma genes identified [4-6].
Elevated intraocular pressure (IOP) is one of the major risk factors for the development  and progression [8,9] of glaucoma. Glaucomatous IOP elevation is due to increased aqueous humor outflow resistance and is associated with morphological and biochemical changes in the trabecular meshwork (TM). The TM is a reticulated tissue at the iridocorneal junction [10,11] that makes intimate contact in the juxtacanalicular region with the Canal of Schlemm for aqueous filtration and drainage. The TM imparts normal aqueous humor outflow resistance to maintain the shape of the globe. Pathogenic changes in primary open-angle glaucoma (POAG) that lead to decreased outflow include compression of the intratrabecular spaces, loss of TM cells from the collagenous beams, thickening of the beams, accumulation of aberrant amounts and forms of extracellular matrix (ECM) material, and overall changes in cellular morphology [12-15]. While many components of the TM ECM have been identified [16-19] only a few studies address the entire TM proteome.
The establishment of a proteomic database for diseased tissue expedites understanding of the comparative metabolic changes that take place with disease onset and therapeutic treatment . Proteins are gene effectors, and therefore the proteome is dynamic, representing the protein equivalent of the transcriptome at a given point in time in the physiological/pathological state. We have begun a proteomic evaluation of the TM as one approach to better understand the pathophysiology of glaucoma. Cultured TM cells are routinely used in a number of laboratories [21,22], although these cells from normal and especially glaucoma donor eyes grow slowly. In the present study, we have utilized a robust, transformed cell strain, GTM3, originally derived from a glaucoma cell strain that has been previously shown to express many of the same proteins and cell signaling pathways found in normal cultured TM cells . In order to characterize this proteome, TM cell proteins were separated by high-resolution, two-dimensional (2D) electrophoresis followed by mass spectrometric identification of in situ trypsin digests of the proteins [24,25].
Trabecular meshwork cell culture (GTM3)
The transformed human trabecular meshwork cell line GTM3 was immortalized by transformation with SV-40 T-antigen as previously documented . GTM3 cells were grown to confluence in flasks at 37 °C and 5% CO2 in Dulbecco's modified Eagle's medium containing L-glutamine (Glutamax I; Invitrogen/Gibco, Grand Island, NY), 50 μg/ml gentamicin (Invitrogen/Gibco), and 10% fetal bovine serum (Hyclone, Logan, UT). The cells were rinsed with Dulbecco's PBS (Invitrogen/Gibco), removed from the flask with a rubber policeman, and pelleted at 400x g. Cell pellets were flash-frozen and stored at -80 °C.
Solubilized extracts of protein were prepared as previously described in the literature . Each pellet was gently extracted with low-speed vortexing every 10 min on ice for 1 h with 9 M urea containing 4% Nonidet NP-40 (Sigma, St. Louis, MO). The extract was assayed for total protein content using the bicinchoininic acid (BCA; Pierce, Rockford, IL) method of Brown et al. .
First dimension: Isoelectric focusing
Identical, immobilized isoelectric gradients were prepared as follows. For each gradient, 600 μg of protein in urea/Nonidet was mixed with rehydration buffer and loaded into 18 cm, nonlinear gradient DryStrips (pH 3-10; Amersham Biosciences, 17-1235-01, Piscataway, NJ) by rehydration uptake for 16 h at RT [28,29]. Following rehydration, proteins within the strips were focused for a total of 50,800 Vh, with 8,000 V reached in the last 2.5 h of the run. To release proteins with higher mass, the isoelectric focusing (IEF) gels were cracked by flash freezing in liquid nitrogen for 30 s and then stored at -80 °C.
Second dimension: sodium dodecyl sulfate- polyacrylamide electrophoresis
Separation of proteins by mass was accomplished by application of the focused strips to 10% sodium dodecyl sulfate (SDS) polyacrylamide slabs using an Ettan Dalt six apparatus (80-6485-08; Amersham). The slabs were prepared as described by Hochstrasser et al.  using piperazine diacrylamide as the cross-linker. The focused isoelectric strips were first reduced with DTT and then alkylated by exposure to SDS equilibration buffer containing 13.5 mM iodoacetamide (I-6125, Sigma). The strips were immediately applied to the slabs and cemented in place with agarose (pH 8.8). Assembled slabs were electrophoresed at 18 W per gel at 18 °C. Following electrophoresis, the gels were fixed overnight in 40% ethanol/10% acetic acid.
Staining, imaging, and spot detection
The fixed gels were each washed with H2O and stained with colloidal Coomassie Blue  (Gelcode Blue, catalog number 24592; Pierce, Rockford, IL). Destaining was accomplished with distilled H2O using several water changes over a 2 h period. Individual gels were imaged with a Microtek 9800XL scanner (Microtek, Carson, CA). Gray-scale image data from the gels were acquired by reflectance against a white background and saved using full, linear thresholds. Protein spots and spot contours in images of the stained gels were determined using the Gellab II+ 2D image analysis program  by Scanalytics (Billerica, MA). Individual spots within the gel images were subsequently calibrated for mass and isoelectric point by Gellab II+ using the NCBI mass and pI data of selected proteins that were judged to be reliable identifications. Spot coordinates are listed as "estimated" molecular weight and pI data within the protein spot table and are interpolated from the spots used for calibration (Table 1).
Gel excision and treatment
Gel pieces from the gel locations indicated in Figure 1 were extracted manually using a hand-held pipette with a trimmed polypropylene tip. The gel pieces were transferred to a Millipore ZipPlate (Billerica, MA), which enabled subsequent treatment to be done in batches. Destaining of the gel pieces and reduction/alkylation/digestion of the contained proteins were done by adapting published procedures [33,34] for use with the ZipPlate. All solvents were HPLC grade, and reagents were obtained from Sigma-Aldrich (St. Louis, MO). Destaining and cleanup was accomplished by shaking the gels with water, 10% aqueous acetic acid, acetonitrile, methanol, and 1:3:2 formic acid/water/isopropanol mixture. After reduction with dithiothreitol and derivatization with iodoacetamide, the gel pieces were infused with freshly prepared trypsin (Trypsin Gold; Promega, Madison, WI) solution (20 μg/ml in 40 mM ammonium bicarbonate/10% acetonitrile) and incubated at 37 °C overnight. The gels were extracted three times with 5% formic acid in 1:1 water/acetonitrile, and two times with 1% formic acid in 1:1 water/methanol. All extracts for each gel piece were pooled, lyophilized, and stored at -20 °C until analysis.
In cases where subsequent LC/MS/MS analysis led to only a tentative protein identification (three or fewer peptides), reanalysis utilizing a sample pooling procedure was undertaken. Corresponding gel pieces from the remaining gels were excised and pooled, then treated as previously described and reanalyzed by LC/MS/MS. Using this approach, the number of peptides identified typically doubled.
Immediately prior to analysis, extracted peptides were thawed and reconstituted in aqueous 0.1% formic acid (FA)/0.005% heptafluorobutyric acid (HFBA)/2% acetonitrile. Electrospray  LC/MS and LC/MS/MS analysis was performed with a Finnigan LCQ "Classic" (Thermo Electron Corporation, San Jose, CA). The electrospray (ES) interface was built in-house, and featured a 35-gauge stainless steel needle positioned 3 mm directly opposite the entrance to the heated capillary. A 50 μm idx50 mm long C18 column (Michrom BioResources, Auburn, CA) was connected directly to the ES needle via a zero-dead-volume stainless steel fitting, and high voltage (2.4 kV) was applied through this fitting. A ballast LC column (2 mm idx250 mm long C18) was connected in parallel to split the mobile phase flow and reduce flow to the ES interface to 1 μl/min (measured). At this flow rate and with a capillary temperature of 150 °C (indicated), a stable electrospray could be maintained without drying gas.
The LC mobile phases used for separation of the peptide mixtures consisted of 0.1% FA/0.005% HFBA/2% acetonitrile in water (A) and 0.1% FA/0.005% HFBA/5% water in acetonitrile (B). Following sample injection, the mobile phase composition was maintained at 100% A for 5 min then adjusted linearly to 40:60 A/B over the next 45 min.
The mass spectrometer was operated in data-dependent "double play" mode where the two most intense ions in each survey scan were subjected to collisionally induced dissociation and their MS/MS spectra were recorded.
Mass spectral data analysis
Protein identification from the MS/MS data was performed using the TurboSEQUEST  algorithm in BioWorks v. 3.1 (Thermo Electron Corporation, San Jose, CA) to correlate the data against a recent version of the NCBI nonredundant human protein database (National Center for Biotechnology Information, Bethesda, MD). The charge dependent thresholds set for the TurboSEQUEST peptide correlation scores were as follows: χcorr>1.8, 2.5, and 3.5 for single, double, and triple charged ions, respectively. Only peptide ions having χcorr values equal to the indicated values or higher were considered in this study.
A graphical user interface (TM proteome) was developed using HTML and JAVA programming to facilitate access to the data for collaborators at separate locations and to provide access to the ophthalmic research community. The interface displays and integrates the information for all protein spots on a 2D gel. Protein identities and associated annotation are stored in a MySQL database called SeeSpot. SeeSpot is written in Java and is deployed by Java WebStart for portability and accessibility. The software creates a thumbnail of each gel image and provides it as a reference in the gel selection interface. The underlying SeeSpot database contains the peptide identification information and gel image coordinates, which enables the recording, uploading, editing, and annotation of proteomic data. When a user selects a spot with the "cross" icon cursor, all the collected annotations are displayed for the identified protein: the name of the identified protein, gel spot number (or identifier), stated mass, pI, fragment mass profile, coverage percentage of the identifying peptide fragments, and accession number.
Results & Discussion
Conventional biochemical, immunoblotting, and immunochemical analyses of TM cells and tissue have documented the expression of many cytoskeletal and extracellular matrix proteins as well as growth factors and their receptors [26,37-49]. An early investigation of protein expression in GTM3 cells using immunohistochemistry, ELISA assays and 2D PAGE, demonstrated many similarities to nontransformed TM cells . However, there have been few comprehensive proteomic studies of the human TM. Recently, a proteomic examination of normal and glaucomatous TM tissue utilizing one-dimensional PAGE and MS gave many more protein IDs, although far fewer tryptic peptide masses were used for verification of protein identifications . A recent genomic and proteomic investigation of the effects of TGFβ1 and TGFβ2 on cultured TM cells included 2D gel proteomic images and spot identifications . Analytical 2D autofluorograms of radiolabeled cultured TM cells from our laboratory also gave similar protein spot patterns, but at lower resolution . Although our long-term goal has been to characterize the trabecular meshwork proteome, there were several limitations to the present study. We chose to use the GTM3 cell line since it afforded us the ability to extract proteins from cultured cells in sufficient quantity to generate a number of identical preparative, proteomic 2D gels. GTM3 is a transformed cell line, and the level of expression for any given protein in the GTM3 proteome may vary from normal TM cell lines and TM tissue. Solubilization of cells with urea and nonionic detergents does not extract all proteins, particularly membrane proteins , and we expect that these proteins will be under-represented in our analysis. However, the use of stronger chaotropic agents and subcellular fractionation can improve this situation. These studies are in progress. In the present survey, we did not further characterize post-translational modifications of proteins such as phosphorylation or glycosylation. In addition, our identification of proteins was limited due to our constraint to spots stainable by colloidal Coomassie blue.
In this study, we identified 87 "primary" proteins, and the positions of these proteins in the gel are marked on the image (Figure 1). Additional "spots" were analyzed and the proteins identified were found to be isoforms, modifications, or fragments of these 87 proteins. Table 1 contains a listing of the 87 primary proteins identified together with associated data and annotations. All of the proteins listed in Table 1 contain at least three peptide matches, as well as acceptable agreement between the expected and experimental molecular weight and pI values. Moreover, reproducibility of 2D protein patterns within the GTM3 proteome is remarkable, primarily due to the quality control of commercial, immobilized isoelectric focusing gradient strips.
The molecular weight and pI values in Table 1 estimated from gel position are approximations limited by the known nonlinear characteristics of these gels. Therefore, we considered the experimental molecular weight and pI values listed to be accurate to no better than ±10% and ±0.5 units, respectively. Agreement between the expected and experimental molecular weight and pI values constitutes an independent test of the validity of each TurboSequest result as TurboSequest does not use molecular weight and pI information in its search algorithm. The data in Table 1 shows agreement between the expected and experimental molecular weight and pI values was generally good, although proteins on the right (basic) side of the gel had apparent molecular weights that were consistently several kDa lower than expected.
In addition to the spots marked in Figure 1, many additional spots were analyzed and were also found to contain variants of the proteins listed in Table 1 (Figure 2). In these cases, the TurboSequest search result was the same as one of the proteins in Table 1, but the proteins had unexpected molecular weight and/or pI gel positions. Such positional changes may represent post-translational protein modifications and as such may entail significant changes in molecular weight or in pI, particularly if the protein is glycosylated or proteolytically cleaved. For brevity, specific information on these modified proteins is not detailed in this paper, but is available on a website to be described later in this paper.
Consistent with earlier studies, the most striking feature of the protein map is the complex cluster of highly expressed proteins in the upper left quadrant. Variants of the cytoskeletal proteins vimentin, α-tubulin, β-tubulin, and β-actin were the prominent components of the cluster. Lower abundances of lamin and desmin variants were also identified in this region. It is remarkable that approximately 30 of the proteins observed were variants derived from these six "core" proteins. Many of the variants appear at molecular weights well below the core protein, suggesting that cleavage and/or processing of cytoskeletal proteins may be prevalent. Since no protease inhibitors were used in this study to inactivate cellular proteases, some proteolysis may have occurred prior to 2D PAGE separation, although such proteolysis is expected to be minimized in the presence of 9 M urea and high concentrations of non-ionic detergents at 4 °C. Even higher levels of cleaved forms of vimentin in TM cells treated with TGF-β have been noted .
The actins identified by TurboSEQUEST are exclusively β- or γ-variants. As these two variants differ by only one expected peptide, it is not possible to distinguish them on the basis of our MS/MS results. This predominance of the β-/γ-actin variants is significant because expression of α-actin has been noted in some TM cells [37,38] and in TM tissue . Where evidence of the specific peptides characteristic of the α- and β-/γ-actin isoforms was sought, we verified that the β-/γ-actin variants or modifications of these variants were indeed the predominant component in all of the actin spots identified. However, expression of α-actin is not seen in all TM cells or tissue [37,38]. Since GTM3 is a clonal cell line generated by transformation of a standard glaucomatous cell strain, it is probable that the clone was derived from a single cell that does not express α-actin. Low levels of α-actin expression have also been documented in another transformed TM cell strain .
The metabolic enzymes, glyceraldehyde-3-phosphate dehydrogenase, enolase 1, pyruvate kinase 3, phosphoglycerate kinase 1, and triosephosphate isomerase 1 dominated the basic side of the gel. Again, the variation was remarkable with over 40 distinct proteins arising from changes to these five core primary proteins. Several of these enzymes may play a role in glaucoma. Marked downregulation of a number of variants of pyruvate kinase and enolase has been noted in TM cells treated with TGF-β .
Apart from vimentin and the metabolic enzymes previously mentioned, a number of other proteins identified in the current work are also found to be altered in TGFβ-treated TM cells . Protein disulfide isomerase, calumenin, and eukaryotic translation elongation factor (ETEF) 1δ are upregulated by TGFβ treatment, while annexins 1 and 2 as well as peroxireductase 1 (Thioredoxin peroxidase 2) are downregulated.
The NCBI accession numbers listed in Table 1 were converted to Gene Link IDs and submitted to DAVID 2.0, a relational database of gene ontology and functional annotation at the National Institute of Allergy and Infectious Diseases , in order to evaluate the functional categories of the proteins identified. DAVID analysis identified 75 proteins by their Gene IDs and assigned 70 to varying molecular functional categories. The resulting functional distribution of the proteins is given in Table 2. Seventeen (20%) of the original 87 proteins identified by mass spectrometry in Table 1 were unspecified as to functional class while those remaining reflected predictable metabolic categories at p values less than 0.05.
We have established an interactive website (TM proteome; Figure 3) designated "The Human Ocular Proteome". The concept for this sharing is similar to that seen for "SWISS-2DPAGE Map Selection" at the ExPASy proteomics user site (Swiss Prot). It is expected that laboratories investigating the proteomes of trabecular meshwork cell strains will be able to confirm their protein identifications and use the resource to add additional information regarding proteins expressed in the human TM. Currently the site contains the proteome of a single cultured human TM cell line (GTM3), but the authors hope the site will evolve to include additional ocular cells and tissues. Each proteomic image has interactive annotation. The website is intended to be dynamic, and protein spot information will be periodically updated. Additional background information is also given as regards the proteomic project descriptions and methods utilized, as well as other useful links. After selecting a thumbnail image, the user is able to view the information for each annotated spot, as indicated by a red and white cross (Figure 4). A link is provided to the NCBI record for each protein based on the provided accession number. SeeSpot also queries a local mirror of the UCSC Human Annotation database for a Swiss-Prot identifier, and if successful, provides a respective link (Figure 5).
In this study, we did not detect the presence of known glaucoma-associated proteins such as myocilin , optineurin , WRD-36 , or opticin . Myocilin expression decreases or disappears when TM cells are cultured , a fact that is consistent with our results. Only proteins with sufficient concentration as to have been stained by Coomassie blue could be detected by mass spectrometry using the methodology previously described. Moreover, not all proteins, particularly membrane proteins, are soluble in urea and nonionic detergents. Lastly, the GTM3 cell strain may be derived from a glaucomatous cell stain that did not express these known glaucoma proteins in an aberrant fashion.
However, the GTM3 proteome did display proteins that may be related to the etiology of glaucoma. For example, two significant spots derived from calreticulin were noted to the left (acidic side) of the cytoskeletal proteins. The most prominent of these has an apparent molecular weight of 63.6 kDa, well above the expected calreticulin mass of 48.113 kDa, and is consistent with extensive glycosylation of the core protein. Glycosylation of calreticulin has been reported in other cell types , and the expression of specific carbohydrate ligands may influence both the calcium-signaling role and chaperonin activity of calreticulin . A model of calreticulin-binding to hemagglutinin shows that it binds primarily to incompletely disulfide-bonded folding intermediates and conformationally misfolded forms . It is possible that modified chaperonin activity in glaucomatous cell strains may play a role in the expression and aberrant processing of disulfide-containing proteins such as mutant myocilin [63,64].
Numerous heat shock family proteins (HSPs) were identified in this study, including variants of 27 kDa, 60 kDa, and 70 kDa HSPs, as well as related proteins including stress-induced phosphoprotein (HSP70/HSP90-organizing protein) and chaperonin-containing TCP1 subunits. The trabecular meshwork is located in an environment with relatively high oxidative and mechanical stress. These stress-related proteins, such as the heat shock proteins, may allow the TM to resist such stresses. In addition, certain heat shock proteins play an important role in glucocorticoid receptor function . TM cells are sensitive to the effects of glucocorticoids, which alter TM cell biochemistry and can induce ocular hypertension and glaucoma [22,66].
In summary, we have documented protein expression in a cultured human TM cell strain using high-resolution 2D electrophoresis to resolve urea-and-detergent soluble proteins and to identify those proteins by LC/MS/MS. These data are available through an internet website to foster communication between laboratories regarding proteomic information as it relates to glaucoma. In order to improve spot resolution and the number of proteins identified, future studies will incorporate the use of a new, Fourier-transform spectrometer to increase MS sensitivity at lower levels of protein concentration in gels stained with fluorescent probes such as Sypro Ruby . It is hoped this initial step toward documenting the proteome of the TM will begin the process of elucidating the molecular defects responsible for, or resulting from, glaucoma.
The authors wish to acknowledge the kind help extended to us by Dr. Debra Fleenor at Alcon Laboratories in the supply and maintenance of the GTM3 cells that made this project possible.
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