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The technique of fluorescent two-dimensional (2D) difference gel electrophoresis for differential protein expression analysis has been evaluated using a model breast cancer cell system of ErbB-2 overexpression. Labeling of paired cell lysate samples with N-hydroxy succinimidyl ester-derivatives of fluorescent Cy3 and Cy5 dyes for separation on the same 2D gel enabled quantitative, sensitive, and reproducible differential expression analysis of the cell lines. SyproRuby staining was shown to be a highly sensitive and 2D difference gel electrophoresis-compatible method for post-electrophoretic visualization of proteins, which could then be picked and identified by matrix-assisted laser-desorption ionization mass spectroscopy. Indeed, from these experiments, we have identified multiple proteins that are likely to be involved in ErbB-2-mediated transformation. A triple dye labeling methodology was used to identify proteins differentially expressed in the cell system over a time course of growth factor stimulation. A Cy2-labeled pool of samples was used as a standard with all Cy3- and Cy5-labeled sample pairs to facilitate cross-gel quantitative analysis. DeCyder (Amersham Biosciences, Inc.) software was used to distinguish clear statistical differences in protein expression over time and between the cell lines. The technique of fluorescent two-dimensional (2D) difference gel electrophoresis for differential protein expression analysis has been evaluated using a model breast cancer cell system of ErbB-2 overexpression. Labeling of paired cell lysate samples with N-hydroxy succinimidyl ester-derivatives of fluorescent Cy3 and Cy5 dyes for separation on the same 2D gel enabled quantitative, sensitive, and reproducible differential expression analysis of the cell lines. SyproRuby staining was shown to be a highly sensitive and 2D difference gel electrophoresis-compatible method for post-electrophoretic visualization of proteins, which could then be picked and identified by matrix-assisted laser-desorption ionization mass spectroscopy. Indeed, from these experiments, we have identified multiple proteins that are likely to be involved in ErbB-2-mediated transformation. A triple dye labeling methodology was used to identify proteins differentially expressed in the cell system over a time course of growth factor stimulation. A Cy2-labeled pool of samples was used as a standard with all Cy3- and Cy5-labeled sample pairs to facilitate cross-gel quantitative analysis. DeCyder (Amersham Biosciences, Inc.) software was used to distinguish clear statistical differences in protein expression over time and between the cell lines. The ability to determine statistically significant alterations in protein expression that correlate with disease or occur consequent to experimentally induced changes in cells is fundamental to the exploitation of proteomics. Previous studies from our laboratory have used two-dimensional (2D) 1The abbreviations used are: 2D, two-dimensional; DIGE, differential gel electrophoresis; MALDI, matrix-assisted laser desorption ionization; MS, mass spectroscopy; HRGβ1, heregulin β1; CHAPS, 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid; IPG, immobilized pH gradient; AmBic, ammonium bicarbonate; Hsp, heat shock protein. ) gel analysis of immunomagnetic affinity-sorted primary human breast cells to establish the protein expression profiles of luminal and myoepithelial cells (1Page M.J. Amess B. Townsend R.R. Parekh R. Herath A. Brusten L. Zvelebil M.J. Stein R.C. Waterfield M.D. Davies S.C. O'Hare M.J. Proteomic definition of normal human luminal and myoepithelial breast cells purified from reduction mammoplasties.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12589-12594Google Scholar). These differential expression studies were carried out using replicate 2D gels of each sample and post-staining with a fluorescent protein dye. The subsequent detailed curation and correlation of images was used to derive data sets for statistical analysis. This approach was attractive, because the fluorescent protein stain has a wide linear range of detection, thus giving improved quantification of both high and low abundance proteins. However, in these experiments, many pairs of gels were required to establish statistically significant differences in protein expression as each gel contains inherent experimental variations that limit image superimposition. The introduction of fluorescent 2D differential gel electrophoresis (DIGE) by Unlu et al. (2Unlu M. Morgan M.E. Minden J.S. Difference gel electrophoresis: a single gel method for detecting changes in protein extracts.Electrophoresis. 1997; 18: 2071-2077Google Scholar) 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 (3Tonge R. Shaw J. Middleton B. Rowlinson R. Rayner S. Young J. Pognan F. Hawkins E. Currie I. Davison M. Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology.Proteomics. 2001; 1: 377-396Google Scholar). 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 (4Harris R.A. Eichholtz T.J. Hiles I.D. Page M.J. O'Hare M.J. New model of ErbB-2 over-expression in human mammary luminal epithelial cells.Int. J. Cancer. 1999; 80: 477-484Google Scholar). The ErbB-2 receptor tyrosine kinase (also known as neu/HER2) is overexpressed in 25–30% 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. The parental HB4a cell line was established from flow-sorted normal human breast luminal epithelial cells by immortalization with a non-DNA binding, temperature-sensitive mutant of SV40 large T-antigen (U19tsA58) (5Stamps A.C. Davies S.C. Burman J. O'Hare M.J. Analysis of proviral integration in human mammary epithelial cell lines immortalized by retroviral infection with a temperature-sensitive SV40 T-antigen construct.Int. J. Cancer. 1994; 57: 865-874Google Scholar). The ErbB-2-overexpressing variant HBc3.6 clone was derived from HB4a by stable co-transfection with a full-length normal human ErbB-2 cDNA under control of the murine mammary tumor virus long terminal repeat promoter and an SV40 polyadenylation signal. The HBc3.6 clone was selected, because it overexpresses a level of ErbB-2 similar to that seen in many breast carcinomas of luminal cell origin (4Harris R.A. Eichholtz T.J. Hiles I.D. Page M.J. O'Hare M.J. New model of ErbB-2 over-expression in human mammary luminal epithelial cells.Int. J. Cancer. 1999; 80: 477-484Google Scholar). 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) HRGβ1 (R 1: 377-396Google Scholar). 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 3–10 (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 HRGβ1 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 HRGβ1 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. Immobilized non-linear pH gradient (IPG) strips, pH 3–10 (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 18× 20-cm 9–16% 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 manufacturer's 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 at 30 gel at the dye run the of the 2D gels were directly between glass using a (Amersham Biosciences, This two and at the different for each of the Cy dyes and for SyproRuby fluorescent protein An image is and to images were by the according to the The images generated were as image for further protein profile analysis. Gels were in 30% (v/v) (v/v) and washed in and total protein was detected by post-staining with SyproRuby dye (Molecular Probes) for h at room dye was removed by twice in and gels were using the 2D at the excitation and emission for the Gels were also with according to the protocol of et al. A. M. M. of proteins polyacrylamide Scholar). were and using of or with DeCyder software from Amersham Biosciences, were also detected by of images using For DeCyder image the differential analysis of DeCyder was first used to the Cy2, Cy3, and Cy5 images for each gel and to detect spot for the of spot this resulting from and were The analysis was used to abundance differences between samples run on the same gel. The variation analysis of DeCyder was then used to all image from difference analysis for a comparative cross-gel statistical analysis. was required at this point to set on gels for more accurate cross-gel image superimposition. of Cy3 and Cy5 spot with the Cy2 standard spot each gel a This was compared across all gels for each and a statistical analysis was performed using the triplicate from each experimental by 2D-DIGE were with SyproRuby protein and were for according to this of were from 2D gels using a automated spot (Amersham Biosciences, Inc.) following the manufacturer's were in μl of in and at for protein by were washed twice in m m ammonium in and in a for 10 min. were reduced in 10 mm mm for min at and then in 50 mm mm for 1 h at room temperature in the were then washed twice in mm AmBic, and were with 30 of modified in mm for h or at was and were further in were and in of were onto a in 1 μl of was performed using a mass in the with mass were with mass was carried out using the of we to compare the sensitivity of protein using the N-hydroxy succinimidyl ester derivatives of the Cy3 and Cy5 dyes with post-staining that are with subsequent In of a standard protein were labeled with Cy and the sensitivity of was compared with that of staining A. M. M. of proteins polyacrylamide Scholar). This analysis that Cy dye labeling was in more sensitive we could detect 1 of standard protein by Cy dye labeling compared with 5 with the staining protocol (data not shown). approach required of gels to glass for automated spot picking. This was to gel and of the gel but also because fluorescent used for the are on the We that staining was sensitive on the bonded gels and staining from gel to thus making it for 2D-DIGE The fluorescent protein stain SyproRuby was much more sensitive uniform staining from gel to and its ability to detect proteins was by the of gels to glass plates. SyproRuby was also more sensitive Cy3 and Cy5 because a number of proteins by SyproRuby were not in the images this effect be because of the sensitivity of SyproRuby we out the that proteins not be modified by the Cy dyes as as the software A. two-dimensional gel using the II Sci. we detected an of more gel using SyproRuby versus Cy dye Thus, in SyproRuby to be an that is with 2D-DIGE of images with Cy3 or Cy5 images from the same gel that many not exactly This was particularly in the molecular weight range of the gels This is because proteins are minimally labeled, and the labeled protein has the mass of a covalently dye However, this was not uniform for all proteins, that dye labeling could the or that more one lysine is labeled on that particular protein. For this post-electrophoretic staining was and of were prior to picking. The Cy dye labeling experiments differences in protein expression between the parental and ErbB-2-overexpressing cell with of these proteins by SyproRuby post-staining For of the of we that the volume of the from Cy dye images with the amount of protein and the ability to identify by low volume that were by SyproRuby staining could not be identified by and it will be to these from gels on which more protein is We tested the reproducibility of Cy dye the same sample an HB4a or an HBc3.6 was labeled with Cy3 and Cy5, and the mixed labeled samples were run on the same we detected only differences in the abundance of proteins (data not shown). These differences were more for the low abundance proteins. In a further replicate samples of HB4a and HBc3.6 lysates were labeled with Cy3 and Cy5, and run on different We were to detect the same differences in the expression of particular proteins from gel to gel These differences were also the were (data not shown). these are in with the from the more of the technique carried out by et al. (3Tonge R. Shaw J. Middleton B. Rowlinson R. Rayner S. Young J. Pognan F. Hawkins E. Currie I. Davison M. Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology.Proteomics. 2001; 1: 377-396Google Scholar) and that this technique is both sensitive and reproducible and can be used for the rapid of differences in the protein of two separate cell lysate identify differences in protein expression resulting from ErbB-2 we four sets of gels of HB4a and HBc3.6 lysates labeled with Cy3 and Cy5 in both We to compare the expression profiles of only the serum-starved cell to experiments and to the of sample with serum proteins. these the two cell lines differences in and (4Harris R.A. Eichholtz T.J. Hiles I.D. Page M.J. O'Hare M.J. New model of ErbB-2 over-expression in human mammary luminal epithelial cells.Int. J. Cancer. 1999; 80: 477-484Google differential protein expression in the of The protein image sets were and using of the differences were also detected by directly the Cy3 and Cy5 images using We detected distinct protein that differences in expression between the two cell which were in all gel images and were by SyproRuby post-staining These were for automated spot and of were identified with by and mass The and differential labeling of the identified proteins are shown on the Cy dye images in DeCyder software was used to the difference in expression under serum-starved conditions between the two cell lines of these proteins a or in expression in the ErbB-2-overexpressing cell the in expression This molecular protein is involved in and but more it has been shown to be overexpressed in human of the breast in shock proteins in human Scholar). of the heat shock protein in cancer shock proteins in human was in the ErbB-2-overexpressing cell of differentially expressed proteins in cells identified by difference or in expression in HBc3.6 to in for the identified of matching the from mass the total number of or in expression in HBc3.6 to in for the identified of matching the from mass the total number of in a gel of differentially expressed protein identified by and mass of Cy dye images from samples run on pH 3–10 non-linear gradient IPG strips and 9–16% gradient polyacrylamide gels are proteins in the HBc3.6 cell line are shown in in the HBc3.6 cells are shown in to for changes in expression using DeCyder of the identified proteins have also been in transformation and These are the protein M. J. and of a human protein expressed in human A. E. A. L. S. of in normal and breast the J. I. of expression by growth and of tumor J. and the cell protein J. of the of proteins in and Scholar). Thus, the of these proteins to be involved in ErbB-2-mediated and transformation of this cell and that this cell system is a cell model of breast cancer. We also that expression of the and in the HBc3.6 cells is a of by ErbB-2 overexpression. changes in the expression of the identified proteins protein, and could at in for the different by the two cell The identified were all from gels with only μg) of Cy samples that were with This that Cy dye labeling and SyproRuby post-staining not the ability to identify proteins by unlabeled protein is picked for using this labeling it is that Cy dye of lysine residues the of The proteins are either by mass or of be with statistical because of the low abundance of of the low abundance proteins will of of will be for of differentially expressed protein that be identified by mass We further our studies to the of HRGβ1 over time on the expression profiles of the parental and ErbB-2-overexpressing cells We have recently differences in the of these cells to HRGβ1, F. L. J. and for which is a of the of ErbB-2 in the Scholar). DeCyder analysis of protein expression in to as as differences in expression between the two cell lines at each time point of the proteins previously identified by were also detected by DeCyder software analysis The data were to statistically changes in protein abundance (i.e. of were protein with a difference in abundance between the two cell lines at the time point these proteins, were in the parental cell line were in the ErbB-2-overexpressing cell line a difference were differentially proteins. The number of significant differences between the cell lines was following stimulation. the HBc3.6 cell line a number of proteins with with its in to HRGβ1 A comparison of the in expression over time that were more proteins at a number of proteins were at 24 h in both cell lines of protein with a significant difference in abundance between cell in in in a of protein with a significant in abundance over in a The abundance of of the identified proteins not with HRGβ1 were protein and 2 expression in HB4a cells in to growth its expression in HBc3.6 cells. is a that an associated kinase and M.J. of human as a with associated kinase F. J. J. The a of proteins from to its is and 2 were in the HBc3.6 and expression was further reduced by HRGβ1 that ErbB-2 is to on our using both and to further the of the identified proteins in ErbB-2-mediated transformation. We are also to identify the proteins expression is by and ErbB-2 as by DeCyder analysis. In we that 2D-DIGE and DeCyder image analysis is a sensitive, and reproducible technique for statistically significant differences in the protein expression profiles of multiple This approach was more rapid that compare post-electrophoretic gels and thus more for statistical this methodology we have identified proteins that are now in ErbB-2-mediated transformation and for breast cancer We L. and for of the and for and Currie and of Amersham Biosciences, Inc. for DeCyder and
Gharbi et al. (Fri,) studied this question.