胃癌及癌旁组织定量比较蛋白质组学研究

为寻找胃癌特异的肿瘤标记物,用于胃癌临床诊断及药物治疗靶点的选择,本研究采用荧光差异显示凝胶电泳（DIGE）技术分离并筛选 Cy3、Cy5 及 Cy2 荧光素标记的胃癌及对应癌旁组织差异表达蛋白质,用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）或串联质谱技术进行鉴定并分析。结果共筛选出 33 个差异表达蛋白质点,其中 9 个蛋白质点在胃癌组织中上调,24 个蛋白质点下调。对 22 个蛋白质点采用质谱技术成功鉴定,突变结蛋白、锰超氧化物歧化酶、热休克蛋白 60等在胃癌中高表达,热休克蛋白 27、前列腺素 F 合酶、硒结合蛋白 1、锌指蛋白 160、微管蛋白 α6、真核生物翻译延伸因子 1 α1 等在胃癌组织中低表达,并筛选出 5 个未知蛋白。这些差异表达蛋白可望成为胃癌诊断的特异标记物,并与胃癌的发生、发展及预后等有关,为胃癌的诊断、发生机制的研究提供了新的思路。     

双向凝胶电泳中三种蛋白质检测方法的比较

高通量双向电泳是蛋白质组学的核心 ,双向电泳凝胶上蛋白质点的检测方法应具有灵敏度高、线性范围宽和兼容质谱鉴定等优点 .采用差异凝胶电泳 (differencegelelectrophoresis ,DIGE)技术以Cy3(1 (5 carboxypentyl) 1′ propylindocarbocyaninehalideN hydroxysuccinimidylester)和Cy5 (1 (5 carboxypentyl) 1′ methylindodicarbocyaninehalideN hydroxysuccinimidylester)荧光分别标记正常和TNF α处理细胞的蛋白质 ,用Cy2 (3 (4 carboxymethyl)phenylmethyl) 3′ ethyloxacarbocyaninehalideN hydroxysuccinimidylester)荧光标记正常和TNF α处理细胞蛋白质的等量混合样品作为内标 ,混合 3种荧光标记的蛋白质后 ,在同一等电聚焦胶条进行聚焦 ,然后在聚丙烯酰胺凝胶上进行第二向电泳 ,用 3种波长的激光激发扫描得到凝胶图象 ,DIGE中多个样品在同一条件下电泳 ,因而匹配率高 ,且引入内标使蛋白质点的检测与定量更为准确 .DIGE技术与质谱相结合 ,实现了高通量和相对准确定量 .与硝酸银和考马斯亮蓝染色结果相比较 ,DIGE技术具有灵敏度高、线性范围宽和不影响后续质谱鉴定等优点     

Database of two-dimensional polyacrylamide gel electrophoresis of proteins labeled with CyDye DIGE Fluor saturation dye

CyDye DIGE Fluor saturation dye (saturation dye, GE Healthcare Amersham Biosciences) enables highly sensitive 2-D PAGE. As the dye reacts with all reduced cysteine thiols, 2-D PAGE can be performed with a lower amount of protein, compared with CyDye DIGE Fluor minimal dye (GE Healthcare Amersham Biosciences), the sensitivity of which is equivalent to that of silver staining. We constructed a 2-D map of the saturation dye-labeled proteins of a liver cancer cell line (HepG2) and identified by MS 92 proteins corresponding to 123 protein spots. Functional classification revealed that the identified proteins had chaperone, protein binding, nucleotide binding, metal ion binding, isomerase activity, and motor activity. The functional distribution and the cysteine contents of the proteins were similar to those in the most comprehensive 2-D database of hepatoma cells (Seow et al.., Electrophoresis 2000, 21, 1787-1813), where silver staining was used for protein visualization. Hierarchical clustering on the basis of the quantitative expression profiles of the 123 characterized spots labeled with two charge- and mass-matched saturation dyes (Cy3 and Cy5) discriminated between nine hepatocellular carcinoma cell lines and primary cultured hepatocytes from five individuals, suggesting the utility of saturation dye and our database for proteomic studies of liver cancer.     

Proteomic analysis of individual variation in normal livers of human beings using difference gel electrophoresis

Normal Chinese Liver Proteome Expression Profile is one of the major parts of Human Liver Proteome Project. Before starting the studies, it is necessary to examine the interindividual variation of normal liver proteome and evaluate the minimal size of samples for proteomic analysis. In this study, normal liver samples from ten individual volunteers were collected and the proteome profiles of these samples were analyzed using 2-D difference gel electrophoresis (DIGE) combined with MALDI-TOF/TOF MS. The individual liver tissue lysates were labeled with Cy3 and Cy5 while the pooled sample was labeled with Cy2 as an internal standard, which minimized gel-to-gel variation. After analysis by the DeCyder software, up to 2056 protein spots were detected on the master gel. The CV of standardized abundance was calculated for the protein spots that were matched across all ten gels. The CV values of these protein spots ranged from 6.4 to 108.5% and the median CV was approximately 19%, which demonstrated that the protein expression of normal liver among different individuals was relatively stable. The eight proteins with CV values over 50% were identified which would be a caveat when considering these proteins as potential disease-related markers. Moreover, the one-way ANOVA feature showed a correlation between sample size and individual variations. The results showed that when the sample size exceeded 7, the individual variations were not significant to the whole pool. Our results are an important basis for liver protein expression profiles and comparative proteomics of liver disease.     

Evaluation of two-dimensional difference gel electrophoresis for protein profiling. Soluble proteins of the marine bacterium Pirellula sp. strain 1

Two-dimensional gel electrophoresis (2DE) is a central tool of proteome research, since it allows separation of complex protein mixtures at highest resolution. Quantification of gene expression at the protein level requires sensitive visualization of protein spots over a wide linear range. Two-dimensional difference gel electrophoresis (2D DIGE) is a new fluorescent technique for protein labeling in 2DE gels. Proteins are labeled prior to electrophoresis with fluorescent CyDyes trade mark and differently labeled samples are then co-separated on the same 2DE gel. We evaluated 2D DIGE for detection and quantification of proteins specific for glucose or N-acetylglucosamine metabolism in the marine bacterium Pirellula sp. strain 1. The experiment was based on 10 parallel 2DE gels. Detection and comparison of the protein spots were performed with the DeCyder trade mark software that uses an internal standard to quantify differences in protein abundance with high statistical confidence; 24 proteins differing in abundance by a factor of at least 1.5 (t test value <10(-9)) were identified. For comparison, another experiment was carried out with four SYPRO-Ruby-stained 2DE gels for each of the two growth conditions; image analysis was done with the ImageMaster trade mark 2D Elite software. Sensitivity of the CyDye fluors was evaluated by comparing Cy2, Cy3, Cy5, SYPRO Ruby, silver, and colloidal Coomassie staining. Three replicate gels, each loaded with 50 microg of protein, were run for each stain and the gels were analyzed with the ImageMaster software. Labeling with CyDyes allowed detection of almost as many protein spots as staining with silver or SYPRO Ruby.     

Two-dimensional fluorescence difference gel electrophoretic analysis of Streptococcus mutans biofilms

Compared with traditional two-dimensional (2D) proteome analysis of Streptococcus mutans grown as a biofilm from a planktonic culture at steady state (Rathsam et al., Microbiol. 2005, 151, 1823-1837), the use of 2D fluorescence difference gel electrophoresis (DIGE) led to a 3-fold increase in the number of identified protein spots that were significantly altered in their level of expression (P < 0.050). Of the 73 identified proteins, only nine were up-regulated in biofilm grown cells. The results supported the previously surmised hypothesis that general metabolic functions were down-regulated in response to a reduction in growth rate in mature S. mutans biofilms. Up-regulation of competence proteins without any concomitant increase in stress-responsive proteins was confirmed, while the levels of glucosyltransferase C (GtfC), involved in glucan formation, O-acetylserine sulfhyrylase (cysteine synthetase A; CsyK), implicated in the formation of [Fe-S] clusters, and a hypothetical protein encoded by the open reading frame, SMu0188, were also up-regulated.     

采用定量蛋白质组学技术筛选小鼠肝癌淋巴道转移相关蛋白

淋巴道转移是上皮来源恶性肿瘤转移的早期阶段,其发生机制不清,一直是肿瘤学研究面临的难题,为寻找淋巴道转移相关蛋白,以一对来源于同一亲本细胞,且淋巴道转移潜能显著不同的小鼠肝癌腹水型细胞株为研究对象,其中Hca-F为高淋巴道转移力细胞株,Hca-P为低淋巴道转移力细胞株,采用定量蛋白质组学技术——荧光差异双向凝胶电泳,建立了高低淋巴道转移力小鼠肝癌细胞荧光差异蛋白表达图谱,高通量筛选与肿瘤淋巴道转移相关的蛋白质.经DeCyde软件分析,共得到163个有统计学差异的蛋白质点,选择2倍以上的差异性蛋白质点23个,经质谱鉴定得到17个蛋白质,在Hca-F中高表达的蛋白质有7个:转羟乙醛酶、波形蛋白、肌酸激酶(脑)、膜联蛋白7、膜联蛋白5、烯酰辅酶A水合酶1(过氧化物酶体)、核内异质核糖核蛋白A2/B1异构体1.而在Hca-F中低表达的蛋白质有10个:真核翻译延长因子2、Ero1样蛋白、乙醛脱氢酶2(线粒体)、苹果酸盐脱氢酶2(NAD)、β-内酰胺酶2、谷胱甘肽S转移酶"1、泛素C末端水解酶同工酶L3、内质网蛋白29(前体)、溶血磷脂酶1、微管不稳定蛋白.这些差异性蛋白质的功能涉及到代谢、蛋白质分泌、蛋白质结合、核苷酸结合,钙离子结合、凋亡和调节生长等过程.对这些蛋白质功能的进一步验证,将有助于解析肿瘤淋巴道转移的分子机制.     

A nascent proteome study combining click chemistry with 2DE

To investigate the dynamic cellular response to a condition change, selective labeling of the nascent proteome is necessary. Here, we report a method combining click chemistry protein labeling with 2D DIGE. To test the relevance of the method, we compared nascent proteomes of actively growing bacterial cells with that of cells exposed to protein synthesis inhibitor, erythromycin. Cells were incubated with methionine analog, homopropargyl glycin, and their nascent proteome was selectively labeled with monosulfonated neutral Cy3 and Cy5 azides specially synthesized for this purpose. Following fluorescent labeling, the protein samples were mixed and subjected to standard 2D DIGE separation. The method allowed us to reveal a dramatic reduction of newly synthesized proteins upon erythromycin treatment, while the total proteome was not significantly affected. Additionally, several proteins, whose synthesis was resistant to erythromycin, were identified.     

Serum differential protein identification of Xinjiang Kazakh esophageal cancer patients based on the two-dimensional liquid-phase chromatography and LTQ MS

The aim of this study was to investigate the impact of chemo-radiotherapy on serum protein expression of the esophageal cancer patients and discover potential biomarkers by detecting serum proteins mass spectrometry of the healthy Kazakh people in Xinjiang as well as the patients before and after their chemo-radiotherapy. In order to separate and compare the three serum samples (the healthy group’s, the patients’ before and after chemo-radiotherapy) with two-dimensional protein liquid chromatography system (Proteome LabTM PF-2D), then detect the differential protein spots with linear trap quadruple mass spectrometer (LTQ MS/MS). (1) The Kazakh esophageal cancer patients got 21 expressed protein spots peaks with significant difference after chemo-radiotherapy compared with before; before the treatment there were 10 different expressed protein spots compared with the healthy group, and after it there were four peaks in the expression of protein spots compared with the healthy group. (2) After LTQ mass spectrometric detection, 22 proteins were up-regulated in serum samples of the healthy group, 22 were up-regulated of the patients before medical treatment and 5 were up-regulated after chemo-radiotherapy. (3) 8 proteins including APOA1 can be served as serum markers in Kazakh esophageal cancer diagnosis, and proteins like CLU can be served as serum markers in judging the resistance and sensitivity towards chemo-radiotherapy. (4) The abnormal expressions of APOC2, APOC3, Antithrombin-III in esophageal cancer were discovered for the first time. Specific protein spots related to Xinjiang Kazakh esophageal cancer diagnosis and chemo-radiotherapy can be identified in the serum, which will probably become a maker in Kazakh esophageal cancer diagnosis and therapeutic evaluation.     

Proteomics strategy based on liquid-phase IEF and 2-D DIGE: application to bone marrow mesenchymal progenitor cells

Global comparative proteomics is a promising new approach with broad application in basic and clinical biological science. Recent advances include the development of 2-D DIGE, a proteomic equivalent to mRNA differential display, in which differentially labeled samples are multiplexed and analyzed by high-resolution 2-DE. This study presents a new 2-D DIGE protocol, in which complex protein samples from normal and leukemic human bone marrow mesenchymal progenitor cells were used as model samples for a novel combination of liquid-phase IEF with 2-D DIGE. Using liquid-phase IEF, the normal and leukemic cells were pre-fractionated into five subproteomes after multiplexing but prior to DIGE. Under these conditions, 2-D DIGE resolved >5000 protein-containing spots within the pH range 4.6-7.0. Differential labeling combined with subsequent MALDI-MS/MS identified proteins that were differentially expressed in leukemic cells. This analysis mapped protein identities to 128 mesenchymal progenitor cell proteins with at least one unique peptide match at >95% confidence. Of these proteins, 72 (56%) were expressed more than 1.25-fold higher or lower in leukemic cells compared with normal cells (p<0.05). These data were used to infer gene ontology biological processes that may be altered in leukemic bone marrow mesenchymal progenitor cells.     

Proteomic analysis of primary esophageal squamous cell carcinoma reveals downregulation of a cell adhesion protein, periplakin

Recent advances in two-dimensional electrophoresis (2-DE) such as fluorescent 2-D differential gel electrophoresis (2-D DIGE) has made it possible to detect and quantitate the critical changes involved in disease pathogenesis. We have previously identified novel proteins with altered expression in primary colorectal cancer using agarose 2-DE that has a higher loading capacity than immobilized pH gradient gel. The aim of this study is to identify novel proteins with altered expression in primary esophageal cancer using the powerful method of agarose 2-DE and agarose 2-D DIGE. Excised tissues from 12 patients of primary esophageal cancer were obtained. Proteins with altered expression between cancer and adjacent non-cancer tissues were analyzed by agarose 2-D DIGE and identified by mass spectrometry. Thirty-three proteins out of 74 spots with altered expression in tumors were identified. Among them, a 195-kDa protein, periplakin, was significantly downregulated in esophageal cancer, which was confirmed by immunoblotting. Immunohistochemistry showed that periplakin was mainly localized at cell-cell boundaries in normal epithelium and dysplastic lesions, while it disappeared from cell boundaries, shifted to cytoplasm, in early cancers and scarcely expressed in advanced cancers. These results suggest that periplakin could be a useful marker for detection of early esophageal cancer and evaluation of tumor progression.     

Analysis of DIGE data using a linear mixed model allowing for protein-specific dye effects

Differential in-gel electrophoresis (DIGE) experiments allow three protein samples to be run per gel. The three samples are labeled with the spectrally resolvable fluorescent dyes, Cy2, Cy3, and Cy5, respectively. Here, we show that protein-specific dye effects exist, and we present a linear mixed model for analysis of DIGE data which takes dye effects into account. A Java implementation of the model, called DIGEanalyzer, is freely available at http://bioinfo.thep.lu.se/digeanalyzer.html. Three DIGE experiments from our laboratory, with 173, 64, and 24 gels, respectively, were used to quantify and verify the dye effects. DeCyder 5.0 and 6.5 were used for spot detection and matching. The fractions of proteins with a statistically significant (0.001 level) dye effect were 19, 34, and 23%, respectively. The fractions of proteins with a dye effect above 1.4-fold change were 1, 4, and 6%, respectively. The median magnitude of the dye effect was 1.07-fold change for Cy5 versus Cy3 and 1.16-fold change for Cy3 versus Cy2. The maximal dye effect was a seven-fold change. The dye effects of spots corresponding to the same protein tend to be similar within each of the three experiments, and to a smaller degree across experiments.     

Proteomic search for potential diagnostic markers and therapeutic targets for ovarian clear cell adenocarcinoma

Clear cell adenocarcinoma (CCA) has a highly malignant potential in human epithelial ovarian cancer. The serum CA-125 is widely used as a marker for ovarian cancer, but the level is relatively low in CCA. Therefore, new sensitive biomarkers are required. In this report, we describe a promising proteomic analysis that is differentially expressed in CCA when compared to mucinous adenocarcinoma, using the ovarian cultured cell lines OVISE, OVTOKO, and MCAS. The disease-associated proteins were identified by 2-D differential gel electrophoresis (2-D DIGE) and MS. In this analysis, 18 up-regulated and 31 down-regulated spots were observed that had at least two-fold differences in the two CCA cell lines than in MCAS as control cells. Some of the proteins differentially expressed in CCA were previously observed as alternative expression levels in ovarian and/or other cancers in clinical samples. In a subsequent preliminary differential study using surgical specimens from patients with CCA, it was demonstrated that the identified proteins were expressed differentially in actual tissues, as well as in the CCA culture cells. The results from this investigation show the potentiality of a proteomic approach for identifying disease-associated proteins, which may eventually serve as diagnostic markers or therapeutic targets in CCA.     

Variation in the monocyte proteome

We have determined the variability of the monocyte proteome and identified those proteins that demonstrate the greatest variation in the general control population. Monocytes were isolated from 18 healthy (9 male and 9 female) donors ages 18-50 and with no known genetic or blood disorder. A combination of Ficoll-Paque PLUS density centrifugation of cells found in the buffy coat and positive selection with monoclonal antibodies against CD14, coupled to magnetic beads, led to >98% purity of monocytes. A 100,000 g microsomal membrane fraction or 100,000 g supernatant fraction from a control subject was compared to the equivalent fractions from a distinct control subject by two-dimensional differential gel electrophoresis (2D DIGE). Those protein spots that demonstrated Cy3-/Cy5- ratios greater than 2.5-fold in at least one experiment were selected for further statistical analysis. We determined variability for 31 cytosolic and 12 membrane protein spots. Proteins have been identified for 27 of the cytosolic protein spots and 9 of the microsomal membrane protein spots by in-gel digestion with trypsin followed by reverse-phase high-performance liquid chromatography in line with tandem mass spectrometry. We identified 24 distinct monocyte proteins that demonstrated the greatest variability in this general control population. The proteins demonstrating the greatest variance in the cytosolic fraction were enolase-1 and WD (tryptophan-aspartate) repeat-containing protein 1, and in the membrane fraction they were lamin B1 and L-plastin. This study demonstrates the importance of considering variance in the control population when performing future protein profiling comparisons of monocytes derived from disease versus control populations.     

Discovering robust protein biomarkers for disease from relative expression reversals in 2-D DIGE data

This study assesses the ability of a novel family of machine learning algorithms to identify changes in relative protein expression levels, measured using 2-D DIGE data, which support accurate class prediction. The analysis was done using a training set of 36 total cellular lysates comprised of six normal and three cancer biological replicates (the remaining are technical replicates) and a validation set of four normal and two cancer samples. Protein samples were separated by 2-D DIGE and expression was quantified using DeCyder-2D Differential Analysis Software. The relative expression reversal (RER) classifier correctly classified 9/9 training biological samples (p<0.022) as estimated using a modified version of leave one out cross validation and 6/6 validation samples. The classification rule involved comparison of expression levels for a single pair of protein spots, tropomyosin isoforms and alpha-enolase, both of which have prior association as potential biomarkers in cancer. The data was also analyzed using algorithms similar to those found in the extended data analysis package of DeCyder software. We propose that by accounting for sources of within- and between-gel variation, RER classifiers applied to 2-D DIGE data provide a useful approach for identifying biomarkers that discriminate among protein samples of interest.     

Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry

Two-dimensional difference gel electrophoresis (2-D DIGE) coupled with mass spectrometry (MS) was used to investigate tumor-specific changes in the proteome of human colorectal cancers and adjacent normal mucosa. For each of six patients with different stages of colon cancer, Cy5-labeled proteins isolated from tumor tissue were combined with Cy3-labeled proteins isolated from neighboring normal mucosa and separated on the same 2-D gel along with a Cy2-labeled mixture of all 12 normal/tumor samples as an internal standard. Over 1500 protein spot-features were analyzed in each paired normal/tumor comparison, and using DIGE technology with the mixed-sample internal standard, statistically significant quantitative comparisons of each protein abundance change could be made across multiple samples simultaneously without interference due to gel-to-gel variation. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and tandem (TOF/TOF) MS provided sensitive and accurate mass spectral data for database interrogation, resulting in the identification of 52 unique proteins (including redundancies due to proteolysis and post-translationally modified isoforms) that were changing in abundance across the cohort. Without the benefit of the Cy2-labeled 12 sample mixture internal standard, 42 of these proteins would have been overlooked due to the large degree of variation inherent between normal and tumor samples.     

2-D DIGE analysis of butyrate-treated HCT-116 cells after enrichment with heparin affinity chromatography

Butyrate, a 4-carbon short chain fatty acid, is responsible for the protective effects of fiber in colorectal cancer prevention. To better understand the 'blueprint' of butyrate's chemopreventive role in this disease, we performed 2-dimensional difference gel electrophoresis (2-D DIGE) of butyrate-treated HCT-116 colorectal cancer cells after pre-fractionation using heparin affinity chromatography. A combination of this enrichment step with overlapping narrow range IPGs (pH 4-7 and pH 6-11) in 2-D DIGE resulted in the detection of 46 differentially expressed spots. Twenty-four of these were identified by MS analyses, and 5 spots were found to be heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). Three isoforms of 38 kDa were down-regulated while two with Mr approximately 26 kDa were up-regulated. These represent phosphorylated isoforms of hnRNP A1 as verified by immunoblotting with anti-phosphotyrosine and anti-phosphoserine antibodies. Using 2-DE, subcellular fractionation and western blot analysis, we further showed that full-length hnRNP A1 underwent down-regulation, cleavage and cytoplasmic retention upon butyrate treatment. These indicate that modulations of hnRNP A1 may play a significant role in the mediation of growth arrest and apoptosis by butyrate.     

Proteome analysis of hepatocellular carcinoma by two-dimensional difference gel electrophoresis: novel protein markers in hepatocellular carcinoma tissues

Hepatocellular carcinoma (HCC) is a highly malignant tumor, and chronic infection with hepatitis B virus is one of its major risk factors. To identify the proteins involved in HCC carcinogenesis, we used two-dimensional fluorescence DIGE to study the differentially expressed proteins in tumor and adjacent nontumor tissue samples. Samples from 12 hepatitis B virus-associated HCC patients were analyzed. A total of 61 spots were significantly up-regulated (ratio >/= 2, p 相似文献   

Proteome analysis of gastric cancer metastasis by two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-mass spectrometry for identification of metastasis-related proteins

A well-described animal model was used to understand the molecular mechanisms of carcinogenesis and metastasis of gastric cancer at the protein level. Gastric cancer was induced in 12 Wistar rats by oral administration of N-methyl-N'-Nitro-N-Nitrosoguanidine (MNNG). The protein expression patterns of normal gastric tissue, gastric cancer, and corresponding metastases were analyzed by proteomics in matched tissues of 3 rats. Proteins in the region of molecular masses of 15-75 kDa and an isoelectric point of 3-7 were separated by two-dimensional electrophoresis (2-DE) and identified by peptide fingerprinting with matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS). Twenty-seven spots corresponding to 25 different proteins served as landmarks for comparison between tissues. The identified proteins included cytoskeletal proteins, stress associated proteins, proteins involved in signal transduction, cell proliferation and differentiation, and metabolism. Eleven proteins were up-regulated and 2 proteins were down-regulated in tumor tissue when compared with normal tissue. Twelve proteins were up-regulated and 8 proteins were down-regulated in the metastases when compared with the primary tumor. The overexpression of HSP27 in gastric cancer was confirmed by immunohistochemical analysis of human gastric cancer specimens. Combining well-defined animal models with proteome analysis will improve our understanding of the fundamental changes that contribute to the process of carcinogenesis and the formation of metastases in gastric cancer.     

Proteomic analysis of infiltrating ductal carcinoma tissues by coupled 2-D DIGE/MS/MS analysis

There is a growing interest in protein expression profiling aiming to identify novel diagnostic markers in breast cancer. Proteomic approaches such as two-dimensional differential gel electrophoresis coupled with tandem mass spectrometry analysis (2-D DIGE/MS/MS) have been used successfully for the identification of candidate biomarkers for screening, diagnosis, prognosis and monitoring of treatment response in various types of cancer. Identifying previously unknown proteins of potential clinical relevance will ultimately help in reaching effective ways to manage the disease. We analyzed breast cancer tissues from five tumor and five normal tissue samples from ten breast cancer subjects with infiltrating ductal carcinoma (IDC) by 2-D DIGE using two types of immobilized pH gradient (IPG) strips: pH 3-10 and pH 4-7. From all the spots detected, differentially expressed (p < 0.05 and ratio > 2) were 50 spots. Of these, 39 proteins were successfully identified by MS, representing 29 different proteins. Ten proteins were overexpressed in the tumor samples. The 2-D DIGE/MS/MS analysis revealed an increase in the expression levels in tumor samples of several proteins not previously associated with breast cancer, such as: macrophage-capping protein (CAPG), phosphomannomutase 2 (PMM2), ATPase ASN1, methylthioribose-1-phosphate isomerase (MRI1), peptidyl-prolyl cis-trans isomerase FKBP4, cellular retinoic acid-binding protein 2 (CRABP2), lamin B1 and keratin, type II cytoskeletal 8 (KRT8). Ingenuity Pathway Analysis (IPA) revealed highly significant (p = 10(-26)) interactions between the identified proteins and their association with cancer. These proteins are involved in many diverse pathways and have established roles in cellular metabolism. It remains the goal of future work to test the suitability of the identified proteins in samples of larger and independent patient groups.