Two-Dimensional Electrophoresis in Prostate Cancer Research and Diagnostics

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1 Received Revisions Received Accepted Two-Dimensional Electrophoresis in Prostate Cancer Research and Diagnostics Glòria Tabarés, 1 Wolfgang Hoesel, 1 Klaus Jung 2 ( 1 Protein Chemistry Department, Roche Diagnostics GmbH, Penzberg, Germany, 2 Department of Urology, University Hospital Charité, Humboldt University, Berlin, Germany) DOI: /9TMWPME1Q1MK3N39 Abstract Two-dimensional electrophoresis (2-DE) is the most commonly-used technique for large-scale identification of proteins in clinical samples. Here, we review the use of this method to improve prostate cancer diagnostics, especially on the analysis of prostate-specific antigen Proteomics refers to all techniques and procedures applied to analyze the proteome, which is defined as all proteins encoded by a genome. In cancer research, proteomics is used to identify proteins involved in carcinogenesis and cancer progression. Novel targets, as well as diagnostic and prognostic or predictive markers, can result from these investigations. 1 Proteomics has distinct advantages over genomic and ribonucleic acid expression studies because proteins are ultimately responsible for the malignant phenotype. 2 A proteomic analysis usually comprises 2 steps: protein separation and protein identification, which also includes the characterization of the post-translational protein modifications. 3 Currently, the most commonly-used technique for large-scale identification of proteins in clinical samples is two-dimensional electrophoresis (2- DE) in combination with image analysis or mass spectrometry (MS). In the following, we critically review the use of 2-DE for prostate cancer diagnostics; therefore, we discuss the advantages and limitations of this technique with special emphasis on its applicability to prostate cancer diagnostics and describe the analysis of free PSA (fpsa) subforms by 2-DE in sera from prostate cancer patients. Two-Dimensional Electrophoresis Two-dimensional electrophoresis separates proteins according to their molecular weight (size) and charge (ie, isoelectric point [pi]). Following electrophoresis, the pattern of protein spots from 1 type of sample (eg, serum from men with BPH) can be compared qualitatively with that obtained from another type of sample (eg, serum from men with prostate cancer [PCa]) or quantitatively based on the (PSA). Two-dimensional electrophoresis is a powerful method, which makes possible the detection and analysis of thousands of proteins and their post-translational modifications. However, it is a sophisticated technique, which does not resolve all kind of proteins. Some groups were able to improve prostate cancer diagnostics using the 2-DE pattern of free PSA isoforms on prostate cancer and non-prostate cancer sera. To this end, it would be very interesting to further investigate and develop these new diagnostic approaches. densitometric intensity of the spots observed in 1 type of sample versus another. In the first dimension, protein separation occurs by isoelectric focusing, usually with precasted immobilized phgradient (IPG) strips. 4,5 The second dimension uses linear gradient SDS-polyacrylamide gel electrophoresis (PAGE) for the separation of proteins according to their molecular weights. To visualize and quantify proteins after separation in 2-DE gels, it is necessary to stain them. The traditional staining methods are Coomassie Blue and silver staining. Recently, more sensitive fluorescent dyes are also applied for 2-DE visualization. 6,7 To detect proteins specifically, a transfer of the separated proteins from the gel to a nitrocellulose or polyvinylidine difluoride membrane is performed and the protein of interest is visualized with a specific antibody. This technique is known as Western blotting. The intensities of the corresponding protein spots can be quantified by image analysis. Advantages and Limitations Two-dimensional electrophoresis is a well-established, robust, and extensively-used approach in proteomics of different research fields, 3 and it is presently the most powerful method to analyze the protein phenotype of a cell and the expressions of genes that cannot be detected on a genetic level. 8,9 Two-dimensional electrophoresis has the advantage of making possible the simultaneous visualization of thousands of protein spots, the quantification of their levels, and the detection of post-translational modifications. The main limitation of 2-DE, however, is its expense with regard to human labor and consumables, as it needs several steps from the careful sample preparation (tissue or body fluid) to the electrophoresis and the detection and qualitative labmedicine.com December 2007 Volume 38 Number 12 LABMEDICINE 735

2 or quantitative evaluation of spots, which requires a lot of experience. There are some inherent analytical problems since 2- DE does not resolve highly-basic proteins, those smaller than 10 kda, or very large protein molecules. 2 Very hydrophobic proteins are underrepresented, and the most acidic or basic proteins remain undetected. 9 2-DE Results for the Analysis of the Prostate Cancer Proteome Several groups of researchers identified proteins that appeared to be differentially expressed in prostate cancer and non-cancerous prostate tissue when comparing the corresponding proteomes from tissue extracts by 2-DE. 10 For example, Ahram and colleagues 11 compared normal and tumor cells from prostate tissues of prostate cancer patients. They found 40 tumor-specific protein alterations, although the majority of these changes were not shared among all the neoplasm samples. So far, most proteomic studies have used tumor tissue as the source for biomarker discovery. 12 With advances in proteomic technologies and the realization that the molecular milieu of even localized cancer is often reflected in circulating fluids, serum has become an increasingly-used source for biomarker discovery. 2 In as much, our studies were focused on the analysis of prostate-specific antigen (PSA) and its molecular forms, as PSA is the most used tumor marker in serum. Prostate-Specific Antigen Prostate-specific antigen is a 28 kda glycoprotein exhibiting a serine-protease activity similar to chymotrypsin and belonging to the kallikrein family. 13 Prostate-specific antigen in serum is the leading marker for the early detection of prostate cancer 14,15 ; however, it is not fully specific for prostate cancer, as other prostate pathologies (like benign prostatic hyperplasia or inflammations) can also induce serum PSA elevations. 16,17 Several concepts, such as PSA density, PSA velocity, and age- or race-specific reference limits, have been recommended for improving the limited diagnostic specificity of PSA as a cancer marker. 18,19 The measurements of the molecular forms of PSA have been shown to be the most promising for improving the limited diagnostic specificity of PSA. 20 Total PSA (tpsa) circulates in serum in complexed forms bound to protease inhibitors and in a non-complexed, free form. 21 Approximately 70% to 90% of tpsa is bound mainly to α1-antichymotrypsin, and a small amount to α1-protease inhibitor and inter-αtrypsin inhibitor; the remaining 10% to 30% of tpsa is not bound to serum proteins and is called fpsa. The percentage of fpsa to tpsa (%fpsa) is lower in patients with prostate cancer than in those without. The exact reason for this phenomenon has not yet been elucidated, but numerous reports demonstrated the high diagnostic validity of %fpsa for differentiating between malignant and benign prostate diseases. 22 It has also been shown that the fpsa fraction is heterogeneous and occurs in several subforms. 23,24 Analysis of these fpsa subforms (eg, propsa, benign prostatic hyperplasia [BPH]-associated PSA, nicked PSA) has shown promising results to be useful for diagnostic purposes DE Results for PSA Characterization In order to assess the diagnostic suitability of the major electrophoretic subforms of fpsa with the aim to improve prostate cancer diagnostics, different 2-DE analyses were performed, some of them focusing on the glycosylation structures of fpsa as post-translational modification. The 2-DE pattern of fpsa forms from the sera of men with and without prostate cancer has recently been characterized in detail. It has been demonstrated that such analysis could improve both sensitivity and specificity in prostate cancer diagnostics. 23,28-31 In addition, comparing the 2-DE pattern of fpsa from seminal plasma and conditioned media from the prostate cancer cell line LNCaP, many differences in the glycosylation of PSA have also been found. 32 In a study in 1999, Charrier and colleagues 23 observed some differences between standard-molecular-weight and low-molecular-weight fpsa, according to its moiety in a 2-DE followed by Western blot detection. Further 2DE-analyses confirmed these results and described the subforms with regard to molecular weight and pi in more detail. Using a special immunosorption protocol 33,34 to isolate serum PSA from men with and without prostate cancer, followed by 2-DE and detection by Western blotting, 15 different immunoreactive fpsa subforms of different concentrations were detected. Figure 1 displays these main fpsa subforms observed at MW 28 to 35 kd. Two forms, named F2 and F3, appeared in all samples. The pattern of the serum fpsa subforms were similar in men with and without prostate cancer, but the ratios between spots F2 and F3 were different to a certain extent, in as much as F2 was lower in non-cancer than in cancer serum samples and F3 behaved inversely. These results provide an option for differentiating men with prostate cancer from men with benign prostate diseases, specifically in the range of 2 to 4 ng/ml tpsa. 29 The lower-molecular-weight fpsa spots observed by these authors, corresponding to spots J to M in Figure 1, could represent nicked fpsa isoforms, but further investigations must be done in order to characterize these low-molecular-weight fpsa spots. The 2-DE fpsa pattern of seminal plasma was similar to that of serum, while the patterns of protein spots from nonmalignant and malignant prostate tissues were similar to each other and to the pattern observed in serum from men without (eg, BPH; Figure 1A) and with (Figure 1B) PCa, respectively. Figure 1 demonstrates that spots corresponding to the F2 and F3 isoforms of fpsa are the predominant spots in pooled serum from men with BPH or with PCa; however, the ratio (F2/F3) of the intensity of isoforms F2 and F3 was qualitatively lower in the pooled serum from men with PCa compared with those with BPH. Spots F1 to F4 appear in all the human samples analyzed; therefore, it was more useful to compare different samples than other less frequently detected spots. 28 The fpsa subforms pattern, which differs completely from the other samples described, is fpsa secreted by the prostate cancer cell line LNCaP, whose main subforms are not F2 and F3, but forms with a more basic pi due to the lack of sialic acid in its glycans. 32,36 This indicates that this cell line is a rather inappropriate model to study PSA characteristics in human serum. All these results prove the usefulness of the 2-DE method for the characterization of fpsa isoforms. A further study 29 compared the use of tpsa and the %fpsa with the ratio of the 2 main fpsa subforms, F2 and F3. An improved 736 LABMEDICINE Volume 38 Number 12 December 2007 labmedicine.com

3 (-5/-7)proPSA by 2-DE. In a study by our group, 32 a mouse monoclonal antibody for immunosorption, which specifically recognized the (-5/-7)proPSA forms, was used. From the results, it was concluded that (-5/-7)proPSA forms are present in all major spots that are observed in the fpsa serum pattern of prostate cancer, but there were no differences observed which could be used for diagnostic purpouses. Figure 1_Analysis of immunopurified free PSA (fpsa) by two-dimensional electrophoresis and Western blotting with chemiluminescence detection. The separation is based on the protein molecular weight in kilodalton (kda) and on its isoelectric point (pi). A) Spot pattern corresponding to fpsa immunosorbed from a serum pool of benign prostatic hyperplasia (BPH) patients. B) Spot pattern corresponding to fpsa immunosorbed from a serum pool of prostate cancer patients. All of the spots correspond to different isoforms from fpsa, and the most frequently detected are named to facilitate the comparison between both patterns. Modified from Tabarés and colleagues. 31 differentiation between men with and without PCa for tpsa levels up to 10 µg/l could be obtained and showed this parameter as a potential novel tool in the tpsa gray zone. Detection of Glycosylated fpsa Isoforms Generally, N-glycans are more branched and more sialylated in tumors. 37,38 Differences in the sialic acid content can be a reason for the presence of diverse glycoprotein forms, and a decrease in pi often reflects an increase in the sialic acid content of a glycoprotein. 8,39 The isoform pattern of serum proteins, regarding its post-translational modifications, can be characterized by Western blot detection following 2-DE. Only a few reports on the glycosylation of fpsa from serum have been described so far and some are contradictory. Therefore, studies on the glycosylation of PSA were undertaken to clarify conflicting results and to find whether glycan features could be useful for improving the diagnostic value of PSA. 32 Removal of sialic acid was performed with a sialidase pretreatment of samples before 2-DE protein separation resulting in a complete desialylation of glycoproteins. It was observed that the fpsa pattern from men with and without prostate cancer, after sialic acid release, resembled the fpsa pattern secreted by LNCaP cells, which does not contain sialic acid. 31 By 2-DE, combined with sialidase treatment, the heterogeneity of fpsa as a glycoprotein was confirmed. Thus, this technique is very useful to investigate post-translational modifications of proteins. Other Proteomic Techniques The results described suggest that subforms of fpsa are able to improve the diagnostic differentiation between benign and malignant prostate disease. Therefore, it would certainly be interesting to be able to assign structural features to the different spots which have been detected in these complex patterns, allowing the development of specific diagnostic tests. However, taking into account that a 2-DE spot contains only a total amount of 1 ng or less fpsa, this would not be an easy task. In order to improve the specific analysis of the different fpsa subforms in low amounts of sample, other proteomic techniques, like the combination of 2-DE with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), can be applied, or the analysis of fpsa by liquid chromatography followed by electrospray ionization mass spectrometry (LC-ESI-MS). Non-2-DE methods generally require smaller sample sizes, and some of the techniques are more automated than 2-DE; thus, they are suitable for the analysis of a large number of samples. However, limitations pertain to protein identification and data validation. Moreover, most of these methods detect only a limited set of proteins in each assay. 9 Mass Spectrometry Several studies are currently being carried out to identify proteins by mass spectrometry (MS). Liquid chromatography- MS instruments are often used, where the sample is injected into the mass spectrometer, often by split flow from an HPLC through capillary reversed-phase columns or by nanoflow devices that allow a long sample analysis time. With MALDI- TOF mass spectrometers, the sample is deposited on a target with a crystallized matrix and introduced into the flight tube of the mass spectrometer, then a laser pulse is delivered onto the sample, and the sample and the matrix enhance desorption of the substances. 8 Surface-enhanced laser desorption ionization (SELDI-TOF) mass spectrometry is a relatively new technology developed to facilitate high-throughput protein profiling. It is a method that combines ProteinChip and MS technologies. 2 Detection of (-5/-7)proPSA Isoforms ProPSA is a precursor of PSA, normally carrying a 7 amino acid pro-leader peptide at the N-terminal end of PSA. However, propsa forms with truncated pro-leader peptides have also been identified in sera from prostate cancer patients, and it has been shown that they might have potential for improving prostate cancer diagnostics. 27,40,41 To this end, it was of interest to see if it was possible to detect differences in the spot pattern of serum samples regarding MS Application for Prostate Cancer Diagnostics A few reports describe the use of MS for prostate cancer reasearch and diagnostics. Regarding prostate cancer, some studies have shown that information generated by MS-based protein separation, like SELDI-TOF, can help to distinguish men with PCa from those with benign prostate diseases. 2 Some recent reports describe the analysis by LC-MS of the serum proteome in a prostate cancer xenograft model with labmedicine.com December 2007 Volume 38 Number 12 LABMEDICINE 737

4 human prostate cancer cells. 42 Another interesting technique is the glycomic profiling by MALDI-MS. 43 The authors compared the profiles of blood samples from healthy men and prostate cancer patients and found structures that can be considered as cancer-specific. Only a few studies describe the analysis of PSA by MS. Satterfield and Welch 44 compared 6 commercially-available PSA samples purified from seminal plasma by LC-MS and MALDI-MS and concluded that these techniques are sensitive enough to detect impurities in some of these samples, which should be taken into account before using them as authentic PSA standards for calibrants for in vitro diagnostics. Final Remarks and Future Perspectives The understanding of the role of post-translational modifications in biologic processes and disease pathology will very likely increase. Identification of such modified proteins will require further development of both 2-DE and MS-based technologies. 9 It has been concluded that there is no substitute in sight for 2-DE, but that there are a number of areas of 2-DE technology that can be improved, like sample preparation or detection methods. 45 The combination of 2-DE with MS analysis could be another option for improvement. In contrast to genomic analyses, 2-DE has the potential to separate post-translational modifications. Therefore, it would be very interesting to further investigate all these new approaches with the focus of improving prostate cancer diagnostics. LM Acknowledgements: This work was supported by grants from the Deutsche Forschungsgemeinschaft (JU 365/5-1) and from Roche Diagnostics GmbH. 1. Veenstra TD, Prieto DA, Conrads TP. Proteomic patterns for early cancer detection. Drug Discov Today. 2004;9: Ornstein DK, Tyson DR. Proteomics for the identification of new prostate cancer biomarkers. 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