Comprehensive proteome analysis of ovarian cancers using liquid phase separation, mass mapping and tandem mass spectrometry: a strategy for identification of candidate cancer biomarkers

A two-dimensional (2-D) liquid phase separation method, liquid isoelectric focusing followed by nonporous reversed-phase high performance liquid chromatography (HPLC), was used to separate proteins from human ovarian epithelial whole cell lysates. HPLC eluent was interfaced on-line to an electrospray ionization (ESI) time of flight (TOF) mass spectrometer to obtain accurate intact protein molecular weights (Mr). 2-D protein expression maps were generated displaying protein isoelectric point (pI) versus intact protein Mr. Resulting 2-D images effectively displayed quantitative differential protein expression in ovarian cancer cells versus non-neoplastic ovarian epithelial cells. Protein peak fractions were collected from the HPLC eluent, enzymatically digested, and analyzed by matrix-assisted laser desorption/ionization (MALDI) TOF-mass spectrometry (MS) peptide mass fingerprinting and by MALDI-quadrupole TOF tandem mass spectrometry peptide sequencing. Interlysate comparisons of differential protein expression between two ovarian adenocarcinoma cell lines, ES2 and MDAH-2774, and ovarian surface epithelial cells was performed. Five pI fractions from each sample were selected for comparative study and over 300 unique proteins were positively identified from the 2-D liquid expression maps using MS, which covered around 60% of proteins detected by on-line ESI-TOF-MS. This represents one of the most comprehensive proteomic analyses of ovarian cancer samples to date. Protein bands with significant up- or down-regulation in one cell line versus another as viewed in the 2-D expression maps were identified. This strategy may prove useful in identifying novel ovarian cancer marker proteins.     

Classification of cancer cell lines using an automated two-dimensional liquid mapping method with hierarchical clustering techniques

A two-dimensional liquid mapping method was used to map the protein expression of eight ovarian serous carcinoma cell lines and three immortalized ovarian surface epithelial cell lines. Maps were produced using pI as the separation parameter in the first dimension and hydrophobicity based upon reversed-phase HPLC separation in the second dimension. The method can be reproducibly used to produce protein expression maps over a pH range from 4.0 to 8.5. A dynamic programming method was used to correct for minor shifts in peaks during the HPLC gradient between sample runs. The resulting corrected maps can then be compared using hierarchical clustering to produce dendrograms indicating the relationship between different cell lines. It was found that several of the ovarian surface epithelial cell lines clustered together, whereas specific groups of serous carcinoma cell lines clustered with each other. Although there is limited information on the current biology of these cell lines, it was shown that the protein expression of certain cell lines is closely related to each other. Other cell lines, including one ovarian clear cell carcinoma cell line, two endometrioid carcinoma cell lines, and three breast epithelial cell lines, were also mapped for comparison to show that their protein profiles cluster differently than the serous samples and to study how they cluster relative to each other. In addition, comparisons can be made between proteins differentially expressed between cell lines that may serve as markers of ovarian serous carcinomas. The automation of the method allows reproducible comparison of many samples, and the use of differential analysis limits the number of proteins that might require further analysis by mass spectrometry techniques.     

Two-dimensional reference map of Agrobacterium tumefaciens proteins

Proteomics based on two-dimensional (2-D) gel electrophoresis of proteins followed by spot identification with mass spectrometry is a commonly used method for physiological studies. Physiological proteomics requires 2-D reference maps, on which most of the main proteins are identified. We present a reference map for the bacterial plant pathogen Agrobacterium tumefaciens proteins, which contains more than 300 entries with an isoelectric point (pI) between 4 and 7. The quantitative study of the proteins in the analytical window of the master gel demonstrated unique features, in comparison with other bacteria. In addition, a theoretical analysis of several protein parameters was performed and compared with the experimental results. A comparison of the theoretical molecular weight (MW) of the proteins and their theoretical pI with their vertical and horizontal migration distances, respectively, pointed out the existence of several proteins that strongly diverted from the graph trend-line. These proteins were clearly subjected to post-translational modifications, which changed their pI and/or MW. Additional support for post-translational modifications comes from the identification of multiple spots of the same gene products. Post-translational modifications appear to be more common than expected, at least for soluble proteins, as more than 10% of the proteins were associated with multiple spots.     

Glycoproteomic analyses of ovarian cancer cell lines and sera from ovarian cancer patients show distinct glycosylation changes in individual proteins

Ovarian cancer is difficult to diagnose in women because symptoms of the disease are often not noticed until the disease has progressed to an advanced untreatable stage. Although a serum test, CA125, is currently available to assist with monitoring treatment of ovarian cancer, this test lacks the necessary specificity and sensitivity for early detection. Therefore, better biomarkers of ovarian cancer are needed. A glycoprotein analysis approach was undertaken using high resolution Fourier transform ion cyclotron resonance mass spectrometry to analyze glycosylated proteins present in the conditioned media of ovarian cancer cell lines and in sera obtained from ovarian cancer patients and normal controls. In this study, glycosylated proteins were separated by gel electrophoresis, and individual glycoproteins were selected for glycosylation analysis and protein identification. The attached glycans from each protein were released and profiled by mass spectrometry. Glycosylation of a mucin protein and a large glycosylated protein isolated from the ES2 ovarian cancer cell line was determined to consist of mostly O-linked glycans. Four prominent glycoproteins of approximate 517, 370, 250, 163 kDa from serum samples were identified as two forms of apolipoprotein B-100, fibronectin, and immunoglobulin A1, respectively. Mass spectrometric analysis of glycans isolated from apolipoprotein B-100 (517 kD) showed the presence of small, specific O-linked oligosaccharides. In contrast, analysis of fibronectin (250 kD) and immunoglobulin A1 (163 kD) produced N-linked glycan fragments in forms that were sufficiently different from the glycans obtained from the corresponding protein band present in the normal serum samples. This study shows that not only a single protein but several are aberrantly glycosylated, and those abnormal glycosylation changes can be detected and may ultimately serve as glycan biomarkers for ovarian cancer.     

Identification of metastasis-associated proteins in a human tumor metastasis model using the mass-mapping technique

For most cancer cell types, the acquisition of metastatic ability leads to clinically incurable disease. The identification of molecules whose expression is specifically correlated with the metastatic spread of cancer would facilitate the design of therapeutic interventions to inhibit this lethal process. In order to facilitate metastasis gene discovery we have previously characterized a pair of monoclonal cell lines from the human breast carcinoma cell line MDA-MB-435 that have different metastatic phenotypes in immune-compromised mice. In this study, serum-free conditioned media was collected from the cultured monoclonal cell lines and a mass mapping technique was applied in order to profile a component of each cell line proteome. We utilized chromatofocusing in the first dimension to obtain a high resolution separation based on protein pI, and nonporous silica reverse-phase high performance liquid chromatography was used for the second dimension. Selected proteins were identified on the basis of electrospray ionization time of flight mass spectrometry (ESI-TOF MS) intact protein mapping and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting. Using this approach we were able to map over 400 proteins and plot them as a 2-D map of pI versus accurate M(r). This was performed over a pI range of 4.0-6.2, and a mass range of 6-80 kDa. ESI-TOF MS data and further analysis using MALDI-TOF MS confirmed and identified 27 differentially expressed proteins. Proteins associated with the metastatic phenotype included osteopontin and extracellular matrix protein 1, whereas the matrix metalloproteinase-1 and annexin 1 proteins were associated with the non-metastatic phenotype. These findings demonstrate that the mass mapping technique is a powerful tool for the detection and identification of proteins in complex biological samples and which are specifically associated with a cellular phenotype.     

A method for three-dimensional protein characterization and its application to a complex plant (corn) extract

Knowledge of host protein properties is critical for developing purification methods for recombinant proteins from a specific host, or for choosing suitable hosts and targeted expression tissues for a specific recombinant protein. A method to obtain a three-dimensional (3D) map (surface hydrophobicity (SH), isoelectric point (pI), and molecular weight (MW)), of a host's aqueous soluble protein properties was developed. The method consists of hydrophobic partitioning in a PEG 3350 (15.7%)-Na(2)SO(4) (8.9%)-NaCl (3%) aqueous two-phase (ATP) system followed by quantitative, 2D-electrophoretic characterization of the proteins of each equilibrium phase and the original extract. The pI and MW of host proteins were obtained directly through 2D electrophoresis. The partition coefficients of individual proteins were obtained by quantitative matching of protein spots in the top and bottom phase gels and calculating the protein partition coefficients from this information. Correlation of the partition coefficient to a SH scale was established by partitioning several model proteins with known surface hydrophobicities in the same ATP system. The inclusion of the extract gel provided for a spot selection criterion based on satisfactory mass balance closure. The method is illustrated by application to a mixture of model proteins and to complex mixtures, that is, corn germ proteins extracted at pH 7 and pH 4.     

枇杷成花过程叶片蛋白质变化动态

研究了温室内水分胁迫下盆栽枇杷和大田枇杷的成花和未成花枝梢叶片可溶性蛋白质含量在花芽分化过程中的变化动态,同时对成花和未成花枝梢顶芽进行特异蛋白双向凝胶电泳研究。结果表明,枇杷成花诱导需经历可溶性蛋白质含量一定程度的升高,然后急剧下降的过程,即可溶性蛋白质的升高对应成花诱导,而蛋白质的下降与形态分化紧密相关。成花植株枝梢顶芽与未成花植株枝梢顶芽的2-DE图谱总模式相同,但前者比后者多了两种蛋白质,其分子量和等电点一为MW 14110.5±110.8、pI 5.350±0.008,另一为MW 66446.3±260.9、pI 4.730±0.032,两种蛋白质均呈酸性,可能与枇杷成花密切相关。     

A critical assessment of SELDI-TOF-MS for biomarker discovery in serum and tissue of patients with an ovarian mass

ABSTRACT: BACKGROUND: Less than 25% of patients with a pelvic mass who are presented to a gynecologist will eventually be diagnosed with epithelial ovarian cancer. Since there is no reliable test to differentiate between different ovarian tumors, accurate classification could facilitate adequate referral to a gynecological oncologist, improving survival. The goal of our study was to assess the potential value of a SELDI-TOF-MS based classifier for discriminating between patients with a pelvic mass. METHODS: Our study design included a well-defined patient population, stringent protocols and an independent validation cohort. We compared serum samples of 53 ovarian cancer patients, 18 patients with tumors of low malignant potential, and 57 patients with a benign ovarian tumor on different ProteinChip arrays. In addition, from a subset of 84 patients, tumor tissues were collected and microdissection was used to isolate a pure and homogenous cell population. RESULTS: Diagonal Linear Discriminant Analysis (DLDA) and Support Vector Machine (SVM) classification on serum samples comparing cancer versus benign tumors, yielded models with a classification accuracy of 71-81% (cross-validation), and 73-81% on the independent validation set. Cancer and benign tissues could be classified with 95-99% accuracy using cross-validation. Tumors of low malignant potential showed protein expression patterns different from both benign and cancer tissues. Remarkably, none of the peaks differentially expressed in serum samples were found to be differentially expressed in the tissue lysates of those same groups. CONCLUSION: Although SELDI-TOF-MS can produce reliable classification results in serum samples of ovarian cancer patients, it will not be applicable in routine patient care. On the other hand, protein profiling of microdissected tumor tissue may lead to a better understanding of oncogenesis and could still be a source of new serum biomarkers leading to novel methods for differentiating between different histological subtypes.     

Looking for cancer clues in publicly accessible databases

What started out as a mere attempt to tentatively identify proteins in experimental cancer-related 2D-PAGE maps developed into VIRTUAL2D, a web-accessible repository for theoretical pI/MW charts for 92 organisms. Using publicly available expression data, we developed a collection of tissue-specific plots based on differential gene expression between normal and diseased states. We use this comparative cancer proteomics knowledge base, known as the tissue molecular anatomy project (TMAP), to uncover threads of cancer markers common to several types of cancer and to relate this information to established biological pathways.     

Protein microarrays using liquid phase fractionation of cell lysates

We describe an approach in which protein microarrays are produced using a two-dimensional (2-D) liquid phase fractionation of cell lysates. The method involves a pI-based fractionation using chromatofocusing in the first dimension followed by nonporous reversed-phase high-performance liquid chromatography (HPLC) of each pI fraction in the second dimension. This allows fractionation of cellular proteins in the liquid phase that could then be arrayed on nitrocellulose slides and used to study humoral response in cancer. Protein microarrays have been used to identify potential serum biomarkers for prostate cancer. It is shown that specific fractions are immunoreactive against prostate cancer serum but not against serum from healthy individuals. These proteins could serve as sero-diagnostic markers for prostate cancer. Importantly, this method allows for use of post-translationally modified proteins as baits for detection of humoral response. Proteins eliciting an immune response are identified using the molecular mass and peptide sequence data obtained using mass spectrometric analysis of the liquid fractions. The fractionation of proteins in the liquid phase make this method amenable to automation.     

A wide range of protein isoforms in serum and plasma uncovered by a quantitative intact protein analysis system

We have implemented an orthogonal 3-D intact protein analysis system (IPAS) to quantitatively profile protein differences between human serum and plasma. Reference specimens consisting of pooled Caucasian-American serum, citrate-anticoagulated plasma, and EDTA-anticoagulated plasma were each depleted of six highly abundant proteins, concentrated, and labeled with a different Cy dye (Cy5, Cy3, or Cy2). A mixture consisting of each of the labeled samples was subjected to three dimensions of separation based on charge, hydrophobicity, and molecular mass. Differences in the abundance of proteins between each of the three samples were determined. More than 5000 bands were found to have greater than two-fold difference in intensity between any pair of labeled specimens by quantitative imaging. As expected, some of the differences in band intensities between serum and plasma were attributable to proteins related to coagulation. Interestingly, many proteins were identified in multiple fractions, each exhibiting different pI, hydrophobicity, or molecular mass. This is likely reflective of the expression of different protein isoforms or specific protein cleavage products, as illustrated by complement component 3 precursor and clusterin. IPAS provides a high resolution, high sensitivity, and quantitative approach for the analysis of serum and plasma proteins, and allows assessment of PTMs as a potential source of biomarkers.     

Increased WD-repeat containing protein 1 in interstitial fluid from ovarian carcinomas shown by comparative proteomic analysis of malignant and healthy gynecological tissue

We aimed to identify differentially expressed proteins in interstitial fluid from ovarian cancer employing multiple fractioning and high resolution mass spectrometry-based proteomic analysis, and asked whether specific proteins that may serve as biomarker candidates or therapeutic targets could be identified. High throughput proteomics was conducted on immunodepleted and fractioned interstitial fluid from pooled samples of ovarian carcinomas, using endometrial carcinomas and healthy ovarian tissue as controls. Differential analysis revealed the up-regulation of extracellular proteasomes in tumor interstitial fluid compared to the healthy control. Moreover, a number of differentially expressed proteins in interstitial fluid from ovarian carcinomas compared with control tissues were identified. Detection of proteasome 20S related proteins in TIF compared to IF from healthy tissue indicates that the 20S proteasome can have a role in the tumor microenvironment. Six selected proteins, CEACAM5, FREM2, MUC5AC, TFF3, PYCARD and WDR1, were independently validated in individual tumor lysates from ovarian carcinomas by multiple reaction monitoring initiated detection and sequence analysis, Western blot and/or selected reaction monitoring. Quantification of specific proteins revealed substantial heterogeneity between individual samples. Nevertheless, WD repeat-containing protein 1 was confirmed as being significantly overexpressed in interstitial fluid from ovarian carcinomas compared to healthy ovarian tissue by Orbitrap analysis of individual native interstitial fluid from ovarian and endometrial carcinomas and healthy ovarian tissue. We suggest that this protein should be explored as a therapeutic target in ovarian carcinomas. This article is part of a Special Issue entitled: An Updated Secretome.     

Protein expression analysis using quantitative fluorescence image analysis on tissue microarray slides

We have developed a tissue microarray (TMA)-based quantitative fluorescence image analysis (QFIA) method in which protein markers on TMA sections were labeled by immunofluorescence using tyramide signal amplification and a quantitative fluorescence detection system. Using this method, BRCA1 protein expression patterns were studied in the TMA sections of cell lines with known levels of BRCA1 expression and in a small group of human tissue samples obtained from sporadic epithelial ovarian cancers, their corresponding adjacent dysplastic fields, and distant non-tumor areas. We detected distinctive BRCA1 expression patterns in high-grade and low-grade sporadic epithelial ovarian cancers and their associated adjacent dysplastic fields. However, such patterns of expression could not be adequately detected by traditional immunohistochemical staining methods. TMA-QFIA provides a sensitive, automated, and quantitative measurement of protein expression on archived tissue and cell samples and will be a useful tool for protein-level molecular profiling analyses.     

Two-dimensional liquid chromatography protein expression mapping for differential proteomic analysis of normal and O157:H7 Escherichia coli

A multidimensional chromatographic method has been applied for the differential analysis of proteins from different strains of Escherichia coli bacteria. Proteins are separated in the first dimension using chromatofocusing (CF) and further separated by nonporous reversed-phase high-performance liquid chromatography (NPS-RP-HPLC) in the second dimension. A 2-dimensional (2-D) expression map of bacterial protein content is created for virulent O157:H7 and nonvirulent E. coli strains depicting protein isoelectric point (pI) versus protein hydrophobicity. Differentially expressed proteins are further characterized using electrospray/ionization time-of-flight mass spectrometry (ESI-TOF-MS) for intact protein molecular weight (MW) determination and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mass fingerprinting for protein identification. Using this method, no significant differential protein expression is exhibited between the two O157:H7 strains examined over a pH range of 4.0-7.0, and O157:H7 strains could be distinguished from nonvirulent E. coli. Several proteins differentially expressed between O157:H7 and nonvirulent E. coli are identified as potential markers for detection and treatment of O157:H7 infection.     

Expression of EMSY gene in sporadic ovarian cancer

The majority of familial breast and ovarian cancers arise from mutations in the BRCA1 and BRCA2 genes. Amplification at the 11q13.5 locus is commonly observed in breast and ovarian cancers. In 2003, Hughes-Davies et al. identified a novel gene (EMSY) at this locus which binds BRCA2 within a region deleted in some cancers. Although little is known about the cellular role of EMSY, indirect evidence suggests that this nuclear protein is capable of silencing the activation potential of BRCA2. In this study we aimed to investigate expression of the EMSY gene and its protein product in sporadic ovarian cancer. Real-time quantitative RT-PCR was performed in 50 ovarian cancer and 17 normal ovarian tissue samples. Overexpression of the EMSY gene was found in 6/50 cases (12%), but in none of the control samples. To determine the EMSY protein by Western blotting, semi-quantitative analysis of the EMSY protein was performed using the Scion Image Gel Analysis Program. Statistical analysis was performed using SPSS 11.5. All patients having EMSY overexpression also displayed increased levels of the EMSY protein. Sporadic ovarian cancer shows overexpression of EMSY at a prevalence similar to that found in breast cancer and the overexpression is correlated with the protein level.     

Coupling of native liquid phase isoelectrofocusing and blue native polyacrylamide gel electrophoresis: a potent tool for native membrane multiprotein complex separation

In this study, a new 3D native electrophoretic protocol is proposed for an exhaustive separation and identification of membrane proteins. It is based on native liquid phase isoelectrofocusing (N-LP-IEF) of protein complexes in the first dimension, followed by blue native polyacrylamide gel electrophoresis (BN-PAGE) in the second dimension, where both the pI and the molecular masses of protein complexes (2D N-LP-IEF-BN) were used to separate them in their native form. Finally, each single component can be resolved using denaturing electrophoresis (3D N-LP-IEF-BN-SDS-PAGE). The thylakoid membrane of spinach which contains four big protein complexes was chosen as a model for setting up analytical methods suitable for any membrane proteins. The pI-based MicroRotofor has a number of advantages over BN-PAGE: it does not require the addition of any chemicals, and separation of complexes is based on the protein's real physicochemical properties which inevitably change when dye is added. Results were more easily reproduced than with BN, and the pI of each native complex was also determined. Although some fractions still contained comigrating complexes after MicroRotofor, these were subsequently separated by BN for further analysis. Thus, highly hydrophobic complexes, such as ATP-synthetas and Cyt b6/f, were separated in native form as were various complexes of LHCII trimers, which have different pI but similar molecular masses. SDS-PAGE revealed almost all the subunits from the four photosynthetic complexes, indicating that by using 3D N-LP-IEF-BN-SDS-PAGE it is possible to achieve a greater degree of component identification than with 2D BN-SDS-PAGE.     

A decomposition model to track gene expression signatures: preview on observer-independent classification of ovarian cancer

MOTIVATION: A number of algorithms and analytical models have been employed to reduce the multidimensional complexity of DNA array data and attempt to extract some meaningful interpretation of the results. These include clustering, principal components analysis, self-organizing maps, and support vector machine analysis. Each method assumes an implicit model for the data, many of which separate genes into distinct clusters defined by similar expression profiles in the samples tested. A point of concern is that many genes may be involved in a number of distinct behaviours, and should therefore be modelled to fit into as many separate clusters as detected in the multidimensional gene expression space. The analysis of gene expression data using a decomposition model that is independent of the observer involved would be highly beneficial to improve standard and reproducible classification of clinical and research samples. RESULTS: We present a variational independent component analysis (ICA) method for reducing high dimensional DNA array data to a smaller set of latent variables, each associated with a gene signature. We present the results of applying the method to data from an ovarian cancer study, revealing a number of tissue type-specific and tissue type-independent gene signatures present in varying amounts among the samples surveyed. The observer independent results of such molecular analysis of biological samples could help identify patients who would benefit from different treatment strategies. We further explore the application of the model to similar high-throughput studies.