From the Ludwig Institute for Cancer Research, 91 Riding House Street, London W1W 7BS, United Kingdom
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ABSTRACT |
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The introduction of fluorescent 2D differential gel electrophoresis (DIGE) by Unlu et al. (2) has now made it possible to detect and quantitate differences between experimental pairs of samples resolved on the same 2D gel. The basis of the technique is the use of two mass- and charge-matched N-hydroxy succinimidyl ester derivatives of the fluorescent cyanine dyes Cy3 and Cy5, which possess distinct excitation and emission spectra. These are used to differentially label lysine residues of two protein samples for comparative analysis of the mixed sample on one gel. The ability to directly compare two samples on the same gel not only avoids the complications of gel-to-gel variation but also enables a more accurate and rapid analysis of differences and reduces the number of gels that need to be run. This procedure has been further developed by Amersham Biosciences, Inc. and has been evaluated recently in an in vivo mouse toxicology study (3).
Labeling reactions are carried out under conditions where proteins are "minimally" labeled, such that only 20% of molecules of a particular protein are covalently modified with one Cy dye molecule. Detection of proteins for excision and mass spectroscopy requires post-staining of gels with a general protein stain, because the unlabeled majority of a protein will not exactly co-migrate with the labeled protein, particularly in the low molecular weight range. A third fluorescent Cy dye (Cy2) has also been introduced, making it possible to compare three samples on one gel. An experimental design that should allow a much more accurate statistical analysis of expression differences across multiple gels can now be developed, because different Cy3- and Cy5-labeled samples can be compared with a Cy2-labeled "standard" run on every gel.
In this paper we describe further evaluation of the 2D-DIGE technique and seek to extend our protein expression studies in breast cancer. Specifically we investigated ErbB-2-mediated transformation in a model cell line system comprised of an immortalized luminal epithelial cell line and a derivative stably overexpressing ErbB-2 at a similar level to that seen in breast carcinomas (4). The ErbB-2 receptor tyrosine kinase (also known as neu/HER2) is overexpressed in 2530% of breast cancers and is often associated with poor prognosis. Differentially expressed proteins detected in this cell system are likely to be involved in the processes of ErbB-2-mediated transformation. In the first experiments we evaluated the feasibility of the technique for monitoring protein expression changes in a model cell line system and tested its utility for high sensitivity, high throughput differential expression proteomics. We have examined the sensitivity and reproducibility of Cy3/Cy5 dye labeling and employed SyproRuby (Molecular Probes) gel staining to visualize proteins for automated spot picking. We have identified a number of differentially expressed proteins resulting from ErbB-2 overexpression using matrix-assisted laser-desorption ionization (MALDI) mass spectroscopy (MS). In a second set of experiments, we introduced Cy2 dye labeling of a standard pooled sample for linking gel images of pairs of samples from differentially treated cells. In this case we have employed proprietary software from Amersham Biosciences, Inc. (DeCyder) to collect, process, and derive statistical data in an experiment that examines the effect of a growth factor on the expression profile of a normal, mammary luminal epithelial cell line versus its ErbB-2-overexpressing derivative.
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EXPERIMENTAL PROCEDURES |
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Cells were routinely maintained in RPMI 1640 with 10% (v/v) fetal calf serum, 2 m M glutamine, 100 IU/ml penicillin, and 100 µmg/ml streptomycin (all from Invitrogen) and 5 µmg/ml hydrocortisone and 5 µmg/ml insulin (both from Sigma) at 37°C in a 10% CO2 humidified incubator. Cells were starved for 48 h in insulin-free medium containing 0.1% fetal calf serum prior to harvesting. Starved cells were also stimulated with 1 nM (8 ng/ml) HRGß1 (R & D Systems) for 4, 8, and 24 h. The concentration of HRGß1 used was determined to be the minimum required to induce maximal extracellular signal-regulated kinase 1/2 and Akt phosphorylation after 10 min of stimulation as determined by immunoblotting (data not shown).
Sample Preparation and Protein Labeling
Cells at 80% confluence were washed twice in 0.5x phosphate-buffered saline, lysed in lysis buffer (4% (w/v) CHAPS, 2
M thiourea, 8 M urea, 10 mM Tris-HCl, pH 8.5), and then homogenized by passing through a 25-gauge needle six times. Insoluble material was removed by centrifugation at 14,000 rpm for 20 min at 10°C. Protein concentration was determined using the Coomassie protein assay reagent (Pierce).
Cell lysates were labeled with N-hydroxy succinimidyl ester-derivatives of the cyanine dyes Cy2, Cy3, and Cy5 (Amersham Biosciences, Inc.) following the protocol described previously (3). Typically, 100 µg of lysate was minimally labeled with 400 pmol of either Cy3 or Cy5 for comparison on the same 2D gel. Labeling reactions were performed on ice in the dark for 30 min and then quenched with a 50-fold molar excess of free lysine to dye for 10 min on ice. Differentially labeled samples were mixed and reduced with 65 m M dithiothreitol for 15 min. Ampholines/pharmalytes, pH 310 (1% (v/v) each; Amersham Biosciences, Inc.), and bromphenol blue were added, and the final volume was adjusted to 350 µl with lysis buffer. For the HRGß1 stimulation experiments, lysates from three separate time course experiments were run in parallel. The triplicate sets of cells were serum-starved for 48 h and then stimulated for 4, 8, and 24 h with 1 nM HRGß1 or left unstimulated (0 h). The 24 lysates generated were labeled with Cy3 (for HB4a) and Cy5 (for HBc3.6). A pool of all samples was also prepared and labeled with Cy2 to be used as a standard on all gels to aid image matching and cross-gel statistical analysis. The Cy3 and Cy5 labeling reactions (100 |µg of each) from each time point were mixed and run on the same gels with an equal amount (100 µg) of Cy2-labeled standard. Thus, the triplicate samples and the standard were run on 12 gels (i.e. three gels with two cell lines from each of the four time points), to generate 36 images.
Protein Separation by 2D Gel Electrophoresis and Gel Imaging
Immobilized non-linear pH gradient (IPG) strips, pH 310 (Amersham Biosciences, Inc.), were rehydrated with Cy-labeled samples in the dark at room temperature overnight, according to the manufacturers guidelines. Isoelectric focusing was performed using a Multiphor II apparatus (Amersham Biosciences, Inc.) for a total of 80 kV-h at 20°C, 10 mA. Strips were equilibrated for 15 min in 50 m
M Tris-HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 1% (w/v) SDS containing 65 mM dithiothreitol and then for 15 min in the same buffer containing 240 mM iodoacetamide. Equilibrated IPG strips were transferred onto 18x 20-cm 916% gradient or 12% uniform polyacrylamide gels poured between low fluorescence glass plates. Gels were bonded to the inner plate using bind-saline solution (PlusOne) according to the manufacturers protocol. Strips were overlaid with 0.5% (w/v) low melting point agarose in running buffer containing bromphenol blue. Gels were run in Protean II gel tanks (Bio-Rad) at 30 mA per gel at 10°C until the dye front had run off the bottom of the gels.
2D gels were scanned directly between glass plates using a 2920 2DMaster Imager (Amersham Biosciences, Inc.). This charge-coupled device-based instrument possesses two six-position filter wheels (excitation and emission) enabling scanning at the different wavelengths specific for each of the Cy dyes and for SyproRuby fluorescent protein stain. An image is built up and converted to gray scale pixel values. Gel images were normalized by adjusting the exposure times according to the average pixel values observed. The images generated were exported as tagged image format (.tif) files for further protein profile analysis.
Post-staining, Image Analysis, and Spot Picking
Gels were fixed in 30% (v/v) methanol, 7.5% (v/v) acetic acid overnight and washed in water, and total protein was detected by post-staining with SyproRuby dye (Molecular Probes) for 3 h at room temperature. Excess dye was removed by washing twice in water, and gels were imaged using the 2920 2D Imager at the appropriate excitation and emission wavelengths for the stain. Gels were also post-stained superficially with silver according to the protocol of Shevchenko et al. (6). Images were curated and analyzed using Melanie III (Swiss Institute of Bioinformatics, Geneva, Switzerland) or with DeCyder software (on trial from Amersham Biosciences, Inc.). Differences were also detected visually by direct overlay of images using Adobe PhotoShop (Adobe Systems Incorporated).
For DeCyder image analysis, the differential in-gel analysis mode of DeCyder was first used to merge the Cy2, Cy3, and Cy5 images for each gel and to detect spot boundaries for the calculation of normalized spot volumes/protein abundance. At this stage, features resulting from non-protein sources (e.g. dust particles and scratches) were filtered out. The analysis was used to rapidly calculate abundance differences between samples run on the same gel. The biological variation analysis mode of DeCyder was then used to match all pairwise image comparisons from difference in-gel analysis for a comparative cross-gel statistical analysis. Operator intervention was required at this point to set landmarks on gels for more accurate cross-gel image superimposition. Comparison of normalized Cy3 and Cy5 spot volumes with the corresponding Cy2 standard spot volumes within each gel gave a standardized abundance. This value was compared across all gels for each matched spot, and a statistical analysis was performed using the triplicate values from each experimental condition.
Changes observed by 2D-DIGE analyses were aligned with SyproRuby protein patterns, and spots were selected for picking according to this post-stained image. Spots of interest were excised from 2D gels using a Syprot automated spot picker (Amersham Biosciences, Inc.) following the manufacturers instructions. Spots were collected in 200 µl of water in 96-well plates and kept frozen at -20°C for protein identification by MALDI-MS.
Protein Identification by MALDI-MS
Gel pieces were washed twice in 25 m
M ammonium bicarbonate (AmBic) in 50% acetonitrile and dried in a SpeedVac for 10 min. Samples were reduced in 10 mM dithiothreitol, 25 mM AmBic for 45 min at 50°C and then alkylated in 50 mM iodoacetic acid, 25 mM AmBic for 1 h at room temperature in the dark. Gel pieces were then washed twice in 25 mM AmBic, 50% acetonitrile and vacuum-dried. Proteins were proteolysed with 30 ng of modified trypsin (Promega, Southampton, United Kingdom) in 25 mM AmBic for 16 h or overnight at 37°C. Supernatant was collected, and peptides were further extracted in 5% trifluoroacetic acid, 50% acetonitrile. Peptide extracts were vacuum-dried and resuspended in 3 |gml of water. Digests (0.5 µl) were spotted onto a MALDI target in 1 µl of matrix (2,5-dihydroxybenzoic acid). MALDI-MS was performed using a Reflex III reflector time-of-flight mass spectrometer (Bruker Daltonik, Bremen, Germany) in the reflector mode with delayed extraction. All mass spectra were internally calibrated with trypsin autolysis peaks. Peptide mass mapping was carried out using the MS-Fit program (Protein Prospector; University of California, San Francisco, CA).
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RESULTS AND DISCUSSION |
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Our planned high-throughput approach required bonding of gels to glass plates for automated spot picking. This was to prevent gel shrinkage upon fixation and movement of the gel during picking but also because fluorescent markers used for coordinating the picking process are mounted on the plate. We found that superficial silver staining was even less sensitive on the bonded gels and staining varied considerably from gel to gel, thus making it unsuitable for 2D-DIGE post-staining. The fluorescent protein stain SyproRuby was much more sensitive than silver, consistently gave uniform staining from gel to gel, and its ability to detect proteins was unaffected by the process of bonding gels to glass plates. SyproRuby was also slightly more sensitive than Cy3 and Cy5 labeling, because a number of proteins detectable by SyproRuby were not easily visualized in the Cy3/5 images (Fig. 1A). Although this effect may be because of the enhanced sensitivity of SyproRuby staining, we cannot rule out the possibility that some proteins may not be modified by the Cy dyes as efficiently as others. Using the Melanie III software (7), we detected an average of 1.4 ± 0.1 times more gel features using SyproRuby versus Cy dye labeling. Thus, in practice, SyproRuby appears to be an ideal post-stain that is compatible with 2D-DIGE labeling.
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The preliminary Cy dye labeling experiments revealed obvious differences in protein expression between the parental and ErbB-2-overexpressing cell lines, with most of these proteins detectable by SyproRuby post-staining (Fig. 1A). For most of the spots of interest, we found that the volume of the spots from Cy dye images correlated with the amount of protein and hence the ability to identify them by MALDI-MS. Several low volume spots that were barely detectable by SyproRuby staining could not be identified by MALDI-MS, and it will be necessary to pick these spots from preparative gels on which more protein is loaded.
We next tested the reproducibility of Cy dye labeling. When the same sample (either an HB4a or an HBc3.6 urea-solubilized lysate) was labeled with Cy3 and Cy5, and the mixed labeled samples were run on the same gels, we detected only very minor differences in the abundance of some proteins (data not shown). These differences were more apparent for the very low abundance proteins. In a further experiment, replicate samples of HB4a and HBc3.6 lysates were labeled with Cy3 and Cy5, respectively, and run on different gels. We were able to detect the same differences in the expression of particular proteins from gel to gel (Fig. 2). These differences were also detectable when the sample-dye combinations were reversed (data not shown). Taken together, these results are in agreement with the conclusions drawn from the more rigorous validation of the technique carried out by Tonge et al. (3) and show that this technique is both sensitive and reproducible and can be used for the rapid identification of differences in the protein content of two separate cell lysate samples.
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We detected 35 distinct protein spots that showed consistent differences in expression levels between the two cell lines, which were present in all gel images and were detectable by SyproRuby post-staining (Fig. 3). These spots were selected for automated spot picking, and 18 of them were identified with confidence by MALDI-MS and peptide mass fingerprinting (Table I). The migration patterns and differential labeling of the identified proteins are shown on the Cy dye images in Fig. 4. DeCyder software was used to calculate the average -fold difference in expression under serum-starved conditions between the two cell lines (see below). Six of these proteins showed a 2-fold or greater increase in expression in the ErbB-2-overexpressing cell line. Hsp27 showed the greatest increase in expression Table I). This molecular chaperone protein is involved in various cellular stress responses, apoptosis and actin reorganization, but more significantly, it has been shown to be overexpressed in numerous human cancers, including invasive ductal carcinoma of the breast (reviewed in Ref. 8). Interestingly, Hsp70, another member of the heat shock protein family dysregulated in cancer (8), was down-regulated in the ErbB-2-overexpressing cell line.
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Effects of HRGß1 Treatment and ErbB-2 Overexpression on Protein Expression in Mammary Luminal Epithelial Cells: Statistical Analysis of Differential Expression
We further extended our studies to examine the effects of HRGß1 treatment over time on the expression profiles of the parental and ErbB-2-overexpressing cells (see "Experimental Procedures"). We have recently observed differences in the responsiveness of these cells to HRGß1,2 which is a ligand of ErbB-3, the preferred heterodimerization partner of activated ErbB-2 (reviewed in Ref. 13).
DeCyder analysis showed trends of protein expression in response to HRG|gb1 treatment, as well as differences in expression between the two cell lines at each time point (Fig. 5). All of the proteins previously identified by MALDI-MS were also detected by DeCyder software analysis (Table I). The data were filtered to reveal statistically relevant -fold changes in protein abundance (i.e. p values of <0.05). There were 135 protein spots with a greater than 1.3-fold average difference in abundance between the two cell lines at the zero time point Table II). Of these 135 proteins, 34 were higher in the parental cell line (HB4a), whereas 101 were higher in the ErbB-2-overexpressing cell line (HBc3.6). At a 2-fold difference cut-off, there were 23 differentially regulated proteins. The number of significant differences between the cell lines was increased following HRG|gb1 stimulation. Overall, the HBc3.6 cell line had a greater number of proteins with increased expression, perhaps correlating with its increased proliferation in response to HRGß1 treatment. A comparison of the change in expression over time indicated that there were more proteins down-regulated than up-regulated at 4 h, whereas a higher number of proteins were up-regulated than down-regulated at 24 h in both cell lines Table III).
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In conclusion, we show that 2D-DIGE and DeCyder image analysis is a sensitive, MS-compatible, and reproducible technique for identifying statistically significant differences in the protein expression profiles of multiple samples. This approach was more rapid than conventional analyses that compare multiple, post-electrophoretic stained gels and thus require more runs for statistical certainty. Using this methodology we have identified numerous proteins that are now implicated in ErbB-2-mediated transformation and may represent future targets for breast cancer therapies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This manuscript is dedicated to the memory of Craig A. Brooks, who is greatly missed.
1 The abbreviations used are: 2D, two-dimensional; DIGE, differential gel electrophoresis; MALDI, matrix-assisted laser desorption ionization; MS, mass spectroscopy; HRGß1, heregulin ß1; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; IPG, immobilized pH gradient; AmBic, ammonium bicarbonate; Hsp, heat shock protein.
2 John F. Timms, Sarah L. White, Michael J. OHare, and Michael D. Waterfield, submitted for publication.
* The cost of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1743 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 44-207-8784126; Fax: 44-207-8784040; E-mail: jtimms{at}ludwig.ucl.uk.
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REFERENCES |
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