Multivariate statistical tools applied to the characterization of the proteomic profiles of two human lymphoma cell lines by two-dimensional gel electrophoresis

Mantle cell lymphoma (MCL) cell lines have been difficult to generate, since only few have been described so far and even fewer have been thoroughly characterized. Among them, there is only one cell line, called GRANTA-519, which is well established and universally adopted for most lymphoma studies. We succeeded in establishing a new MCL cell line, called MAVER-1, from a leukemic MCL, and performed a thorough phenotypical, cytogenetical and molecular characterization of the cell line. In the present report, the phenotypic expression of GRANTA-519 and MAVER-1 cell lines has been compared and evaluated by a proteomic approach, exploiting 2-D map analysis. By univariate statistical analysis (Student's t-test, as commonly used in most commercial software packages), most of the protein spots were found to be identical between the two cell lines. Thirty spots were found to be unique for the GRANTA-519, whereas another 11 polypeptides appeared to be expressed only by the MAVER-1 cell line. A number of these spots could be identified by MS. These data were confirmed and expanded by multivariate statistical tools (principal component analysis and soft-independent model of class analogy) that allowed identification of a larger number of differently expressed spots. Multivariate statistical tools have the advantage of reducing the risk of false positives and of identifying spots that are significantly altered in terms of correlated expression rather than absolute expression values. It is thus suggested that, in future work in differential proteomic profiling, both univariate and multivariate statistical tools should be adopted.     

Identification of the regulatory proteins in human pancreatic cancers treated with Trichostatin A by 2D-PAGE maps and multivariate statistical analysis

In this paper, principal component analysis (PCA) is applied to a spot quantity dataset comprising 435 spots detected in 18 samples belonging to two different cell lines (Paca44 and T3M4) of control (untreated) and drug-treated pancreatic ductal carcinoma cells. The aim of the study was the identification of the differences occurring between the proteomic patterns of the two investigated cell lines and the evaluation of the effect of the drug Trichostatin A on the protein content of the cells. PCA turned out to be a successful tool for the identification of the classes of samples present in the dataset. Moreover, the loadings analysis allowed the identification of the differentially expressed spots, which characterise each group of samples. The treatment of both the cell lines with Trichostatin A therefore showed an appreciable effect on the proteomic pattern of the treated samples. Identification of some of the most relevant spots was also performed by mass spectrometry.     

人卵巢癌耐药细胞株的比较蛋白质组分析

本文采用高分辨二维凝胶电泳分离技术对人卵巢癌细胞株COC1及其耐药细胞株COC1/DDP中的蛋白质进行分离和差异表达分析, 应用基质辅助激光解吸电离-飞行时间质谱对酶解多肽进行测定[即测定蛋白质的肽质量指纹图(Peptide mass fingerprinting, PMF)], 并通过相应的数据库搜索来鉴定蛋白质. 为获得更准确的检索结果, 采用串联质谱技术对各肽段进行氨基酸测序, 并应用IPI-HUMAN数据库对上述检索结果进一步加以确认.      

Analysis of mouse beta-haptoglobin chain by lectin affinoblotting detection

Two different human mammary carcinoma cell lines were xenotransplanted into nude mice. Serum samples were obtained prior to and after transplantation and investigated by two-dimensional electrophoresis (2-DE). By comparison of these silver-stained patterns additional protein spots were detected resulting either from proteins secreted or shed by the tumor itself or from mouse proteins induced by the tumor or the transplantation procedure. One group of spots detectable in post-transplantation serum as well as in control serum after mock-transplantation but not in pretransplantation serum was microsequenced and identified as mouse beta-haptoglobin. The carbohydrate structures of beta-haptoglobin were characterized by two different immunochemical glycoprotein staining procedures to detect differential terminal glycan modifications.     

Proteome study of colorectal carcinogenesis

Development of cancer is a complex process involving multiple changes in gene expression. To unravel these alterations, a proteome approach aimed at the identification of qualitative and quantitative changes in protein composition, including their post-translational modifications, attracts great attention. Our study was focused on the identification of proteins whose amount is altered in the course of malignant transformation of colon mucosa. Proteins extracted from tissue specimens or cell lysates were separated by two-dimensional gel electrophoresis (2-DE). Comparative analyses of 2-DE protein patterns were done using computerized image analysis. Selected proteins exhibiting statistically significant abundance alterations comparing healthy and diseased tissues were identified by mass spectrometry. Globally, we have found 57 proteins that exhibited either a significant decrease or increase in amount in pathological tissues, and 18 of these were annotated by mass spectrometry. The alterations in the expression of nine proteins were common for both precancerous and neoplastic tissues suggesting their role in colon tumorigenesis. The epithelial origin of all identified spots was checked in two cell lines Caco-2 and DLD-1 originating from well-differentiated and poorly differentiated colon carcinoma, respectively.     

Proteomic analysis of pancreatic ductal carcinoma cells treated with 5-aza-2'-deoxycytidine

A pancreatic adenocarcinoma cell line (PaCa44), which contains, among other alterations, a methylated p16 promoter, was treated with a chemoterapeutic agent, 5-aza-2'-deoxycytidine (DAC), in order to evaluate the effect of this drug on cell growth and protein expression. Cell proliferation was strongly inhibited by a 24 h DAC treatment and this inhibition lasted for at least 10 days. Master maps of control and treated PaCa44 cells were generated by analysis with the PDQuest software. The comparison between such maps showed up- and downregulation of 45 polypeptide chains, of which 32 were downregulated and 13 upregulated, out of a total of 700 spots detected by a medium-sensitivity stain, micellar Coomassie Brilliant Blue. Fingerprinting by mass spectrometry analysis enabled the identification of 36 of these spots. Among the major changes in DAC-treated cells: cofilin and profilin 1 are silenced; coactosin, peptidyl-propyl cis-trans isomerase A and cystatin B are decreased by 22, 16- and 15-fold, respectively; stress-70 protein, superoxide dismutase and protein disulfide isomerase A3 are increased by 13-, 11-, and 5-fold, respectively. The significance of some of these major changes is discussed.     

Identification of differentially expressed proteins between human hepatoma and normal liver cell lines by two-dimensional electrophoresis and liquid chromatography-ion trap mass spectrometry

In the previous study, the proteomes of the human hepatoma cell line BEL-7404 and the normal human liver cell line L-02 were separated by high resolution two-dimensional electrophoresis (2-DE). Image analysis revealed that 99 protein spots showed quantitative and qualitative variations that were significant (P < 0.01) and reproducible. Here we report the identification results of some of these protein spots. Protein spots excised from 2-D gels were subjected to in-gel digestion with trypsin, and the resulting peptides were measured by microbore high performance liquid chromatography - ion trap - mass spectrometry (LC-IT-MS) to obtain the tandem mass (MS/MS) spectra. Twelve protein spots were identified with high confidence using SEQUEST with uninterpreted MS/MS raw data. Besides inosine-5'-monophosphate dehydrogenase 2, heat shock 27 kDa protein, calreticulin and calmodulin, whose expression was elevated in hepatoma cells, glutathione-S-transferase P was identified from hepatoma cells in which its level was 18-fold higher compared to human liver cells. Two spots were identified as the homologs of reticulocalbin for the first time in hepatoma cells and their expression increased compared to liver cells. However, tubulin beta-1 chain and natural killer cell enhancing factor B were downregulated in hepatoma cells. A tumor suppressing serpin, maspin precursor, was identified from one spot whose quantity was much higher in the normal liver cell line. More interestingly, epidermal fatty acid-binding protein (E-FABP) and fatty acid-binding protein, adipocyte-type (A-FABP), were detected in liver cells but not in hepatoma cells. The functional implication of the identified proteins was discussed.     

Two-dimensional electrophoresis reveals differential protein expression in high- and low-secreting variants of the rat basophilic leukaemia cell line

The aim of this investigation was the identification of cellular proteins that confer a high secretory phenotype on subclones of the rat basophilic leukaemia (RBL) cell line as a model of mast cell regulated degranulation. Following protein separation by two-dimensional (2-D) electrophoresis and silver staining, more than 2000 polypeptide "spots" were resolved reproducibly. Higher sample loads and Coomassie blue staining facilitated the identification by delayed extraction-matrix-assisted laser desorption/ionization (DE-MALDI) mass spectrometry of several polypeptides that were differentially expressed in the high- and low-secreting clones. Several proteins were identified whose expression could contribute to the difference in secretory phenotype. Furthermore, silver-stained 2-D gel patterns suggested differential expression of proteins in the 20-25 kDa and the pI 4.5-7.5 range, characteristic of small guanosine 5'-triphosphate (GTP)-binding proteins. By a combination of "GTP overlay" and immunoblotting, we were able to demonstrate differential expression of small GTP binding-proteins, including Rab3 proteins, in high-and low-secreting clones. The sensitivity of this complementary approach facilitated the detection of some GTP binding and Rab3 proteins, whose expression was not evident in silver-stained 2-D gels.     

Proteomic evaluation of cell preparation methods in primary hepatocyte cell culture

In vitro liver preparations are being used increasingly to study various aspects of chemical hepatotoxicity and thus have become powerful alternatives to in vivo toxicologic models. Primary hepatocyte culture systems are especially useful in screening cytotoxic and genotoxic compounds and assessing biochemical lesions associated with chemical exposure. We have begun to use this approach in combination with proteomic analysis to construct a molecular "toxicoproteomic" test system for a broad range of relevant and potentially toxic chemicals. Using a highly parallel two-dimensional electrophoretic (2-DE) protein separation system to analyze cells from culture systems, we previously observed significant variations in protein expression that were unrelated to chemical exposure. We hypothesized these artifactual protein alterations were the result of the variations in the culture conditions or cell manipulations, or both. Therefore, we conducted a study to assess the expression of hepatocyte proteins cultured on 6-well plates and recovered for analysis either by scraping/pelleting or direct in-well solubilization. Following incubation of 1.2 x 10(6) hepatocytes in six-well plate, recovery and solubilization of the cells and 2-DE of the solubilized lysates of 100 000 cells, we detected 1388 proteins in the in-well solubilized samples compared to 899 proteins in the washed/scraped/pelleted cell samples, a loss of 35%. Based on protein identification by peptide mass fingerprinting, the subcellular location of nearly all of the proteins whose abundance decreased were cytosolic and those few that increased were either microsomal, mitochondrial, or cytoskeletal proteins. These results emphasize the variation introduced by cell-handling during recovery of hepatocytes from culture plates and may explain at least some of the artifactual differences observed in earlier in vitro experiments.     

Monitoring of protein profiles for the optimization of recombinant fermentation processes using public domain databases

The expression of human superoxide dismutase in fed-batch fermentation of E. coli HMS174(DE3)(pET3ahSOD) was studied as model system. Due to the frequently used strong T7 promoter system a high metabolic load is exerted, which triggers stress response mechanisms and finally leads to the differentiation of the host cell. As a consequence, host cell metabolism is partly shifted from growth to survival accompanied by significant alterations of the protein pattern. In terms of process optimization two-dimensional electrophoresis deserves as a powerful tool to monitor these changes on protein level. For the analysis of samples derived from different states of recombinant protein production wide-range Immobiline Dry Strips pH 3-10 were used. In order to establish an efficient procedure for accelerated process optimization and to avoid costly and time-consuming analysis like mass spectrometry (MS), a database approach for the identification of significant changes of the protein pattern was evaluated. On average, 935 spots per gel were detected, whereby 50 are presumably stress-relevant. Out of these, 24 proteins could be identified by using the SWISS-2DPAGE database (www.expasy.ch/ch2d/). The identified proteins are involved in regulatory networks, energy metabolism, purine and pyrimidine nucleotide synthesis and translation. By this database approach, significant fluctuations of individual proteins in relation to recombinant protein production could be identified. Seven proteins show strong alterations (>100%) directly after induction and can therefore be stated as reliable marker proteins for the assessment of stress response. For distinctive interpretation of this highly specific information, a bioinformatic and statistic tool would be essential in order to perceive the role and contribution of individual proteins in stress response.     

A first step towards practical single cell proteomics: a microfluidic antibody capture chip with TIRF detection

We have developed a generic platform to undertake the analysis of protein copy number from single cells. The approach described here is 'all-optical' whereby single cells are manipulated into separate analysis chambers using an optical trap; single cells are lysed by a shock wave caused by laser-induced microcavitation, and the protein released from a single cell is measured by total internal reflection microscopy as it is bound to micro-printed antibody spots within the device. The platform was tested using GFP transfected cells and the relative precision of the measurement method was determined to be 88%. Single cell measurements were also made on a breast cancer cell line to measure the relative levels of unlabelled human tumour suppressor protein p53 using a chip incorporating an antibody sandwich assay format. These results suggest that this is a viable method for measuring relative protein levels in single cells.     

Two-dimensional molecular profiling of mantle cell lymphoma

The present research establishes standard two-dimensional (2-D) maps for control, reactive lymph node and non-Hodgkin's lymphoma (mantle cell lymphoma, MCL). Medium sensitivity, mass spectrometry compatible colloidal Coomassie has revealed a total of ca. 750 spots in each of the maps. Comparison of 2-D maps by statistical packages, such as the PDQuest, established up- and downregulation of a total of ca. 145 spots, with positive variations of up to 10-folds and negative variations of up to 13-folds in both MCL biopsies' protein extracts. Qualitative and quantitative variations in the two lymphoma samples are consistent. More than 20 proteins have been so far identified by matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF)-mass spectrometry, with an additional five spots, which gave very good spectra but could not be matched to any of the presently available databases. Some of the spots, such as the 78 kDa glucose-regulated protein precursor and the glutathione S-transferase P, appear to be in common with other tumors, such as lung adenocarcinoma. Others may simply reflect overall changes in cellular metabolism and growth rate that occur during malignancy and thus might turn out to be in common with any cell population receiving any kind of stress. Some (notably T-cell leukemia/lymphoma protein 1A, TCL1, found to be 10-fold overexpressed) appear to be specific of the non-Hodgkin's lymphoma here studied. Western blot and immunohistochemical analyses were applied to obtain further information about stathmin (Op18) and TCL1, respectively.     

质谱法分析2型糖尿病大鼠神经视网膜差异表达蛋白

采用二维电泳(2DE)分离了正常SD大鼠和2型糖尿病模型大鼠神经视网膜组织总蛋白, 并用Image Master 5.0软件分析比较了正常组和糖尿病组2DE图像, 正常组检测到 3122±37(n=3)个蛋白质点; 糖尿病组检测到2702±21(n=3)个蛋白质点. 约150个蛋白质点的表达水平在两组之间存在明显差异(P<0.05). 在糖尿病组中表达上调的点68个, 下调的点82个. 选择20个差异表达蛋白质点进行肽质量指纹谱(PMF)或串联质谱鉴定, 其中7个蛋白已有报道与糖尿病视网膜病变(DR)相关, 10个蛋白尚未见有报道.     

Cell surface receptors and their dynamics on toxin-treated malignant cells.

The binding, mobility, and mode of cell entry of the plant toxin ricin (or RCAII) were investigated on susceptible and partially resistant murine cell lines. When susceptible cells (SV40-transformed 3T3 fibroblast cells and BW5147 lymphoma cells) were examined, ricin bound rapidly, induced endocytosis, and entered the cell cytoplasm via broken endocytotic vesicles to inhibit cell protein synthesis, as found previously (1). Addition of lactose within 15 min after initial ricin binding prevented toxicity. After this time lactose addition no longer blocked the inhibition of protein synthesis. In a partially resistant lymphoma (BW5147/RCA3) that shows only a slight reduction in the total number of ricin-binding sites, ricin bound rapidly to the cell surface, but was endocytosed significantly less at low ricin doses compared to its parental line, indicating a possible difference in cell surface behavior. The exposed surface proteins on the BW5147 parental and BW5147/RCA3 resistnat lines were examined by 125I-labeling utilizing lactoperoxidase-catalyzed iodination. The radiolabeled components were solubilized and separated by slab electrophoresis in sodium dodecyl sulfate. Autoradiograms of the slab gels indicated that two surface components of approximately 80,000 and 35,000 mol wt were much less exposed or were missing on the resistant line.     

From proteomics to genomics

Presently, science is moving from genomics to proteomics in order to get insight into the functional network of gene expression. Actually however, proteomics is much older than genomics and dates back to the introduction of the two-dimensional gel electrophoresis technique (2-DE) independently by Klose and O'Farrell. Based on this approach almost all cellular proteins can be separated. New developments in mass spectrometry allowed identification of single spots in the 2-DE protein pattern, including the underlying genes. Joachim Klose has focused his pioneering 2-DE studies on mouse models with special emphasis on quantitative protein variants. According to him, proteins are living molecules exhibiting a characteristic protein phenotype.     

Deceptive responsive genes in gel-based proteomics

The standard method of the global quantitative analysis of gene expression at the protein level combines high-resolution two-dimensional gel electrophoresis (2DE) with mass spectrometric identification of protein spots. One of the major concerns with the application of gel-based proteomics is the need for the analytical and biological accuracy of the datasets. We mathematically and empirically simulated the possibility of the technical regulations of gene expression using 2DE. Our developed equation predicted a detectable alteration in the quantity of protein spots in response to a new protein added in, with various amounts. Testing the predictability of the developed equation, we observed that a new protein could form deceptive expression profiles, classified using prevalent tools for the analysis of 2DE results. In spite of the theoretically predicted overall reduction of proteins that resulted from adding the new protein, the empirical data revealed differential amount of proteins when various quantities of the new protein were added to the protein sample. The present work emphasize that employment of 2DE would not be a reliable approach for biological samples with extensive proteome alterations such as the developmental and differentiation stages of cells without depletion of high abundant proteins.     

High-throughput screening of protein binding by equilibrium dialysis combined with liquid chromatography and mass spectrometry

A new approach for screening plasma protein binding is presented. The method is based on equilibrium dialysis combined with rapid generic LC-MS bioanalysis by using a sample pooling approach enabling high-throughput screening of protein binding in the drug discovery phase. The method is evaluated by a comparison of measured unbound free fractions f(u) (%) between single and pooled compounds for a test set of structurally diverse compounds with a wide range of unbound fractions. Test compounds include 1 acidic and 10 basic drug standards along with 36 new chemical entities. A good correlation (R2>0.95) of f(u) (%) between the single and pooled compounds is found, suggesting that at least 10 compounds can be simultaneously measured with acceptable accuracy. A simplified drug-protein binding model is applied to calculate the f(u) (%) of drugs at various drug and protein concentrations and this is applied to elucidate the applicability of the sample pooling approach from a theoretical standpoint. Moreover, pH shifts in the plasma were observed after dialysis when using different types of buffers and the impact of that on the f(u) is illustrated in association with their physicochemical properties, in particular the ionization state of compounds by the profile of effective mobility as a function of pH. A new buffer is proposed being able to minimize the pH shift of plasma during the dialysis. In addition, the application of the proposed buffer does not necessarily require adjusting plasma pH before the dialysis and utilizing a CO2 incubator during the dialysis. The effect of the ionic strengths of different buffers on MS signals is investigated with regard to ion suppression. The sample pooling method not only significantly reduces the plasma volume required but also the number of bioanalysis samples as compared to the single compound measurements by a conventional approach. The new proposed approach is especially beneficial for measuring in vitro protein binding in matrices such as mouse plasma where plasma is available only in limited amounts. The current new development will facilitate the drug discovery process by more rapidly assessing the protein binding potential of drug candidates.     

Proteomic profiling of pancreatic ductal carcinoma cell lines treated with trichostatin-A

A pancreatic adenocarcinoma cell line (Paca44) was treated with trichostatin-A (TSA), a potent inhibitor of histone deacetylases, in order to evaluate the effect of this drug on protein expression. Master maps of control and treated Paca44 cells were generated by analysis with the PDQuest software. The comparison between such maps showed up- and downregulation of 51 polypeptide chains, out of a total of 700 spots detected by a medium-sensitivity stain, micellar Coomassie Brilliant Blue. Fingerprinting by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry analysis enabled the identification of 22 of these spots. Among these proteins, of particular interest are the two downregulated proteins nucleophosmin and translationally controlled tumor protein, as well as the upregulated proteins programmed cell death protein 5 (also designated as TFAR19) and stathmin (oncoprotein 18). The modulation of these four proteins is consistent with our observation that TSA is able to inhibit cell growth of Paca44 by causing cell cycle arrest at the G2 phase and apoptotic cell death.     

Two-dimensional electrophoretic analysis of transformation-sensitive polypeptides during chemically, spontaneously, and oncogene-induced transformation of rat liver epithelial cells.

Recently, we described the establishment of a computerized database of rat liver epithelial (RLE) cellular polypeptides (Wirth et al., Electrophoresis, 1991, 12, 931-954). This database has now been expanded to include the analysis of cellular polypeptide alterations during chemically (aflatoxin B1; AFB), spontaneously, and oncogene (v-Ha-ras, v-raf, and v-myc/v-raf)-induced transformation of RLE cells. Two-dimensional mapping of [35S]methionine-labeled whole cell lysate, cell-free in vitro translation products and [32P]orthophosphate-labeled polypeptides revealed subsets of polypeptides specific for each transformation modality. A search of the RLE protein database indicated the specific subcellular location for the majority of these transformation-sensitive proteins. Significant alterations in the expression of the extracellular matrix protein, fibronectin, as well as tropomyosin- and intermediate filament-related polypeptides (vimentin, beta-tubulin, the cytokeratins, and actin) were observed among the various transformant cell lines. Immunoprecipitation and Western immunoblot analysis of tropomyosin expression in four individual AFB-, as well as four spontaneously induced, and each of the oncogene-transformed cell lines indicated that five major tropomyosin (Tm 1-5) isoforms were variably expressed in the various cell lines, including one polypeptide tentatively identified as Tm6. Whereas alterations in tropomyosin expression appeared to be transformation-specific, alterations in the individual intermediate filament polypeptides were related more to the differentiation state of the individual cell lines rather than to the transformation phenotype. These studies extend our earlier efforts toward the establishment of a comprehensive computerized database of RLE cellular proteins and demonstrates how such a database may serve as a useful source for studies concerning the regulation of growth and differentiation as well as transformation of RLE cells.