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.     

Profiling the progression of cancer: separation of microsomal proteins in MCF10 breast epithelial cell lines using nonporous chromatophoresis

The heterogeneity of cellular protein expression has stimulated development of separations targeting smaller groups of related proteins rather than entire proteomes. The following work describes the development of a technique for the characterization of membrane subproteomes from five different breast epithelial cell lines. Intact membrane proteins are separated by hydrophobicity in the first dimension using nonporous reversed-phase high-performance liquid chromatography (RP-HPLC) to generate unique chromatographic profiles. Fractions of eluent are further separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to create distinct banding patterns. This hybrid liquid phase/gel phase method circumvents issues of membrane protein precipitation and provides a simple strategy aimed at isolating and characterizing a traditionally underrepresented protein class. Membrane protein profiles are created that discriminate between microsomal fractions of breast epithelial cells in different stages of neoplastic progression. Proteins are subsequently identified using matrix-assisted laser desorption/ionization - mass spectrometry (MALDI-MS) mass fingerprinting and MALDI-quadrupole time of flight - tandem mass spectrometry (QTOF-MS/MS) peptide sequencing. Furthermore, as this strategy preserves intact protein structure, further characterization can be performed on proteins producing mass fingerprint spectra and fragmentation spectra that did not result in database protein identifications. The coupling of nonporous RP-HPLC with SDS-PAGE provides a useful alternative to two-dimensional PAGE (2-D-PAGE) for membrane protein analysis.     

Web-based two-dimensional database of Saccharomyces cerevisiae proteins using immobilized pH gradients from pH 6 to pH 12 and matrix-assisted laser desorption/ionization-time of flight mass spectrometry

An image based two-dimensional (2-D) reference map of very alkaline yeast cell proteins was established by using immobilized pH gradients (IPG) up to pH 12 (IPG 6-12, IPG 9-12 and IPG 10-12) for 2-D electrophoresis and by using matrix-assisted laser desorption/ionization-time of flight mass spectrometry peptide mass fingerprinting for spot identification. Up to now 106 proteins with theoretical isoelectric points up to pH 11.15 and molecular mass between 7.5 and 115 kDa were localized and identified. Additionally, due to the improved resolution of steady-state isoelectric focussing with IPGs, even low copy number proteins with codon bias below 0.02 were detected and identified.     

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.     

Separation and identification of soybean leaf proteins by two-dimensional gel electrophoresis and mass spectrometry

To establish a proteomic reference map for soybean leaves, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 260 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. Fifty-three of these protein spots were identified by searching NCBInr and SwissProt databases using the Mascot search engine. Sixty-seven spots that were not identified by MALDI-TOF-MS analysis were analyzed with liquid chromatography tandem mass spectrometry (LC-MS/MS), and 66 of these spots were identified by searching against the NCBInr, SwissProt and expressed sequence tag (EST) databases. We have identified a total of 71 unique proteins. The majority of the identified leaf proteins are involved in energy metabolism. The results indicate that 2D-PAGE, combined with MALDI-TOF-MS and LC-MS/MS, is a sensitive and powerful technique for separation and identification of soybean leaf proteins. A summary of the identified proteins and their putative functions is discussed.     

Setting up standards and a reference map for the alkaline proteome of the Gram-positive bacterium Lactococcus lactis

Despite the fact that almost 39% of the theoretical expressed proteins of Lactococcus lactis have a predicted isoelectric point above 7, these proteins have not been studied in previous proteome analyses. In the present study, we set up a reference map of alkaline lactococcal proteins by using immobilized pH gradients (IPG) spanning pH 6 to 12 and 9 to 12, and protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Different electrophoresis systems for isoelectric focusing were evaluated to optimize the first dimension. Best results were obtained by sample application using cup-loading at the anodic side and increasing the final voltage up to 8000 V for IPGs, using N,N-dimethylacrylamide as monomer. After two-dimensional gel electrophoresis of extracts obtained from exponentially growing cells, about 200 protein spots were selected for identification by peptide mass fingerprinting. With MALDI-TOF MS, 153 proteins were identified that were the products of 85 different genes. Their predicted isoelectric points range from as high as 11.31 to as low as 6.34. Ribosomal proteins, hypothetical proteins and proteins with unknown function represent the largest groups of identified proteins. For further classification, the codon adaptation index (CAI) and grand average of hydropathicity (GRAVY) for each lactococcal protein were calculated. The protein with the lowest CAI identified in this study is the manganese ABC transporter ATP-binding protein. Less than 10% of the alkaline lactococcal proteins have a smaller CAI. The highest GRAVY for an identified protein is 0.26. The complete in silico data of Lactococcus lactis as well as clickable reference maps are available at www.wzw.tum.de/proteomik/lactis.     

Proteome analysis of hairy root from Panax ginseng C.A. Meyer using peptide fingerprinting, internal sequencing and expressed sequence tag data

As an initial step to the comprehensive proteomic analysis of Panax ginseng C. A. Meyer, protein mixtures extracted from the cultured hairy root of Panax ginseng were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). The protein spots were analyzed and identified by peptide finger printing and internal amino acid sequencing by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and electrospray ionization quadrupole-time of flight mass spectrometry (ESI Q-TOF MS), respectively. More than 300 protein spots were detected on silver stained two-dimensional (2-D) gels using pH 3-10, 4-7, and 4.5-5.5 gradients. Major protein spots (159) were analyzed by peptide fingerprinting or de novo sequencing and the functions of 91 of these proteins were identified. Protein identification was achieved using the expressed sequence tag (EST) database from Panax ginseng and the protein database of plants like Arabidopsis thaliana and Oryza sativa. However, peptide mass fingerprinting by MALDI-TOF MS alone was insufficient for protein identification because of the lack of a genome database for Panax ginseng. Only 17 of the 159 protein spots were verified by peptide mass fingerprinting using MALDI-TOF MS whereas 87 out of 102 protein spots, which included 13 of the 17 proteins identified by MALDI-TOF MS, were identified by internal amino acid sequencing using tandem mass spectrometry analysis by ESI Q-TOF MS. When the internal amino acid sequences were used as identification markers, the identification rate exceeded 85.3%, suggesting that a combination of internal sequencing and EST data analysis was an efficient identification method for proteome analysis of plants having incomplete genome data like ginseng. The 2-D patterns of the main root and leaves of Panax ginseng differed from that of the cultured hairy root, suggesting that some proteins are exclusively expressed by different tissues for specific cellular functions. Proteome analysis will undoubtedly be helpful for understanding the physiology of Panax ginseng.     

Improved mass spectrometric identification of gel-separated hydrophobic membrane proteins after sodium dodecyl sulfate removal by ion-pair extraction

Separation and identification of hydrophobic membrane proteins is a major challenge in proteomics. Identification of such sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins by peptide mass fingerprinting (PMF) via matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) is frequently hampered by the insufficient amount of peptides being generated and their low signal intensity. Using the seven helical transmembrane-spanning proton pump bacteriorhodopsin as model protein, we demonstrate here that SDS removal from hydrophobic proteins by ion-pair extraction prior to in-gel tryptic proteolysis leads to a tenfold higher sensitivity in mass spectrometric identification via PMF, with respect to initial protein load on SDS-PAGE. Furthermore, parallel sequencing of the generated peptides by electrospray ionization-mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) was possible without further sample cleanup. We also show identification of other membrane proteins by this protocol, as proof of general applicability.     

Proteomic analysis of progressive factors in uterine cervical cancer

Human papillomavirus (HPV) infections play a crucial role in the progress of cervical cancer. The high-risk HPV types are frequently associated with the development of malignant lesions. Some of the latest studies have demonstrated that the high-risk HPV 16 and 18 are predominantly detected in the more aggressive cancers. In the present study, we aimed to establish the proteomic profiles and characterization of the tumor related proteins by using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). For proteomic analysis, patients infected by HPV 16 or 18 were included in this study. We compared nuclear protein and cytoplasmic protein, separately by using the subcellular fraction. Differential protein spots between cervical cancer with high-risk HPV, HPV 16 or HPV 18, and HaCaT cell lines were characterized by 2-DE. Those proteins analyzed by peptide mass fingerprinting based on MALDI-TOF MS and database searching were the products of oncogenes or proto-oncogenes, and the others were involved in the regulation of cell cycle, for general genomic stability, telomerase activation, and cell immortalization. However, there was no difference in protein characterization for cervical cancer between HPV 16 and HPV 18 infection. Nonetheless, these data are valuable for the mass identification of differentially expressed proteins involved in human uterine cervical cancer. Moreover, the data has enormous value for establishing the human uterine cervical cancer proteome database that can be used in screening a molecular marker for the further study of human uterine cervical cancer, and also for studying any correlation among the cancers induced by HPV.     

Characterization of wheat gliadin proteins by combined two-dimensional gel electrophoresis and tandem mass spectrometry

A proteomics-based approach was used for characterizing wheat gliadins from an Italian common wheat (Triticum aestivum) cultivar. A two-dimensional gel electrophoresis (2-DE) map of roughly 40 spots was obtained by submitting the 70% alcohol-soluble crude protein extract to isoelectric focusing on immobilized pH gradient strips across two pH gradient ranges, i.e., 3-10 or pH 6-11, and to sodium dodecyl sulfate-polyacrylamide electrophoresis in the second dimension. The chymotryptic digest of each spot was characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and nano electrospray ionization-tandem mass spectrometry (MS/MS) analysis, providing a "peptide map" for each digest. The measured masses were subsequently sought in databases for sequences. For accurate identification of the parent protein, it was necessary to determine de novo sequences by MS/MS experiments on the peptides. By partial mass fingerprinting, we identified protein molecules such as alpha/beta-, gamma-, omega-gliadin, and high molecular weight-glutenin. The single spots along the 2-DE map were discriminated on the basis of their amino acid sequence traits. alpha-Gliadin, the most represented wheat protein in databases, was highly conserved as the relative N-terminal sequence of the components from the 2-DE map contained only a few silent amino acid substitutions. The other closely related gliadins were identified by sequencing internal peptide chains. The results gave insight into the complex nature of gliadin heterogeneity. This approach has provided us with sound reference data for differentiating gliadins amongst wheat varieties.     

Proteomic analysis of the human pathogen Trypanosoma cruzi

Trypanosoma cruzi, the protozoan that causes Chagas disease, possesses a complex life cycle involving different developmental stages. Experimental conditions for two-dimensional electrophoresis (2-DE) analysis of T. cruzi trypomastigote, amastigote and epimastigote proteomes were optimized. Comparative proteome analysis of the cell-cycle stages were carried out, revealing that few proteins included in the 2-DE maps displayed significant differential expression among the three developmental forms of the parasite. In order to identify landmark proteins, spots from the trypomastigote 2-DE map were subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry peptide mass fingerprinting, resulting in 26 identifications that corresponded to 19 different proteins. Among the identified polypeptides, there were heat shock proteins (HSP; chaperones, HSP 60, HSP 70 and HSP 90), elongation factors, glycolytic pathway enzymes (enolase, pyruvate kinase and 2,3 bisphosphoglycerate mutase) and structural proteins (KMP 11, tubulin and paraflagellar rod components). The relative expression of the identified proteins in the 2-DE maps of the T. cruzi developmental stages is also presented.     

Proteomic profiling identifies breast tumor metastasis-associated factors in an isogenic model

A combination of LC and MS was applied to an isogenic breast tumor metastasis model to identify proteins associated with a cellular phenotype. Chromatofocusing followed by nonporous-RP-HPLC/ESI-TOF MS was applied to cell lysates of 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. This method was developed to separate proteins based on pI and hydrophobicity. The high resolution and mass accuracy of ESI-TOF measurements provided a good correlation of theoretical MW and experimental Mr values of intact proteins measured in mass maps obtained in the pH range 3.8-6.4. The isolated proteins were digested by trypsin and analyzed by MALDI-TOF MS, MALDI-QIT-TOF MS, and monolith-based HPLC/MS/MS. The unique combination of the techniques provided valuable information including quantitation and modification of proteins. We identified 89 selected proteins, of which 43 were confirmed as differentially expressed. Metastasis-associated proteins included galectin-1, whereas annexin I and annexin II were associated with the nonmetastatic phenotype. In this study, we demonstrate that combining a variety of MS tools with a multidimensional liquid-phase separation provides the ability to map cellular protein content, to search for modified proteins, and to correlate protein expression with cellular phenotype.     

Cellular stress-related protein expression in Helicobacter pylori-infected gastric epithelial AGS cells

Helicobacter pylori infection leads to gastroduodenal inflammation, peptic ulceration, and gastric carcinoma. Moreover, H. pylori may induce disease-specific protein expression in gastric epithelial cells. The present study was aimed at determining differentially expressed proteins in H. pylori-infected gastric epithelial AGS cells. AGS cells were treated with H. pylori at a bacterium/cell ratio of 300:1 for 12 h. Altered protein patterns as separated by two-dimensional electrophoresis using pH gradients of 4-7 were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. Four differentially expressed proteins, whose expression levels were increased by more than two-fold in H. pylori-infected cells, were analyzed. These proteins (14-3-3 protein alpha/beta, cullin homolog 3, alpha-enolase, ezrin) are known to be related to cell proliferation, cell adhesion, and carcinogenesis, and may be mediated by cellular stress, such as reactive oxygen species. In conclusion, the identification of these differentially expressed proteins provide valuable information for the understanding of the pathophysiologic mechanisms of H. pylori-induced gastric diseases, and may be useful as prognostic indices of H. pylori-related gastric disorders.     

Establishment of a two-dimensional electrophoresis map for Neospora caninum tachyzoites by proteomics

Expressed proteins and antigens from Neospora caninum tachyzoites were studied by two-dimensional gel electrophoresis and immunoblot analysis combined with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Thirty-one spots corresponding to 20 different proteins were identified from N. caninum tachyzoites by peptide mass fingerprinting. Six proteins were identified from a N. caninum database (NTPase, 14-3-3 protein homologue, NcMIC1, NCDG1, NcGRA1 and NcGRA2), and 11 proteins were identified in closely related species using the T. gondii database (HSP70, HSP60, pyruvate kinase, tubulin alpha- and beta-chain, putative protein disulfide isomerase, enolase, actin, fructose-1,6-bisphosphatase, lactate dehydrogenase and glyceradehyde-3-phosphate dehydrogenase). One hundred and two antigen spots were observed using pH 4-7 IPG strips on immunoblot profiles. Among them, 17 spots corresponding to 11 antigenic proteins were identified from a N. caninum protein map. This study involved the construction of in-depth protein maps for N. caninum tachyzoites, which will be of value for studies of its pathogenesis, drug and vaccine development, and phylogenetic studies.     

Identification of human whole saliva protein components using proteomics

The determination of salivary biomarkers as a means of monitoring general health and for the early diagnosis of disease is of increasing interest in clinical research. Based on the linkage between salivary proteins and systemic diseases, the aim of this work was the identification of saliva proteins using proteomics. Salivary proteins were separated using two-dimensional (2-D) gel electrophoresis over a pH range between 3-10, digested, and then analyzed by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-TOF mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Proteins were identified using automated MS and MS/MS data acquisition. The resulting data were searched against a protein database using an internal Mascot search routine. Ninety spots give identifications with high statistical reliability. Of the identified proteins, 11 were separated and identified in saliva for the first time using proteomics tools. Moreover, three proteins that have not been previously identified in saliva, PLUNC, cystatin A, and cystatin B were identified.     

Comparative proteomic analyses of the yeast Saccharomyces cerevisiae KNU5377 strain against menadione-induced oxidative stress

The Saccharomyces cerevisiae KNU5377 strain, which was isolated from spoilage in nature, has the ability to convert biomass to alcohol at high temperatures and it can resist against various stresses. In order to understand the defense mechanisms of the KNU5377 strain under menadione (MD) as oxidative stress, we used several techniques for study: peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) followed by two-dimensional (2D) gel electrophoresis, liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), and surface-enhanced laser desorption ionization-time of flight (SELDI-TOF) technology. Among the 35 proteins identified by MALDI-TOF MS, 19 proteins including Sod1p, Sod2p, Tsa1p, and Ahp1p were induced under stress condition, while 16 proteins were augmented under normal condition. In particular, five proteins, Sod1p, Sod2p, Ahp1p, Rib3p, Yaf9p, and Mnt1p, were induced in only stressed cells. By LC-ESI-MS/MS analysis, 37 proteins were identified in normal cells and 49 proteins were confirmed in the stressed cells. Among the identified proteins, 32 proteins were found in both cells. Five proteins including Ye1047cp and Met6p were only upregulated in the normal cells, whereas 17 proteins including Abp1p and Sam1p were elevated in the stressed cells. It was interesting that highly hypothetical proteins such as Ynl281wp, Ygr279cp, Ypl273wp, Ykl133cp, and Ykr074wp were only expressed in the stressed cells. SELDI-TOF analysis using the SAX2 and WCX2 chips showed that highly multiple-specific protein patterns were reproducibly detected in ranges from 2.9 to 27.0 kDa both under normal and stress conditions. Therefore, induction of antioxidant proteins, hypothetical proteins, and low molecular weight proteins were revealed by different proteomic techniques. These results suggest that comparative analyses using proteomics might contribute to elucidate the defense mechanisms of KNU5377 under MD stress.     

Snake venomics: characterization of protein families in Sistrurus barbouri venom by cysteine mapping, N-terminal sequencing, and tandem mass spectrometry analysis

The protein composition of the crude venom of Sistrurus barbouri was analyzed by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were separated by reversed phase high-performance liquid chromatography and characterized by N-terminal sequence analysis. The molecular mass and number of cysteine residues of the purified proteins were determined by matrix-associated laser desorption/ionization-time of flight mass spectrometry. Selected protein bands were subjected to in-gel tryptic digestion and peptide mass fingerprinting. Analysis of the tandem mass spectrometry spectra of selected doubly-charged peptide ions was done by collision-induced dissociation in a quadrupole-linear ion trap instrument. Our results show that the venom proteome of the pigmy rattlesnake S. barbouri is composed of proteins belonging to a few protein families, which can be structurally characterized by their disulfide bond contents.     

Proteome reference maps of Medicago truncatula embryogenic cell cultures generated from single protoplasts

Using a combination of two-dimensional gel electrophoresis (2-DE) protein mapping and mass spectrometry (MS) analysis, we have established proteome reference maps of Medicago truncatula embryogenic tissue culture cells. The cultures were generated from single protoplasts, which provided a relatively homogeneous cell population. We used these to analyze protein expression at the globular stages of somatic embryogenesis, which is the earliest morphogenetic embryonic stage. Over 3000 proteins could reproducibly be resolved over a pI range of 4-11. Three hundred and twelve protein spots were extracted from colloidal Coomassie Blue-stained 2-DE gels and analyzed by matrix-assisted laser desorption/ionization-time of flight MS analysis and tandem MS sequencing. This enabled the identification of 169 protein spots representing 128 unique gene products using a publicly available expressed sequence tag database and the MASCOT search engine. These reference maps will be valuable for the investigation of the molecular events which occur during somatic embryogenesis in M. truncatula. The proteome reference maps and supplementary materials will be available and updated for public access at http://semele.anu.edu.au/.     

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.